Theoretically, elements with hexadecapolar outer shells could also exist, but none of the known elements have filled g-orbitals. On the other hand, the research paradigm of ‘colloidal atoms' displays complexity of particle behaviour exceeding that of atomic counterparts, which is driven by DNA functionalization, geometric shape and topology and weak external stimuli. Here we describe elastic hexadecapoles formed by polymer microspheres dispersed in a liquid crystal, a nematic fluid of orientationally ordered molecular rods. Because of conically degenerate boundary conditions, the solid microspheres locally perturb the alignment of the nematic host, inducing hexadecapolar distortions that drive anisotropic colloidal interactions. We uncover physical underpinnings of formation of colloidal elastic hexadecapoles and describe the ensuing bonding inaccessible to elastic dipoles, quadrupoles and other nematic colloids studied previously.Outermost occupied electron shells of chemical elements can have symmetries resembling that of monopoles, dipoles, quadrupoles and octupoles corresponding to filled  Colloidal systems can form bulk phases such as liquid and crystals, but they also exhibit interesting behaviours that have no atomic analogues. Here, by dispersing solid polymer microspheres in a nematic liquid crystal, Senyuk et al. demonstrate spontaneous formation of hexadecapolar nematic colloids. Using a combination of holographic optical tweezers (HOT)1n(r)-distortions and quantify colloidal pair interactions by measuring distance and angular dependencies of elastic potentials, demonstrating relations between the director structure and medium-mediated inter-particle forces. Finally, we explain our findings using a model based on elastic multipole expansion and discuss how the experimental framework we have developed may enable colloidal self-assembly into novel forms of tunable pre-engineered matter without known atomic analogues.In this work, we describe colloidal elastic hexadecapoles that spontaneously form around solid microspheres immersed in a uniformly aligned NLC. The unusual symmetry of elastic distortions arises from conically degenerater0 locally distort n(r), which is manifested by eight bright lobes around the particle perimeter seen in POM between the crossed polarizer and analyser (n(r) at the particle's perimeter is parallel to polarizer or analyser. Using a phase retardation plate and interference of polarized light propagating through the particle-distorted structure, we reveal that n(r) tilting away from the far-field director n0 switches between clockwise and counterclockwise directions (corresponding to the blue and yellow colours in the micrograph) eight times as one circumnavigates the sphere 9n(r) distortions schematically shown in n(r) with respect to PSM's local surface normals s, which were previously demonstrated for NLCs at flat polystyrene-coated surfacesψe  depicted in nx of n(r) onto the x-axis orthogonal to n0 can be visualized around PSM using colours that highlight positive, near zero and negative nx (n(r) at the NLC-PSM interface and correspond to the boojum and Saturn ring topological defects, respectively. Away from the particle surface and these topological singularities, the experimentally reconstructed n(r) is continuous  of a radius analyser . These be sphere . Bright-rfacesψe . To miniative nx . The blantinuous  and consR orientations with respect to n0 as well as at different separation distances. Using video-microscopy, we track the ensuing particle motions, which result from a combination of Brownian jiggling and elasticity-mediated interactions. The submicron waist and relatively low power (∼10 mW) of a focused trapping beam allow us to avoid the influence of the trapping on the measurementsR are shown in 7815r0. However, the angular dependence of these forces is very rich, with eight angular segments of inter-particle attractions separated by eight angular segments of repulsions, with the intermediate angular sectors within which particles move sideways as the inter-particle elastic forces are orthogonal or at large angles to R  and the simplified equation of motion 0≈−ξFint yield the pair interaction potential Uint r0 and stable R orientations with respect to n0 within one of the two angular sectors of assembly in each quadrant dependent on ψe: θ1≈22°-26° or θ2≈64°-75°  from n0= are small. Assuming n(r)≈, the NLC elastic free-energy reads7823To model experiments, we exploit the electrostatic analogy of the far-field director distortions because of a colloidal sphere that can be represented as elastic multipoles789101112232425K is an average Frank elastic constantnμ=0, with solutions expanded into multipoleswhere al=blr0l+1 is the elastic multipole moment of the lth order (2l-pole)22232425bl from exact solutions for n(r) or from relevant experiments (n(r) is symmetric about the particle centre, similar to analogous electrostatic charge distributions that have no dipole or octupole electric moments22232425n(r) shown in b2, b4 and b6, which also determine the colloidal pair-interaction energy24where eriments  and 3a. Pl+l′(cosθ) are the Legendre polynomials. For colloidal quadrupoles, b2 dominates and b4 and b6 can play a role only at smallR. For our particles (n(r) can be qualitatively understood as a superposition of configurations of two separate quadrupoles, one with the Saturn-ring and one with boojums910111622232425R. Fitting experimental R(t) within different angular sectors of pair interaction with the corresponding theoretical predictions yields a unique set of parameters b2, b4 and b6  that is found playing a dominant role, and even the higher-order term (64-pole) plays a detectable role at relatively small R -distortions of quadrupolar configuration1624272316232728293031n0 =Ncos(mϕ)Plm(cosΘ) with =,  and , respectively, where N is a normalization constant, Θ is a polar and ϕ is an azimuthal angles, Plm(cosΘ) is the associated Legendre polynomial; the constant l determines the order of a multipole  and –l≤m≤l. This close analogy between electrostatic and elastic colloidal multipoles may help devising approaches for self-assembly of colloidal mesostructured composite materials.It is instructive to compare hexadecapolar NLC colloids to other known elastic multipoles and their electrostatic analogues. Colloidal spheres with strong tangential anchoring at their surface form 293031n0 . Such a To conclude, we describe hexadecapolar NLC colloids with unusual field configurations, highly anisotropic elastic interactions, and versatile forms of self-assembly. Our findings pose a challenge of realizing pure NLC colloidal octupoles, lower order multipoles that have not been observed so far, and provide the means of probing the role of hexadecapolar moments in inter-molecular interactions using colloidal model systems. Self-assembly of hexadecapolar NLC colloids is expected to yield a diverse family of two- and three-dimensional low-symmetry crystal lattices. Beyond the rich experimental platform for fundamental studies, our colloids have potential uses in designing and realizing reconfigurable ‘soft' photonic crystals383926812r0≈2.05, 2.65 and 4 μm and with conically degenerate surface boundary conditions were prepared using one of two different methods as follows43434445D=2r0≈4.1 μm) colloidal spheres, which were used as PSMs in our experiments, intrinsically provide conic degenerate surface boundary conditions for the NLC director. In the second preparation method, we used polystyrene divinylbenzene spherical particles PC06N  of D≈8 μm and DC-05 (Thermo Scientific) of D≈5.3 μm dispersed in 5CB. As dispersed, these particles provide tangential alignment for 5CB molecules and the director field n(r). However, after keeping these colloidal dispersions in 5CB at the elevated temperature of ≈100 °C for about 12 h, the alignment at the surface of these polymer particles changed to a conical degenerate 2/2 costs (ref. n(r) to a polar angle ψ away from the easy axis at ψe relative to the bulk elastic energy density is characterized by the so-called conical anchoring extrapolation length K/Wc∼(50-500) nm <<r0, whereWc∼10−5 J m−2. Since K/Wc<<r0, one can assume that the tilted conically degenerate boundary conditions are rigid with respect to changing ψ. To prepare samples with NLC dispersions in a uniformly aligned host, dry PSMs were added to 5CB. The dilute NLC colloidal dispersions were then filled into the cells comprised of two glass plates separated by glass spacers defining the cell gap in the range 15-30 μm, which were sealed at all edges using fast-setting epoxy glue after the filling process. The inner surfaces of cell substrates were treated with polyimide PI2555 (obtained from HD Microsystems) for in-plane homogeneous alignment of the far-field director n0 defined by unidirectional rubbing.Colloidal PSM particles of radii generate , similarsts ref.  for deviPOM , nonline19n(r) and form elastic hexadecapoles in the aligned NLC .Supplementary Figures 1-6, Supplementary Note 1-2 and Supplementary References


CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration–response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC50  or by response to low nicotine  when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted . These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the  CHRNA2-A7, CHRNA9-10) and the β subunits by three genes (CHRNB2-B4). The α7, α9 and α10 subunits can form functional receptors without incorporating a β subunit, while a minimum of two α and two β subunits  are required to form functional heteromeric receptors. Certain combinations of receptor subunits are more common in the central nervous system (CNS), and there is regional specificity with regard to subtype expression in the mammalian CNS Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels formed from numerous combinations of receptor subunits, each encoded by a separate gene. Neuronal nAChR α subunits are encoded by eight genes in mammals (CHRNA5 is the most strongly associated single nucleotide polymorphism (SNP) in several genome-wide association studies (GWAS) of smoking quantity CHRNA5, that alter CHRNA5 mRNA expression in vivo, alter risk for both nicotine and alcohol dependence CHRNB3-CHRNA6 gene cluster also are associated with cigarette consumption in a recent GWAS CHRNA3, CHRNA4 and CHRNB4, with the risk for nicotine dependence Variants in or near nicotinic acetylcholine receptor genes have been found to be associated with nicotine related behavior in humans. A non-synonymous change  in CHRNB4 identified in a deep resequencing study of a cohort of nicotine-dependent and control subjects were associated with reduced risk of developing nicotine dependence in vitro tests of functional consequences of non-synonymous coding variants can significantly improve the association between genotype and phenotype. We report the functional impact of rare variants in CHRNB4, and results that demonstrate that incorporating information on the functional consequences can improve the association between genotype and the complex behavioral phenotype of nicotine dependence. Furthermore, the results suggest that an increased response to low concentrations of nicotine