Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS)  Therefore, these data are consistent with the region near S48 playing a role in assembly.Inspired by the observation that pentamerization of nucleophosmin is regulated by phosphorylation  and sedimentation equilibrium analytical ultracentrifugation (AUC). The results from both methods support a model of weak infinite linear self‐association, specifically isodesmic assembly resulting in unbranched linear chains. CG‐MALS data were collected for wild‐type TDP‐43 NTD in a wide range of concentrations up to more than 300 μM. The mass average molecular weight increases continuously as a function of the concentration of the wild‐type NTD Fig E. ModelsKD ~ 40 μM (We observe evidence of the same self‐association behavior for TDP‐43 NTD in AUC experiments. In sedimentation velocity (SV) experiments, at least three species are observed when the data are modeled to determine the sedimentation coefficient distribution of the present species, and their fractional populations shift to larger species in a concentration‐dependent manner . However ~ 40 μM . The diset al, in vitro  cells, TDP‐43RRM‐GFP localizes to the nucleus and concentrates into a single large liquid–liquid phase separated assembly per cell over time . These findings suggest that the details of the NTD structure and interactions in the region surrounding S48, not just phosphorylation at S48, are critical for mechanisms that mediate TDP‐43 control of splicing. To further determine the requirement for NTD oligomerization in TDP‐43 splicing regulatory function, we studied NTD Y4R and E17R, two other self‐assembly deficient variants (see below), in our cellular assays. We observed a dramatic decrease in TDP‐43 activity upon introduction of both mutations  TDP‐43 NTD wild‐type titrated with increasing concentrations of NMR‐invisible  S48E TDP‐43 NTD would show chemical shift deviations only near S48. Indeed, chemical shift deviations are only observed in the C‐terminal half (residues 40–80) of wild‐type  TDP‐43 NTD S48E shows chemical shift deviations at residues in the N‐terminal half (residues 1–35) of the S48E protein  may be the consequence of the conformational change caused by the interaction occurring in the N‐terminal interface. These residues are the only outliers from the single interface behavior observed for other residues in the domain. Collectively, these data immediately point to a head‐to‐tail assembly  that are high for positions in close proximity to a stabilized nitroxide spin label, even in transiently populated states. Assuming a two‐state monomer–dimer equilibrium, the relative orientation of two NTD monomers in the assembled dimer state can be discerned given a sufficient number of samples with conjugated nitroxide radical spin labels at single positions spatially separated across the monomer surface. Therefore, MTSL spin labels were conjugated to the NTD at engineered cysteine variants at positions 2 or 29 or at native cysteine positions 39 or 50 and mixed with 15N isotopically enriched NTD C39S/C50S in order to measure 1HN transverse paramagnetic relaxation enhancements, Γ2, arising only from intermolecular contacts  S48E resulted first in disappearance of peaks due to the kinetics of exchange between monomer and complex  would inhibit self‐assembly as potently as S48E. In order to test this hypothesis, we generated NTD variants E17R and Y4R and examined their effects on self‐assembly. When the NMR chemical shift deviations for increasing concentrations of each mutant alone were compared to wild‐type, we found that E17R and Y4R NTD variants do not disrupt folding  but do d–30 would of S48E . The res13C/15N‐edited and 13C/15N‐filtered/edited NOESY measurements using the 13C/15N‐labeled S48E (or Y4R) complexed with natural abundance Y4R (or S48E)  . The filor S48E) . With thet al, et al, The structure of each subunit is largely consistent with the previously published NTD monomer structure of 2N4P  alter the interface dramatically, it is also likely that dimer (and higher‐order structures) is not saturated in their samples of polymerization competent wild‐type, as evidenced by the smaller chemical shift differences compared to our NMR titration, complicating interpretation of filtered NOESY experiments which can contain spectral artifacts from incomplete filtering  that the NTD oligomerization is important for function, (ii) that phosphosite S48  tag, and TEV cleavage site  fusion tag (designated pJ4M) was constructed using overlap extension PCR (Horton The TDP‐43‐MBP expression construct (designated pJ4M/TDP‐43) was created by subcloning the DNA sequence for TDP‐43, which was amplified from pJ411/TDP‐43 by PCR using primers 5‐TAATACGACTCACTATAGGG‐3 and 5‐TCATCACTCGAGCATGCCCC‐3, into pJ4M via restriction sites NdeI and XhoI.Escherichia coli BL21 Star DE3 (Life Technologies). Cells were grown in LB media for natural abundance isotope labeling. For expression of  isotope labeling, cells were grown in M9 minimal media supplemented with 13C‐glucose and/or 15NH4Cl (Cambridge Isotope Laboratories). In order to purify the TDP‐43 NTD, cell cultures were grown at 37°C to an OD600 of 0.6–0.9. Protein expression was induced by addition of isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) (GoldBio) to 1 mM, followed by incubation at 37°C for 4 h. Cells were harvested by centrifugation, resuspended in NTD binding buffer  supplemented with complete EDTA‐free protease inhibitor cocktail (Roche), and lysed using an EmulsiFlex C3 (Avestin). Cell lysates were clarified by centrifugation and applied to a 5 ml Histrap HP column . The column was washed with five volumes of binding buffer, and TDP‐43 NTD was eluted with a linear gradient of NTD elution buffer . Cleavage and removal of the N‐terminal Thio‐His tag was achieved by incubation with 0.03 mg/ml TEV protease concomitant with dialysis into NTD dialysis buffer , followed by a subsequent purification with the 5 ml Histrap HP column. TDP‐43 NTD was further purified by SEC over a Superdex 75 26/60 column  with NTD SEC buffer . Purified TDP‐43 NTD was concentrated to ~500 μM, flash frozen, and stored at −80°C.Point mutations were introduced either by Quikchange mutagenesis or by overlap extension PCR with mutagenic primers. Proteins were overexpressed in For purification of full‐length TDP‐43‐MBP fusion protein, cell cultures were grown at 37°C to an OD600 of 0.6–0.9. Cell cultures were then cooled to 16°C, after which protein expression was induced with addition of IPTG to 1 mM, followed by incubation at 16°C overnight. Cells were harvested by centrifugation, resuspended in TDP‐43 binding buffer  glycerol, 1 mM DTT) supplemented with complete EDTA‐free protease inhibitor cocktail, and lysed with the EmulsiFlex C3. Cell lysates were clarified by centrifugation, applied to the 5 ml Histrap HP column, and washed with five volumes of TDP‐43 binding buffer. TDP‐43‐MBP was eluted with a linear gradient of TDP‐43 elution buffer  glycerol, 1 mM DTT).Eluant was further purified by SEC over a Superdex 200 26/60 column  with TDP‐43 SEC buffer . Purified TDP‐43‐MBP was concentrated to 250 μM, flash frozen, and stored at −80°C.et al, NMR spectra were processed with NMRPipe CO, CBCA(CO)NH, and HNCACB.15N and 1H chemical shifts were measured from the crosspeaks on the HSQCs of 5, 20, 100, 200, 350 and 500 μM WT and 5, 100 and 200 μM variants in the condition of 20 mM HEPES pH 6.8, 1 mM DTT, and 10% D2O. Chemical shift deviations for 1H and 15N were quantified by subtracting the chemical shift of the 5 μM sample from the respective value of each other samples. The average chemical shift deviations (CSDs) were calculated by the equation: 15N S48E was titrated by 50 μM, 100 μM, 400 μM, 1 mM, 1.6 mM, and 2 mM of natural isotopic abundance Y4R. 15N Y4R was titrated by 50 μM, 100 μM, 400 μM, 800 μM, 1 mM, and 2 mM of natural isotopic abundance S48E. The CSDs for 15N and 1H were quantified by subtracting the chemical shifts of 100 μM 15N S48E (or 15N Y4R) from the respective value of each mixture samples and then calculated by the equation above.For the titrations of S48E and Y4R, the 100 μM of either 1H‐15N HSQCs were acquired at 500 MHz 1H Larmor frequency with 32 transients per data point with 64* and 1,536* complex pairs in the indirect 15N and direct 1H dimensions, with corresponding acquisition times of 35 and 219 ms, and sweep widths of 36.0 and 14.0 ppm centered at 116.5 and 4.7 ppm, respectively.15N‐labeled C39S/C50S in 20 mM HEPES pH 6.8, 10% D2O.Since TDP‐43 1–80 contains two intrinsic cysteines, C39 and C50, either cysteine was substituted by serine to generate the variants C39S and C50S, respectively. Other PRE variants including single cysteine substitution at S2 and S29 were engineered from the construct of C39S C50S TDP‐43 1–80. One mM MTSL  or 1 mM AcMTSL  was added into 50 μM of each variant protein under the condition of 20 mM Tris–Cl pH 8.0, 150 mM NaCl. After incubating 1 h at room temperature, the excess label was removed by HiPrep desalting column and the protein was exchanged to the buffer of 20 mM HEPES pH 6.8. Finally, the paramagnetic probe MTSL  was conjugated at C2, C29, C39, and C50, respectively. The samples were concentrated and flash frozen. For the intermolecular PRE measurement, 100 μM MTSL (or AcMTSL)‐conjugated PRE variant was mixed with 100 μM 1HNR2, were measured at 500 MHz 1H frequency for paramagnetic and diamagnetic samples, with 192* and 1,536* complex pairs in the 15N indirect and 1H direct dimensions, corresponding acquisition times of 105 and 21.9 ms, and sweep widths of 36 and 14 ppm centered at 116.5 and 4.7 ppm, respectively. Each 1HNR2 experiment comprised three interleaved 1HNR2 relaxation delays: 0.1, 2.1, 5.1 ms. PRE values (Γ2 = 1HNR2para − 1HNR2dia) were obtained from the data collected with 0.1‐ and 2.1‐ms delays using the two time point method as described previously . Distance restraints were derived from sets of 3D‐ NOESY experiments acquired with mixing time of 120 ms. Isotope‐filtered NOESY experiments  were performed to identify intermolecular NOE, which were further classified in strong, medium and weak restrains corresponding to 3.5, 4.5, and 6.0 Å upper limits. Dihedral angle restraints were calculated by using software TALOS+ , which uses a software‐controlled multiple syringe pump to create a concentration gradient, and a DAWN‐HELEOS multi‐angle light‐scattering photometer to collect data from the incoming sample stream. Static light‐scattering data were collected at 14 scattering angles as a function of protein concentration. For each injection, the solution was allowed to come to equilibrium for 60 s. Equilibrium MALS data as a function of concentration were analyzed as described by Attri et al , and datMx