ATG9A is essential during autophagosome biogenesis; however, the function of this multispanning membrane protein is not well defined. Judith et al. report that ATG9A vesicles deliver PI4KIIIβ to the autophagosome nucleation site to produce PI4P and initiate autophagosome formation. ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid–starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes.  Macroautophagy, referred here as autophagy, is a dynamic, highly conserved, lysosomal-mediated degradative process necessary for eukaryote development, survival, and homeostasis. Autophagy can selectively eliminate damaged organelles, protein aggregates, and viral and bacterial pathogens. Autophagosome formation involves a cytoplasmic protein machinery acting on a membrane source to nucleate and form a phagophore, which will close to become a double-membrane autophagosome, which is then degraded after fusion with endolysosomes. The molecular machinery driving autophagy is composed of ATG proteins . While dATG9 is the only multispanning ATG membrane protein essential for autophagy. In yeast, Atg9 vesicles are implicated in the delivery of membrane components to the initiation site or the preautophagosomal compartment . LikewisUnder nutrient-rich conditions, ATG9A is mainly located in the perinuclear region, colocalizing with medial and TGN markers of the Golgi complex, and partially with early and recycling endosomes . During ATG9A vesicles are highly mobile, and their trafficking is controlled by nutrient-regulated signaling: in fed and starved cells, ATG9A trafficking from the Golgi complex is controlled by the ULK1/2 complex . The ULKIn amino acid starvation, ATG9A localizes to tubular-vesicular structures adjacent to nascent phagophores, called the ATG9 compartment . Also foThe relationship of the ATG9 compartment and the highly mobile vesicles seen in both yeast and mammalian cells to the phagophore is not known. Although several properties of ATG9A are similar to those reported in yeast, in mammalian cells, ATG9A undergoes a complex trafficking pathway through the endocytic and secretory pathways . Our worUsing an antibody to endogenous ATG9A, we immunoisolated ATG9A-positive membranes and determined their composition by proteomics. We found Arfaptins (ARFIP1 and ARFIP2) to be components of ATG9A-positive membranes. ARFIPs are Bin/Amphiphysin/Rvs (BAR) domain–containing proteins, proposed to be required for sensing and generation of membrane curvature . The ARFDespite their similar domain structure and subcellular localization, there are notable differences between ARFIP1 and 2. ARFIP1, shown to be required for secretory granule biogenesis , is reguIn addition, we show that the phosphatidylinositol 4 kinases, PI4KIIα and PI4KIIIβ, are components of ATG9A-positive membranes. As PI4KΙΙα acts at autophagosome–lysosome fusion , we studTo identify the protein composition of ATG9A vesicles, nutrient-rich and amino acid–starved HEK293A cells were mechanically lysed, and ATG9A-positive membranes were immunoisolated using a hamster monoclonal antibody raised against human ATG9A or, as a control for specificity, hamster IgM . Stable BIF-1, ARFIP1, and ARFIP2 are BAR domain–containing proteins. Whereas BIF-1 is required for trafficking of ATG9A , 2016, lConsistent with previous reports , ARFIP1 Next, we determined the effect of ARFIP2 depletion during autophagy. Analysis of the localization of ARFIP2 relative to ATG9A revealed an association in the perinuclear region in FM . In ES, Thus, as ARFIP2 is present on ATG9A vesicles, we asked if it behaved like ATG9A and is found associated with forming autophagosomes. We performed live-cell imaging of cells under amino acid starvation stably expressing GFP-ATG13 (a member of the ULK1/2 complex) and mRFP-ATG9A, and transiently expressed iRFP-ARFIP2  cells , quantifARFIP2 contains an AH adjacent to the BAR domain . The AH As our data suggest that ARFIP2 modulates ATG9A trafficking, we assessed whether depletion of ARFIP2 would modify the composition of the ATG9A vesicles upon amino acid starvation. Using immunoisolation-SILAC coupled with liquid chromatography/tandem mass spectrometry, we compared immunoisolated ATG9A-positive membranes from CTRL and CrARFIP2 KO cells in ES . Among tThese data and the fact that ARFIP2 localized at the Golgi apparatus led us to investigate the two major PI4-kinases present on the Golgi apparatus: PI4KIIα and PI4KIIIβ . We confLegionella pneumophila, which labels PI4P . A target sequence in the fifth exon of human ARFIP2 was selected . The appropriate oligonucleotide was cloned into Bbs1 site of pSpCas9(BB)-2A-GFP plasmid obtained from the laboratory of F. Zhang  according to the cloning protocol provided by the laboratory. Following single-cell sorting on GFP-positive cells, colonies were screened for ARFIP2 deficiency by immunoblot.To induce autophagy, cells were washed three times with PBS and incubated in ES for 2 h. Where indicated, cells were treated with 100 nM bafilomycin A1  for 2 h or VPS34 IN1  for 1 h.The antibodies used in this work are as follows. Mouse antibodies: anti-WIPI2 , anti-GMSecondary antibodies for immunofluorescence (from Life Technologies unless otherwise specified) were anti-rabbit IgG Alexa Fluor 488, 555, and 647; anti-mouse IgG Alexa Fluor 488, 555, and 647; and anti-hamster Cy3 (Jackson ImmunoResearch). HRP-conjugated secondary antibodies used for Western blotting were from GE Healthcare.GFP-ARFIP2 was a gift from K. Nakayama  was used for transient transfection of cells according to the manufacturer’s instructions. DNA plasmids were used at 1 µg/ml. For RNAi, cells were transfected with the relevant siRNA oligonucleotide using oligofectamine , followed by a second transfection using Lipofectamine 2000 the following day, both according to the manufacturer’s instructions. Cells were harvested 72 h after transfection. The final concentration of siRNA oligonucleotides was 100 nM. siRNAs used (Dharmacon): D-001220-01 , D-020256-01 (ARFIP1), D-012820-01 (ARFIP2-01), D-012820-02 (ARFIP2-02), D-014294-02 (ATG9A), and D-006777-02 (PI4KIIIβ).Cells were lysed in ice-cold TNTE buffer  containing EDTA-free Complete Protease Inhibitor cocktail  and phosphatase inhibitor cocktail . Lysates were cleared by centrifugation for 15 min at 13,000 rpm, heated at 65°C for 15 min, and resolved on NuPAGE BIS-Tris 4–12% gels (Life Technologies) followed by transfer onto a PVDF membrane (Millipore). Following incubation with primary and secondary antibodies, the Western blots were developed by enhanced chemiluminescence  on Hyperfilm MP  or by chemiluminescence detection using Amersham Imager 680 . Densitometry was performed by using ImageJ software .Cells were lysed using IP buffer  and 1× EDTA-free complete protease inhibitor and phosphatase inhibitor cocktail, and the clarified lysates used for immunoprecipitation were incubated with either rabbit anti-ATG9A  or rabbig at 4°C. Pellets were resuspended using ice-cold isotonic buffer A  supplemented with EDTA-free complete protease inhibitor cocktail and phosphatase inhibitor cocktail and incubated for 15 min on ice. The resuspended pellet was then passed through a 27G needle 15 to 20 times for homogenization before clarification by centrifugation at 3,000 g at 4°C. Supernatants were used for incubation overnight at 4°C with hamster anti-ATG9A or hamster IgM CTRL coupled with protein A Dynabeads . The ATG9A-positive membranes on the beads were washed three times with isotonic buffer supplemented with 150 mM NaCl. The ATG9A-positive membranes on the beads were then eluted by competition with ATG9A peptide (HPEPVPEEGSEDELPPQVHK). The eluted ATG9A-positive membranes were then pelleted by centrifugation at 47,000 rpm for 1 h at 4°C. The pellet membranes were resuspended in 2× Laemmli sample buffer before being resolved by SDS-PAGE and Western blotting. For confocal microscopy, mRFP-ATG9A–positive membranes were immunoisolated following the protocol described above and then fixed with 2% PFA after the washing step.For immunoisolation of ATG9A-positive membranes, HEK293 cells were treated with FM or ES for 2 h. Cells were then washed in ice-cold PBS and harvested by centrifugation at 200 For immunoisolation of GFP-P4MX2–, GFP-ATG13–, GFP-DFCP1–, and GFP-LC3B–positive membranes, mouse anti-GFP antibody or mouse anti-FLAG M2 (CTRL) coupled to protein A Dynabeads was used. After the overnight incubation of the supernatants with the antibody coupled to protein A Dynabeads, GFP-positive membranes were washed with the isotonic buffer supplemented with 75–150 mM NaCl and eluted in 2× Laemmli sample buffer before being resolved by SDS-PAGE and Western blotting.14N, 12C) or heavy  lysine and arginine . ATG9-positive membranes were isolated from SILAC HEK293 cells either from nutrient-rich condition or amino acid starvation, using a hamster monoclonal antibody specific to the C-terminal domain of ATG9 conjugated to magnetic beads. Eluted proteins were subjected to SDS-PAGE until the running front had migrated 2–3 cm into the gel. Proteins were in-gel digested using trypsin, and peptides were analyzed using a Q Exactive mass spectrometer coupled to an Ultimate3000 HPLC equipped with an EASY-Spray source (Thermo Fisher Scientific). Raw data were processed using MaxQuant v1.3.0.5, with SILAC selected as the quantification algorithm. The proteingroup.txt output table was imported into Perseus software for further processing, statistical analysis, and visualization. Data were analyzed to screen for proteins enriched on ATG9A-positive membrane in nutrient-rich and amino acid starvation conditions and filtered as follows. Of all proteins identified, only those that were detected with a higher intensity in the specific ATG9A immunoisolation compared with IgM CTRL immunoisolation were considered as candidates. For each of these proteins, the log2 values of the normalized SILAC ratios were plotted against the log10 of the intensity to generate the scatterplot depicted in http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository.For SILAC experiments, HEK293 cells were grown in DMEM supplemented with 10% dialyzed FCS , and growth medium was supplemented with a combination of either 100 mg per liter of light , 0.2% Triton X-100 (ULK1 staining), or room temperature methanol (endogenous LC3 staining) for 5 min. Coverslips were blocked with 5% BSA  for 1 h, incubated with primary antibody in 1% BSA for 1 h, washed with PBS, and incubated with secondary antibody in 1% BSA for 30 min, before final washing with PBS and MilliQ water. Cells were stained with Hoechst 33258 solution  and mounted with Mowiol 4-88 .Cells were grown to subconfluence on coverslips and treated with FM or ES for 2 h. Cells were fixed by addition of 1 volume of prewarmed 4% PFA to the medium for 15 min at room temperature. Cells were then washed three times using PBS containing 50 mM NH4Cl and once with MilliQ water, and mounted.For PI4P staining, cells were permeabilized for 5 min with 20 µM digitonin in buffer A containing 20 mM Pipes, pH 6.8, 137 mM NaCl, and 2.7 mM KCl. Cells were washed three times in buffer A and then blocked for 45 min in buffer A containing 5% BSA. Anti-PI4P antibody was added in buffer A for 60 min at room temperature. After three washes with buffer A, secondary fluorophore-labeled antibodies were added in buffer A. Cells were postfixed for 5 min with 2% PFA, washed three times with PBS containing 50 mM NHFor conventional confocal microscopy, a confocal laser microscope  with a 63× NA 1.46 plan-Apochromat objective was used. Superresolution microscopy was performed with Zeiss LSM 880, Airyscan-equipped confocal microscope with a 63× NA 1.4 plan-Apochromat objective. After acquisition, images were processed using an Airyscan processing tool on the ZEN software provided by Zeiss. Image