may reduce the risk of developing nicotine dependence.We have provided evidence that rare variants in De-identified data from the Collaborative Genetic Study of Nicotine Dependence (COGEND) were used. All participants in COGEND provided written informed consent for genetic studies and agreed to share their DNA and phenotypic information for research purposes. The Washington University Human Research Protection Office granted approval for data to be used for this study.Full length coding sequences for the human nicotinic α3 (NP_000734.2) and β4 (NP_000741.1) subunits were kindly provided by Dr. J. Lindstrom . Subunits were sub-cloned into the pcDNA3 vector . The FLAG epitope [DYKDDDDK] was introduced into α4 between the 6 and 7 positions of the mature polypeptide using QuikChange . Mutations that produced the variants were also introduced using the QuikChange kit. For each construct the entire subunit coding region was sequenced to verify that only the desired mutation had been introduced.2 at 37°C. Cells were re-plated in the same medium the day before transfection.HEK 293 cells  were maintained in a mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 , with 10% fetal bovine serum , penicillin (100 units/ml) and streptomycin (100 ug/ml) in a humidified atmosphere containing 5% COFor physiological and cell surface ELISA studies, subunits were transfected at a 1∶1 mass ratio using Effectene  according to the manufacturer’s instructions. Briefly, 3 µg of cDNA per well of a 24-well dish was mixed with the Enhancer and the Effectene Transfection Reagent. The cells were incubated with the mix for 6 to 18 h. Electrophysiological experiments were performed on either the second or third day after transfection, while ELISAs were performed on the third day after transfection.2 at 37°C.For mRNA expression and protein expression experiments, cells were seeded into 6-well polylysine-coated plates and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin/streptomycin. Cells were transfected using Lipofectamine 2000  according to the manufacturer’s protocol incubated in a humidified atmosphere containing 5% COCHRNB4 mRNA production HEK 293 cells were transiently transfected with a negative control (empty pcDNA3), or wild-type α3 plus wild-type or variant β4 constructs. After two days of growth, RNA was extracted from cell lysates with an RNeasy kit (Qiagen). Extracted RNA (10 ug) was then converted to cDNA using the High Capacity cDNA Reverse Transcriptase kit (Life Technologies). CHRNB4 mRNA expression was then measured using SYBRgreen  using one primer spanning the boundary between exons 3 and 4 and another primer spanning the boundary between exons 4 and 5 to ensure only cDNA was amplified.To measure 2HPO4, and 1.4 mM KH2PO4, pH 7.3) containing 1% Triton-X for 5 min, incubated with a horseradish peroxidase conjugated secondary antibody (Thermo Fisher Scientific), washed 3× with PBS containing 1% Triton-X for 5 min and finally incubated with the horseradish peroxidase substrate 3, 3, 5, 5, 0-tetramethylbenzidine. Images were taken allowing for 5 min of exposure using a Syngene western blot imager .To measure β4 protein levels in transiently transfected HEK293 cells, we performed western blots on cell lysates using a poly-clonal β4 antibody generously provided by Dr. Cecilia Gotti . After two days of growth, each well of the 6-well plate was used to create a cell lysate. Cells were lysed with 150 µl of lysis buffer (50 mM Tris (pH 6.8), 150 mM NaCl, 2 mM EDTA, 0.25% NP40, 1% TritonX). Total protein concentration was then measured by the bicinchoninic acid (BCA) method  for each cell lysate. 20 µg of protein from each lysate was incubated at 95°C for 5 min in 1× Laemmli buffer (0.25 M Tris (pH 6.8), 8% SDS, 40% glycerol, 0.01% bromophenol blue dye and 20% β-mercaptoethanol). Denatured samples were loaded onto a 4–20% Criterion  gel in TG-SDS buffer ) and run at 125 V for 90 min. Protein in the gel was transferred to a nitrocellulose membrane in TG-SDS buffer containing 20% methanol overnight at 4°C. Blots were incubated in TG-SDS containing 4% powdered milk for 25 min at room temperature, then incubated at room temperature with a primary β4 polyclonal antibody for 90 min. The blots were then rinsed 3× with phosphate-buffered saline  were then added to reach a total reaction volume of 20 µl. This reaction was then incubated at 37°C for 1 h. Upon completion of the PNGase reaction, the resultant solution was run on western blots as described above.For digestion with PNGase, 1 µl of 10× glycoprotein denaturing buffer was added to 20 µg of protein from each lysate and incubated at 100°C for 10 min. Subsequently, 2 µl 10× G7 Reaction Buffer , 2 µl 10% NP40 (NEB), HSurface ELISA assays were performed as described previously The surface ELISA data were analyzed as follows. The machine background was subtracted from each OD reading, then the OD readings were divided by the protein for that subunit combination. The normalized value for the negative control (pcDNA3) for that experiment was then subtracted from all values. Finally, to control for variation in expression between experiments, the subtracted expression levels were normalized to the positive control (wild-type) value for that experiment. The final value gives an estimate of the relative expression where a value of 1 indicates identical expression to wild-type subunits.2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4) and cells expressing high levels of surface receptors were identified by a bead-binding technique Cells were plated in 35 mm tissue culture dishes, and maintained and transfected as described. For recordings, cells were rinsed with recording bath solution . Currents were recorded using an Axopatch 200 amplifier . The cells were clamped at −60 mV and all experiments were carried out at room temperature (20–23°C). The current traces were low-pass filtered at 2 kHz and digitized at 10 kHz. The analysis of whole-cell currents was carried out using the pClamp 9.0 software package (Molecular Devices).Macroscopic currents were recorded using whole-cell voltage clamp as described 50)n)) where Y(X) is the response to a concentration X, and the parameters a , EC50  and n (Hill coefficient) were determined by non-linear regression using SigmaPlot . To combine data, the relationship for each cell was scaled by the fit maximum. In some cases, it was clear that high concentrations of agonist produced a reduced response, likely due to open-channel block by the agonist The basic parameter measured was the peak response to a given concentration of acetylcholine (ACh) or nicotine. Four sec pulses of agonist were applied at intervals of 30 sec with continuous application of bath solution between pulses to allow recovery from desensitization. Concentration-response relationships were obtained by applying pulses of different agonist concentration. The data from a cell were analyzed by fitting with the Hill equation: Y(X) = a. Accordingly, the measurements were performed using the concentrations closest to the half-maximal concentration of agonist to avoid channel block. Neuronal nicotinic receptors show evidence for multiple, kinetically distinct, forms of desensitization The cells used for physiological studies all were selected on the basis that they expressed receptors on the cell surface. Accordingly the average maximal response to acetylcholine will not be representative of the total cell population. Therefore, we adopted the relative cell surface ELISA signal as the assay for numbers of surface receptors for the different receptors expressed.Unless otherwise noted all chemicals used were obtained from Sigma-Aldrich.50 or relative maximal response).Data are presented as mean ± SE (number of observations). For ELISA studies results from each experiment constituted one observation; data were obtained in triplicate in each experiment. For physiological results each cell constituted an observation  was 60% or more of the best fitting value. Data from ELISA experiments were excluded if the expression normalized to protein for the positive control (wild-type subunits) did not differ from that for the negative control (pcDNA3) with a probability of less than 0.05 (two-tailed t-test), indicating a failed transfection.Basic statistical computations were made with Excel . ANOVA was performed using Stata . Figures were prepared with SigmaPlot .C. elegans glutamate-activated Cl- channel X-ray crystal structure (http://swissmodel.expasy.org/). Structures were visualized and displays generated using Chimera 1.6.2 (http://www.cgl.ucsf.edu/chimera).The homology model was made by threading the α3 and β4 subunits onto the C and D subunits respectively of the DNA samples were collected as part of the Collaborative Genetic Study of Nicotine Dependence (COGEND). All individuals were current or former smokers who had smoked at least 100 cigarettes in their lifetime and underwent a semi-structured interview, which assessed smoking behavior, other substance use and comorbid psychiatric conditions. The COGEND sample includes 710 African Americans (461 nicotine dependent (ND) cases and 249 smokers with no symptoms of dependence (controls). Nicotine dependent cases have a Fagerström Test of Nicotine Dependence (FTND) We focused our attention on the African-American population for the following reasons. In our initial report http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000092.v1.p1). Subject collection was supported by NIH grant P01 CA89392 (PI - L Bierut) from the National Cancer Institute. Phenotypic and genotypic data are stored in the NIDA Center for Genetic Studies (NCGS) at http://zork.wustl.edu/under NIDA Contract HHSN271200477451C (PIs J Tischfield and J Rice).The Collaborative Genetic Study of Nicotine Dependence is a collaborative research group and part of the National Institute on Drug Abuse (NIDA) Genetics Consortium (De-identified data from the Collaborative Genetic Study of Nicotine Dependence (COGEND) were used. All participants in COGEND provided written informed consent for genetic studies and agreed to share their DNA and phenotypic information for research purposes.www.r-project.org) using linear regression incorporating age and sex as covariates. In order to ensure that associations were not affected by population stratification, we calculated principal components (PCs) with ∼3700 SNPs spread out across the genome CHRNB450, nicotine EC50, nicotine efficacy, response to low nicotine and cell-surface expression) for the particular variant the individual carried. These values were then used as the predictor in a linear regression using age, sex, and the first two principal components (PC1 and PC2) as covariates. As described in the Results, data for variants occurring in only one individual were included by weighting them by the parameter value of the non-singleton variant or wild-type with the closest parameter estimate.Association analyses were performed in R . Accordingly, we generated expression constructs containing the variants . Lastly, this receptor subunit combination is expressed in the interpeduncular nucleus, the medial habenula and the ventral tegmental area, brain regions believed to be involved in addictive behavior, as