is the molar mass of protein X, and [Xi] is the concentration of the species Xi. R is normalized to an optical constant K* defined asin which n0 denotes the refractive index of the solvent, λ0 the vacuum wavelength of incident light (690 nm), NA, Avogadro's number, and dn/dc is the refractive index increment of the sample . The concentrations of each species are related to the equilibrium constants and total protein concentration, resulting in the following equations for typical monomer–dimer, monomer–trimer, monomer–i‐mer equilibria:where i = 1 represents the free monomer, the total molar concentration [X]total is known at each gradient injection, and R(0)/K* is measured. Non‐linear least square optimization is used to obtain a single KA value that fits the data across the entire concentration range of interest.Above, et al, To describe isodesmic self‐association, we used equations previously described  and the molecular weight of the building block, which was in agreement with a TDP‐43 NTD monomer within error.Fitting parameters were The SEC multi‐angle light‐scattering (SEC‐MALS) experiments were carried out using a WTC‐0150S5  size‐exclusion column  with three detectors connected in series: an Agilent 1200 ultraviolet (UV) detector , a Wyatt DAWN‐HELEOS‐II multi‐angle light‐scattering (MALS) detector, and a Wyatt Optilab T‐rEX differential refractive index (RI) detector . The column was equilibrated with 20 mM HEPES pH 6.8, 1 mM DTT, and either 0 or 150 mM NaCl. All data were collected at 25°C. A 100 μl sample was injected into the column using an auto‐sample injection method with a flow rate of 0.5 ml/min. Protein in the eluent was detected via UV absorbance at 280 nm, light‐scattering, and refractive index detectors. The data were recorded and analyzed with the Wyatt Astra software (version 7.0).Protein samples of TDP‐43 NTD WT and S48E were prepared at 400 μM in the following buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM DTT. The amine cross‐linker BS3 (bis(sulfosuccinimidyl)suberate, Fisher Scientific) was added to create a molar ratio of 50. Reactions were incubated at room temperature for 30 min. The reactions were quenched by the addition of 100 mM Tris pH 7.5 and were incubated at room temperature for at least 15 min prior to taking samples for analysis on a gel.et al, https://sedfitsedphat.nibib.nih.gov/software/default.aspx).The cell assembly, containing identical sample and reference buffer volumes of 360 μl, was placed in a rotor and temperature equilibrated at rest at 20°C for 2 h before it was accelerated from 0 to 50,000 rpm. Rayleigh interference optical data were collected continuously for 12 h separately. The velocity data were modeled with diffusion‐deconvoluted sedimentation coefficient distributions c(s) in SEDFIT, using algebraic noise decomposition and with signal‐average frictional ratio and meniscus position refined with non‐linear regression  following standard protocols unless mentioned otherwise  using the reversible one‐step self‐association model. The association scheme used in this analysis was [A] ↔ [A2] ↔ [A3] ↔ [A4] ↔ …. [isodesmic] with A being the TDP‐43 NTD monomer, and KDiso the single dissociation constant for the association steps were obtained from the analysis. Errors of the fits represent the 68.3% confidence interval (CI) using an automated surface projection method , 25,000 (for 24 h), and 43,000 rpm (for 24 h) (Zhao Oligopeptides and phospho‐specific rabbit antibodies against TDP‐43 amino acids 40–53 (GLRYRNPVSQCMRG) were custom produced by GenScript . Confirmation of antibody specificity was performed by serially diluting TDP‐43 oligopeptides in 8 M urea and spotting to nitrocellulose membranes, which were then blocked in 6% milk in Tris‐buffered saline (TBS). Membranes were then probed with polyclonal antibodies specific to the phospho‐peptide (α‐TDP‐43 pSer48) or non‐specific to the phosphorylated state (PAN antibody). Primary antibodies were detected with IRDye conjugated secondary antibodies , and their fluorescence was measured and imaged using the Odyssey LI‐COR Imaging System.HEK293T cells were obtained from ATCC (CRL‐11268) and maintained under standard conditions at 37°C in DMEM‐based media (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% P/S/G (penicillin–streptomycin–glutamine). Lysates were prepared by suspension and incubation of cells in nondenaturing lysis buffer  100 μl phosphatase inhibitor cocktail (Sigma‐Aldrich P0044), 1,250 units Benzonase nuclease (Sigma‐Aldrich E1014).® Precast AnyKD™ acrylamide gels (Bio‐Rad 456‐9034) and nitrocellulose membranes (Bio‐Rad 162‐0146). Immunoblotting with custom and commercial α‐TDP‐43 antibodies (CST 3449) and detection with the LI‐COR imaging system were performed as described above. Immunoprecipitation of HEK293T cell lysates was performed according to the manufacturer's protocol with Protein G Dynabeads (Invitrogen 10007D) and mouse monoclonal α‐TDP‐43 antibody (Proteintech 60019‐2).Western blotting was performed using standard laboratory procedures with Mini‐PROTEAN2, 1 mM DTT) at 37°C. The membranes were then probed as described above.Phosphatase treatment of nitrocellulose membranes was performed using calf intestinal phosphatase . Following detection of the 43 kDa species by Western blotting, membranes were stripped of primary and secondary antibodies using stripping reagent (Thermo Fisher 46430). Membrane sections containing putative TDP‐43 species were cut and blocked with 5% bovine serum albumin and 0.1% Triton X‐100 in TBS. Identical membrane pieces were treated with and without CIP in reaction buffer . Mammalian expression constructs were cloned into modified pCS‐DEST backbones (Addgene 22423).2 in DMEM high glucose  supplemented with 10% FBS, 2 mM l‐glutamine (Gibco), 1 mM non‐essential amino acids (Gibco), and 1% penicillin/streptomycin (Gibco), unless otherwise noted.All TDP‐43 phase separation reporter assays were performed in HEK‐293T cells obtained from the American Tissue Culture Consortium . Cells were maintained and imaged at 37°C and 5% COMammalian TDP‐43 expression constructs (500 ng) were transfected into HEK‐293T cells grown on tissue culture‐treated μ‐slides (ibidi) with X‐tremeGENE 9 (Roche). To compare expression levels, cells were harvested 24 h after transfection and analyzed by (i) flow cytometry with a BD Accuri C6 instrument  or (ii) immunoblotting using anti‐GFP (Novus NB600‐308) and anti‐alpha‐tubulin (Sigma T6199) primary antibodies. Blots were developed with a LiCor Odyssey imager using appropriate dye‐conjugated secondary antibodies. To follow TDP‐43 reporter phase formation over time, cells were imaged in L15 medium (Gibco) + 10% FBS at 37°C every hour for a total of 24 h after transfection with a Leica DMI‐6000B microscope equipped with an Andor Yokogawa spinning disk confocal unit. A 488‐nm laser was used for GFP excitation and an Andor iXon Ultra EMCCD camera for signal detection.RRM‐GFP nuclear and droplet signals in live cells, each confocal image was taken with identical detector settings. Mathematica (Wolfram Research) was used to outline nuclei based on the total nuclear GFP signal, and to quantify the fluorescence signal of different TDP‐43RRM‐GFP variants in the nucleoplasm versus the droplet phase.A Leica SP8 confocal laser scanning microscope equipped with a 64× glycerol (NA 1.4) objective (Leica) and a temperature‐ and gas‐controlled incubation chamber (Life Imaging Services) was used for image acquisition. The 488‐ and 561‐nm laser lines were used for excitation of GFP (green channel) or mCherry (red channel), respectively. For quantification of TDP‐43t), the fluorescence intensities within the bleached droplet hemisphere were integrated and normalized to the fluorescence intensity of the corresponding unbleached droplet hemisphere. These normalized, time‐dependent fluorescence intensities It were then used to calculate the fluorescence recovery (FR) according to the following formula: FR(t) = (It − It0)/(Ibefore bleaching − It0), with t0 being the first time point observed after photobleaching.Half‐bleach experiments were performed 24 h after transfection of the indicated constructs using the FRAP module of the Leica SP8 microscope. The Leica Application Suite X software was used for quantification and GraphPad Prism to plot and analyze replicate half‐bleach experiments. For each recorded time point . This served as template for site‐directed mutagenesis using the QuikChange Site‐Directed Mutagenesis Kit (Agilent Technologies) to generate TDP‐43 mutants for mammalian expression. Oligonucleotides used for mutagenesis: Y4R_FW, gacgacaagctttctgaacgtattcgggtaaccgaagatg; Y4R_RV, catcttcggttacccgaatacgttcagaaagcttgtcgtc; E17R_FW, agaacgatgagcccattcgaataccatcggaagacgatg; E17R_RV, catcgtcttccgatggtattcgaatgggctcatcgttct; S48A_FW, cgctacaggaatccagtggctcagtgtatgagaggtg tc; S48A_RV, gacacctctcatacactgagccactggattcctgtagcg; S48E_FW, cgctacaggaatccagtggaacagtgtatgagaggtgtc; S48E_RV, gacacctctcatacac tgttccactggattcctgtagcg.l‐glutamine, and sodium bicarbonate and supplemented with filtered fetal bovine serum at 10%. RNAi‐mediated downregulation of TDP‐43 was carried out as previously described (1) and siCONTROL Nontargetting siRNA#1 (Dharmacon) was used as control. TDP‐43 downregulation was confirmed by immunoblotting.Human cell lines were grown in growth media‐Dulbecco's modified Eagle's medium, 4,500 mg/l glucose, et al, Splicing assays were carried out in HeLa cells transiently transfected with Lipofectamine 2000 and Oligofectamine in the case of siRNA (Life Technologies) according to manufacturer protocols. Splicing assays were performed using the CFTR reporter minigene as previously described  was used to detect endogenous and tagged TDP‐43. GAPDH was used as the loading control (Abcam ab181602).NLF, AW, AEC, DRM, MTN, and VHR designed and conducted the gel filtration, protein expression and purification, and structural NMR experiments. AEC developed the C‐terminal MBP vector and phase separation assay. MTN solved the structure of the dimer from samples made by AW and data collected together with AW. NLF, AW, and AEC wrote the manuscript with input from the authors. TM and EWM conceived and analyzed the CG‐MALS and cross‐linking experiments, and EWM carried them out. TM, EWM, and AN conceived the AUC experiments, and AN carried out and analyzed them. HBS conceived, performed, and analyzed the phase separation cell culture experiments, with input and resources provided by RR. YMA and ANR conceived and analyzed the splicing experiments, and ANR carried them out. FS and SNR conceived of, performed, and analyzed the immunoblotting experiments.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Review Process FileClick here for additional data file.


