Innovative methods producing transparent and flexible electrodes are highly sought in modern optoelectronic applications to replace metal oxides, but available solutions suffer from drawbacks such as brittleness, unaffordability and inadequate processability. Here we propose a general, simple strategy to produce hierarchical composites of functionalized graphene in polymeric matrices, exhibiting transparency and electron conductivity. These are obtained through protein-assisted functionalization of graphene with magnetic nanoparticles, followed by magnetic-directed assembly of the graphene within polymeric matrices undergoing sol–gel transitions. By applying rotating magnetic fields or magnetic moulds, both graphene orientation and distribution can be controlled within the composite. Importantly, by using magnetic virtual moulds of predefined meshes, graphene assembly is directed into double-percolating networks, reducing the percolation threshold and enabling combined optical transparency and electrical conductivity not accessible in single-network materials. The resulting composites open new possibilities on the quest of transparent electrodes for photovoltaics, organic light-emitting diodes and stretchable optoelectronic devices. Transparent and electrically conducting flexible films are in high demand but production can be both time-consuming and expensive. Here, the authors report a method for assembling modified graphene flakes in controlled distributions within polymeric matrices by use of magnetic fields. However, bulk materials containing mixtures of the conductive elements and the flexible matrix present the advantage to provide more mechanical integrity over sandwich or layered structures, as well as the possibility to create more complex three-dimensional (3D) circuits. In addition, direct ink writing or 3D printing techniques can be applied to build on-demand materials with inks containing both the transparent support and the conductive elements78−1 for films of 1 μm thicknessThe increasing demand for optoelectronic tools from tissue-compatible biomedical devices for health monitoring to indium–tin-oxide-free electrodes for flexible solar cells has recently stimulated the search for cost-effective materials with comparable performance, but improved flexibility to replace the commonly used stiff and brittle components1235−1 in cellulose nanofibrils aerogel membranes while maintaining 90% in transparency and 5% in strain at rupture−1 in 81% transparent polyethylene terephthalate-congo red single-wall carbon nanotube composites1819202223242530Recent efforts have focused on employing conductive anisotropic particles exhibiting high aspect ratio to reduce the amount of opaque material needed to reach the percolation threshold in the composite11121314One enticing approach to further decrease the amount of conductive particles needed to achieve the percolation threshold is to fabricate hierarchical composites exhibiting multiple percolating networks at different length scales32In this study, we describe a simple processing route to fabricate transparent and conducting polymer-based composite films using external magnetic fields to assemble magnetized graphene flakes into hierarchical networks of predefined features. Using virtual magnetic moulds, transparency is obtained by locally concentrating the dark conductive flakes into continuous networks. When large micrometric mesh sizes are used as moulds, such control is achieved over areas spanning over several centimeters, resulting in double-percolating hierarchical networks. In addition to spatial control, tuning the orientation of the functionalized graphene flakes within the microscopic pattern further enhances the electrical conductivity. By carefully engineering the mesh size of virtual magnetic moulds, we demonstrate how this new strategy can lead to transparent, electrically conducting polymer–graphene composites with great potential for applications in advanced compliant optoelectronics.Hydrophilic magnetically responsive reduced graphene oxide (m-rGO) flakes are synthesized through decoration of exfoliated micrometric graphene oxide nanosheets (GO) with 10 nm superparamagnetic iron oxide nanoparticles . The at37xy-plane is achieved by applying a horizontally rotating magnetic field394041z axis  compared with a horizontal configuration is the first evidence of the successful alignment of the flakes in the intended direction  patterns through magnetic manipulation of diluted dispersions enables the creation of hierarchical double-percolating networks . At the ϕi is calculated from the density of the m-rGO flakes and the coverage of iron oxide nanoparticles and BSA . Such a small resolution has not yet been reported in stretchable and transparent strain sensors used for health-monitoring devices where high strains, typically up to 300%, is the main property targeted5051A possible further advantage of the proposed strategy is that of allowing the production of conductive and stretchable functional composites serving as strain sensors of high precision. Indeed, thanks to the intrinsic flexibility of polymers, we conducted in-plane confined compression experiments on polydimethylsiloxane substrates covered by the gelatin m-rGO film while recording the change in electrical conductivity  were dissolved in 25 ml water and vigorously stirred. After heating to 60 °C, 10 ml NaOH solution at 2 wt% was added with 250 mg of hexadecylpyridinium bromide (Fluka).Spherical magnetite nanoparticles were synthesized by co-precipitation of FeSOA measure of 18 ml of the GO stock solution was mixed with 2 ml of 0.5 wt% of the BSA protein (Sigma-Aldrich). In a following step, this suspension was stirred with 250 μl of 4.4 wt% SPIONs