channels were acquired sequentially using appropriate filter sets for DAPI, EGFP, mRFP, iRFP, and Alexa Fluor 555, 488, and 647 and combined in the corresponding microscope software. All imaging was performed at room temperature.For conventional confocal microscopy, a confocal laser microscope  with a 63× oil-immersion lens was used. High-resolution confocal microscopy was performed with a Zeiss LSM 880, Airyscan-equipped confocal microscope with a 63× oil-immersion lens. After acquisition, images were processed using an Airyscan processing tool on the ZEN software provided by Zeiss.The experiments were repeated as indicated in the figure legends, and representative images are shown. The level of colocalization was determined by acquiring confocal images from ∼30 cells per condition. Pearson’s coefficient was then determined using Fiji software. LC3, WIPI2, and ULK1 puncta formation was quantified by Imaris image analysis software. Line scans have been made by using Fiji software. Association between GFP-ATG13 spots with the different markers , GFP-LC3 spots with PI4P, and endogenous LC3 spots with GFP-PI4KIIIβ and with WIPI2 were analyzed with Fiji software based on the Manders’ overlap coefficient. Additionally, lines were drawn crossing GFP-ATG13, GFP-LC3B, or LC3 spots associated with the different markers, and line plots of the fluorescence intensity were used to confirm the association. In all experiments, images shown in individual panels were acquired using identical exposure times or scan settings and adjusted identically for brightness and contrast using Photoshop CS5 (Adobe).2 and immediately imaged using a Zeiss LSM 880, Airyscan-equipped confocal microscope with a 63× NA 1.4 plan-Apochromat objective. Images were processed using an Airyscan processing tool on the ZEN software provided by Zeiss.Live-cell imaging was performed on HEK293A cells stably expressing GFP-ATG13 (HEK293A-GFP-ATG13) and mRFP-ATG9A cotransfected with iRFP-ARFIP2 or GFP-ATG13 and mRFP-ATG9A cotransfected with iRFP-PI4KIIIβ or on HEK293 cells stably expressing mRFP-ATG9A cotransfected with GFP-P4MX2 seeded on glass-bottom microwell dishes . Cells were placed into ES at 37°C and 5% COn numbers. Statistics were performed using GraphPad Prism 6 software, as detailed in the figure legends.The statistical details of all experiments are reported in the figures, including statistical analysis performed, error bars, statistical significance, and exact Fig. S1 provides evidence that in contrast to ARFIP2, ARFIP1 is not required for autophagy. Fig. S2 provides evidence that ARFIP2 is essential for autophagy. Fig. S3 shows the relationship between ATG9A and the PI4P pathway. Fig. S4 shows that the different stages of autophagosomes contain PI4P and PI4KIIIβ. Video 1 provides evidence that ATG9A vesicles containing ARFIP2 interact transiently with ATG13 vesicles. Video 2 shows that ATG9A vesicles contain PI4P. Video 3 shows that ATG13 vesicles interact with ATG9A vesicles containing PI4KIIIβ. Table S1 is a list of proteins associated with ATG9A-positive membranes under nutrient-rich and starvation conditions. Table S2 is a list of Golgi proteins associated with ATG9A-positive membranes. Table S3 is a list of proteins associated with, or depleted from, ATG9A-positive membrane upon ARFIP2 depletion.


By calculating the one-body carrier density probability measure of the graphene sheet, we show that the density fluctuation ( The understanding of the origin and the influence of disorder, as well as interactions in graphene seems to be essential in understanding of the experiments and also in designing graphene-based electronic devices. The effect of these quantities and the resulting screening are not as easygoing as ordinary simple metals5. There is a huge literature concerning the interplay of particle interactions and disorder in mono and multilayer graphene13 which cause variety of phenomenons. Examples of the effect of disorder on the properties of graphene are its effect on: the compressibility14, the electron-phonon interaction15, the Fano factor and conductivity5, the magnetoresistance of bilayer graphene16, the graphene Hall bars17, the optical properties of graphene quantum dots18 and the effective electronic mass in bilayer graphene19. Disorder-based spintronics in graphene20, and Levi-flight transport caused by anisotropically distributed on-site impurities21 are other examples for which the disorder plays a dominant role. The disorder-mediated Kondo effect22 and the formation of electron-hole puddles (EHPs) are examples for which the disorder and the inter-particle interactions play vital roles simultaneously. EHPs are believed to be responsible for the observed minimum conductivity of graphene and was predicted theoretically by Hwang et al.23 and Adam et al.24 and was also confirmed in experiments in the vicinity of the Dirac point34. They are characterized by the state in which some strong carrier density inhomogeneities with density fluctuations much larger than the average density (for low densities) emerge35. In this case  and the similar situations, a many body treatment is needed in which the effect of interaction and the disorder enter to the play with the same footing. Such an investigation can be found in36 in which it has been shown that for the gauge field randomness, related to the formation of ripples characterized by a disorder strength 36. This shows the vital role of disorder in electronic properties of graphene which even can change the role of interaction and cause the system to exhibit the non-Frmi liquid behaviors6.Since the discovery of graphene as a hybrid between metal and insulator, this material with relativistic energy spectrum of zero-gap Dirac Fermions38. For the undoped graphene in one loop approximation of the polarization function (for which the intra-band excitations are forbidden due to the Pauli principle), it is shown that the static dielectric function 39 and no collective modes are allowed within the PRA approximation. For finite μ however, the more general solution of Shung40 is applicable which predicts that the potential is screened with the screening length vF ≡ Fermi velocity, kF ≡ Fermi momentum, 40. Based on these results, in low-densities (in intrinsic or weakly disordered graphene) due to the lack of screening one should take into account the long-range part of the potential which leads to the issue of logarithmic phase shifts for Coulomb scatterers6. These results were obtained for disorder-free system and within the RPA approach which is questionable for the graphene as pointed out by Mishchenko41. Despite of this huge literature, there is a limited understanding concerning the properties of the polarization function in the general form and also the behavior of multi-body charge density probability measures, especially in the low-density case in which EHPs appear.The polarization function of the graphene as an important quantity which is sensitive to both the inter-particle interaction and the disorder has been obtained and analyzed in the cone approximation in the bubble expansion42. To obtain disorder corrections to the dielectric function we develop a diagramatic technique for the TFD theory. We obtain the two-body correlation function using some stochastic analysis and find the equation governing it. We show that, in addition to the screening length found by others, there is a characteristic length scale for the screening, namely qch which is related to the disorder strength and inter-particle interactions via ni is the disorder strength, rs is the dimensionless interaction factor to be defined in the text and d is the substrate distance. For small densities we show that there occurs an instability in which the imaginary part of the dielectric function becomes negative and some charge density oscillations with disorder-dependent wave length emerge. At these densities screening-anti-screening transition occurs along with changing sign of the real screened potential. The disorder significantly changes the Fourier component of the screened potential for large wave lengths showing the fact that the large-scale behaviors of the system is mostly affected by the disorder. We characterize the system in the vicinity of this transition in detail. In addition to the one- and the two-body (static) correlation functions, the equation governing the total probability measure of the system is introduced and analyzed and a closed expression is proposed for it. The proposed expression is valid for large densities.A more reliable approach should involve both interaction and disorder simultaneously which is the aim of the present paper. For this, the Thomas-Fermi-Dirac (TFD) is a proper candidate which has the capacity to bring the effect of all LDA energies in the calculations as well as the tunable disorder in the same time. We employ this approach involving exchange-correlation energies and white noise out-plane disorder. For the EHPs, TFD techniques have proved to be very useful and are widely usedThe paper has been organized as follows: In the next section we present the general construction of the problem and introduce the TFD theory for determining the ground state of graphene. The one-body probability measure is obtained in SEC. 2. Section 3 is devoted to finding the two-body probability measure and the dielectric function. The screened potential as well as the charge density oscillations are argued in this section. The total probability measure of the system is analyzed in SEC. 4. In the conclusion section we close the paper by highlighting the main findings of the paper. Appendix 41. The renormalization group analysis in the weak coupling limit in the first order approximation shows that the Coulomb interactions are marginally irrelevant due to the logarithmically divergent velocity6. In the strong interaction side however, it is shown that the graphene has non-Fermi liquid behaviors with power-law quasiparticle dispersion43, the limit which cannot be reached for finite densities away from the Dirac point. In the weak coupling side, it is known that in contrast to ordinary metals in which the screening makes the interactions short-ranged, in low-density (intrinsic or weakly disordered) graphene due to the lack of screening one should take into account the long-range part of the potential which leads to the issue of logarithmic phase shifts for Coulomb scatterers6. The effect of disorder is however of special importance since, as stated in the introduction, it may completely change the behavior of the Dirac Fermions36. TFD theory, as an approach which has the potential to bring the interaction and disorder in the calculations in the same footing is introduced and analyzed in this section. We mention some points concerning the TFD theory as a coarse grained method and the perturbation diagrams which are used in our analysis. The multi-point probability measures and their relations to the many body disordered response functions are of special importance in this paper which is described in the next sections.The effect of interaction and disorder in graphene has a long story in the literature. It is known that the random-phase approximation (RPA) fails to describe the interaction effects in graphene which was firstly pointed out by MishchenkoT is the time ordering operator, 44:Let us start by analyzing the zero-temperature polarization operator which is defined by 45. In this figure the single-line circles show the carrier density at the point x, i.e. n(x) and the shaded area shows all interaction effects, such as Coulomb lines and external (disordered) interaction lines. In the present paper we mainly deal with r and r′ are r are the full electronic density Consider the ground state, and define mentclass2pt{minimmentclass2pt{minimmentclass2pt{minim44  