A study on graphene that was synthetized by means of a novel transfer-free fabrication approach and is employed as sensing material is herein presented. Multilayer graphene was deposited by chemical vapour deposition (CVD) mediated by CMOS-compatible Mo. The utilized technique takes advantage of the absence of damage or contamination of the synthesized graphene, because there is no need for the transfer onto a substrate. Moreover, a proper pre-patterning of the Mo catalyst allows one to obtain graphene films with different shapes and dimensions. The sensing properties of the material have been investigated by exposing the devices to NO2, NH3 and CO, which have been selected because they are well-known hazardous substances. The concentration ranges have been chosen according to the conventional monitoring of these gases. The measurements have been carried out in humid N2 environment, setting the flow rate at 500 sccm, the temperature at 25 °C and the relative humidity (RH) at 50%. An increase of the conductance response has been recorded upon exposure towards NO2, whereas a decrease of the signal has been detected towards NH3. The material appears totally insensitive towards CO. Finally, the sensing selectivity has been proven by evaluating and comparing the degree of adsorption and the interaction energies for NO2 and NH3 on graphene. The direct-growth approach for the synthesis of graphene opens a promising path towards diverse applicative scenarios, including the straightforward integration in electronic devices.The sp Due to its extraordinary electronic, chemical, mechanical, thermal and optical properties, graphene has been defined as the “wonder material” of the 21st century with plentiful applications in several fields such as energy storage, biomedical, electronics –6. HowevAnother issue that needs to be addressed is the problem of slow and incomplete recovery in the operative regime of standard temperature and pressure, which can be solved by providing the sensors with appropriate electronics, able to perform data analysis  or a ref2 interface. More recently, Lukosius et al. have shown the successful growth of a graphene layer underneath Ni bars on insulating SiO2 layers, so avoiding the metal contamination problems and complexity associated to graphene transfer [All that said, the development of new approaches enabling the direct growth of large-area graphene layers on arbitrary insulating substrates, compatible with conventional Si technology, is of crucial importance –15. A potransfer .2 substrates by means of a transfer-free CVD method mediated by Mo. The pre-patterning of the Mo layer allows the graphene to be shaped in the desired form by means of standard lithography techniques, following a completely Si-technology-compatible approach. Chemiresistive devices have been realized by shaping the sensing layer in form of rectangular strips.In the present work, we show the results of the sensing performances of conductometric devices in which multilayer graphene films have been directly synthesized on insulating SiO2, NH3 and CO. In particular, NO2 and NH3 are known to exhibit an electron acceptor- and donor-like nature respectively, as extensively shown in the literature [In order to probe the gas-sensing capabilities of the fabricated devices, three standard pollutants have been chosen, namely NOterature ,22–23. T2 and NH3.Finally, the sensing specificity of the material has been probed by evaluating and comparing the degree of adsorption and the interaction energies for NOThe material, prepared according to the procedure discussed in detail in the Experimental section, has been morphologically and structurally characterized in a previous work , from whIn −1) and D′ (ca. 1600 cm−1) bands is related to presence of defects and in particular the D intensity, roughly 5-fold lower than that of the G peak, attests a low density of defects. This evidence confirms the non-degradation of the material even during ageing.Starting from this premise, we have further deepened the analysis of the material by performing a Raman analysis in several different sites of the graphene film. As can be seen in I(D)/I(D′) ratio allows one to discriminate the type of defects in the film. Indeed, as reported by Eckmann et al., the value of the slope of the I(D)/I(G) vs I(D′)/I(G) curve, namely I(D)/I(D′), can be related to the preponderant typology of defects in the different graphene samples [I(D)/I(D′) ratio exhibits its maximum value, around 13, for sp3-like defects, it decreases down to about 7 in the case of vacancies and it is minimum for edge defects (ca. 3.5). In our case the estimated I(D)/I(D′) value is around 4.4, indicating that defects in the material are reasonably ascribed to the boundaries.The analysis of the  samples . In partl = 206 μm, w = 5 μm  was chosen because of the undesired influence of oxygen. The number of active sites on the graphene surface decreases due to binding of O2 molecules via weak physisorption [In order to investigate the reliability of the fabrication process in terms of gas-sensing behaviour, two identical devices have been considered, labelled with A and B. As described below in the Experimental section, the sensing layer has a rectangular geometry: w = 5 μm  and t = sorption . This ph2. As can be observed, the base conductance values are comparable (GA ≈ GB ≈ 22 µS). During exposure, both chemiresistors exhibit an identical percentage variation of the conductance without reaching a plateau. In the figures the asymptotic values of conductance are also displayed. Such quantities have been evaluated by extrapolating the final conductance value by fitting each curve, within the time window of exposure to the analyte, with the exponential functionGMIN/MAX represents the asymptotic value of the conductance after exposure to the analyte. When the gas inlet is stopped, a slow decrease of the device conductance and thus an incomplete recovery follows. This is not surprising and can be explained in terms of interaction energy between graphene and analyte, as will be explained later. Since NO2 acts as an electron acceptor, during interaction the electronic charge is transferred from graphene to the analyte. Thus, the observed increase in the device conductance is naturally expected because of the interaction of the molecule with a p-type material.where 3. Even in this case both chemiresistors exhibit a comparable percentage variation of the conductance, as well as similar kinetics  has been converted into a number of analyte molecules per unit time, , by knowing the incoming concentrations (1 ppm for NO2 and 250 ppm for NH3). The total pressure in the testing chamber (pTOT = 1 atm) has been taken into account. Furthermore, a uniform molecule density dV = Nt/Vc in the chamber (Vc = 0.4 dm3) has been assumed:The absolute number of analyte molecules approaching the devices has been firstly estimated, referring to the responses reported in t = 600 s has finally been evaluated as:The effective number of colliding analyte molecules during the exposure in the time interval Δ2 and NH3 is 250−2/3. This factor will be used later to normalize the number of adsorbed molecules to the same amount of incoming analyte.It is worth noting that the ration of these quantities for NOG0 of the devices  obeys Ohm’s law:The initial conductance value The conductivity can be expressed in terms of volumetric density and mobility of the charge carriers:2 and 103 cm2/V·s), in the calculations, a carrier mobility of μ ≈ 1000 cm2/V·s has been assumed [G/G0 in 10 min. This quantity is linked to the number of adsorbed molecules via the virtual charge-transfer amount resulting from the interaction between graphene and analyte molecules, Δq. Hence, the effective number of analyte-induced charge carriers NS onto the devices surface can be expressed asAccording to the mobility values reported in literature for CVD graphene  >> 1, it is possible to writeAnalogously to the conventional treatment of doped semiconductors, after having estimated the maximum number of adsorbed analyte molecules, they have been assimilated as punctual impurities in the crystal lattice. Herein the Maxwell–Boltzmann approximation for the statistical description of charge carriers can be justified by virtue of the semi-classical regime of the system, with low induced charge density, of the order of 10graphene ) and a t2, both for devices A and B (kBT ≈ 25 meV) is not sufficient to “escape” from the potential well associated to the adsorption.In particular, the estimated characteristic energies for NO A and B , appear G/G0, NS, , α,  and Eint) calculated for devices A and B, for NO2 and NH3, are summarized in The primary quantities  Mo target. The metal film thickness can be made as thin as 50 nm without any segregation at the graphene CVD temperature (≈1000 °C), because of the higher melting point of Mo (2623 °C) compared to that of other conventional catalysts such as Cu (1085 °C) or Ni (1455 °C). Moreover, this allows a pre-patterning the film for the selective growth of graphene down to micrometre-size dimensions. A photo-lithographic process, combined with SF6 dry etching, has been used to shape the Mo in the desired form.4″ Si wafers coated by 90 nm thick thermal SiO2/Si substrate by means of AIXTRON BlackMagic Pro equipment, setting the temperature at 1000 °C, the pressure at 25 mbar and using Ar/H2/CH4 as feedstock. At the end of the CVD growth, the Mo has been selectively etched away underneath graphene employing phosphoric acid. Due to anchoring at the sides of the patterned catalyst, the graphene layer sticks directly to the underlaying SiO2. Cr/Au metal contacts have finally been patterned by a lift-off process to design a four-point probe structure. The final chemiresistive devices consist of a material strip 206 µm long and 5 µm wide, placed between two couples of metal electrodes , placed in a thermostatic box. Precise amounts of analyte were let into the chamber via a system of programmable mass flow controllers (MKS) and electro-pneumatic valves. Relative humidity can be adjusted as required through a water bubbler placed in a thermostatic bath. The bias voltage is provided by TTi QL355T Precision Power Supply.The chemiresistors have been tested in a Gas Sensor Characterization System  consisting of a stainless steel testing chamber  20 min carrier gas flow (baseline), (2) 10 min exposure to the analytes at the chosen concentrations, (3) 20 min carrier gas flow (recovery). The measurements have been carried out in inert NG0 is the unperturbed conductance value and Gf is the conductance value at the end of the exposure.where