suspension for 1 h to allow for the physical adsorption of the SPIONs at the surface of the graphene oxide sheets through interactions with the BSA. Volume of 100 μl of reducing agent hydrazine monohydrate (Sigma-Aldrich) was added at 80 °C for 20 h under continuous stirring. After cooling to room temperature, the mixtures were kept at 4 °C.z axis, whereas the samples with biaxially aligned flakes were obtained by rotating the magnetic field on the x–y plane. The samples were afterwards cooled down to room temperature and dried for 24 h in ambient conditions to consolidate the gelatin and fix the designed architecture  aqueous solution of 20 wt% gelatin  was mixed with the corresponding volume of the suspension of m-rGO in water and stirred at 55 °C. For the experiments with controlled orientation, the mixture was cast into small polyethylene moulds. A static vertical magnetic field of 50 mT was used for the uniaxial alignment of the graphene flakes along the itecture . Samplesitecture . To demoitecture . The welPAMPS hydrogel was synthesized according to a modified processCharacterization of the m-rGO and the composites film is detailed in the The data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Le Ferrand, H. et al. Magnetic assembly of transparent and conducting graphene-based functional composites. Nat. Commun. 7:12078 doi: 10.1038/ncomms12078 (2016).Supplementary Figures 1-8, Supplementary Tables 1-2, Supplementary Note 1, Supplementary Discussion, Supplementary Methods and Supplementary References


Compared with the general population, an excess of rare copy number variants (CNVs) has been identified in people with schizophrenia. Females with neurodevelopmental disorders and in the general population have been reported to carry more large, rare CNVs than males. Given that many schizophrenia datasets do not have equal gender ratios in cases and controls, sex differences in CNV burden might have impacted on estimates of case-related CNV burden and also on associations to individual loci. In a sample of 13,276 cases and 17,863 controls, we observed a small but significant excess of large (≥500 Kb), rare (<1%) CNVs in females compared with males in both cases and controls . The burden of 11 schizophrenia-associated CNVs was significantly higher in female cases compared with male cases , but after accounting for the rates of CNVs in controls, we found no significant gender difference in the risk conferred by these loci. Controlling for gender had a negligible effect on the significance of association between specific CNVs and schizophrenia. The female excess of large CNVs in both cases and controls suggests a female protective mechanism exists for deleterious CNVs that may extend beyond neurodevelopmental phenotypes. Schizophrenia has a strong genetic component involving rare and common alleles distributed across many genes14A gender-bias in prevalence has been repeatedly observed in neurodevelopmental disorders. In autism spectrum disorder (ASD), the mean male-to-female ratio is about 4:110111213de novo CNVs and genes disrupted by de novo CNVs than males with ASD16171519A recent study found an excess of deleterious autosomal CNVs in females compared with males in a neurodevelopmental disorders cohort and in an ASD cohortGiven the excess burdens of CNVs in females in both neurodevelopmental and control samples, we tested whether similar differences in burden exist in schizophrenia using a large case control dataset . We evaluated the CNV burden in females and males at a genome-wide level and for 11 strongly associated schizophrenia risk loci. We also tested whether association between specific CNVs and schizophrenia are robust to adjusting for gender. Consistent with earlier findings, we found an increased CNV burden for large (≥500 Kb), rare (<1%) CNVs in female controls and now report an excess in female cases with the disorder. Although female cases also had an excess burden compared with males for 11 CNVs that have been implicated in schizophrenia, the association between those CNVs and schizophrenia was not diminished after controlling for gender.−5) and in males  did not statistically differ (Z-test P = 0.19).Females had a genome-wide excess of large (≥500 kb) and rare (<1%) CNVs in cases  and controls   althoughWhen deletions and duplications were analysed separately , we foun−18) and males , we found no significant difference in effect size (Z test P = 0.14). Hence, the non-significant test by sex in the controls might reflect lower power given that (by definition) there are fewer observations of these pathogenic CNVs in controls.The burden of 11 schizophrenia risk CNVs was significantly higher in female cases than male cases  but not in female controls compared with male controls  . HoweverWe have carried out an analysis of gender stratified CNV burden in schizophrenia at both a genome-wide level and for previously associated loci. We restricted our analysis to the autosome as sex chromosomal CNVs were not available for all samples. We found a genome-wide excess of large, rare CNVs in females compared with males, irrespective of disease status. Similar observations have been reported in ASD and general population cohorts15At a genome-wide level, an excess female CNV burden was only observed for large CNVs (≥500 kb). However, we cannot confidently exclude a similar effect from smaller CNVs given the limited resolution to which microarrays can accurately call CNVs. When deletions and duplications ≥500 Kb were analysed separately, we found that deletions primarily accounted for the excess genome-wide CNV burden seen in female controls.Recent findings of an excess mutational burden in females provide support for the view that females are relatively