term of the Fig. mentclass2pt{minimThe determination of r and r′), the extra contribution comes from the quantities like f is an arbitrary function). In such cases we define the pair (or two-body) distribution measure46:n at r and n′ at r′. It is notable that For the more general case in which we are interested in calculating multi-point correlation functions with two insertion points . We turn back to this point at the end of this section.In this section we concentrate on calculating one-body probability density The differential form of the charge profile is:n(r). We consider a one-body function P be the probability measure of charge density to be n at the position r, so that f is defined as Now let us perform some Ito calculations to obtain the probability measure of Let us consider the above expression term by term. Firstly we note that fs, we can demand that the equality holds for the integral kernels and obtain:If we note that this equation should hold for all arbitrary Now let us concentrate on mentclass2pt{minimmentclasspt{minimar, the left hand side of the above equation equals to zero which results to:Therefore for the homogeneous system, which is independent of the observation point n(r) as follows:Fns. In fact Fn is everywhere non-zero and well-defined and finite except at n = 0 and n = 0 for which, as we will see, becomes divergent and n) |n|3/2 whose derivative is not well-behaved in n = 0.By equating the expression inside the bracket to a constant (which is zero for the symmetry considerations) we obtain the equation governing the distribution of ζ0 limit. In this limit, and considering S is the area of the sample and P0 is the normalization constant. This relation may seem to be unsuited, since it grows unboundedly for negative values of n. Actually there is no contradiction, due to the presence of Gn whose amount grows negatively for negative n values, which returns the above equation to the expected form. In fact the original charge equation has electron-hole symmetry for the case Pn. Our approximation (considering Gn as a constant) violated this symmetry. Re-considering this quantity as a dynamical variable retains the mentioned symmetry. It is also notable that the second term in the exponent Pn.With the above limitation in mind, let us first turn to the asymptotic behaviors of Eq. μ have been actually coded in ζ′. Large amounts of G favors large charge fluctuations. To see how G controls the charge fluctuations, we note that the limit G. In fact μ controls 〈n〉 which directly affects G. In the limit G becomes vanishingly small, so that In the above equation, the effects of disorder and interaction and The total solution of Eq. rsFn and Pn for positive ns for Pn approaches continuously to n〉. Before closing this section, let us turn to calculating the compressibility (κ). Knowing that the compressibility is proportional to 〈n2〉 − 〈n〉2 we can easily do the integrals and show that, for the un-gated intrinsic graphene n〉 → 0, In the above equation f caused by the infinitesimal growth of the charge density is In this section we consider a two-body functional. i.e. Pn,n′:Just like the calculations of the previous section we write . The result has been shown in Fig. ζ (Rmax is the amount of R in which Gx(R) shows a peak). It can be shown that Gx(R) has some interesting features. Firstly the tail of this function (for large R’s) behaves in a logarithmic fashion. To show this we have shown R/Rmean in a semi-logarithmic graph in the inset of Fig. ζs (ζ = 1 in this graph), the graphs fit properly to each other with a nearly the same slope. Clearly this behavior can’t remain for very large Rs, since Gx(R) should be positive in all R range. The logarithmic behavior in graphene has been observed in many aspects, e.g. the logarithmic enhancement of Fermi velocity39, or exchange-driven singularity in the Fermi velocity in intrinsic graphene49, but up to the author’s knowledge such a behavior in two-point correlation functions has not been reported before. The logarithmic behavior is the characteristics of free Boson systems that our effective model for intermediate spatial scales corresponds to. The other feature of Gx(R) is the narrowing of its peak as ζ increases, and that Rmax and Gx(Rmax) are decreasing functions of ζ. The change of these quantities has been shown in the Fig. ζ. The power-law behavior arises from the scale-invariance of the TFD equation which was analyzed at the end of SEC. 1. To characterize the behavior of Gx(R) let us analyze the width of the Gx(R) defined by Gx(Rmax) and Rmax in terms of ζ. The behaviors are A very important quantity is the density-density correlation function x〉 is small enough, i.e. ζ is small, Rmax becomes large and therefore δ increases unboundedly which leads to large density inhomogeneity, i.e. formation of EHPs.In the limit in which the interaction is strong enough and (or) the fraction 〈Gx(R), i.e. G(q) which is shown to be:Jn(x) is the Bessel function of the first kind, G(q) vanishes. The presence of disorder induces a characteristic wave vector ζ in a semi-logarithmic scale. For large ζ values this function is nearly constant for small values of q value which we name q0. For q values in the order of (and larger than) qch, we enter the phase in which q0 is ζ-dependent and increases as ζ increases. It is interestingly seen that q0 show power-law behaviors in terms of ζ with exponents shown in the figure, i.e. The other important quantity is the Fourier transform of n(r), i.e. Eq. To understand where these power-law behaviors come from, we should look at the main equation of G(q) on the screened potential is very interesting. The Fourier component of the screened potential (vsc(q)) in long wave lengths  is crucially changed, so that vsc is not changed with respect to the bare interaction potential for large and intermediate wave numbers. It has been shown in the inset of Fig. q for large q’s. The cross-over between these two behaviors occur in some q interval. It is seen that there is a characteristic q there (the peak), which results to some oscillations in the real space. The potential in the real space v(R) has some interesting features. For large R’s and large ζ’s this quantity falls off just like the bare potential, i.e. 1/R with some oscillations which is evident in Fig. R limit the potential tends to a constant value, which cause ζ increases, ζ decreases  shows a peak. The emergent oscillatory behavior is the effect of characteristic wave number for which the screened potential shows peak.The effect of G(q). It is well-known that for zero temperature case the polarization given by the RPA for mono-layer garphene is constant for q with a discontinuity in the derivative at 5. G(q)  for which Before closing this section, it is worth mentioning some points concerning the form of mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimP({n}) which is the probability measure for the charge configuration {n}. This function actually depends not only to n configuration, but also to its gradient: f is an arbitrary local or non-local smooth function of the density n, i.e. r0 is some reference point. Without loose of generality we set n  shows a peak. It was found that Gx(R) falls off logarithmically for large R’s, i.e. kF a disorder-driven characteristic momentum (qch) emerges in the system that controls the behaviors of the screened potential. The Fourier component of screened potential changes significantly for small wave numbers. for small enough ζ’s  we have observed an instability in which the imaginary part of the dielectric function becomes negative and some oscillations are observed for the charge density δn(r). The oscillations are absent for larger ζ values . Along with this change, a screening-anti-screening transition occurs in which δn(0) changes the sign. The observed instability is certainly affected by the higher order contribution, but we think that it should show itself for small enough densities, since the effect increases in a power-law fashion with ζ.In this paper we have investigated the properties of the disorder-averaged dielectric function by analyzing the one- and two- body charge density distribution functions of mono-layer graphene. For calculating them, we have used the Thomas-Fermi-Dirac (TFD) approach, taking into account the tunable disorder and inter particle interactions on an equal footing. To use the TFD theory for calculating the linear screening, we developed a diagrammatic technique and in the first order approximation in disorder strength, the polarization operator and charge modulation due to external potential have been obtained. The one- and the two- body charge density distribution functions were obtained using some stochastic analysis which carries both the effect of white-noise out-plane disorder and the interaction. The closed form of the functions were obtained for high density and low inter-particle interaction limits. By analyzing the one-body distribution function we found that the charge density fluctuations is proportional to ζ limit and was shown to be of the quadratic form. For smaller ζ’s we think that derivatives of charge densities should come to calculations which is the subject of our future research.In the last part of the paper we have calculated the total distribution measure of the mono-layer graphene sheets. The resulting equation has been analytically solved for large Interaction-disorder-driven characteristic momentum in graphene, approach of multi-body distribution functions


P = 0.110) and was significantly lower at high ambient humidity . The multivariable analysis also showed that high ambient humidity was independently associated with low sensitivity of FIT . In conclusion, high ambient humidity decreased the sensitivity, while high ambient temperature along with high ambient humidity decreased the positivity rate of FIT.Although it is known that ambient temperature can affect the diagnostic performance of the fecal immunochemical test (FIT), the impact of other weather parameters, including humidity, on the sensitivity of FIT remains to be further investigated. We aimed to evaluate the impact of ambient temperature and humidity on the performance of FIT for screening for advanced colorectal neoplasia (ACRN). We included asymptomatic individuals who had undergone both screening colonoscopy and FIT. The diagnostic performance of FIT, including its sensitivity, was analyzed according to the ambient temperature and humidity on the day that FIT was performed. Temperature and humidity were divided into five levels. Among 35,461 participants, 589 (1.7%) had ACRN. The positivity rate of FIT was lower at ≥24 °C (3.1%) than at <0 °C (3.9%), 0–8 °C (4.3%), and 8–16 °C (3.9%). It was also lower at 80–90% humidity (3.1%) than at < 60% humidity (3.9%). Multivariable analysis showed that high ambient temperature (≥24 °C) with high ambient humidity (≥80%) was associated with a low positivity rate of FIT . Sensitivity tended to decrease at high ambient temperature (<24 °C vs. ≥24 °C; 20.8% vs. 14.6%,  The US Preventive Services Task Force recommends screening for CRC starting at 50 years of age and continuing until age 752. In addition, the US Multi-Society Task Force strongly recommends colonoscopy every 10 years or an annual fecal immunochemical test (FIT) as first-tier options for screening individuals at average risk of colorectal neoplasia (CRN)3. Although colonoscopy can effectively prevent CRC through resection of colorectal adenoma, colonoscopic screening has not been popular worldwide owing to limited medical resources5. FIT is now regarded as the best noninvasive tool for CRC screening6. The Asia Pacific guideline indicates FIT as the first choice for CRC screening in resource-limited countries7. FIT has been adopted as a population-based screening program in many countries, including Korea8.Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related mortality in the world11. The sensitivity and specificity of FIT are maintained across individuals with different clinical risk factors12. However, there is concern that the performance of FIT is influenced by seasonal variations. Several studies from different countries, including Korea, France, Italy, and Netherlands, reported that the positivity rate of FIT is lower in summer16. High ambient temperature is suggested as a possible factor that lowers the positivity rate of FIT17. This is an important issue, because the exposure of fecal samples to a high ambient temperature may be unavoidable in population-based screening13.It is well known that FIT is superior to the guaiac test in terms of efficacy in preventing CRC, through enhanced detection of advanced adenomas15. In addition, a contradictory study shows that ambient temperature alone does not affect the performance of FIT18. Not only ambient temperature but also other weather parameters, such as humidity, may affect seasonal variation in the performance of FIT. In an experimental study, high humidity was associated with an increased rate of hemoglobin degradation19. In this study, we aimed to evaluate the impact of weather parameters, including ambient humidity as well as ambient temperature, on the performance of FIT using a large cohort of asymptomatic individuals who underwent both colonoscopy and FIT. The positivity rate, sensitivity, and specificity of FIT for ACRN were assessed according to ambient temperature and humidity.However, the impact of high ambient temperature on the performance of FIT is yet to be further investigated. The positivity rate of FIT depends on the prevalence of CRC or advanced CRN (ACRN); therefore, it is better to evaluate the performance of FIT in terms of sensitivity and specificity rather than its positivity rate. In most studies, however, sensitivity and specificity could not be assessed because only participants who showed positive results for FIT were offered colonoscopyThe medical records of 46,654 participants who underwent both screening colonoscopy and FIT were reviewed. Of these participants, 5,544 were excluded because they had previously undergone a colonic examination, colorectal surgery, or surgery for CRN. In addition, 74 participants with inflammatory bowel disease were excluded. Of the remaining 41,036 participants, 3,171 were excluded due to poor bowel preparation and 2,404 because of incomplete data. A final total of 35,461 participants was included in this study.Table The monthly average temperature and relative humidity during the study period (from 2004 to 2015) are shown in Fig. Figure The positivity rate, sensitivity, and specificity of FIT for ACRN were assessed according to ambient temperature and humidity Fig. . The posSensitivity tended to decrease at temperatures above 24 °C, although no statistical difference was identified. Moreover, sensitivity decreased at 80–90% humidity compared to 60–70% humidity. Although there was no statistical difference, ≥90% humidity tended to be related with low sensitivity.In contrast, specificity increased at high ambient temperature and humidity. Specificity at an ambient temperature ≥24 °C was higher than at 0–8 °C or 8–16 °C. Specificity at 80–90% ambient humidity was higher than at <60% ambient humidity.To assess the impact of ambient temperature and humidity on the sensitivity of FIT, we further performed a subgroup analysis of participants with CRC. Among 27 participants with CRC, 21 showed a positive result on FIT, and the sensitivity was 77.8%. The sensitivity was 100% in 3 participants who underwent FIT at a high ambient temperature (≥24 °C), while it was 50% in 4 participants who underwent FIT at high humidity (≥80%).The results of subgroup analyses according to the type of FIT device (HM-JACK and OC-SENSOR DIANA) are shown in Table Results of logistic regression analysis for the positivity rate, sensitivity, and specificity of FIT for ACRN are presented in Table In the univariable analysis, high ambient humidity was associated with low sensitivity, but high ambient temperature was not. However, in the multivariable analysis, high ambient humidity was also associated with low sensitivity .In the univariable logistic regression model for specificity, both high ambient temperature and high ambient humidity were significant factors. In the multivariable analysis, high ambient temperature and humidity (temperature ≥24 °C and humidity ≥80%) were associated with high specificity  of FIT. Neither high ambient temperature alone nor high ambient humidity alone was associated with specificity in the multivariable analysis.Figure 20. The sensitivity and specificity of FIT are unaffected by the characteristics of the individuals undergoing screening. Our previous study on the performance of FIT showed that the sensitivity was not affected by age, sex, smoking history, or obesity12. Although specificity decreased in individuals with diverticula or hemorrhoids, it was not influenced by clinical risk factors for ACRN, including male sex, obesity, and smoking12. Owing to these characteristics, FIT can be used for population-based screening regardless of the characteristics of the individuals being screened.FIT is a widely accepted noninvasive tool for screening for CRC20. Several studies have reported that the positivity rate of FIT was relatively low in the summer months16. In addition, a recent study by Cha et al. showed that the risk of interval cancer was 16% higher in individuals who underwent FIT in summer as opposed to in winter13.However, there is concern regarding the performance of FIT when fecal samples are obtained during the warm summer months15. Although the sensitivity and specificity of FIT were assessed in the study by Cha et al., only individuals who were diagnosed with CRC within 1 year after undergoing FIT were regarded as true-positive cases13. Given that some of the individuals with false-negative results on FIT may not be diagnosed within 1 year, the sensitivity of FIT may have been overestimated in that study. In the current study, the sensitivity and specificity of FIT could be calculated accurately, because our cohort underwent both colonoscopy and FIT. In addition, our study showed that the sensitivity and specificity as well as the positivity rate of FIT were influenced by high ambient temperature and high humidity. The multivariable analysis demonstrated that high ambient temperature (≥24 °C) and humidity (≥80%) decreased the positivity rate and increased the specificity of FIT. In addition, high ambient humidity decreased the sensitivity of FIT. This is the first clinical study to show that high humidity and high temperature can reduce the quality of FIT. Our findings suggest that optimal humidity and temperature are important in maintaining the quality of FIT.Whether ambient temperature actually affects sensitivity and specificity remains unclear, because most previous studies evaluated the performance of FIT based on its positivity rateet al.21, in which degradation of the fecal hemoglobin level was facilitated when samples were stored at 28 °C rather than at 4 °C and 20 °C. In addition, delayed return of fecal samples may cause false-negative results owing to hemoglobin degradation22. Another experimental study showed that minimizing both temperature and humidity is essential to maintain the integrity of the hemoglobin tetramer molecules19. When fecal samples were stored for 1 month at 37 °C, almost 100% of hemoglobin degraded at high humidity (>50%), whereas only 33–36% of hemoglobin degraded at low humidity (<30%)19. This result implies that not only temperature but also humidity is very important in maintaining the performance of FIT.The impact of ambient temperature on the performance of FIT is supported by a previous experimental study by Vilkin et al. from Germany reported that the sensitivity and specificity of FIT did not differ between low-temperature and high-temperature groups18; this might be attributable to the different geographic areas, with different climate signatures, where the studies were conducted. In southern Germany, where the study by Niedermaier et al. was conducted, the summers are not very humid23. Relatively low humidity in summer might prevent deterioration of the sensitivity of FIT. In contrast to the study from Germany, a study performed in Florence, Italy and a nationwide Korean study showed seasonal variation in the diagnostic performance of FIT14, and Italy and Korea are not only very hot, but also very humid in summer25. In particular, Korea has a regional climate characteristic of the rainy season, and typhoons are frequent from July to September; therefore, there are many hot and humid days in summer. In summary, the diagnostic performance of FIT may be influenced by humidity as well as by temperature. This impact of humidity on the sensitivity of FIT seemed to be seen even in participants diagnosed with CRC. Although tests of statistical significance were not conducted due to the very small sample size, sensitivity for CRC tended to decrease at high humidity.In addition, the impact of humidity on the performance of FIT may explain the contradictory results in previous studies focusing on the impact of ambient temperature on FIT. For example, Niedermaier Considering that the hemoglobin level might be decreased on humid days, it may be reasonable to lower the cut-off values of FIT on such days. We therefore investigated the changes in the diagnostic performance of FIT by reducing the cut-off values in humid environments, and we found that the sensitivity of FIT increased from 12.5% to about 20% by reducing the cut-off value to 10 μg hemoglobin/g feces . However, at the same time, the specificity decreased from about 97% to 95.5–95.9%. Given that the specificity was 96.3–96.8% at low humidity, 95.5–95.9% specificity seemed to be relatively poor. Therefore, lowering the cut-off value for FIT performed on humid days is not the perfect solution. Performance of FIT should preferably be avoided on humid days, such as those in the rainy season in Korea. However, if carrying out the test is unavoidable on humid days, it should be kept in mind that the sensitivity of FIT may be decreased. In this situation, reinterpretation of the results of FIT based on the reduced cut-off value may be considered.21. Van Rossum et al. reported that delays of more than 5 days in returning fecal samples increased false-negative results22. In our study, however, fecal samples were usually collected on the day before FIT was carried out or on the day of FIT. Second, two different devices were used for FIT, depending on the study period, and the performance of FIT may differ according to the type of device used. In the subgroup analyses, however, similar tendencies were observed in the diagnostic performance of FIT. In addition, there has been no evidence that the impact of seasonal variation differs according to the FIT device. The Korean study by Cha et al. showed that both types of tests  had lower positivity rates in the summer months than in winter13. The type of buffer formulation used in the FIT may be an additional issue. Recently, a novel buffer formulation for the OC-Sensor FIT has been developed, to minimize hemoglobin degradation of fecal samples exposed to high temperatures. An in vitro study showed that the new formulation buffer enhanced hemoglobin-stabilizing properties in high temperature environments (>22 °C)26. However, the clinical implications of the use of this new buffer on CRC screening should be further evaluated in a subsequent study. Third, the number of participants with ACRN was relatively small, although our study included a large number of