Two central dimensions in psychotherapeutic work are a therapist’s empathy with clients and challenging their judgments. We investigated how they influence psychophysiological responses in the participants. Data were from psychodynamic therapy sessions, 24 sessions from 5 dyads, from which 694 therapist’s interventions were coded. Heart rate and electrodermal activity (EDA) of the participants were used to index emotional arousal. Facial muscle activity (electromyography) was used to index positive and negative emotional facial expressions. Electrophysiological data were analyzed in two time frames: (a) during the therapists’ interventions and (b) across the whole psychotherapy session. Both empathy and challenge had an effect on psychophysiological responses in the participants. Therapists’ empathy decreased clients’ and increased their own EDA across the session. Therapists’ challenge increased their own EDA in response to the interventions, but not across the sessions. Clients, on the other hand, did not respond to challenges during interventions, but challenges tended to increase EDA across a session. Furthermore, there was an interaction effect between empathy and challenge. Heart rate decreased and positive facial expressions increased in sessions where empathy and challenge were coupled, i.e., the amount of both empathy and challenge was either high or low. This suggests that these two variables work together. The results highlight the therapeutic functions and interrelation of empathy and challenge, and in line with the dyadic system theory by  Psychotherapy is done through social interaction. While the form and content of therapists’ responses to their clients’ experience may vary greatly, among other possible distinctions, psychotherapist’s attitudes toward the client can be divided to two basic orientations or facets: empathy and challenge . To empaWe presume that the therapist’s empathetic and challenging behaviors are linked to the mental and bodily emotion systems of the participants. This paper will focus on that linkage. We ask how empathy and challenge in the therapists’ interventions connect to emotion-related physiological responses  in the participants during these interventions, and more globally during the therapy sessions.The dyadic systems theory by The structure of the paper is as follows: we will next discuss earlier research on psychophysiology and social interaction in psychotherapy, present our micro-analytical approach [conversation analysis (CA)], and hypotheses. In the method section we discuss the data collection, interaction variables, and the method of the analysis. We will then present our results from two time frames: (1) across the sessions and (2) during interventions. Finally, we will discuss the results with regard to earlier research.valence and arousal . Some take a different direction, focusing on shorter events within larger encounters: participants’ psychophysiological responses during interventions , during As whole, the picture provided by earlier research is not quite unified. For example, empathy has been variably connected to an increase and decrease in arousal, and in a divergence in arousal in the participants. In a recent systematic review of the literature, In the current study, we will investigate the interplay of empathy, challenge, and psychophysiological responses in therapists and clients, leaving aside the complex questions of synchrony. We draw our hypotheses from earlier research that suggests that empathy and challenge connect to dyadic internal regulation of emotion. More specifically, as in Besides autonomic arousal, we will analyze facial muscle activation (EMG) associated with the valence of emotional expression (smile and frown). Earlier experimental research using EMG has linked increase in frown activation to empathy  and mentIn his recent review,  suggesteOur micro-analytical approach on psychotherapy interaction is based on CA. CA is a social scientific and linguistic method for the study of the practices and processes of social interaction see . It giveformulations  during the interventions, and (2) during the whole therapy sessions(1)Empathy, viewed from the formulations by observers, decreases the clients’ and increases the therapists’ physiological arousal (as indexed by EDA and heart rate).(2)Empathy increases positive emotional facial expressions (as indexed by orbicularis oculi muscle activity) and decreases negative emotional expressions (as indexed by corrugator supercilii muscle activity) in both participants.(3)Challenge, viewed from the formulations by CA coders, increases physiological arousal (EDA and heart rate) in both participants.(4)Challenge decreases positive emotional expressions (orbicularis oculi muscle activity) and increases negative emotional expressions (corrugator supercilii muscle activity) in both participants.The data were recorded from psychodynamic psychotherapy sessions. The participants were five dyads. The therapists were experienced private psychotherapists in their fifties and sixties with advanced level psychotherapy training  and long experience in psychodynamic psychotherapy. They all had post-graduate degrees (from medicine or psychology) alongside their psychotherapy training. The participants were sitting during the sessions, the frequency of which was once or twice a week. Two clients suffered primarily from depression, one from anxiety disorder, one from eating disorder and one from obsessive-compulsive disorder. Four of the clients were in their twenties, one in her sixties. The data include one dyad of a male therapist and a male client, two dyads of a female therapist and a female client and two dyads of a male therapist and a female client.Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. They reviewed oral and written information about the research, and they were given the opportunity to cancel the consent for any reason. When the data was collected, there was no relevant ethics committee in Finland for this study, because the study was done at a social science department and did not involve patients in public health care . However, another study  that was part of the same project used identical procedure of data collection, data management and publication practices. This study involved patients in public health care, and was thus under the auspices of the Ethical Board of Helsinki University Hospital, which gave its permission to the study on 21.09.2011. From each dyad six subsequent sessions were recorded. The sessions were from different phases of the therapies: one from the early phase, three from the middle and one in the ending phase of the therapy. From these 30 sessions, six sessions were used in a preliminary data-driven analysis of the interaction, building of coding schemes, and in the training of coders. This training set was excluded from the final coding and analysis of the data. For building the coding scheme, we selected first two sessions from one of the dyads and then the first session from the other dyads. The rest of the sessions (24) were used in the actual coding and analysis.orbicularis oculi and corrugator supercilii muscle locations on the left side of the face. Physiological data were synchronized with the audiovisual recording using digital markers from the computer running the video collection.Data were recorded with two Nexus-10 amplifiers . EDA signal was recorded at 32 Hz with two electrodes affixed to middle phalanges of the non-dominant hand. ECG signal was recorded at 512 Hz using a modified Lead II electrode placement. Facial EMG data were recorded at 2048 Hz with bipolar electrodes placed at Data preprocessing was conducted with Matlab 8.5. The EDA data were smoothed with a low-pass filter at 1 Hz. ECG signal was filtered and R-peaks were detected using the ECGLAB  toolbox.Conversation analysis is mostly a qualitative research method, but it can be applied also in quantitative research, as in the current study. The method is context-sensitive in terms of the actual interaction, but independent from any theories of psychotherapy. The basic principle in CA is to observe the relation between adjacent turns at talk; the social actions are not identified as separated from their context, but in relation to what has been said before, most importantly in the just preceding turn. This was done in this study too. This kind of context-sensitivity to the previous utterance sets challenges to the reliability of coding the interaction, but on the other hand it leaves freedom for the coders to interpret the situation from the perspective of the participants see . We findApplying CA, we coded all interventions in which the therapists communicate to the client what they have heard the client said in his/her previous talk. The formulations can have declarative or interrogative forms but their primary function is not to elicit information but to offer a rephrased version of the client’s talk. Coders of the data (Voutilainen and Kahri) are conversation analysts with 5–10 years’ experience of the method. The coders did not have clinical background, and the coding scheme was independent from clinical reasoning.Challenge was coded from all formulations. We assessed whether a formulation is challenging or not. On the basis of qualitative analysis of the data, we built a coding scheme that identified three basic ways in which the psychodynamic therapists challenge the clients’ preceding talk: (1) therapist can formulate the problematic emotion as more intense than expressed by the client, (2) therapist can shift the focus from an external referent to an internal referent (experience) of the client, or (3) therapist can formulate the content of the clients’ experience as different than the client had described it as. If the therapist’s formulation performed one or more of these it was coded as challenging. The challenge can be very discreet and overlap with empathetic understanding of the client. Obviously, interpretation by the coders was needed. This interpretation, however, was based on CA , not clinical reasoning. Coders (Voutilainen and Kahri) were the same as in coding of formulations. First the coders coded sessions of the training set  and discussed cases of disagreement before coding independently the rest of the sessions.From the 24 coded sessions, 694 formulations were coded. Of the formulations, 455 were coded as non-challenging (“benign”) and 239 as challenging. Ten of the 24 sessions were double coded. Unlike the identification of formulations and challenge, the measurement of empathy was not based on CA coding but on the judgment of naïve raters. We considered rating, rather than coding, as apt for measuring empathy, because of the multimodality of empathetic displays e.g.,  and undeFor the analysis of physiological responses to the whole session (tonic responses), we calculated the mean skin conductance level (SCL), heart rate [beats per minute (bpm)], and root mean square (RMS) value of EMG activity for each session. In addition, we quantified changes in arousal (as indexed by SCL and heart rate) across the session. The EDA signal was downsampled (by factor of 8) to 4 Hz and cardiac  signal was interpolated to form a 4-Hz uniformly sampled time series and converted to heart rate (bpm). Coefficient of the least squares regression line  during session, multiplied by time and thus resulting in difference between the start and end point values in original units, was used as an index of change across a session. Logarithmic transformations were conducted for the data to normalize the distributions where appropriate. The interactional variables were the ratio of challenging formulations for a session and mean empathy of the formulations during a session.When analyzing physiological responses to interventions (phasic responses), as the length of the interventions differed, we focused on the last part of an intervention where the content of the intervention is apparent and the client begins to respond to the possible challenge. We calculated the mean physiological values for 10-s epochs ranging from 5 s before to 5 s after the end of an intervention. The interactional variables were the type of formulation  and empathy score of the formulation.Physiological