well as being the dominant form expressed in peripheral ganglia The β4 subunit can assemble with different α subunits to produce functional receptors. We focused on the properties of receptors containing the α3 and β4 subunits for several reasons. First, the genes encoding these subunits are located proximally in the human genome and have been shown previously to be highly correlated in expression patterns across brain regions CHRNB4 and one rare missense variant was identified in the CHRNA3 as part of the previous sequencing project. In this study we did not examine the properties of the variant CHRNA3 subunit, so all experiments were done with wild-type α3 and α4 subunits. Analysis of the sequencing results reveals that several variants show linkage disequilibrium and thus cosegregate non-randomly in the population. Upon phasing of individual haplotypes, we observed that everyone  carrying β4(R136W) also carried β4(M467V) on the same allele, so we created an expression construct both β4(R136W) and β4(M467V) in the same subunit and used this double variant in functional studies rather than β4(R136W). In addition, a subset of individuals carrying β4(S140G) (32 out of 63) also carried β4(M467V) on the same allele. Accordingly, we created expression constructs containing both β4(S140G) and β4(M467V) and used this construct in studies, as well as β4(S140G) and β4(M467V).A total of 10 rare missense variants were identified in CHRNA3 using qRT-PCR. We observed no significant differences between the mRNA levels produced from each variant-containing plasmid compared to wild-type CHRNB4 containing plasmids (data not shown). These data suggest that any observed differences in total or cell-surface protein levels are not the result of altered initial mRNA levels.We first measured mRNA expression levels produced by plasmids containing each of the tested variants when expressed together with wild-type Next, to determine if overall β4 protein, both intra-cellular and cell-surface, was altered by the introduction of variants, we performed western blots on total cell lysates from HEK 293 cells transiently transfected with wild-type α3 and either wild-type or mutant β4 containing plasmids. Analysis of the band intensity for each mutant protein provided no evidence of altered total β4 expression for any of the variant plasmids (data not shown). The S140G variant is predicted to abolish glycosylation at amino-acid position 138. The S is the third amino acid in the consensus glycosylation signal NXS, where X can be any amino acid. We hypothesized that the absence of glycosylation at this site should result in both faster migration on a western blot and low cell-surface protein expression. Both predictions were observed. Furthermore, treatment with PNGase to remove all glycosylation of wild-type β4 and β4(S140G) resulted in indistinguishable and more rapid migration (data not shown).CHRNB4 alter the steady-state level of protein expressed on the cell-surface, we performed cell-surface ELISAs on cultured HEK 293 cells two days after transient transfection with constructs expressing either wild-type β4 or variant β4 in conjunction with wild-type α3 or wild-type α4. When the α3 or α4 subunits were transfected in the absence of a β4 subunit the surface ELISA signal was indistinguishable from that of cells transfected with empty vector .To determine whether mutations in 2 = 0.76, P = 0.01). The correlation suggests that the defect in surface expression results from some inability of the β4 variants to fold or traffic appropriately, rather than a specific defect in the ability to interact with a particular α subunit. Further experiments will be required to determine which mechanism(s) is more likely to explain the reduced surface expression for each variant . An elegant study of a homologous mutation in the mouse β4 subunit (β4(R348C); Cell-surface expression was decreased for nearly all variants tested  and 3. I50). A smaller EC50 value corresponds to a higher potency. To measure the relative efficacy of nicotine we determined the relative maximal response of a cell to nicotine compared to the maximal response to ACh. We also measured the relative response at the end of a 4 sec application of agonist, compared to the peak, to estimate the overall accumulation of receptors in a non-responsive (“desensitized”) state. Finally, we measured the relative response to a low concentration of nicotine (1 µM for α3-containing receptors and 0.3 µM for α4-containing receptors) to estimate the response to a more pharmacologically-relevant concentration of nicotine.To test the electrophysiological properties, receptors containing wild-type and variant β4 subunits were co-expressed with α subunits in HEK 293 cells. Whole-cell voltage-clamp experiments were performed to examine the basic functional properties of receptors containing variant subunits. Cells were selected using a bead-binding assay (see Methods) to increase our chance of obtaining measurable responses. To estimate the potency of ACh and nicotine we determined the concentration that produced a half-maximal response (ECThe results are summarized in 50), and results are summarized in 50 values for ACh (1 of 19 combinations) or nicotine (3 of 19).We obtained concentration-response relationships for activation by ACh and nicotine. Graphs of typical relationships are shown in The magnitude of the current elicited by a maximally effective concentration of agonist gives a measure of the efficacy of the agonist . We do not know this value in absolute terms, for example from single-channel studies that would directly measure the probability a channel is open. However, we can estimate the relative efficacy of nicotine compared to ACh from a ratio of the estimated maximal responses from a cell tested with both agonists. None of the combinations tested resulted in a significant change.50 concentration of agonist, to avoid possible complications due to channel block by high concentrations. A value of 1 for sag indicates that there was no decline in response, while a value of 0 indicates that the response declined completely to baseline. Most variants showed no significant difference from wild-type in sag. The β4(R136Q) variant showed greatly increased sag in the presence of either ACh or nicotine, but only when expressed with the α4 subunit.We obtained an estimate of the overall accumulation of receptors in non-responsive states from the amount of decrement (“sag”) from the peak response to the end of a 4 sec application of agonist. As discussed in Methods, we used responses elicited by an ∼ECFinally, we estimated the response to a low concentration of nicotine, as a fraction of the maximal response to ACh. The brain concentration of nicotine for a smoker is estimated to reach 100 to 1000 nM 50 and nicotine EC50). Our motivation was to determine whether there were indications that variants had similar effects on functional responses to both ACh and nicotine (possibly suggesting an agonist-independent effect on activation) or on functional responses when expressed with the two α subunits. We did not correct the results for multiple comparisons . There was a significant association between response to low nicotine when expressed with an α3 subunit and response to low nicotine when expressed with an α4 subunit  and between the amount of sag when expressed with α3 and α4 . Overall, the functional effects of the variants did not fall into a simple pattern, such as both ACh and nicotine having reduced potency (increased EC50) when a variant is expressed with a given α subunit, nor a pattern in which similar effects occurred when a variant is expressed with different α subunits.In addition, we tested for correlations between parameters  as the response variable and including age and sex as covariates. Using all missense variants, there is no significant association between carrier status at CHRNB4 and logCPD    . We have 0.0009)  that the 0.0009) . This re = 0.01) . These o50 for variants expressed with α3  or by relative response to low nicotine when expressed with either α3  or α4  explained more phenotypic variance and produced a significant association. The results of this weighting procedure for these significant parameters can be seen in To perform parameter-weighted analyses of these data, we first had to decide which parameters to use for individuals harboring multiple variants. As mentioned, we observed that all individuals harboring β4R136W) also harbored β4(M467V) on the same allele and that all individuals harboring both β4(S140G) and β4(M467V) harbored these variants on the same allele. As a result, we used parameters estimated from constructs harboring both variants. Every individual  carrying β4(T91I) also carried α3(R37H). However, we had elected to study properties of receptors with wild-type α subunits, and individuals with α3(R37H) also carried a wild-type α3 allele. We found (data not shown) that receptors containing the α3(R37H) variant expressed poorly on the cell surface and so it is likely that most functional receptors in the brain would contain wild-type α3 subunits. Finally, variants that occurred in only one individual were grouped with the non-singleton variant or wild-type with the nearest parameter estimate because values for the average CPD for singleton variants would be poorly estimated due to sample size . These parameters are the ones that resulted in significant improvement in the association when functional weighting was used, and the functionally-weighted association indicated that large values were protective. We then examined correlations between parameters and PhyloP score for all variants examined including singletons, using 2 metrics. The first was the relative parameter value, and the second was the absolute value of the logarithm of the relative parameter value. The second metric was used as a measure of effect size irrespective of direction, since relative values of 2-fold and one half would receive equal treatment. With a total of 32 regressions, 3 showed a P value less than 0.05: for “sag” for variants expressed with α3 the linear parameters had P = 0.015 and the logarithmic parameters P = 0.037, while for the ACh EC50 for variants expressed with α3 the logarithmic parameters had P = 0.039 (the linear parameters had P = 0.5) (results not corrected for multiple comparisons). These observations indicate that the PhyloP score is not a strong predictor of functional effect. Our observation that weighting variants based on occurrence at a conserved position improved association appears to be happenstance, and based on the particular distribution of functional effects with respect to PhyloP scores.It is interesting to consider why selecting variants based on PhyloP score improved the association, in the absence of any functional weighting. A possible explanation is suggested by considering CHRNB4 variants weighted by functional consequence could be due to chance, we performed permutation tests. For each of the significant parameters  we randomly assigned parameter values from the set of measured values we obtained to each of the variants without replacement and performed 10,000 or 20,000 permutations. The number of permutations performed