participants. Therefore, the statistical power in the analysis of sensitivity was relatively low, and we could not identify the impact of high ambient temperature on the sensitivity of FIT. Although ambient temperature was not independently associated with low sensitivity in our study, the influence of ambient temperature may not be completely ruled out, considering the results of previous studies. Larger studies may be required for reaching a definitive conclusion. Finally, this study was performed in Korea, which has a characteristic climate; therefore, it is difficult to generalize our findings worldwide. However, our results can be generalized to countries or regions with a climate similar to that of Korea.Although our study demonstrated the impact of ambient temperature and humidity on the performance of FIT for ACRN, it has several limitations. First, we could not investigate the return time of fecal samples, and it is well known that degradation of hemoglobin is influenced by timeDespite the limitations, our research enhances the understanding of the impact of weather parameters on the diagnostic performance of FIT. Ambient temperature and humidity may affect the performance of FIT for ACRN. High ambient humidity decreased the sensitivity of FIT. In addition, a high ambient temperature with high ambient humidity decreased the positivity rate of FIT. These results have important implications for FIT-based screening programs in countries where temperature and humidity vary widely by season. In countries with such a climate, FIT-based screening may have to be avoided during hot and humid seasons.The Kangbuk Samsung Health Study is a prospectively established cohort study of South Korean men and women aged ≥18 years, who underwent a comprehensive annual or biennial health examination at two large health checkup centers, namely the Kangbuk Samsung Hospital Total Healthcare Center in Seoul and in Suwon, South Korea. The current study participants are a subset of the population in the Kangbuk Samsung Health Study conducted between 2004 and 2015, and included participants who underwent both screening colonoscopy and FIT as part of a comprehensive health checkup.27. In brief, according to the Industrial Safety and Health Law in Korea, employees should undergo a health checkup every one or two years. In addition, as part of the welfare policy, Korean companies often support the cost of health examinations, including CRC screening programs, regardless of the guidelines27. At our health checkup centers, a comprehensive health examination program includes screening colonoscopy and FIT. Although some participants chose either FIT or colonoscopy as a CRC screening modality, others prefer to undergo both examinations. The examinations carried out  depend on individual preference.Reasons for the large number of participants in this cohort who underwent both colonoscopy and FIT have been described previously28.Among participants aged ≥30 years, those who met the following criteria were excluded: (i) previous colonic examination, colorectal surgery or CRN; (ii) history of inflammatory bowel disease; (iii) poor bowel preparation; and (iv) incomplete data for analysis. Poor bowel preparation was defined as “large amounts of solid fecal matter found, precluding a satisfactory study; unacceptable preparation; <90% visible mucosa”This study was approved by the institutional review board of Kangbuk Samsung Hospital. The requirement for informed consent was waived since only de-identified data were retrospectively assessed. All experiments were performed in accordance with relevant guidelines and regulations.29. Body mass index (BMI) was calculated as measured weight (kg) divided by the square of the height (/m2), and obesity was defined as a BMI ≥ 25 kg/m2, which is the proposed cut-off value for Asian populations31.Demographic data, including age, sex, height, weight, family history of CRC, smoking habits, underlying diseases, and medications, were retrieved from an electronic medical database. Data on family history of CRC, smoking habits, and underlying diseases had been collected based on a self-administered questionnaire method. A family history of CRC was defined as the presence of the disease in first-degree relatives at any age. Participants were considered as having a history of smoking if they were former or current smokers who had smoked ≥100 cigarettes in their lifeOnly board-certified endoscopists were involved in carrying out the colonoscopic examinations, which were performed using an EVIS LUCERA CV-260 colonoscope . Four liters of polyethylene glycol solution were used for bowel cleansing. Suspicious neoplastic lesions were biopsied or removed by polypectomy or endoscopic mucosal resection.32.CRN was defined as cancer or adenoma, and ACRN was defined as cancer or advanced adenoma. Advanced adenoma was defined as the presence of one of the following features: tumor diameter ≥10 mm, tubulovillous or villous structure, and high-grade dysplasiaA single fecal sample was collected at home, using a sampling tube containing buffer designed to minimize hemoglobin degradation, on the day before FIT was performed or on the day of FIT. After being sealed in a plastic bag, the fecal sample in the tube was hand-delivered to the laboratory on the day of FIT. Date of FIT was defined as the date on which the fecal sample arrived in the laboratory. The fecal hemoglobin level was determined using the HM-JACK test  from 2004 to 2009, and the OC-SENSOR DIANA test  from 2010 to 2015. The cut-off value of FIT was 20 μg hemoglobin/g feces.www.weather.go.kr/weather/climate/past_table.jsp). As two centers, one in Seoul and the other in Suwon, were included in this study, weather information was collected for each of these locations. In this study, ambient temperature was defined as the average temperature of the day. The daily weather information was then merged into the clinical dataset of each participant, based on the date of FIT.Data about daily air temperature and relative humidity during the study period were collected from the official website of the Korea Meteorological Administration  was also included in the model, in order to consider the rainy season in Korea , with high temperatures and high humidity.Finally, to evaluate whether poor sensitivity of FIT in summer (or on hot and humid days) would improve by adjusting the cut-off value of FIT, we assessed changes in sensitivity and specificity when the cut-off value was reduced.P-value < 0.05 was regarded as being significant in group comparisons. Statistical analyses were performed using statistical software R .A Table S1. Diagnostic performance of the fecal immunochemical test according to the type of device used, ambient temperature, and ambient humidity.


Nanog, are dependent on the direct association of Sin3 for active transcription to occur. Sin3 appears to have properties of a co-repressor, co-activator and general transcription factor, and has thus been termed a co-regulator complex. Through a series of unique domains, Sin3 is able to assemble HDAC1/2, chromatin adaptors and transcription factors in a series of functionally and compositionally distinct complexes to modify chromatin at both gene-specific and global levels. Unsurprisingly, therefore, Sin3/HDAC1 have been implicated in the regulation of numerous cellular processes, including mammalian development, maintenance of pluripotency, cell cycle regulation and diseases such as cancer.At face value, the Sin3 histone deacetylase (HDAC) complex appears to be a prototypical co-repressor complex, that is, a multi-protein complex recruited to chromatin by DNA bound repressor proteins to facilitate local histone deacetylation and transcriptional repression. While this is almost certainly part of its role, Sin3 stubbornly refuses to be pigeon-holed in quite this way. Genome-wide mapping studies have found that Sin3 localises predominantly to the promoters of actively transcribed genes. While Sin3 knockout studies in various species result in a combination of both up- and down-regulated genes. Furthermore, genes such as the stem cell factor,  One of  I) . The ELM2-SANT domain of MTA1 (part of the NuRD complex) contains specific and highly conserved basic residues which help co-ordinate the negatively charged phosphates in InsP6 [in vitro [The highly related deacetylases, HDAC1 and HDAC2 (HDAC1/2) share 82% amino acid identity and form the catalytic core of numerous co-repressor complexes, which account for ∼50% of all cellular deacetylase activity in embryonic stem (ES) cells  and T-cein InsP6 . These rin InsP6 . The Sinin vitro . NonetheIn many regards, Sin3 is the prototypical co-repressor complex, that is, a multi-protein complex recruited to chromatin by DNA bound repressor proteins to facilitate local histone deacetylation and transcriptional repression –23. WhilO-GlcNAc transferase, OGT [The central platform of the complex is the Sin3 protein, which binds directly to HDAC1/2 and many other adaptors ,21,22, tase, OGT ; althougase, OGT ,40,42–44Understanding how different SAPs co-operate to recruit the Sin3 complex to chromatin is a crucial step towards understanding its functions in cells. With a dozen or more different SAPs it raises the question: are they all bound at the same time? And if not, how many different variants of the Sin3 complex are there? To address these questions Streubel et al.  employedOne well-studied sub-division among Sin3 complexes comes from work in yeast. Purification of Sin3/Rpd3(HDAC1) complexes identified two distinct biochemical entities of different apparent molecular mass, termed Rpd3 large  and Rpd3 small  ,53. Rpd3The domain structure of Sin3A/B is highly conserved from yeast to man . From N-The three PAH domains are imperfect ∼100 amino acid repeats with a conserved fold, consisting of four α-helices separated into pairs by a central loop ,61,64–66Kd in the sub-micromolar region, 50–200 nM) are guided by long-range electrostatic interactions. van Ingen et al. [Kd from 1.4 µM (Mxd1 5–20aa) to 0.4 µM (Mxd1 5–24aa). The increased affinity was the result of a long-range electrostatic attraction between Sin3B Lys165 (which sits above the hydrophobic cleft) and Mxd1 Glu23. An N:O bridge forms through hydrogen bonding which orients the SID for binding and brings it close enough to the PAH domain for the relatively short range hydrophobic interaction to occur. Sin3A-PAH2 contains an equivalent Lys residue, Lys315, whose charge also contributes to the binding of the Mxd1-SID [All three PAH domains show a significant degree of similarity and yet the specificity of interacting partners is quite distinct. The Mxd1-SID binds PAH2 but does not interact with PAH1 or 3 ,68; whiln et al.  observedMxd1-SID , suggestMxd1-SID ,64,70.in silico screen of small molecule inhibitor mimetics identified FDA-approved avermectin derivatives as PAH2 binders [In closing this section, it may be useful to reflect on the usage of the PAH domains. Data from Streubel et al. show that Sap30 is present in a 1 : 1 complex with Sin3A suggesting that PAH3 may be permanently occupied . This wo binders  which ph binders .Saccharomyces cerevisiae, which contains a single Suds3 gene, deletion causes dissociation of Sin3 and HDAC1 (Rpd3), and the remaining HDAC1 is enzymatically inactive [The binding site for HDAC1 in Sin3A was initially mapped between PAH3 and PAH4 (still a domain in 1997) and duly named the HID . This isinactive . Deletioinactive , indicatinactive . Dimerisinactive , which mA solution structure of the Suds3-SID bound to a portion of the HID (Sin3A 601–742 aa) has been described with a sub-micromolar affinity . CharactThe Sin3/HDAC complex is abundant, stable and ubiquitously expressed in mammalian tissues and cells. The temporal and cell type-specific nature of its activity, in