data were analyzed by the linear mixed models (LMM) procedure in SPSS with restricted maximum likelihood estimation. When analyzing physiological responses to the whole session, role nested within dyad was specified as the subject variable and session number was specified as the repeated variable, and AR(1) was specified  as the covariance structure for the residuals. When analyzing physiological responses to interventions, role nested within dyad and session were specified as the subject variables and intervention number was specified as the repeated variable, and ARMA was specified as the covariance structure for the residuals. Role , challenge , therapist empathy (rating), and Role × Challenge, Role × Empathy, and Challenge × Empathy interactions were specified as fixed effects.The data supported many of our hypotheses but also provided unexpected results. Most importantly, the change in the participants’ physiological activation across a session was as we expected: the therapist’s empathy increased the therapist’s physiological activation and decreased the client’s physiological activation. We also found that the therapist’s arousal increased during the challenging interventions. Furthermore, there was an interaction effect of empathy and challenge, suggesting that these variables work together. Besides the statistically significant results, we also report trends in the data, considering also them of importance given the small database and the natural  setting of the research.F = 14.68, p < 0.01] and Role × Empathy  significantly predicted SCL change. Larger mean therapist empathy led to an increase in SC levels in therapists, whereas in patients it led to decrease of EDA. Figure 1 illustrates the effect of empathy on the change in SCL across a session for therapists and clients.Our hypothesis on empathy and physiological arousal was confirmed regarding the change in SCL across a session. Role of the participant . This appeared to be the case primarily for the clients (four of the five dyads as shows in the figure 2 below) rather than the therapists.Regarding our hypothesis on challenge, a higher ratio of challenging interventions tended to be associated with an increase in SCL across a session, but this effect failed to reach statistical significance . There was no significant effect of empathy or challenge on the mean SCL across a session .In addition, a higher ratio of challenging interventions tended to lead to a larger increase in SCL across the session especially when empathy was low, but the interaction between challenge and empathy failed to reach statistical significance  and high mean heart rate . Also empathy increased heart rate of the participants across a session ; thus, the hypothesis that empathy would decrease the client’s heart rate was not supported. Unexpectedly, empathy and challenge also interacted. In sessions with low empathy and a low ratio of challenging interventions, and sessions with high empathy and a high ratio of challenging interventions, the heart rate of the participants decreased across the sessions . On the other hand, in these sessions the mean heart rate of both participants was higher . This suggests that empathy and challenge are connected also at the physiological level. We will return to this in the discussion.As we hypothesized, challenge increased heart rate of the participants. Challenge was associated with both an increase in heart rate across a session , whereas challenge was negatively associated with it . Also the Challenge × Empathy interaction was significant . As was the case for heart rate, empathy was related to high orbicularis oculi activity across sessions with a high ratio of challenging responses, but the reverse was true for sessions with low challenge.As hypothesized, empathy was positively associated with orbicularis oculi EMG activity across sessions . Also empathy tended to be positively associated corrugator activity, but the effect was non-significant .Although challenge tended to be associated with higher corrugator EMG activity across sessions, the association was non-significant . Our hypothesis that challenge would increase SCL in both participants was partly supported. Challenge and role interacted significantly . A post hoc comparison showed that challenging interventions increased SCL in therapists (p < 0.001), but not in clients. Empathy was not related to SCL elicited by interventions.Therapists had a significantly lower SCL  compared to clients . The positive association of empathy with heart rate failed to reach statistical significance . However, the Empathy × Role interaction was significant , indicating that empathy increased heart rate of therapists, but not of clients. The main effect of challenge was a non-significant trend , but the Challenge × Role interaction was significant . Challenging interventions increased heart rate in therapists (p < 0.001) and decreased it in clients (p < 0.05). Thus, both empathy and challenge seem to increase the therapist’s, but not client’s, physiological arousal in response to interventions (formulations).Therapists had a significantly lower heart rate  = 9.86, p < 0.001]. Empathy tended to increase orbicularis oculi activity more in response to non-challenging interventions, but the Challenge × Empathy interaction failed to reach significance . The interaction of challenge and role was significant ; challenging interventions decreased orbicularis oculi activity only in clients. Thus, although empathy and challenge did not influence a client’s arousal during interventions, they appear to influence the client’s positive emotional expressions.Empathy was positively associated with orbicularis oculi EMG responses  to interventions . Also, empathy tended to increase corrugator activity in therapists, whereas the reverse tended to be the case in clients, although the Empathy × Role interaction failed to reach significance .Corrugator Supercilii activity indexing negative emotional expressions tended to lower in the therapists compared to clients, although the difference was not significant [Figure 5 shows these results on responses to interventions.empathy increased the therapist’s and decreased the client’s skin conductance over the session, (b) empathy increased orbicularis oculi EMG activity indexing positive emotional expressions  in both the therapists and the client across the sessions, (c) challenge increased heart rate of both the therapist and client across the sessions, and (d) challenge increased the therapist’s skin conductance during the very intervention. Although the hypotheses were not confirmed in all respects, the results support our general hypothesis that the therapist’s empathy is connected to positive valence and decrease in arousal in the client, and that the therapist’s challenge is connected to increase in physiological arousal in the participants. This is in line with how clinical literature describes the effect of these two basic orientations of therapeutic work (The results confirmed our hypotheses in four respects: (a) tic work . Our stuIn particular, the result on the effect of empathy at the session level suggests that the phenomenon of “sharing the emotional load” that has been found from everyday storytelling  is also Given the lack of earlier research, we did not have specific hypotheses regarding the differences between the physiological responses in the two time frames: during whole sessions and during interventions. However, we made some intriguing findings regarding the time frame of the physiological correlates of challenge that call for further work, theoretical and empirical. While in terms of heart rate, both participants respond to challenge at the session level (heart rate increases), in terms of EDA a trending effect suggests that only clients respond to challenge at the session level. However, phasically, during and immediately after the very formulations, only the therapists’ arousal—both EDA and heart rate—increased, whereas clients did not show a phasic physiological response to challenge. This is different to what was found in the earlier studies on confrontation , and mayFurthermore, the results suggest that the “professional specificity” of the therapist’s emotion regulation pertains to challenge more than to empathy: in the therapists, the regulation of empathy, unlike challenge, seems to come closer to the regularities of generic human interaction—what above was depicted as the sharing of the emotional load. In other words, the therapist’s own emotion system seems to react more “profoundly” to being empathetic than to being challenging. On the other hand, the therapists’ arousal may be connected to the effort in reflective, “controlled” type of empathy that Another effect that we did not predict but that calls for further research is the interaction effect that empathy and challenge had in predicting heart rate and facial muscle activation. In sessions where the therapist shows much empathy and challenge, along with sessions with low empathy and low challenge, heart rate was higher and positive facial muscle activity (smile) stronger than in sessions where these variables were less balanced. On the other hand, heart rate decreased over the sessions where empathy and challenge were in balance. This suggests that the close proximity of empathy and challenge in therapeutic work found in earlier interaction research  is refleThis study has demonstrated the effort and sophistication in the psychotherapists’ professional work with their clients’ emotion. The study confirms that the therapists responses to their clients have immediate effects in both the therapist’s and the clients’ emotion system. Recognizing the physiological load and its successful regulation in standards sessions contributes to understanding of the psychotherapist’s professional role, and highlights the importance of the therapist’s self-care.A limitation of the current study is the small amount of data and that some of the effects were found only as trending. Our physiological data shows that the client’s autonomic activation is constantly relatively high during the sessions, and this can amount to a roof-effect that prevents activation related to subtle differences from showing in the statistics. Data from more dyads would be helpful in solving this problem. Another limitation of this study considers the “fair to good” inter-coder reliability for challenge, and the “fair” inter-rater reliability for empathy. In our view, the common disagreement between coders and raters reflects the nature of the data , and that the coding and rating schemes allowed context-sensitive interpretation. We acknowledge that more coders and raters would probably improve the reliability. Another methodological aspect in the coding is that our coding scheme is applicable especially in the type of psychodynamic therapy that our data represents. To study challenge in other types of therapies, a coding scheme sensitive to that context would be needed. Finding a best solution considering both good reliability and context-sensitivity in the coding of interactional phenomena remains as a challenge for further research.AP, LV, NR, MS, and PH: conception and design of the study. LV, PH, MK, AP, and NR: acquisition, analysis, and interpretation of data. LV, PH, and AP: drafting the manuscript. AP, LV, NR, MS, PH, and MK: revising the manuscript, 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.