was selected to provide a reasonable chance of obtaining a probability comparable to the experimentally observed one - for example with experimental P = 6×10−5, observing one more significant association in 20,000 permutations would give an estimate of 1/20,000 (5×10−5). In the case of response to low nicotine when expressed with α3 (association P of 6×10−5) none of 20,000 permutations had a lower P value, indicating that the association is likely significant at P<5×10−5. For ACh EC50 when expressed with α3 (P = 2×10−4) 48 of 10,000 had a lower P value, suggesting the association is likely significant at P<5×10−3. However, for response to low nicotine when expressed with α4 (P = 4×10−4) 960 of 10,000 permutations had a lower P value, suggesting that this association could readily arise by chance.In order to determine if the association we observed between CPD and CHRNB4 are associated with reduced nicotine dependence risk. We extended this analysis by including all variants and incorporating the experimentally determined functional consequences of the incorporation of subunits harboring rare variants. The mechanism underlying an association, however, can be unclear. The complexity of the effects of nicotine in the brain, its metabolism and the neuronal networks that lead to addiction make prediction of the relationship between genetic variants and behavior difficult. Even in the specific case of variants in the neuronal nicotinic receptors it is difficult to reach definitive conclusions regarding the role of variants in these genes in vivo in the absence of knowledge of the functional effects. This is largely due to two factors: (1) the role of neuronal nicotinic receptors in the brain is not well understood and (2) these receptor subunits combine in many different combinations, forming sub-types expressed in various patterns with indeterminate functional redundancy. Even when functional effects are identified, it can be difficult to directly connect the functional change to the behavioral phenotype and, at present, it is not possible to a priori associate a receptor functional phenotype with the risk of developing nicotine dependence. We have taken a different approach, which is to measure a number of basic functional properties and to determine which properties improve the association between genotype and phenotype, with the idea that this will provide insights into possible underlying functional changes.Common variants only account for a small fraction of the risk of developing nicotine dependence, suggesting that a major portion of the genetic contribution to the risk of developing nicotine dependence might result from many rare variants. We previously showed CHRNB4 gene observed from sequencing a cohort of nicotine dependent individuals and non-dependent smoking controls. These parameters were then used to weight variants in a gene-based association test to test the hypothesis that relevant parameters will improve the observed association at this locus.Accordingly, we characterized the functional consequences of rare variants in the The majority of variants significantly reduced cell-surface protein expression. Despite this, cell-surface expression was not significantly associated with CPD in this dataset. This is, perhaps, expected as mice lacking the β4 subunit only show alterations in nicotine withdrawal symptoms and decreased adverse effects at high nicotine doses compared to wild-type, suggesting that even large effects on protein level may have small effects on behavior CHRNB4 variants, response to low concentrations of nicotine and ACh EC50 were able to improve the observed association between rare variants in CHRNB4 and CPD. In each case the association indicated that large values of the parameter is protective - a larger response to low nicotine or a less potent effect of ACh. This suggests that overall an enhanced response to concentrations of nicotine that may be reached in the brain of a smoker may be a protective factor. The other parameters did not result in significant improvements in association, although the overall trends  in comparison to our modest decrease (to 0.7-fold). When expressed with the α4 subunit, the variant increased the EC50 for ACh by 3.3-fold (compared to a decrease to 0.7 in our results) and that for nicotine by almost 17-fold (compared to a decrease to 0.9). However, there did not appear to be any change in nicotine relative efficacy . We do not have an explanation for the difference in results.Few studies have been made of the functional properties of receptors containing variant β4 subunits. In one, 4 variants were expressed in CHRNB4 may decrease craving and withdrawal symptoms Recent work suggests that the α3β4* subtype of nicotinic receptors may contribute significantly to nicotine related behaviors by activating the habenulo-interpeduncular pathway The single variant with the strongest association with nicotine dependence is a nonsynonymous coding variant in the α5 subunit, which results in the replacement of aspartate 398 with asparagine The present work suggests that future analysis of rare variant associations may depend on the development of high-throughput methods of assessing allele function in order to clearly distinguish the alleles with functional effects. This is due largely to three factors. First, genes are capable of harboring alleles with opposing effects and these opposing effects obscure each other in a collapsing approach. For instance, in our data, the T375I variant is protective (lower average logCPD) while the S140G variant is a risk allele, although both have PhyloP score >2. Second, alleles that alter protein function do not necessarily have the same magnitude of effect and as such the variants of lesser effect can obscure the variants of greater effect, particularly if they are of higher frequency. This was the case for the M467V+S140G and T375I variants. Both are protective alleles, but the M467V+S140G variant confers only slightly lower risk than normal while the T375I confers much less risk. Lastly, one technique to gain power is to use only variants with putative higher prior probability of having effects on the function of the gene product . This often greatly reduces the number of variants and the number of carriers available for association testing, thereby reducing power and possibly obscuring associations if variants have opposing effects. When all variants are assessed for function, each can be used in the association test regardless of magnitude or direction of effect.Overall, our results indicate that incorporating functional information into association analyses can improve power to detect associations if relevant parameters are measured, and that methods of assaying the functional impact of variants across the genome will likely greatly improve our ability to understand the genetic basis for diseases in the era of whole-exome and whole-genome sequencing. The reduction in power resulting from large proportions of variants with little or no impact on protein function or mixtures of protective and risk variants being included in gene-based burden tests is substantial and will have to be addressed if we hope to understand the full scope of variation impacting complex diseases and traits. The results can also suggest possible mechanisms for an association, in the present case indicating that a larger relative response to low concentrations of nicotine may reduce the risk of developing nicotine dependence.


Acute promyelocytic leukemia (APL) remains the best example of a malignancy that can be cured clinically by differentiation therapy. We demonstrate that APL may emerge from a dynamical endogenous molecular-cellular network obtained from normal, non-cancerous molecular interactions such as signal transduction and translational regulation under physiological conditions. This unifying framework, which reproduces APL, normal progenitor, and differentiated granulocytic phenotypes as different robust states from the network dynamics, has the advantage to study transition between these states, i.e. critical drivers for leukemogenesis and targets for differentiation. The simulation results quantitatively reproduce microarray profiles of NB4 and HL60 cell lines in response to treatment and normal neutrophil differentiation, and lead to new findings such as biomarkers for APL and additional molecular targets for arsenic trioxide therapy. The modeling shows APL and normal states mutually suppress each other, both in “wiring” and in dynamical cooperation. Leukemogenesis and recovery under treatment may be a consequence of spontaneous or induced transitions between robust states, through “passes” or “dragging” by drug effects. Our approach rationalizes leukemic complexity and constructs a platform towards extending differentiation therapy by performing “dry” molecular biology experiments. Leukemia is a common malignancy that may affect about 1.5 percent of a total population during their lifetimeα gene fusion and chimeric proteinα, β and γ), and the retinoid X receptors (RXRs). RARα interacts with RXR, and the RARα-RXR heterodimer recruits corepressor or coactivator to differentially regulate transcription of its target genes. Retinoic acid releases the nuclear corepressor from the RAR/RXR complex results in eventual transcriptional activation of RAR-target genes. PML/RARα acts as a constitutive repressor that is insensitive to physiological concentrations of retinoic acid. APL patients are treated with pharmacological doses of ATRA to overwhelm the leukemogenic potential of PML/RARα.APL is most frequently characterized by the t translocation, which causes the PML/RARα induced oncogenic transformation has been studied using various APL mouse models56α is the only driving genetic event capable of initiating a typical APL disease when expressed in transgenic mice. However, the emergence of full-blown APL after initiation by PML/RARα requires 12–14 months. This long latency was hypothesized as an indication for additional genetic/epigenetic changes in the progression to the full transformation of APL phenotype, in sequential to PML/RARαα transcript was detected in APL patients under long-term remissionSuch a frequently cited straightforward interpretation of APL development leaves open questionsin vivo synergistic effects of ATRA and ATO should be the collaboration among their distinct targeting molecular pathways. ATO was shown to degrade PML/RARα mediated by sumolationArsenic trioxide (ATO) has been found to be effective in APL both as additive to ATRA, as well as monotherapyα signaling was found to enhance the growth of the granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent colonies derived from normal human bone marrowα simultaneously decreases production of colonies representing other hematopoietic lineages including erythroidα dependent clonal selection is also cell/cell contact dependent. In liquid suspension, ATRA not only enhances the generation of committed myeloid progenitors but also increases the production of more primitive hematopoietic precursorsα itself requires methods to accommodate complexity.Most receptors and molecular pathways are developmentally regulated. RARConsidering these questions