numerous cellular processes, is mediated via transient association with transcription factors. Sin3 can therefore be viewed as a permanent tool in the chromatin toolbox, which can be recruited to specific loci and then applied to regulate chromatin accessibility in particular pathways by tissue-specific factors which interact with PAH, HID or Sin3a_C domains. Since its initial discovery as a transcriptional repressor of Myc target genes, via the association of PAH2 and Mxd1, Sin3 has been associated with numerous cellular processes, including mammalian development, maintenance of pluripotency, cell survival, cell cycle regulation  and Sin3 appears to play a role in each of them. The implication that Sin3 had a role to play in cell cycle was clear from the outset. Mxd1 (formerly Mad1) and Mxi1 are repressor proteins which compete with Myc for their common heterodimeric partner, Max, and form an anti-proliferative counter-weight to the pro-growth role of Myc . Yeast tSchizosaccharomyces pombe [Replication of the genome is an essential and rate-limiting phase of cell cycle. Critically, new DNA requires new chromatin. Histone H4 is initially deposited on nascent DNA in an acetylated form  whices pombe , with boes pombe . In summAlthough Sin3A and Sin3B are 57% identical  and sharConsistent with early embryonic lethality in mice, Sin3A has been reported to be crucial for the maintenance of pluripotency in ES cells ,100,101.Sin3A and Sin3B are highly expressed in all tissues . As permanent members of the chromatin toolbox, they can be recruited by a wide variety of tissue-specific transcription factors during tissue development. It is therefore unsurprising that KO studies have identified key roles for both proteins in a variety of tissue types. Sin3A/B are critical for the maintenance of haematopoietic stem cell homeostasis, with the different isoforms of Sin3 potentially regulating alternative pathways via unique interactions with transcription factors. Loss of Sin3A in the bone marrow of mice results in a reduction in haematopoietic stem cells and subsequent lineages indicating a defect in early haematopoiesis . DeletioAs both global chromatin regulator and gene-specific transcriptional co-regulator, the Sin3 complex, or rather complexes, play roles in all nuclear processes. The flexibility of multiple direct protein–protein interfaces  and a multitude of cofactors  allow its recruitment for the regulation of chromatin homeostasis throughout the cell cycle. Any process which requires access to DNA  will require the manipulation of histone acetylation and as a major HDAC complex, Sin3 will likely play a role. Understanding the complex set of protein–protein interactions, and perhaps teasing these apart as a targeted therapy, will drive Sin3 biology for many years to come.


Case-based learning (CBL) is a highly interactive instructional format widely used in medical education. One goal of CBL is to integrate basic biomedical knowledge and its application to concrete patient cases and their clinical management. In this context, we focus the role of teacher questions as triggers for reproductive vs. elaborative student responses. Specifically, our research questions concern the kinds of questions posed by clinical teachers, the kinds of responses given by students, the prediction of student responses based upon teacher questions, and the differences between the two medical disciplines in focus of our study, internal medicine and surgery.We analyse 19 videotaped seminars  taught by clinicians and attended by advanced medical students. Multiple raters performed a low-inference rating process using a theory-based categorical scheme with satisfactory interrater-reliability.We found that medical teachers mostly posed initial (instead of follow-up) questions and that their questions were more often closed (instead of open). Also, more reasoning (than reproductive) questions were posed. A high rate of student non-response was observed while elaborative and reproductive student responses had a similar prevalence. In the prediction context, follow-up reasoning questions were associated with low non-response and many elaborative answers. In contrast, the highest student non-response rate followed open reproduction questions and initial reasoning questions. Most reproductive statements by students were made following closed reproduction questions.These results deepen our understanding of interactive, questions-driven medical teaching and provide an empirical basis for clinical teachers to use questions in didactically fruitful ways. The application of basic biomedical knowledge to specific patient cases is a core element of medicine, as a discipline and practice. Instructional approaches such as case-based learning (CBL) have hence become essential elements of many medical curricula and they represent “fundamental ways in which future practitioners are educated for their new profession” , p. 52)..2–5], a dialogic teaching. This thriving strand of educational research capitalises upon the idea that didactically orchestrated classroom discussion is a contemporary and powerful pedagogic strategy )Mangold Interact [who was speaking  and what kind of utterance was made by students (reproductive statement / elaborative statement / content-related question / other statement) and by teachers . This means we segmented the entire video into consecutive time intervals of varying length representing specific activities by teachers and students and breaks which occurred between these activities. In coding round two, we further categorized these activities as is elaborated in the following section categorical scheme (whereby we only specify the codes relevant in the present study). For an initial training phase, two videos of case-based seminars were coded by all four raters. These videos had been recorded in a pilot study and were not part of sample for this study. We calculated interrater agreement between pairs of raters and simultaneously visualised different raters’ coding solutions. On this basis, passages in the seminars in which differences between coders had emerged could be identified easily. These passages were discussed, differences in agreement were resolved, and coding rules refined. This was done in an iterative manner until overall interrater agreement between all four coders, measured by Cohen’s Kappa, reached a satisfactory value of .80 . The main study videos were divided between the four raters. Two of the main study videos were analysed by all four coders, resulting in an overall IRR-value of Cohen’s Kappa = 0.65 and pairwise values between .59 and .66, which can be regarded as satisfactory [Coding was done by four researchers  from our group using the software Interact . In rounsfactory .didactically irrelevant teacher questions are unrelated to the learning goals of the seminar and were hence not considered in the further analyses. Didactically relevant teacher questions were further described by the coding schemes type of question, openness of question, and cognitive level of question [type of question, we differentiated initial and follow-up questions  questions. The former are intended to elicit information that should already be known to students, e.g. from pre-clinical medical education. The latter are asked to elucidate cause-effect relationships, differentiate intended from unintended consequences of therapeutic measures, or compare advantages and disadvantages of different therapies and their relevance for the particular case at hand.We analysed the video material using a hierarchical categorical scheme which was based upon published rating schemes , 37. Thequestion . Regardiother. As this was the case in a very small number of instances, we excluded these utterances from all further analyses (cf. results section).Further, we coded different types of student statements made in response to teacher questions. The main categories here were reproductive / elaborative statements and non-response . Boxplots are used to display the distribution of the relative frequencies of the categorised question characteristics within seminars. Corresponding hypothesis testing on the differences between seminars from internal medicine and surgery was performed by two-sided Mann-Whitney U tests on exploratory 5% significance levels. Teacher questions were investigated as predictors of student answers by evolutionary learning of globally optimal classification trees . This maThe current study was approved by the ethics committee of the TUM Rechts der Isar University Hospital (Application code 400/16 S). All teachers and students were informed about the study prior to the seminar via email. One physician refused to take part, so we did not record that respective seminar. At the beginning of each seminar (before starting the video recording), one person from the research team was present to inform all participants about the study, answer open questions, and obtain consent in written from all individuals. We started the video recording only after consent had been declared by all individuals in the room. In one seminar, one student declared unwillingness to be filmed. The student was offered to be seated right beside camera one . In contrast, students made more responses in internal medicine (p = 0.17 for absolute numbers and p = 0.12 for questions per minute).Table To answer this question, we describe the prevalence of the different qualities of teacher questions in our video data. Each single teacher question is characterized by a combination of categories, hence a separate analysis of the prevalence of different combinations is reported below. Figure MD = 6%) or were organizational questions (MD = 7%). However, there were outliers in organizational questions, indicating a relatively high prevalence  of such questions in two seminars. Regarding teacher questions posed with a didactical intention, we first look at the categorical scheme type of question, comprising initial and follow-up questions. As is apparent from Fig. openness of questions, we found that clinical teachers asked much more closed (70%) than open questions (28%). Looking at the cognitive level of teacher questions, the clinical teachers in our sample asked more than two times as many reasoning questions (67%) than reproduction questions (31%). One discipline difference emerged in the area of comprehension questions (p = 0.05) with physicians from internal medicine having asked more questions of this kind than their colleagues from surgery.As was expected, few of the questions posed by clinical teachers were simply focused upon comprehension of student statements , whereas the amount of elaborative and reproductive responses was almost equal (MD = 17%). Very few student responses were concerning organizational matters (3%) or were content-related questions (1%).If looking at the median values reported in Fig. To answer research question 3, we used the evolutionary trees method as explicated in the methods section.cognitive level of teacher questions makes a difference, i.e. whether teacher questions are reproductive or reasoning-oriented. Reproductive questions are associated with a higher amount of reproductive answers (nodes 3 and 4) while reasoning-oriented questions trigger more elaborative answers (nodes 6 and 7). A further difference (node 2) can be found between open and closed reproduction questions. Open vs. closed reproductive questions are associated with a lower degree of reproductive student responses (31% vs. 53%), more elaborative student responses (11% vs. 5%), and a higher student nonresponse (57% vs. 42%). Regarding absolute amounts, closed reproduction questions are posed much more frequent (more than 14 times more often) than open reproduction questions. The right branch originating from node 1, reasoning