Sei-1 is a potential oncogene that plays an important role in promoting genomic instability. Double minute chromosomes (DMs) are hallmarks of gene amplification and contribute to tumorigenesis. Defects in the DNA double-strand break (DSB) repairing pathways can lead to gene amplification. To date, the mechanisms governing the formation of DMs induced by Sei-1 are not fully understood. We established DMs induced by Sei-1 in the NIH-3T3 cell line. RNA-sequencing was used to identify key characteristics of differentially expressed genes. Metaphase spreads were used to calculate DM numbers. Immunofluorescence was employed to detect γH2AX foci. Western blot and Akt pathway inhibition experiments were performed to reveal the role of the PI3K/Akt/BRCA1-Abraxas pathway in Sei-1-induced DMs. Luciferase reporter assay was employed to explore the regulatory mechanisms between Sei-1 and BRCA1. DM formation was associated with a deficiency in DSB repair. Based on this finding, activation of the PI3K/Akt/BRCA1-Abraxas pathway was found to increase the DM population with passage in vivo, and inhibition resulted in a reduction of DMs. Apart from this, it was shown for the first time that Sei-1 could directly regulate the expression of BRCA1. Our results suggest that the PI3K/Akt/BRCA1-Abraxas pathway is responsible for the formation of DMs induced by Sei-1. As an identified oncogene, Sei-1 is located in a frequent amplification region, 19q13.1, in ovarian cancer3 and is associated with mechanisms that lead to chromosomal instability, such as increasing the number of chromosomes, and double minute chromosomes (DMs) and micronuclei formation5. In addition, Sei-1 also inhibits apoptosis by stabilizing the X-linked inhibitor of apoptosis protein6 and by promoting metastasis through AKT or ILK modulation in HER2/neu-suppressed or expressing cancer cells7. However, few reports about the mechanisms that govern the formation of DMs and Sei-1 have been published so far, and as a result, further exploration in this field is needed.Sei-1  is a member of the TRIP-Br family, which acts as a nuclear factor that regulates the cell cycle by interacting with cyclin-dependent kinase 4 (CDK4) and E2F/DP-19. DMs can be detected in many tumors, such as acute myeloid leukemia10, ovarian carcinoma12, glioblastoma multiforme14, and colonic carcinoma16. Moreover, genes located on DMs can play crucial roles in the development of cancer, such as DHFR amplification in methotrexate resistance17 and MYC promotion of proliferation and invasion18. Therefore, the study of the DM formation is important for exploring the mechanisms underlying the development of tumors.DMs are paired, circular, acentric, and self-replicating DNA and are the cytogenetic hallmark of extrachromosomal gene amplification20. When a DSB occurs, it can initiate two main repair mechanisms, homologous recombination (HR) and classical non-homologous end joining. In addition, alternative end joining and single-strand annealing have been found to take part in DSB repair under certain conditions21. Breast cancer type 1 susceptibility protein (BRCA1) is recruited to the sites of DSBs with the help of the BRCA1-Abraxas (BRCA1-A) complex to effectively repair DNA. The BRCA1-A complex is composed of BRCA1 and Abraxas  as well as other components, including Rap80, BRCC36, BRE, and NBA123. Abraxas possesses the domains necessary to interact with the BRCT domains of BRCA1 and other components, which reveals the role of central adaptor protein24. The BRCA1-A complex plays important roles in maintaining genomic integrity during the DNA damage response.DNA double-strand breaks (DSBs) are a form of DNA damage that can lead to genomic instability or apoptosis if not appropriately repaired4. Moreover, DMs can appear during the passages in vivo in this model, which has aroused interest in exploring how these DMs are produced during this process. Although we previously reported the role of the met pathway in the promotion of the formation of DMs induced by Sei-1 in NIH-3T3 murine fibroblasts5, deep-seated and unknown mechanisms are also involved. Therefore, we selected cells from different passages in vivo with high and low DM populations for RNA-sequencing (RNA-seq) to explain the process governing the formation of DMs.Previous studies have shown that Sei-1 not only transforms the NIH-3T3 passage in vivo model to possess characteristics of cancerous formations but it also increases the incidence of genomic instability5. Consistent with previous results, the Sei-1-transfected group appeared to generate xenografts while the vector group failed. The primary cell culture from the original generation in vivo was named CPX1, and the sixth generation in vivo passage was named CPX6. The mean number of DMs for Vec, the control group transfected with null-vector-NIH-3T3 cells, and CPX1 and CPX6 was measured as ~0, 10, and 50, respectively  showed that the number of γH2AX foci increased significantly as the population of DMs increased  were analyzed and shown in supplementary figure 5. All of these suggested that DEGs associated with changes of DNA structures and PI3K/Akt pathway could play essential roles in the process of DM formation. Therefore, many DEGs that were involved in DSB repair . The qPCR was used to verify the accuracy of the RNA-seq data. All genes listed in the table were downregulated and only BRCA1, Rad51, and Chek1 reached the same degree of significance as the original data , and phospho-Akt (Thr308), while there was no change in total Akt, and a significant reduction in BRCA1  and phospho-Akt (Thr308) in addition to significant decreases in total Akt. BRCA1 and Abraxas showed dramatic increases in expression after 10 µM drug treatment for 48 h 8 h Fig. . PI3K wa8 h Fig. . Collect38. In the present study, we aimed to sift through the DEGs related to DM formation, and in doing so identified BRCA1 as an essential gene. This was done using RNA-seq, which differs from the previous study we performed5. In addition, we found that most genes associated with the DSB repair pathway were downregulated, which indicated that a defect existed in repairing DSBs. Gene amplification increases when the factors involved in the occurrence of DSBs, such as IR, DNA synthesis inhibitors, and restriction endonucleases, are added. Defects in either HR-relevant genes such as ATM39, Rad51D40, and Rad5441 or in NHEJ interrelated DNA-PKcs42 can enhance the predisposition of gene amplification mechanisms that include homogeneous staining regions and DMs. Consequently, these phenomena hint that Sei-1 may act as a factor that promotes DSBs and that there is a relationship between the production of DSBs and DMs in the Sei-1-transformed NIH-3T3 model.RNA-seq, which is a deep-sequencing technology, is used to map and quantify transcriptomes and has revealed new transcriptomic insights into gene and exon boundaries, transcript complexity, novel transcription mechanisms, and DEG identification43, but its relationship with the generation of DMs is still unknown. We found that the activation of PI3K/Akt promoted the appearance of DMs in a PTEN-independent manner. Likewise, not only can loss of BRCA1 lead to aneuploidy44 but it also contributes to activating the PI3K/Akt signaling pathway[30]. The BRCA1-A complex is important for genomic stability. However, the detailed mechanisms underlying DMs formation for BRCA1 or the BRCA1-A complex deficiency are not completely known. Our results uniquely support the claim that activating the PI3K/Akt pathway induced loss of function of the BRCA1-A complex to cause the production of DMs under the action of Sei-1 in NIH-3T3 cells, and Sei-1 could also target the promoter and regulate the expression of BRCA1  and authenticated by means of the STR (short tandem repeat) test . The detailed procedures of cell culture, cell passage, and transfection were previously reportedThe following antibodies were used for the experiments: anti-BRCA1 and PTEN antibodies ; anti-Rad51, Chk1, and Abraxas antibodies ; anti-PI3K, pan-Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), and anti-γH2AX antibodies ; IRDye 800DX-conjugated affinity-purified anti-mouse IgG and IRDye 700DX-conjugated affinity-purified anti-rabbit IgG ; and anti-mouse IgG/HRP and anti-rabbit IgG/HRP secondary antibodies . The MK2206 2HCl inhibitor  was also used.Cells in metaphase reached 70–80% confluence and were treated with Colchicine  for 1.5 h. The cells were trypsinized and then washed with phosphate-buffered saline (PBS) and centrifuged. The cell precipitate was re-suspended with 0.075 M KCl and incubated in a 37 °C water bath for 13 min. After undergoing fixation three times, the cell suspension was dropped on an ice-cold clean slide. The drops spread evenly and were stained with Gimsa . The pictures were then photographed using an Olympus BX41 microscope  equipped with a JVC TKC75U color video camera .48.Cell precipitates were treated with Trizol  to extract the RNA. cDNA samples were obtained using the Transcriptor First Strand cDNA Synthesis Kit . Semi-quantitative and quantitative real-time qPCR were performed as previously reportedg at 4 °C for 30 min to extract the total protein. The protein samples were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a PVDF membrane. After blocking with 5% blocking buffer, the membranes were incubated with the appropriate antibodies and then scanned using the Odyssey Imaging system  or the Fluorchem R system . ImageJ software was used for band analysis.Cells were lysed with RIPA buffer and centrifuged at 12,000 × The cells covered 80–90% of the glass coverslips. The cells were washed three times with PBS and fixed in 4% paraformaldehyde . Then, the cells were permeabilized with 0.1% Triton X-100 and blocked in 4% bovine serum albumin. Images were obtained using a DM-RXA2 fluorescence microscope (Leica) after incubation with γH2AX antibody , a fluorescence-conjugated secondary antibody, and DAPI  stain .5. The cells were sent to the Novogene Company  for RNA-seq and analysis. We then independently screened, analyzed the data by R , and validated the DEGs.Primary cells were cultured from mouse xenografts as previously describedThe construction of Sei-1 vectors (EX-A3731-Lv103) and pGL3-BRCA1-promoter luciferase reporter vectors were completed by GeneCopoeia company  and Aizhe Biological Technology Co., Ltd , respectively. The 293T cells were plated in 24-well plates at 50,000/well. The co-transfection of Sei-1 and luciferase reporter vectors were accomplished according to the instruction of HilyMax reagents . The detection of luciferase activity was based on the instruction of Luc-Pair Duo-Luciferase Assay Kits 2.0  and completed by microplate reader .t test and one-way analysis of variance with the Student–Newman–Keuls test were employed for data analysis of DM counts, relative mRNA expression, luciferase reporter assay, and western blots. All data are presented as the mean ± S.D or mean ± S.E.M. Significant differences were defined as P < 0.05.Two-tailed Student’s Figure legends of supplementary materialsSupplementary figure 1Supplementary figure 2