to be clarified, we explored APL in this paper from systems biology viewpoint. We constructed endogenous molecular-cellular networkEndogenous network for APL was constructed with 8 modules, 81 nodes, and 416 interaction based on 357 references listed in In a molecular interaction network, multiple feedbacks coordinate molecular activities. As a result, only limited states of the network (activity profiles of the molecules) determined by network structure are robust ones14We identified attractor S1 as APL-like phenotypic state for the following reasons. We obtained microarray data from Gene Expression Omnibus (GEO) for retinoic acid treated APL cell lines NB4γ and Stat1 activityAttractors S12 and S14 in β up-regulation  and BMP simultaneously, shown in 2930actor S4 .As a structure of dynamical network, attractors are linked via saddle and other unstable fixed points, and are listed in β, NR2F2, SHH pathways and their target genes in the lower panel. Retinoic acid direct target genes and Pu.1 target genes are also included. Since NR2F2 itself is a retinoic acid target, its pathway is excluded from retinoic acid direct target genes. The references for their annotation are given in β, NR2F2, SHH pathways and their switching behavior indicate that they are involved in neutrophil differentiation and ATRA treatment, in agreement with network modeling. The two cell lines, HL-60 and NB4, show some varied gene expression in retinoic acid target genes, but not in BMP, TGF-β, NR2F2, and SHH pathways, indicating retinoic acid pathway might already be partially active in HL-60 cell lines. Nevertheless, a full induction to differentiation requires the switching of BMP, TGF-β, NR2F2, and SHH pathways, as both calculation and analysis of microarray profile suggested.This network is constructed from molecular interactions among each pair of nodes chosen for their involvement in ATRA treatment, normal neutrophil development and APL pathology. If indeed it plays the role of the core endogenous network, it should reproduce major experimental findings for APL as well as neutrophil differentiation, such as molecular profile for major phenotypes. In + T cells35In the process of carcinogenesis, hundreds of molecules and molecular pathways are affectedAnother interesting question is the origin of this APL-like attractor in the molecular interaction network, or equivalently, the mechanism preventing evolution to get rid of this attractor altogether. We found that the APL-like attractor formation may be linked to angiogenesis. Hematopoiesis is a conserved biological process in vertebrates. Given the importance of SHH and NR2F2 in angiogenesis24αBiologically, the most important open question on APL mechanism concerns successful APL treatment in contrast with AMLs, which do not respond to the same differentiation therapy. In microarray data1629302930Network construction and dynamical modeling was performed from the viewpoint of endogenous molecular-cellular network hypothesis on cancerThe construction of network followed an incremental procedure aimed at to capture the essential features of APL leukemogenesis as well as basic cell functions while keeping the network minimal. Retinoic acids are important hormone-like molecules which regulate a wide range of embryonic development processes such as hematopoiesis, central nervous system and early pancreatic bud formation. Under physiological condition, the molecular feedbacks from transcriptional regulations outside hematopoietic lineages need not to be considered. However, it is unclear to what extend these feedbacks participate in leukemogenesis. The network construction started with standard and generic cellular function modules such as cell cycle, apoptosis, stress response, and then myeloid differentiation was added. Next, feedbacks related to embryonic development in endothelial and mesenchymal cell lineagesNote that interactions included in the network are from studies under both physiological and pathological conditions. We assume that most of the molecular regulations are basically intact for the core functions of cells such as cell cycle, Notch pathway etc. In this APL network, the interactions are mostly of direct biochemical type, such as phosphorylation, that may be invariant under both conditions. Nevertheless, there are may be differences in these two conditions. Such differences may be reflected by perturbations on the dynamical equations used. As demonstrated in the βα fusion protein is also identified in APL, we included Stat5 and Stat3 pathways as well as RARsThe nodes presenting the generic features of cellular functions were mostly taken from previous works4245Genes and regulatory molecules affected by ATRA through RARs were selected. Besides Hox family, which are direct retinoic acids targets and participate in vertebrate embryonic developmentin vivo parameters. A semi-quantitative description was thus employed for the core network consisting of a set of nonlinear and coupled differential equations depicting “macroscopic” activation or inhibition between agents, as a refinement of more primitive but useful descriptions such as the Boolean dynamics (Boolean network)A challenge of analyzing endogenous networks is a lack of detailed biological knowledge for modeling the complex interactions between agents, e.g., the absent of xi under the influence of other agents in the network may be written as the form of chemical rate equation including generation rate f and degradation rate g:The dynamical equation for the concentration (activity) of an agent f and g are unknown, we consider a coarse-grained model to catch the “macroscopic” feature: (a) we consider all agents are exponentially self-degrading with gi = xi; and (b) we use the following defined f with an upper bound value 1 for normalizing all concentrations  to vary between ; (c) we employ the commonly used Hill type function52f. If an agent x2 activates agent x1, function f1(x2) is assigned to the formSince generally the expression of n is the Hill coefficient. If there is an inhibition on x1 from x2where x2, x3 and inhibition x4, x5 to x1, we haveIf there are multiple activation A full list of 81 equations can be viewed in 54n may vary from interaction to interaction, we did random parameter tests that allow each interaction  having independent ni and ai, i = 1, …, 416 (ni and ai can be dependent or independent). The statistics on the recurrence rate of each attractor (obtained using the above equations with fixed n and a) demonstrates the robustness of our approach .


Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨ Diabetes is characterized by a constant stage of hyperglycemia leading in the long term to severe damage to several systems , includiMitochondrial dysfunction has been hypothesized to be a key factor in the progression of hyperglycemia-mediated neuronal damage , 5. ThisMitochondrial alterations related to diabetic encephalopathy include increased mitochondrial fission, excessive ROS levels , augmentc + c1 [Further studies are required about these issues for the development of therapeutic strategies to ameliorate the impact of diabetic encephalopathy and other complications of diabetes. In this regard, nutraceuticals with antioxidant properties have been used as alternative treatments to slow and/or prevent the inherent complications of diabetes –14. A cac + c1 .m), lipid peroxidation levels, ROS levels, and (GSH/GSSG) ratios.In this work, we aimed to evaluate the effects of avocado oil on brain mitochondrial function and oxidative status in STZ-induced type I diabetic rats. For this purpose, several parameters of mitochondrial function were analyzed such as respiratory control ratio (RCR), activity of the ETC complexes, transmembrane potential . This research was also approved by the Institutional Committee for Use of Animals of the Universidad Michoacana de San Nicolás de Hidalgo. Diabetes was induced by intraperitoneal administration of STZ (45 mg/kg), in rats subjected to 12-hour fasting conditions. Five days after diabetes induction, the glucose levels were determined and rats exhibiting blood glucose levels higher than 300 mg/dL were included in the experimental trial.Rats were randomly divided in four groups: (1) normoglycemic rats (Control); (2) normoglycemic rats plus avocado oil (Control + AO); (3) diabetic rats (Diabetic); (4) diabetic rats plus avocado oil (Diabetic + AO). Avocado oil was administered orally at a dose of 1 mL/250 g weight in a daily basis for a period of 90 days. A commercial presentation of avocado oil , purchased from a local grocery, was used in the experimental trial.At the end of the treatment, animals were fasted overnight and sacrificed by decapitation. Brain was quickly removed and mitochondria were isolated by differential centrifugation in a Percoll gradient as previously described . Briefly2, and 3 mM KH2PO4 at pH 7.4) in a sealed glass chamber with constant stirring. The rate of oxygen consumption was determined at room temperature using a Clark-type oxygen electrode coupled to an oxygen monitor YSI 5300 and a computer for data acquisition. The determinations started immediately after adding 10 mM glutamate/malate as respiratory substrate for complex I (state 4) and after 3 minutes, 1 mM ADP was added to determine oxygen consumption in the phosphorylating state (state 3). Finally, inhibitors of complex III (1 µg antimycin A) and complex IV (1 mM KCN) were added to inhibit mitochondrial respiration. The respiratory control ratio (RCR) was calculated from the ratio of the state 3/state 4 respiratory rates.To measure mitochondrial respiration, freshly isolated brain mitochondria were resuspended in a final volume of 2 mL of buffer for measuring oxygen consumption  was added to dissipate ΔΨm. The changes in Safranin O fluorescence were measured at λex 495 nm and λem 586 nm in a Shimadzu RF5301PC spectrofluorometer.ΔΨfranin O . 0.5 mg/This determination was carried out in brain mitochondria by measuring the levels of thiobarbituric acid reactive substances (TBARS) . Absorba2PO4 buffer. NADH-oxidoreductase (complex I) activity was assayed in brain mitochondria incubated with 1 µg antimycin A plus 1 mM KCN. After 5 min, 5 mM K3Fe(CN)6 was added and absorbance was followed during 1 min at 340 nm in a Shimadzu UV2550 spectrophotometer. Then, NADH was added and its oxidation was measured during 4 min. The rate of NADH oxidation was calculated using a molar extinction coefficient of 16.3 mM−1 cm−1 for NADH [c oxidoreductase (complex III) activity was followed by measuring at 550 nm the reduction of cytochrome c. Cytochrome c oxidase (complex IV) activity was evaluated by measuring the oxidation of reduced cytochrome c at 550 nm [To determine the activities of the ETC complexes I, II, III, and IV, intact mitochondria were permeabilized with Triton X-100 as previously described . Enzymatfor NADH . Succinat 550 nm .2DCFDA). 