questions, is indirectly connected to nodes 6 and 7 at the bottom. Overall, these are characterized by higher levels of elaborative answers compared to nodes 3 and 4. The highest degree of elaborative student answers—50% in node 6—is associated with follow-up reasoning questions. Node 6 is also characterised by the lowest rate of student non-response in our analysis (30%) and by a comparatively low degree of reproductive student answers (21%). Finally, node 7 is connected to initial reasoning questions and is characterised by a comparatively high degree of student nonresponse (61%). Such questions also trigger elaborative (23%) and reproductive (23%) student answers. As is apparent, node 7 summarises as much as 945 teacher question-student (non-)response sequences. So, the pattern described here is very frequent in our sample of CBL seminars, even more frequently than closed reproduction questions posed by the teachers followed by student (non-)responses. Finally, we found no evidence for a differential functioning of specific question types in the two disciplines.Starting from node 1, it is apparent that the internal medicine and surgery. Thereby, we have analysed how students’ responses to teacher questions (reproductive and elaborative responses vs. student non-response) depend upon different qualities of questions posed by clinical teachers . In the literature, CBL is mostly described as a format which gives students high degrees of freedom to reflect and to engage in clinical thinking . O–6. OinteRegarding teacher questions associated with a high degrees of student elaboration, our results show that reasoning questions are powerful in this respect cf. Fig. . Also, fhow clinical teaching actually works and is practiced by clinical teachers. It can help strengthen a descriptive perspective in clinical teaching research and better understand how certain teaching methods are implemented [In theorizing about what makes CBL effective, authors have provided elaborate, yet often quite general, descriptions of the behaviours of both teachers and learners, e.g. “the facilitator orchestrates the discussion to maximize the breadth and depth of the dialogue” or “advances the learning objectives for which the case was designed by formulating a sequence of trigger questions” lemented  while noout-talked their students [teacher wait time  is a is aout-e . Seconde  clinicae  was rather small in these seminars – which also means that these seminars were not very interactive, but were more like lectures anchored in example cases with occasional episodes of student-teacher interaction. A critical concern which could be connected to this observation is whether it is legitimate to consider the seminars we observed as valid examples of CBL in the first place. We argue that it can be seen as a strength of the present study to include a broad range of ways in which CBL was implemented (from more teacher-guided to more-student centred). Due to this heterogeneity, our study might not reflect the concept of CBL as discussed in the literature in its purest form. However, we argue that our research has high ecological validity in that it reflects that CBL might be interpreted and practiced by different clinical teachers in very different ways.Regarding the comparison between internal medicine and surgery, we only found minor differences. On this basis, it does not seem justified to conclude that CBL works different in various medical disciplines. However, our findings indicate that there is substantial heterogeneity between how different teachers practice case-based teaching (regardless of discipline). This is apparent from several outcomes; overall, the CBL-teachers posed quite few didactically irrelevant questions. However, we had seven seminars in our sample in which between 20% and just over 50% of the questions posed were focused upon organisational aspects response dyads. By pursuing a more high-inference analytic strategy, longer passages and several turns of teacher-student interactions could have been analysed. This surely is a promising idea for future research. In the field of empirical educational research, a certain trend away from low-inference analytic approaches in video studies is apparent . A furthThe present study gives detailed insights into associations between clinical teacher questions and medical student responses in CBL. They are relevant for medical teaching practice and professional development. Some concrete recommendations to clinical teachers can be deducted: When seeking to make students elaborate, teachers should primarily ask reasoning questions or follow-up questions. For eliciting reproductive answers from students, closed reproduction question are suitable. To increase student participation, teachers should strive for low student non-response rates. This can be achieved in different ways. Henning et al.  recommen


We calculated the band structures of a variety of N- and S-doped graphenes in order to understand the effects of the N and S dopants on the graphene electronic structure using density functional theory (DFT). Band-structure analysis revealed energy band upshifting above the Fermi level compared to pristine graphene following doping with three nitrogen atoms around a mono-vacancy defect, which corresponds to p-type nature. On the other hand, the energy bands were increasingly shifted downward below the Fermi level with increasing numbers of S atoms in N/S-co-doped graphene, which results in n-type behavior. Hence, modulating the structure of graphene through N- and S-doping schemes results in the switching of “p-type” to “n-type” behavior with increasing S concentration. Mulliken population analysis indicates that the N atom doped near a mono-vacancy is negatively charged due to its higher electronegativity compared to C, whereas the S atom doped near a mono-vacancy is positively charged due to its similar electronegativity to C and its additional valence electrons. As a result, doping with N and S significantly influences the unique electronic properties of graphene. Due to their tunable band-structure properties, the resulting N- and S-doped graphenes can be used in energy and electronic-device applications. In conclusion, we expect that doping with N and S will lead to new pathways for tailoring and enhancing the electronic properties of graphene at the atomic level. It e [2 g−1 ,5, whiche [2 g−1 . Graphenm2 V s−1 , good th1 [−1K−1  and high1 [−1K−1 . Due to 1 [−1K−1 , optoele1 [−1K−1 , energy-1 [−1K−1 ,13,14, s1 [−1K−1 , and cat1 [−1K−1 . It is e1 [−1K−1 ,31,32,331 [−1K−1 ,29,30,331 [−1K−1 ,23,24. T1 [−1K−1 ,31,32,33−6 eV per atom and the maximum force converged to below 0.05 eV Å−1. The graphene model used in our simulation consisted of a 12.3 × 12.3 × 15.0 Å, 5 × 5 supercell with a vacuum thickness of 15 Å, which avoids interference between adjacent graphene layers. Brillouin-zone integrations were carried out using a 4 × 4 × 1 Monkhorst–Pack Κ-point grid. The effects of van der Waals (vdW) interactions were included using the empirical DFT-D3 correction within the Grimme scheme [First-principles density functional theory (DFT) calculations were carried out using the Vienna Ab Initio Simulation Package (VASP) ,35. Geome scheme . All atoe scheme ,46.2-hybridized N atoms bonded to two neighboring sp2-hybridized C atoms. Using this configuration as the starting point, different configurations of N- and S-doped graphene with mono-vacancy defects were built as simulation models. Three C atoms around a mono-vacancy defect were substituted with different numbers of atomic N and/or S dopants. Regarding model structures in this investigation, research groups successfully reported the synthesis of N- and S-doped graphene [Various configurations exist for the doped and defective graphene chosen as the anode material in a lithium-ion battery (LIB). For instance, N-doped graphene exists in distinct forms that include graphitic, pyridinic, and pyrrolic N atoms . Among tgraphene ,62,63,64graphene .The bandgap clearly opens after doping with N or S, and/or the introduction of a mono-vacancy defect, which is ascribable to the effects of the atomic dopant and/or vacancy defect on the π electrons in the hexagonal rings. Mono-vacancy defects lead to shortages of whole charges compared to pristine graphene, which downshift the Fermi energy, indicating that the mono-vacancy defect acts as a hole dopant with missing π electrons. As shown in On the other hand, N and S atoms have one and two additional valence electrons, respectively; hence, doping with N or S results in an upward shift in the Fermi energy. N1′ and CN1′′ atoms, and +0.143e and +0.082e on the CN2′ and CN2′′ atoms, respectively. The charges on the C atoms on each side of the N atom are almost identical. On the other hand, the C atoms bonded to the S atom exhibit negative charges, at −0.242e on CS1′, and −0.246e on CS1′′. Likewise, the 1N2S-gra system showed a similar trend. As shown in N1′ and CN2′′, bear charges of +0.109e and +0.110e, which are almost identical. The C atoms adjacent to the S atoms exhibit negative charges, with charges of −0.248e and −0.240e on CS1′ and CS1′′, and −0.238e and −0.250e on the CS2′ and CS2′′ atoms, respectively. Finally, the 3S-gra system exhibited charges on the C atoms bonded to the S atoms that were in the −0.245e to −0.233e range. It seems that polarization in the doped region increases with increasing S concentration. The transformed charge-density distribution following doping, as well as the vacancy defect, affects the electronic properties of the graphene system.Finally, we studied the charge-density distribution of each atom around the mono-vacancy defect for each N- and S-doped graphene system by Mulliken population analysis because the charge distributions on the carbon, nitrogen, and sulfur atoms are important for determining the origin of the alternating electronic properties. The present density functional theory study aimed to reveal details of the electronic structures of several N- and S-doped graphenes in order to understand the effects of the N and S dopants on the graphene electronic structure. We found that the band structure of graphene can easily be tuned by doping with N and S atoms. The roles of the atomic N and S dopants on the band energies were clearly revealed; these dopants noticeably perturb the band shapes and open the bandgap at the Dirac point, compared to graphene itself. The band energies of 3N-doped graphene were upward shifted below the Fermi level compared with those of pure graphene, and showed p-type behavior. The band structure exhibits a remarkable electronic transition, from “p-type” to “n-type”, in moving from 3N-gra to 3S-gra (with increasing numbers of S atoms) with a downshifting of the band energy below the Fermi level. Moreover, Mulliken population analysis revealed that the atomic N dopants bear negative charges, whereas the atomic S dopants bear positive charges in N- and/or S-co-doped graphene systems, which is ascribable to differences in the electronegativities and numbers of valence electrons among the C, N, and S atoms. In each N- and/or S-co-doped graphene system, all of the N atoms bear negative charges, whereas all of the S atoms bear positive charges. The average charge on the N atoms gradually increases with decreasing numbers of N atoms in the N- and/or S-co-doped graphene system. In contrast, the average charge on the S atoms decreases with increasing numbers of S atoms in the co-doped graphene system. Due to their tunable band-structure properties, the resulting N- and S-co-doped graphenes can be used in energy and electronic-device applications. In conclusion, we expect that doping with N and S will lead to new pathways for tailoring and enhancing the electronic properties of graphene at the atomic level.