The VACNTs were grown underneath the graphene and lifted up the graphene. The graphene, with its edges anchored on the Al2O3, provided a constrained boundary condition for the VACNTs and hence affected the growth height and mechanical strength of the VACNTs. We prepared three groups of samples: VACNTs without graphene, VACNTs with graphene transferred once (1-Gr/VACNTs), and VACNTs with graphene transferred twice (2-Gr/VACNTs). A nano-indentation system was used to measure the reduced compressive modulus (Er) and hardness (H). The Er and H of Gr/VACNTs increased with the number of transfers of the anchored graphene. The 2-Gr/VACNTs had the largest Er and H, 23.8 MPa and 912 KPa, which are 6.6 times and 5.2 times those of VACNTs without the anchored graphene, respectively. In this work, we have demonstrated a simple method to increase the mechanical properties and suppress the height of VACNTs with the anchored graphene and number of transfers.We fabricated a 3D sandwich hybrid material composed of graphene and vertically aligned carbon nanotube forests (VACNTs) using chemical vapor deposition. The graphene was first synthesized on Cu foil. Then it was transferred to a substrate which had a pre-deposited catalyst Fe film and a buffer film of Al PaulD method . However2 of 30 sccm and argon (Ar) of 600 sccm. The Cu foil was annealed for 30 min at that temperature to remove oxides of Cu in H2 and Ar atmosphere. The mixed reaction gas of CH4, H2, and Ar was admitted into the CVD at flow rates of 10, 30, and 600 sccm for 5 min to grow the graphene sheet. Afterward, the CH4 flow was stopped and the sample was rapidly cooled to room temperature. Next, the as-grown graphene sheet was transferred onto the target substrate with a clean-lifting transfer technique utilizing electrostatic force [2O3 and a 1.9 nm catalyst Fe film. It was defined the deposited zone by the tape as shadow mask pattern and released it after the deposition of buffer layer and catalyst film. In addition, the attached graphene on PET was transferred onto one side of the pre-patterned Fe film for comparison of the mechanical properties with or without graphene, as shown in 2 flow of 400 sccm and C2H4 gas was supplied as the carbon source at a flow rate of 40 sccm for 60 min at 750 °C. The growth processes were conducted under 1 atm. The mechanical properties of the 3D hybrid structure of substrate/VACNTs/graphene were experimentally measured with a nanoindentation instrument.In this work, a 3D hybrid structure with the VACNTs sandwiched between the substrate and graphene was constructed. The graphene was synthesized with a three-temperature-zone CVD (TTZ-CVD) process. The as-grown grapheme was first transferred to the substrate, after which the VACNTs were synthesized by CVD method to form the hybrid structure of substrate/VACNTs/graphene. As-cleaned Cu foil (99.8% purity) was first placed on a quartz boat and inserted into a TTZ-CVD furnace, where it was heated to 1000 °C at 30 min under Hic force . The tra−1) and 2D band (~2700 cm−1) of the graphene [2D/IG ~0.67, and the full width at half-maximum of the 2D band peak was 63.1 cm−1. The results indicated few or more layers of graphene [graphene . The peagraphene ,20. A scgraphene , which rFrom the VACNTs growth process, To further examine the constraining effect of the multi-layer graphene on the mechanical properties, the graphene sheets were transferred once and twice onto different areas of the substrate and VACNTs were grown, as shown in the inset of In this work, a 3D hybrid nanostructure was fabricated with graphene and VACNTs. The mechanical properties of the Gr/VACNTs increased with the number of transfers of the anchored graphene. The height of the VACNTs was affected by the number of times the graphene was transferred. This simple method could be used to fabricate a high strength nano-material. In addition, VACNTs and graphene are the excellent performance of thermal interface materials. This may warrant new applications for the polishing  and micr


P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70,  Human non‐small‐cell lung cancer cell line A549 was purchased from American Type Culture Collections (ATCC). Human normal mesothelial cell line LP‐9 was purchased from the Cell Culture Core Facility at Harvard University . The 5 mesothelioma cell lines and A549 cells were maintained in RPMI‐1640 medium supplemented with 10% heat‐inactivated foetal bovine serum (FBS) and penicillin (100 IU/mL). LP‐9 was maintained in M199 supplemented with 15% heat‐inactivated FBS, 10 ng/mL EGF, 0.4 μg/mL hydrocortisone and penicillin (100 IU/mL). All cell lines were incubated at 37°C in a humid incubator with 5% CO2.2Fresh primary MPM and adjacent normal pleural tissues were obtained from 70 patients with MPM whose primary tumours were surgically resected. There are few exception normal pleural tissues used as normal control from non‐tumour patients on the same array. Primary human MPM samples and normal pleural samples were fixed in formalin and embedded in paraffin in 4‐μm tissue microarray sections. All human tissue samples were obtained and analysed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714–22 942–01).Rabbit anti‐YAP antibody  and rabbit anti‐PD‐L1 antibody  were used for immunohistochemistry (IHC) staining. The tissue microarray slides were deparaffinized and rehydrated and then immersed in boiled 10 mmol/L sodium citrate buffer (pH 6.0) for 10 minutes. After cooling down, slides were incubated in 3% hydrogen peroxide for 10 minutes and washed in PBS 3 times for 5 minutes. Slides were incubated with 10% normal goat serum in PBS for 30 minutes. After being washed in PBS 3 times, the slides were incubated overnight at 4°C with rabbit anti‐YAP antibody  and rabbit anti‐PD‐L1 antibody . The next day, the slides were incubated with biotin‐labelled secondary antibodies and streptavidin‐peroxidase (1:30) for 20 minutes each. Slides were stained for 5 minutes with 0.05% 3,3′‐diaminobenzidine tetrahydrochloride freshly prepared in 0.05 mol/L Tris‐HCl buffer (pH 7.6) containing 0.024% hydrogen peroxidase. The slides finally were counterstained with haematoxylin, dehydrated and mounted in Diatex.The following scoring system was used: −, no stain; +, weak staining (≥10% and <30% stained cellularity considered as positive); ++, moderate staining (≥30% and <50% stained cellularity considered as positive); +++, strong staining (50% or above stained cellularity considered as positive). All scoring was carried out under a low power objective lens (10X) with a Zeiss Axioskop 2 microscope . Images were taken under 10× or 40× objective lens.2.3The SMARTpool siRNA targeting YAP (YAP siRNA‐1) and a non‐targeting siRNA used as control were purchased from Thermo Scientific Dharmacon . Another YAP siRNA (AM16708) targeting the 3′UTR end of the YAP gene (YAP siRNA‐2) was purchased from Life Technologies . The YAP plasmid DNA (pcDNA4/HisMaxB‐YAP) used to overexpress the YAP gene in the cells was purchased from Addgene .The mesothelioma cells were plated in 6‐well plates (for Western blot or PCR) or 24‐well plates (for reporter assay) 24 hours before transfection. Cells were transfected with 100 nmol/L of siRNA using Lipofectamine RNAiMAX . For the YAP rescue procedure with YAP forced overexpression, cells cultured in 6‐well plates were transfected with 4 μg/well of YAP plasmid DNA, and those in 24‐well plates were transfected with 0.8 μg/well. After transfection for 48 hours, cells were harvested for further analysis.2.4The total amount of protein for each sample was 20 μg. The samples were run on 4%‐20% gradient SDS‐polyacrylamide gels  and then were transferred to Immobilon‐P nitrocellulose membranes . The membranes were probed with rabbit anti‐YAP , rabbit anti‐phospho Yap Ser127 , rabbit anti‐PD‐L1 antibody  and mouse anti‐GAPDH  in 4°C overnight after being blocked with 5% non‐fat milk. The membranes were then incubated with species‐specific conjugated secondary antibodies (GE Dharmacon) at room temperature for 1 hour. An ECL blotting analysis system  was used for detecting protein expression.2.5Total RNA was extracted from cells using the High Pure RNA Isolation Kit . RNA was transcribed to the cDNA using the iScript cDNA Synthesis Kit (Bio‐Rad), and the cDNA was used as the template for real‐time PCR. Primers and probe sequences used to detect YAP, CD274, AREG, CTGF and endogenous control gene b‐glucuronidase (GUSB) gene expression were purchased from Life Technologies . Real‐time PCR detection was carried out using TaqMan Technology on an Applied Biosystems 7000 sequence detection system (Applied