0.5 mg/mL intact mitochondria and 1.25 mM H2DCFDA were incubated in a buffer containing 10 mM HEPES, 100 mM KCl, 3 mM MgCl2, and 3 mM KH2PO4 (pH 7.4) during 20 min at 4°C under constant shaking. Later, mitochondrial suspension was placed in a quartz cuvette and basal fluorescence was recorded. After 1 min, 10 mM glutamate/malate was added and the changes in H2DCFDA fluorescence were further followed by 20 min [λex 485 nm; λem 520 nm).ROS levels were determined by measuring the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (Hy 20 min . FluoresμL of the supernatant in 0.1 M sodium phosphate buffer (pH 7.5), 3 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 0.115 unit/mL glutathione reductase in a final volume of 1 mL. After 5 min of incubation at room temperature, 2 mM NADPH was added and the kinetics of the reaction was monitored for 5 min. The increment in absorbance at 412 nm was converted to GSH concentration using a standard curve with known amounts of GSH [Mitochondrial samples were treated with 5% (v/v) sulfosalicylic acid and centrifuged at 7800 g for 10 min to remove denatured proteins, and reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined by an enzymatic method. The total glutathione (GSH + GSSG) content was assayed in a cuvette containing 90 s of GSH . For dett-test using Sigma Plot software v11.0.Results are expressed as the mean ± standard error of at least 3 independent experiments using samples from different animals for each experiment. Statistical differences of the data were determined with Student's The brains used for the experiments of this study were dissected from the same rats used for other study reporting the effects of avocado oil on liver mitochondrial function of diabetic rats . As repoOxygen consumption rates (OCR) in resting state (state 4) and phosphorylating state (state 3) were measured with the objective to evaluate whether diabetes impaired mitochondrial function and determine the protective effects of avocado oil. Diabetes had a notable impact on brain mitochondria respiration Figures  as OCR im. As observed in m. Moreover, ΔΨm remained stable after 2 min and the addition of an uncoupler (CCCP) induced an increase in Safranin fluorescence at initial levels before substrate addition, which is indicative of full dissipation of the ΔΨm. In contrast, the changes elicited by glutamate/malate in Safranin fluorescence were of a considerably lower magnitude in mitochondria from diabetic rats (gray line) and occurs at a slower rate than in mitochondria from control rats (black line), which together indicates that diabetes impaired brain mitochondrial functionality. Notably, avocado oil intake fully prevented the alterations in the ΔΨm observed in diabetic rats (gray pointed line) and did not alter this parameter in the control group (black discontinuous line). Therefore, these results confirm that diabetes induced brain mitochondrial dysfunction and that avocado oil fully prevented this alteration.Further characterization of impaired brain mitochondrial function in diabetic rats was carried out by analyzing ΔΨThe activities of the complexes from the ETC are shown in m and respiration was related to enhanced ROS levels, we evaluated the changes in the fluorescence of H2DCFDA in response to the addition of glutamate/malate. The data presented in In order to explore whether the impairment in both ΔΨLipid peroxidation and GSH/GSSG ratios were analyzed as markers of oxidative stress to test whether enhanced ROS levels in diabetic rats and the protection conferred by avocado oil were parallel to changes in oxidative stress. In comparison to control rats, the levels of TBARS were similar in mitochondria from diabetic rats . HoweverRegarding to the redox status of glutathione, the GSH/GSSG ratio of mitochondria from diabetic rats was 38.3% lower with respect to mitochondria from control animals, indicating a state of higher oxidative stress in mitochondria from diabetic animals. Avocado oil prevents this effect and even augmented by 3.1-fold the GSH/GSSG ratio in comparison to mitochondria from control animals. Besides, avocado oil also produced a more discrete, 1.3-fold increase in this parameter in normoglycemic rats.Diabetes complications are associated with end-stage damage in the eyes, kidneys, peripheral nerves, and the brain –29. In tγ coactivator 1α (PGC-1α), which regulates mitochondrial biogenesis and the expression of several components of the ETC [m to drive the conversion of ADP into ATP. As ΔΨm was severely impaired in mitochondria of diabetic rats . This hypothesis is in fully agreement with ameliorated ROS levels (m observed in mitochondria of the same animals. However, as pointed out by Brand and Nicholls, [1F0-ATP synthase activity. For this reason, altered ETC functionality may indeed have a negligible effect on overall mitochondrial function. Based on these considerations, it may be suggested that mitochondrial dysfunction in brain mitochondria is independent of alterations on the activities of the ETC enzymes. Instead, decreased respiration in state 3 may be due to alterations in ADP phosphorylation as was discussed above.Regarding the activities of the complexes from the ETC, the only significant change during diabetes was an increase in the activity of complex III , which hSTZ rats . AvocadoS levels  and decrS levels  observedicholls, , mitochoMitochondrial ROS levels were higher in the diabetic group . In otheTo estimate the extent of mitochondrial oxidative stress during diabetes and the ability of avocado oil to counteract this process, lipid peroxidation also was determined. According to different reports about the levels of lipid peroxidation in brain mitochondria, there is no consensus about whether this process remains unaltered or increases during diabetes , 41, 53.It is important to note that one important limitation of this work is that cognitive decline due to diabetes and the impact of avocado oil were not assessed in this study, which impedes to confer avocado oil a protective role on diabetic encephalopathy. As a first approach, it was intended in this study to explore whether avocado oil supplementation may protect from diabetes-induced brain mitochondrial dysfunction and exacerbated oxidative stress. However, we thought that the findings from this study pave the road to further research addressing if the administration of avocado oil may have beneficial effects in diabetic encephalopathy, since the attenuation of some features of oxidative stress observed in our study, such as increased lipid peroxidation and glutathione exhaustion, is believed to ameliorate cognitive deficits during diabetes .m induced by diabetes, besides increasing complex III activity. This may be related to decreased ROS levels and improved redox status in diabetic rats as reflected by a higher GSH/GSSG ratio. These effects might delay the onset of diabetic encephalopathy, but this possibility remains to be investigated.Avocado oil improves brain mitochondrial function in diabetic rats by preventing the impairment in mitochondrial respiration and ΔΨ


SLC6A4 promoter genotype.Age at onset (AAO) of bipolar disorders (BD) could be influenced both by a repeat length polymorphism (5HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4) and exposure to childhood trauma. We assessed 308 euthymic patients with BD for the AAO of their first mood episode and childhood trauma. Patients were genotyped for the 5HTTLPR (long/short variant) and the rs25531. Genotypes were classified on functional significance . A sample of 126 Brazilian euthymic patients with BD was used for replication. In the French sample, the correlation between AAO and trauma score was observed only among ‘SS’ homozygotes (p = 0.002) but not among ‘L’ allele carriers. A history of at least one trauma decreased the AAO only in ‘SS’ homozygotes (p = 0.001). These results remained significant after correction using FDR. Regression models suggested an interaction between emotional neglect and ‘SS’ genotype on the AAO (p = 0.009) and no further interaction with other trauma subtypes. Partial replication was obtained in the Brazilian sample, showing an interaction between emotional abuse and ‘LS’ genotype on the AAO (p = 0.02). In conclusion, an effect of childhood trauma on AAO of BD was observed only in patients who carry a specific stress responsiveness-related  In order to identify homogeneous subforms of the disorder, candidate symptom such as age at onset (AAO) of the disorder have been suggested112124117Bipolar disorder (BD) is a heterogeneous and complex disorder with a high heritability close to 60%) and interactions with environmental risk factors  encoding the serotonin transporter, can influence the AAO in BD. An increased presence of the short allele (‘s’) of the 5HTTLPR has been observed in BD patients with an early AAOGenetic factors can also influence AAO in BD. Three independent genetic association studies have concluded that a repeat length polymorphism (5HTTLPR) in the promoter region of the SLC6A4 promoter variants and CTQ  on the AAO of BD.On the basis of such data, this study investigated the interaction between The initial sample consisted of euthymic patients with BD type I, II or not otherwise specified (NOS) who were recruited from three psychiatric departments in France . Patient inclusion criteria were: aged over 18 years; having a diagnosis of BD according to DSM-IV criteriaThe replication sample consisted of patients with BD  who were recruited from the outpatient Bipolar Disorders Program of Hospital de Clínicas de Porto Alegre (HCPA) in Brazil. Patients were diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV-TR)Childhood traumatic events were recorded using the CTQ, a 28-item self-report questionnaire27DNA was prepared from peripheral blood leukocytes or B-lymphoblastoïd cell lines by standard procedures. The genotyping of the 5-HTTLPR, rs25531 as well as the triallelic intron 2 VNTR (Stin2) (not used in this study) were performed by triplex polymerase chain reaction (PCR) followed by restriction endonuclease digestion as described byThe two samples (French and Brazilian) were compared using non-parametric test  for descriptive analysis. Since AAO and CTQ scores were assumed not to have a normal distribution, non-parametric tests were used. In this case, Medians and Median Absolute Deviations (MAD) were given. We tested whether the genotype distributions were in Hardy–Weinberg equilibrium.SLC6A4 promoter genotypes and CTQ subtypes as the predictive variables, and the interactions between CTQ subtypes and genotypes was used as the confounding factors. The model was AAO transformed ~ Genotypes(LL versus LS versus SS) + CTQ subtype + Genotypes × CTQ subtype. AAO was reciprocal square root transformed in the French sample and square root transformed in the Brazilian sample to fulfill the normality assumption required by the parametric procedure.Linear regression analyses were performed to examine the relationship between AAO, We corrected for multiple testing in the French sample, using a FDR  with a p-value = < 0.002 being significant. Statistical analyses were performed with the STATA package (version 12)SLC6A4 promoter variants genotypic frequencies were in Hardy-Weinberg equilibrium (p = 0.48).The French sample comprised 308 patients with BD . All patients completed the CTQ. Three hundred patients were successfully genotyped for the l/s variant (5HTTLPR) and for the rs25531 (8 DNA failed to be amplified by PCR). The The mean AAO of BD was 24.72 ± 9.9 years  and