Moreover, the significant enhancement of photoluminescence can be ascribed to the coupling of the light emitters in multiple quantum wells with the surface plasmon Ag NPs and the EIT effect.In this work, the synthesis and characterization ofgraphene-metal nanocomposite, a transparent conductive layer, is examined. This transparent conductive layer is named graphene-Ag-graphene (GAG), which makes full use of the high electron mobility and high conductivity characteristics of graphene, while electromagnetically induced transparency (EIT) is induced by Ag nanoparticles (NPs). The nanocomposite preparation technique delivers three key parts including the transfer of the first layer graphene, spin coating of Ag NPs and transfer of the second layer of graphene. The GAG transparent conductive nanocomposite layer possess a sheet resistance of 16.3 ohm/sq and electron mobility of 14,729 cm However, the GAG transparent conductive layer would increase the optic power due to plasma-induced enhancement which can be seen in GAG must be GAG (MAX) = 7 × 105, JGA (MAX) = 5.9 × 105)  [A question to consider is why the effective radiated power of GAG is better than GA. If the simple transparency (T) formula (9 × 105) . The ele106 S/m) ,55. This2/(v s),and its sheet resistances are 16.3 ohm/sq. The PL of LED has significantly improved due to the performance of the GAG transparent conductive layer. In conclusion, GAG is a sounded, promising, transparent conductive layer to meet the future demand of light devices.Based on the study of materials of theindex dielectric layer to cover NPs andthe radius of nanoparticles and period of the plasmon, a graphene-metal nanocomposite transparent conductive layer of GAG was designed.Due to the plasma excited effect of Ag NP and double-layer graphene electromagnetically induced effect, the effective radiated power of the GAG transparent conductive layer is reaching 1.33, its carrier motilities are 14,729 cm


Damage to the corticospinal pathway often results in weak dorsiflexion of the ankle, thereby limiting the mobility of people with multiple sclerosis (MS). Thus, strengthening corticospinal connectivity may improve locomotion. Here, we investigated the feasibility of tibialis anterior (TA) motor-evoked potential (MEP) operant conditioning and whether it can enhance corticospinal excitability and alleviate locomotor problems in people with chronic stable MS. The protocol consisted of 6 baseline and 24 up-conditioning sessions over 10 weeks. In all sessions, TA MEPs were elicited at 10% above active threshold while the sitting subject provided 30–35% maximum voluntary contraction (MVC) level of TA background EMG. During baseline sessions, MEPs were simply measured. During conditioning trials of the conditioning sessions, the subject was encouraged to increase MEP and was given immediate feedback indicating whether MEP size was above a criterion. In 3/4 subjects, TA MEP increased 32–75%, MVC increased 28–52%, locomotor EMG modulation improved in multiple leg muscles, and foot drop became less severe. In one of them, MEP and MVC increases were maintained throughout 3 years of extensive follow-up sessions. These initial results support a therapeutic possibility of MEP operant conditioning for improving locomotion in people with MS or other CNS disorders, such as spinal cord injury and stroke. Transcranial magnetic stimulation (TMS) and its motor-evoked potential (MEP) have been widely used to study corticospinal excitability and connectivity noninvasively . GeneralOperant conditioning is a powerful method for modifying a behavior based on its consequences. The past 35 years of spinal reflex operant conditioning studies in monkeys, rats, mice, and humans show that even a simple spinal reflex behavior can be gradually changed through learning and practice –31. AnimThe purpose of this study was to investigate the feasibility and a therapeutic possibility of MEP operant conditioning for improving locomotion in people with chronic stable MS.The MEP operant conditioning protocol used in this study is a modified version of the conditioning protocols developed and used in human subjects with and without spinal cord injury , 37, 38.At the beginning of the study, the subject was familiarized with the protocol and TMS in several preliminary sessions. Then, each subject completed 6 baseline sessions and 24 up-conditioning sessions that occurred at a pace of 3 times per week . In one M-wave threshold to just above the maximum M-wave (Mmax) [Mmax measurement was followed by control MEP trials  were placed over the muscle bellies of TA and soleus for EMG recording , 41. EMGe (Mmax) . This TAP trials . In all P trials . The cri∗; (3) ability to ambulate with or without an assistive device  2 to 6 [∗Except for subject A, who had been taking 4-aminopyridine (Ampyra®) for 5 years at the time of enrollment, none were on neuromodulatory medication . These subjects maintained their long-term MS medication, such as beta interferons , rituximab (Rituxan®), and dimethyl fumarate (Tecfidera®), throughout the 10 weeks of main study period.Four women (age: 47–56 years) with chronic foot drop due to secondary progressive MS participated. Inclusion criteria were (1) neurologically stable for >6 months; (2) medical clearance to participate (with the expectation that current medication and therapy schedule would be maintained without change for at least 4 months)) 2 to 6 ); and  or a bat-wing coil (in subject A) with radii of 9 cm , held over the scalp such that the induced current flowed in the posterior–anterior direction in the brain. When the absolute TA EMG level was maintained within the preset range , anre study , 45–47. Along with control and conditioned MEP size measurements, we also measured the silent period (SP) as the period from the end of the MEP to the recovery of background EMG activity in 50% of the responses , 46, 48.Overground locomotor EMG activity was measured before and after the 24 conditioning sessions. Subject B, who wore an ankle foot orthosis on her unconditioned leg, was asked to remove it for this measurement. Surface EMG was recorded from the TA, soleus, biceps femoris, and vastus lateralis bilaterally. To detect foot contact for each leg, foot-switch cells were inserted between the subject's shoes and feet. For analysis, the foot-contact signal was used to define the beginning of the step cycle. Rectified EMG signals from individual steps were normalized to the mean step cycle time and averaged together to obtain the locomotor EMG activity over the step cycle , 49, 50.For each session of each subject, we calculated average MEP sizes for the 20 within-session control trials and for all three 75-trial blocks together. For these calculations, MEP size was defined as average absolute EMG in the MEP interval minus average background EMG. Changes in these MEP sizes across sessions were quantified in percent of their average values for the 6 baseline sessions. We also determined for each subject the final effects of conditioning on the conditioned MEP by averaging the MEPs for the 75-trial conditioning blocks of conditioning sessions 22–24 and expressing the result in % of the average MEP for the 75-trial blocks of the 6 baseline sessions.  The final effect on the control MEP was calculated by averaging the MEPs for the 20 within-session control trials of conditioning sessions 22–24 and expressing the result in % of the average MEP for the first 20 trials of the 6 baseline sessions (see ).t-test (two-tailed) [Mmax and background EMG levels over all sessions and assess changes in MVC and SP, a factorial ANOVA was used across successive 6-session blocks  [Mmax and TA and soleus background EMG levels in control and conditioning trials remained stable throughout the study . Typically, TA Mmax remained within ±15% to the mean across all the sessions, TA background EMG remained within ±5%, and soleus background EMG remained under 5 μV  throughout the study.To determine for each subject whether MEP up-conditioning was successful, the average conditioned MEPs of the final 6 conditioning sessions were compared to the average MEPs of the 6 baseline sessions by unpaired -tailed) , 37. To In 3 of the 4 subjects, TA MEP increased significantly, together with the TA MVC; and at the end of conditioning, all three spontaneously reported better leg movement in their daily walking. Here, we describe each subject's results individually.Over the course of conditioning, her left TA MEP increased progressively. Her final conditioned MEP size was 175% of the baseline value . This inThere were several noteworthy observations over the course of study. First, this subject had no visible voluntary ankle dorsiflexion at the beginning and needed a cane to compensate for the limited motion in the left leg. After 24 conditioning sessions, she was able to flex the left ankle even against resistance ; and at Mmax remained around 3.8 mV for the entire 172 weeks of her study). An increased MEP and decreased SP duration were also present in most of the follow-up sessions. At the study conclusion , voluntary ankle dorsiflexion remained visible and improved.In this subject, 66 follow-up sessions were performed over 161 weeks (3 years) after the last conditioning session . During The TA MEP increased over the course of study . Her finAfter conditioning, the conditioned leg's TA burst in the late swing phase of walking and soleus burst during the stance phase were clearly increased . LocomotHer final conditioned MEP size was 172% of the baseline value , which cThe TA burst in the swing phase of locomotion increased after conditioning in both the conditioned and the contralateral legs . LocomotMEP up-conditioning did not increase her MEP . Her finIn three of 4 subjects with MS, up-conditioning successfully increased MEP over the course of 24 conditioning sessions, supporting the possibility and feasibility of MEP operant conditioning in people with chronic stable MS.In two of the 3 successfully conditioned subjects, the conditioned MEP increase consisted of within-session task-dependent adaptation and cumulative long-term change, similar to previous studies of H-reflex operant conditioning , 37, 38.B receptor agonist and known to prolong the SP duration [B-mediated intracortical inhibition [In all of these subjects with MS, we noticed that the SP duration was very long  during the baseline sessions. Since baclofen, a common antispastic medication for people with MS, spinal cord injury, and cerebral palsy –53, is aduration , 55, onehibition –58. The hibition  that mayhibition . In subjhibition ). ConsidIn 3 of the 4 subjects, operant up-conditioning of TA MEP was successful and was accompanied by increased MVC, increased TA burst during the swing-phase of locomotion, and improved EMG modulation during locomotion. In 2 of the 3 subjects, the ankle dorsiflexor strength and walking speed also improved; in the third subject, disease progression near the end of the study may have prevented walking speed improvement despite significant increases in MEP, MVC, and locomotor EMG modulation. The increased corticospinal drive to the conditioned TA may explain increases in TA MEP, TA MVC, and TA burst amplitude during the swing phase of walking , 26, butOne of the cases presented here (subject A) has an important implication for future therapeutic applications. Over 3 years of follow-up sessions, her TA MEP, MVC, and SP improvements and the voluntary ankle dorsiflexion that she had not had before conditioning were essentially maintained , despiteThe present study investigated the feasibility of MEP operant conditioning in a small number of individuals with MS. Thus, while the initial results support a therapeutic possibility of this approach for improving locomotion in people with MS , its wider applicability is yet to be confirmed through future clinical studies of larger scales.