Biosystems). Relative Quantification Software (Applied Biosystems) was used for mRNA expression analysis.2.6The 8× GTIIC‐luciferase plasmid (Addgene) and Renilla luciferase pRL‐TK plasmid  were cotransfected into cell lines using the transfection reagent Lipofectamine 2000 (Invitrogen). After 48 hours, cells were lysed and the lysate was transferred into a 96‐well plate for analysis using the Dual‐Luciferase Reporter Assay Kit (Promega). Luminescent signalling was measured on a GloMax‐96 Microplate Luminometer (Promega) according to the manufacturer's instructions.2.7Fragmented chromatin from H2052 cells was incubated with anti‐IgG (negative control), anti‐YAP and anti‐POL‐II (positive control). Recruited DNA was subjected to PCR using the primers for distal enhancer regions of PD‐L1, and PCR products were electrophoresed in agarose gel. The ChIP assay was conducted using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore Corporation). Polyclonal antibodies for YAP  and control rabbit antibody for IgG  were used for ChIP. Two‐pair primers were used for RT‐PCR to amplify the PD‐L1 gene. One pair consisted of 5′‐TCGGTCTGTGAAGGACTGC‐3′ and 5′‐ACCGTTGAGGAATGGATGAA‐3′, resulting in a product size of 203 bp, and the other consisted of 5′‐CCACCACCATTATCTAATTCCA‐3′and 5′‐AAGGAGCCAGACACAAAAGG‐3′, resulting in a product size of 210 bp.2.8Mesothelioma cell lines 211H, H2452, H290 and H2052 were cultured in a 96‐well plate. The cell count placed in each well was 3300. Cells were harvested at the time‐point of 0, 24, 48 and 72 hours. Cells were lysed, and CellTiter‐Glo Luminescent Cell Viability Assay reagent (Promega) was added to generate luminescent signalling. Luminescent signalling was detected using the GloMax‐96 Microplate Luminometer. Proportional cell proliferation was analysed with GraphPad Prism 5.0 software .2.9P value from a 2‐tailed test was <.05.Data are presented as mean ± standard deviation (SD) from 3 independent experiments. All statistical analyses were performed with GraphPad Prism . One‐way ANOVA followed by Scheffe multiple comparisons were used to compare the differences among multiple groups. A significant difference was considered when the 33.1P < .001)   . In 5 mesothelioma cell lines , the p‐YAP S127)/YAP protein expression ratio was low   Figure C.These results suggest that PD‐L1 expression is regulated by YAP expression in H2052, 211H, H2452 and A549 cells.3.4To investigate whether YAP regulates PD‐L1 at the transcriptional level in MPM cells, we examined the PD‐L1 promoter region and found 2 putative TEAD‐binding sites (CATTCC), which are 7911 and 7941 bp upstream of the PD‐L1 transcription start site . However, these patient samples were initially studied 10‐20 years ago, and there was not enough tissue left to evaluate the further molecular status between YAP and PD‐L1. Although PD‐1/PD‐L1 inhibitors can be a new treatment option for unresectable MPM with high PD‐L1 expression, the efficacy of PD‐1/PD‐L1 inhibitors is under investigation in clinical trials currently.Currently, tumour PD‐L1 expression is used as a predictive biomarker for immunotherapy targeting on the PD1/PD‐L1 axis.The regulation of PD‐L1 in tumours is not well understood. Two recent studies reported that the EGFR pathway is involved in the regulation of PD‐L1 expression in EGFR‐mutant human non‐small‐cell lung cancer (NSCLC) cells, whereby inhibition of EGFR signalling decreased PD‐L1 expression in some EGFR‐mutant NSCLC cells, suggesting that tumour PD‐L1 expression may be regulated by cancer‐driven mutation.In our MPM tumour microarray samples, some tumours with weak nucleus YAP staining show PD‐L1 expression . YAP is one of the factors that regulate PD‐L1 expression, and other factors may be involved in regulating PD‐L1 expression in various cancers.To our knowledge, our study is the first to show that inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further investigation of how YAP regulates tumour PD‐L1 expression in various cancers is warranted. In the future, treatments that target YAP may be an alternative to immunotherapy targeting on PD‐1/PD‐L1.All authors have no conflict of interest.PC. Hsu and L. You conceived and designed the study. PC. Hsu, J. Miao, Z. Huang, Z. Xu, J. You, and YL. Yang contributed to the development of methodology. PC. Hsu, J. Miao, WQ. Zhang and D.M. Jablons acquired the data  CW Wang analysed and interpreted the data . PC. Hsu, J. Miao, CT.Yang and L.You wrote, reviewed and revised the manuscript. PC. Hsu, J. Miao, WQ. Chang, Z. Xu and L. You contributed to administrative, technical or material support (organizing data). L. You supervised the study. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


This ensues a new methodological challenge for computational biology which consists in objectively extracting from these data the We deployed the fast multi-scale community mining algorithm based on spectral graph wavelets to characterise the networks of intra-chromosomal interactions in human cell lines. We observed that there exist structural domains of all sizes up to chromosome length and demonstrated that the set of structural communities forms a hierarchy of chromosome segments. Hence, at all scales, chromosome folding predominantly involves interactions between neighbouring sites rather than the formation of links between distant loci.Multi-scale structural decomposition of human chromosomes provides an original framework to question structural organisation and its relationship to functional regulation across the scales. By construction the proposed methodology is independent of the precise assembly of the reference genome and is thus directly applicable to genomes whose assembly is not fully determined.The online version of this article (doi:10.1186/s12859-017-1616-x) contains supplementary material, which is available to authorized users. At smomosomes . Howeveromosomes , 9, our omosomes –18. Hi-Comosomes , 20 is ∼omosomes . Mean inomosomes . Changesomosomes . Neverthomosomes . Principomosomes . Projectomosomes  omosomes –29. In oomosomes , 26–34. omosomes , 27,  and foetal lung fibroblast cell line IMR90 Hi-C data for which TADs are available , allowinMyelogenous leukemia cell line K562 and lymphoblastoid cell line GM06990 Hi-C data  for the Cervical cancer cell line HeLaS3 Hi-C data  where thHere we used Hi-C data obtained in different human cell lines: masked positions.Hi-C intra-chromosomal co-localisation frequency matrices for non-overlapping 100 kb loci correspond to the downloaded matrices that were down-sampled to 100 kb when necessary or were constructed from the alignment files  can be quantified as the correlation , the matrix of correlations between wavelets at scale s is approximated by the correlation between η (≪n) wavelet transforms of random Gaussian functions on the graph ). Sorting the (d has a match in We say that a domain b has a match in bi.e ± 1 pixel away. associated lengths (see below), we consider them in groups We say that a border As discussed in Domain length is an intuitive quantity to order a set of domains. In the same manner, we associated a length with each border of the genomic domains used in this work. TAD borders can be shared by at most 2 consecutive TADs, so we associated them with the length of the shortest TAD they border. At a fixed scale of analysis, a border of the novel interval-communities  delimits two consecutive interval-communities, so  we associated it with the minimum length of the two bordering communities. However these borders also present a strong pattern of conservation from one scale to another . We systematically applied the wavelet-based multi-scale community detection method to all the connected interaction networks scanning 100 scales logarithmically distributed in the range of available scales  reducing them to their main interval. This allowed us to adopt a simple representation of the structural-communities obtained across scales  where the interval-communities reduce to singletons in IMR90 . We also filtered out the communities that more than double in size when reintegrating the masked regions of the genome, e.g. interval-communities spanning the centromers. This leads to 386 (resp. 537) non trivial interval-communities in IMR90 (resp. H1 ES) for the chromosome 12. When applied to the 6 Hi-C datasets considered (Methods), the methodology presented for human chromosome 12 in H1 ES and IMR90 resulted in few thousands interval-communities per dataset . The original study  showed tλ=50  organised in fully connected interval-communities with no specific organisation at scales larger than the community size: the matrix is built as a series of 40 pairs of domains of size 20 nodes and 30 nodes with internal domain interaction set to 60, with the two first (resp. second) sub-diagonals set to 80 (resp. 70) to assure connectivity and with an additive Poisson noise over all interaction pairs of mean value vs distance curves for different interval-community length categories show that on average there are more interactions within the communities than between communities, regardless of the cell line and the community length: the interaction ratio systematically increases to some maximal value at distances ∼ 1–2 Mb, from a maximal value ∼ 1.6 in GM06990 and K562, to ∼ 2.2 in H1 ES and ∼ 3 in IMR90. Over larger distances, the ratio remains rather constant in GM06990 and K562 and decreases to ∼ 1.5 in H1 ES and IMR90  between TADs and interval-communities is slightly higher in H1 ES as compared to IMR90 for all TAD lengths  and a coarser  resolution  Fig. . The prethe TADs  are recomaster replication initiation zones that present similar insulating properties as the ones observed for TADs and interval-communities borders  resolution has provided some refined partitioning of the human genome by TADs of mean size ∼180 kb , much closophila . Interesll types , 20, 59.