the mean CTQ total score was 42.05 ± 12.9 . AAO and CTQ total score did not differ according to genotypes see .SLC6A4 genotypes on non-specific level of trauma .We first tested for an effect of ’SS’ = − 0.32 p = 0.002) and the ‘LS’ genotype (rho’LS’ = − 0.17 p = 0.03) with no correlation observed for those patients carrying the ‘LL’ genotype (rho’LL’ = − 0.02 p = 0.88).The AAO and the CTQ total score were negatively correlated for the whole sample of patients (rho = − 0.16 p = 0.004). However, this correlation was mainly due to patients carrying the ‘SS’ genotype (rhoWe then considered the effect of the absence/presence of any trauma sub-type (regardless of the subtype considered) on the AAO according to genotypes. AAO decreased in the presence of trauma (one or two and more trauma subtype(s)) in the ‘SS’ subgroup (Wilcoxon rank test: p = 0.001) and to a lesser extend in the ‘LS’ subgroup (p = 0.04) but not in the ‘LL’ subgroup (p = 0.44)  with a trend for an interaction also between emotional neglect and ‘LS’ genotype on the AAO (p = 0.09) in the same model see . We founA power calculation with 5 predictors in the regression models showed that our sample size (n = 300) had a power of 0.76 to identify an effect size of 0.04 . A sample size of n = 326 would have been required to reach a power of 0.80 and to identify an effect size of 0.04.SLC6A4 promoter variants genotypic frequencies were in Hardy-Weinberg equilibrium (p = 0.82).The Brazilian sample consisted of 126 patients with BD . All patients completed the CTQ with a mean total score of 52.39 ± 20.4. One hundred and twenty two patients were genotyped for the 5HTTLPR l/s variant and for the rs25531 (4 DNA failed to be amplified by PCR). The distribution of genotypes were the following : 30 patients with ‘LL’ genotype, 63 with ‘LS’ genotype and 29 with ‘SS’ genotype. The The replication sample differed from the initial one for gender distribution (p < 0.001), BD subtypes distribution (p = 0.02) and CTQ total score (p < 0.001); this corresponded to a Brazilian sample consisting of more females, more BD type II and with a higher CTQ total score. The two samples were similar for age at interview (p = 0.99), age at onset (p = 0.59) and genotypes distribution (p = 0.27).Multivariate regression analyses used transformed AAO (sqrt(AAO)) that fulfilled the normality assumption . No significant result was observed when studying physical neglect, physical abuse, nor sexual abuse. We found an interaction between emotional abuse and ‘LS’ genotype on the AAO (p = 0.02) and a trend for an interaction between emotional neglect and ‘LS’ genotype on the AAO (p = 0.08). No interaction was found with ‘SS’ genotype in both models see  and 4.A power calculation with 5 predictors in the regression models showed that this replication sample (n = 122) had a power of 0.34 to identify an effect size of 0.04.SLC6A4 promoter variants and emotional trauma on the AAO of BD. In the French sample AAO significantly decreased in the presence of trauma mainly in ‘SS’ carriers, this being significant after correction for multiple testing. Regression analyses suggested  that emotional traumatic events might be of special interest when studying environmental risk factors associated with an early occurrence of BD. In the replication sample, the results were partly replicated with some nominal signals in favor an interaction between SLC6A4 promoter variants and emotional trauma on the AAO.The identification of factors determining the variability of the AAO in BD is of crucial importance as an early AAO consistently associates with a more severe clinical profile and a poor prognosis12et al.1011131415As previously suggested by Benedetti Firstly, our results are consistent with previous pathophysiological data obtained in various experimental models, including knockout mice, stress-reared rhesus macaques and human functional brain imaging33Second, the mechanisms by which childhood traumatic events decrease the AAO in BD remain unclear. It is likely that this is mediated through the induction of a cascade of neurobiological and neuroendocrine events. Previous studies have suggested that childhood trauma can have long-lasting effects on the catecholamine response to psychological stress353738This causal link (i.e. a greater exposure to trauma leading to an earlier AAO) is not the sole interpretation although above mentioned arguments favor it. Other interpretations that consider ontology, parental psychopathology/psychiatric disorders or gene/environment correlations should also be considered. Patients with a ‘SS’ genotype could present with a more pronounced emotional bias toward negative stimuli2324SLC6A4, focusing only on the promoter region, nor epigenetic mechanisms. Finally, after correction for multiple testing, we also did not reach significance for in-depth investigations of trauma subtypes. We also were not able to totally replicate our results. This issue was possibly related to sample size and reduced power in the replication sample and incomplete comparability between samples. For example, the number of patients with a ‘SS’ genotype in the replication sample was small (n = 29) and had probably reduce our ability to identify an interaction with this particular genotype. Nevertheless, trends for interaction in the larger ‘LS’ genotype group were suggested as shown in the initial sample. Given these limitations, our results will definitively require replication in independent and larger samples of patients with BD. Indeed, the results of gene/environment interaction studies in psychiatry have been a source of controversy, often being hard to replicate or providing mixed results, as in the case of the interaction between 5HTTLPR, stressful life events and the risk of unipolar depression33505152The design of this study has several limitations. First, the retrospective assessment of traumatic events during childhood may be influenced by uncontrolled recall bias4648TLR2) and sexual abuse on the AAO of BD737447879808117Further research is also required on the genetic and environmental factors that modulate the AAO in BD, which is likely to be multifactorial and determined by many genes with small effect sizes or other variants not measured well by SNP arrays, such as rare alleles or copy number variations5455585962676870SLC6A4 promoter variants interact to influence the AAO in BD. Further investigations in larger samples are required to test this hypothesis in order to help guide preventive strategies among subjects at risk of developing a BD (for example offspring of parents with BD and exposed to trauma).Gene-environment interactions represent a new challenge in psychiatric geneticsHow to cite this article: Etain, B. et al. Interaction between SLC6A4 promoter variants and childhood trauma on the age at onset of bipolar disorders. Sci. Rep.5, 16301; doi: 10.1038/srep16301 (2015).


Reference 24 in the original article (reference  and Badjatia [25] with permission” instead of “Anti-shivering protocol example and the bedside shivering assessment scale. IV intravenous, PT per feeding tube, ECG electrocardiogram. Modified from Brophy [25] and Badjatia [24] with permission”.Unfortunately, the original version of this article  containeThe references have been corrected in the original article and are also correctly included in this erratum.


Electrostatic gating using ionic liquid was able to induce superconductivity in MoSe2 but not in MoTe2 because of inefficient electron accumulation limited by electronic band alignment. Alternative gating using KClO4/polyethylene glycol enabled a crossover from surface doping to bulk doping, which induced superconductivities in MoTe2 and WS2 electrochemically. These new varieties greatly enriched the TMD superconductor families and unveiled critical methodology to expand the capability of ionic gating to other materials.Functionalities of two-dimensional (2D) crystals based on semiconducting transition metal dichalcogenides (TMDs) have now stemmed from simple field effect transistors (FETs) to a variety of electronic and opto-valleytronic devices, and even to superconductivity. Among them, superconductivity is the least studied property in TMDs due to methodological difficulty accessing it in different TMD species. Here, we report the systematic study of superconductivity in MoSe Semiconducting transition metal dichalcogenides (TMDs) have attracted considerable interest as typical two-dimensional 2D) materials. Atomically flat and chemically stable thin layers of TMDs can be readily obtained via graphene-like mechanical exfoliationD materia3468910111214 cm−2)1618192032 has revealed enhanced Tc and a dome-like phase diagramRecent advances in the application of the field effect have been achieved using ionic gating by the formation of electrical double layers (EDLs); an EDL consists of narrow (~1 nm) spatial charge doublets that mimic a capacitor capable of accumulating an ultra-dense sheet of carriers . Subsequently, the TMDs showed similar gate-induced insulator–metal transitions but did not all reach the superconducting states. In MoSe2, new gate-induced superconductivity (GIS) was found with a maximum Tc of 7.1 K that follows a dome-shaped phase diagram similar to that of MoS2202 because of the low efficiency of electrostatic electron accumulation that could be achieved in this material using an IL. When the IL was replaced with a KClO4/PEG electrolyte, the electron doping could be significantly enhanced through a crossover to an electrochemical regime beyond the electrostatic limit. As a result, superconductivity in 2H-MoTe2 and WS2 were enabled with observed Tc values of approximately 2.8 K and 8.6 K, respectively. Thus, these results have revealed two series of superconductors in these TMDs. Additionally, this study established new strategies for ionic gating both for electrostatic charge accumulation and electrochemical carrier doping, thereby providing new capability for accessing a wide carrier concentration range and extending superconductivity in other material series.Following the previous work on MoSH-type transition metal dichalcogenides, MX2 , consisting of two-dimensional covalently bonded X−M−X layers. Due to their weak bonds formed through van der Waals interactions between the layers, thin flakes of MX2 can be readily isolated from bulk material via mechanical exfoliation and then fabricated into FET devices (see the Methods section). We used the EDLT structure and employed DEME-TFSI as a gate dielectric; the latter is a widely used IL that has been demonstrated to be capable of accumulating a high density of carriers at the interface even at low bias voltages2 device (MoSe2) prior to the application of the IL. A typical Hall bar geometry was adopted to measure the four-terminal resistance and the Hall carrier density. The thickness of the MoX2 flakes in this work ranged from approximately 20 nm to 100 nm as measured by atomic force microscopy (AFM). We confirmed that the 2H-type crystal structure was maintained in the thin flakes after transport measurements with IL gating as confirmed by synchrotron microbeam X-ray diffraction experiments (see ents see .H-type MX2 with a finite band gap28Eredox) aligns with the Fermi energy of the semiconductor (EF)242526ΦTMD influences the initial charge redistribution and the band bending at the interface before application of gate voltage. Because TMD work functions differ from each other2, MoSe2 and MoTe2 thin flakes for the same IL. Among these three dichalcogenides, MoTe2 exhibits the smallest work function, leading to the weakest initial band bending and the largest carrier injection barrier for electrons. Therefore, a systematic evolution of transistor performance with increasing threshold voltage is expected when the channel material is changed from MoS2 to MoTe2.The gate voltage was applied through a droplet of IL under high vacuum and at a temperature just above the glass transition temperature of DEME-TFSI  to suppress potential chemical reactions between the IL and the film surface2425262  that were all gated using DEME-TFSI. 2, MoSe2 and MoTe2 EDLTs measured with VDS = 0.1 V. All three MoX2 transistors displayed ambipolar behavior with systematic properties: within the same bias range, the two extremes of preferences for electron and hole accumulation were dominated by MoS2 and MoTe2, respectively, whereas MoSe2 exhibited a well-balanced ambipolar transistor performance that was most suitable for light-emitting devices2 to MoTe2. The average values of the threshold voltage Vth versus the MoX2 work function  and by the deep trap states in the energy gap. However, it is noted that both of these factors take only minor roles in EDLTs553233CEDL , the threshold voltage shift ΔVth (=Ntrap/eCEDL) due to the filling of trap states (Ntrap) becomes negligibleBy sweeping the gate voltages at a constant rate of 20 mV/s at 220 K, we measured the transfer curves for the different MoXn2D, which was therefore unambiguously determined through a Hall effect measurement. To eliminate any temporal change in n2D during ionic gating in MoX2 EDLTs, all Hall effect measurements were performed at temperatures below the freezing point of ion movement, where the total number of accumulated carriers is fixed. The Hall coefficient RH was found to change its sign as a function of the gate voltage VG and n2D = 1/|RHe| was linearly proportional to the gate bias VG in the electrostatic region  than MoS2 (8.6 μF/cm2) or MoSe2 (10.6 μF/cm2), indicating that MoTe2 demonstrated the lowest efficiency in electron accumulation.Because our devices were designed to access low-temperature quantum phases, it was critical to quantify the sheet carrier density c region . The EDL2 and MoTe2 thin flake EDLTs, we measured their transport properties down to 2 K by varying VG in the positive direction to access electron transport. The results are shown in the plot of the channel sheet resistance Rs versus T in 2 data are presented in VG . This Tc value was higher than the maximum previously reported for Sr-doped MoSe2 (~5 K)n2D using the Hall effect  with Tc = 0, indicating that no superconducting transitions were observed down to 2 K. We believe that the absence of superconductivity in MoTe2 may be due to the low maximum n2D of approximately 0.7 × 1014 cm−2, which is much smaller than the accumulation on MoS2 or MoSe2. This difference can be attributed to the fact that MoTe2 demonstrated the largest threshold voltage Vth  and the KClO4/PEG electrolyte. For IL gating, an increase in IDS was initiated at a large threshold voltage of approximately 1 V and was no longer observed when VG exceeded 2.5 V, indicating a limit on the formation of a more conductive channel. This peak behavior of IDS versus VG was associated with n2D saturation and was confirmed in multiple devices, implying that the observed behavior was not related to device degradation. For KClO4/PEG electrolyte gating, electrostatic electron accumulation occurred at a much smaller threshold voltage and induced a rapid increase of IDS, reaching a high conducting state at VG approximately 3.5 V. The conducting state was then sustained as VG was continuously increased up to 6 V. This gave rise to the observation of good metallic conduction followed by a superconducting transition at low temperatures, as shown in Rs−T curves . Since superconductivity in MoTe2 has never been reported, the result in H-MoX2 family.To induce superconductivity in MoTeasing VG . We thenT curves  further VG at 300 K for the KClO4/PEG electrolyte; at this temperature, the ions were sufficiently mobile. At such a high temperature, an increase in the leakage current associated with the saturation of IDS was observed above VG = 3.5 V  corresponded to a metallic state that failed to access the superconducting region. This limit was clearly broken when VG exceeded approximately 7 V, where a second rapid upturn of IDS strongly indicated an effective electrochemical doping process, i.e., ion intercalation under high bias. This was supported by the distinctive large hysteresis . Then, a particular KxWS2 line-phase compound that exhibited a superconducting transition with a fixed Tc was formed at approximately 9 V. In this initial state where superconductivity appeared, the sample consists of a mixture of nonsuperconducting phase with lower x and the superconducting phase. As VG further increased, the superconducting phase gradually grew and finally occupied a substantial portion of the whole channel, reaching the “zero resistance” state . This is also supported by the gradual decrease of the normal state resistance with increasing VG . Superconducting transitions were well developed down to “zero resistance” for VG = 8 V. When intercalation further proceeded, an overdoped phase started to grow, forming a mixture of the superconducting phase and the overdoped non-superconducting phase. Finally, zero-resistance disappeared since an overdoped metallic state became dominant at VG = 10 V, where a high nHall of approximately 2.1 × 1016 cm−2 was achieved. With the ability of reaching such a high carrier density, this gate-controllable intercalation process is also applicable to metallic TMD systemVG varies from sample to sample due to the differences in sample thicknesses and device configurations even following the same gating procedure. Here, nHall is important to quantify the doping level of the flake. Assuming that the whole flake was uniformly doped, the bulk carrier density n3D and the doping concentration x can be deduced from the Hall effect and the thickness of the thin flake. Tc versus n3D  and doping concentration x  collected from four different devices with superconducting transitions reaching “zero resistance”, where Tc was defined as the temperature corresponding to 90% of the total transition. The similar Tc values (approximately 8.6 K) within the shaded area support the existence of a line-phase superconducting phase in KxWS2 between x = 0.05 and 0.2. This is consistent with the conventional staging effect frequently observed in the intercalated states of layered systemsx of the superconducting phase should be accurately determined by Raman spectroscopy and X-ray diffraction. When potassium is intercalated beyond the optimal range, superconductivity finally disappeared (Tc = 0), implying a possible phase change to the overdoped non-superconducting compound. 2 exhibited an abrupt superconducting transition at Tc. This result contrasts strikingly with the electrostatically gated MoSe2, for which we observed rather broad superconducting transitions that can be interpreted as a fluctuation phenomenon of the 2D nature of superconductivity32, on the other hand, indicates that this system behaves as a three-dimensional (3D) or at least anisotropic 3D superconductor.Similar superconducting transitions were also observed in other devices. The inset in H-type MoSe2, MoTe2 and WS2 induced by a crossover from electrostatic to electrochemical doping as an exploratory tool to access wide and controllable doping regimes. Following the established electrostatic doping, we discovered the presence of previously unknown gate-induced superconductivity in MoSe2 but failed to find it in MoTe2 because of the lower efficiency of electron accumulation in the latter. By replacing the IL with a KClO4/PEG electrolyte, we accessed the adjacent intermediate doping regime and further electrochemical doping regime beyond the electrostatic limit, where superconductivity was successfully discovered for MoTe2 and WS2. To the best of our knowledge, these two materials are new, unprecedented superconductors. The discovery of a superconductor series in Mo and W chalcogenides proves that superconductivity is a common property for semiconducting TMDs. Additionally, this study provides new strategies and guidelines for the ionic gating technique: optimization of the energy level alignment at the interface for electrostatic carrier accumulation and the use of polymer electrolyte and high gate bias to induce electrochemical doping beyond the electrostatic limit. Combination of these two doping regimes can significantly broaden the applicability of ionic gating, making it a versatile tool for the study of a wide variety of materials. Thus, this study improves the versatility and effectiveness of the ionic gating method, which may play an essential role, in combination with conventional chemical doping, in the discovery of new superconductors.In this study, we report the discovery of superconductivity in 22 single crystals were obtained commercially (SPI Supplies). MoSe2 single crystals were grown via a chemical vapor transport (CVT) technique41TH) at 1050 °C and the lower-temperature side (TL) at 950 °C. MoTe2 and WS2 single crystals were grown using the same method, with chlorine gas as the transporting agent and with a gradient established by maintaining TH/TL at 800 °C/750 °C for MoTe2 and TH/TL at 1000 °C/700 °C for WS2.MoS2 were cleaved from the bulk, as-grown single crystals using the scotch tape method. Subsequently, the flakes were transferred to Nb-doped SrTiO3 substrates covered with a 30 nm HfO2 layer (grown via atomic layer deposition) and SiO2 (300 nm)/Si substrates. We chose atomically flat thin flakes with thicknesses from 20 nm to 100 nm 2 negative mask or a thick electron beam resist (approximately 400 nm ZEP 520A) layer5194/PEG polymer electrolyte, was applied to the top of the thin flake and the side gate electrode for ionic gating. The device configuration is illustrated in 4 in polyethylene glycol  with a [K]:[O] ratio of 1:20. The solution was liquid at 300 K and underwent a glass transition below approximately 250 K.Multilayered thin flakes of MX−5 Torr). To reduce the likelihood of chemical reactions between the IL and the thin flakes, the transfer curves were acquired using a constant source–drain bias voltage of 0.1 V at a gate-voltage sweep rate of 20 mV/s at 220 Ki.e., first performing ionic gating at 220 K, followed by a cooling period under the applied voltage and subsequent warming to 220 K for gating to another VG. When the KClO4/PEG electrolyte was used, VG was applied at 300 K.All transport measurements were performed using a Quantum Design Physical Property Measurement System  in high-vacuum mode 10− Torr. ToHow to cite this article: Shi, W. et al. Superconductivity Series in Transition Metal Dichalcogenides by Ionic Gating. Sci. Rep. 5, 12534; doi: 10.1038/srep12534 (2015).