protected from neurodevelopmental disorders151718192224It is clear that CNV burden differs between males and females in both patients diagnosed with neurodevelopmental disorders, such as schizophrenia, and also in the general population. However, our data indicate that variation in gender CNV burden does not impact on known schizophrenia-CNV associations. We provide further evidence that in both case and control samples, the rate of deleterious CNVs is greater in females than in males, although investigating the reason for this will require large population cohorts.Case and control CNVs were derived from three published samples: CLOZUK46NRXN1 (exonic CNVs only), 3q29, 15q11.2, 15q13.3 and 22q11.2; duplications at 1q21.1, 16p11.2 and 16p13.11, the Prader-Willi/Angelman syndrome (PWS/AS) region and Williams-Beuren syndrome (WBS) region. In our analysis of individual CNV loci, we tested these 11 schizophrenia risk CNVs, along with previously implicated risk CNVs for which the overall evidence is less strongVIPR2, deletions of 17p12, 17q12 and distal 16p11.2. We also tested the impact of gender on protective effects for 22q11.2 duplicationWe tested the burden of 11 schizophrenia risk CNV loci that showed significant association with schizophrenia in our previous publication logit (pr (gender)) ~ number of CNVs + study source + microarray platform logit (pr (gender)) ~ study source + microarray platformWe tested for CNV burden  and burden of 11 schizophrenia risk loci in females versus males by comparing the change in deviance between the following logistic regression models ) using a two-sided test (ANOVA). Odds ratios (ORs) were derived from the correlation coefficients for number of CNVs.To test whether females had more genes disrupted by CNVs than males, a similar analysis was performed where ‘number of CNVs’ in the above model was replaced with ‘number of genes overlapping CNVs’.To test whether the risk of schizophrenia conferred by large CNVs (≥500 kb) or the set of 11 specific schizophrenia CNVs is significantly different for females and males, we compared the effect size in females with the effect size in males using the coefficients and standard errors  derived from the following logistic regression model ):logit (pr(case)) ~ number of schizophrenia CNVs + study source + microarray platformWe note that almost identical P values were obtained when the above model was replaced with Firth’s procedureDifferences in effect size between tests were evaluated with a Z-test, which was constructed using the equation (1)β is the coefficient for the variable of interest taken from the regression model and σ is that variable’s standard error. P values were generated by comparing the absolute of the Z statistic against the cumulative distribution function of the standard normal distribution.where To assess whether a difference in gender proportion between cases and controls confounds association between schizophrenia and specific CNVs, we calculated individual locus association statistics using a Fisher’s Exact test (two-sided) and a Cochran-Mantel-Haenszel Exact test (two-sided) that was stratified by gender.https://www.r-project.org).All statistical tests were conducted using R Statistical Software .


MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease . We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals—16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology—or homeology—driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication—inverted triplication—duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both  MECP2 locus with an unusual pattern consisting of an inverted triplicated segment flanked by duplicated segments of the genome. This complex CGR is facilitated by inverted repeats in a process that mechanistically could occur by two template switches mediated by replicative DNA repair. We now investigate the PLP1 locus and demonstrate that 16/17 CGR independent events present with duplication—inverted triplication—duplication pattern facilitated by two inverted repeats, similar to events involving MECP2. We show that the same inverted repeats facilitating CGR formation are also responsible for an inversion polymorphism observed frequently in the normal population. Intriguingly, one CGR was found to have a quadruplication resulting in the presence of four copies of a genomic segment. Breakpoint studies suggest this quadruplication occurred in a manner consistent with rolling circle amplification as predicted by previously postulated models.Genomic architecture, such as direct or inverted repeats, can facilitate structural variation (SV) of the human genome. SV can consist of deletion, duplication, or inversion of a genomic segment, or combinations thereof, the latter referred to as complex genomic rearrangements (CGR). CGR are defined as requiring two or more novel DNA breakpoint junctions. We described a CGR product at the  Factor VIII gene causes ~45% of severe hemophilia A cases . The79 was noP2 locus .PLP1, the junctional products are not readily amplified and sequenced by long PCR with primers anchored to unique flanking sequence. We adopted an alternative strategy to complement the Southern blotting assay above. Using a semi-quantitative PCR approach, we first confirmed that each of the patients has duplication of A1a and A1b LCRs , overlapping portions of both A1a and A1b LCRs that are implicated in the rearrangements  Figs. 3, S8. ThrIn contrast to the three other individuals for whom we sought to find Jct1 at base pair resolution, in BAB1612/P374 we were able to detect an LCR-mediated breakpoint within 24 bp of microhomology flanked by A1a and A1b sequences . The poiThe DUP-TRP/INV-DUP structure hypothesized for these 16 individuals postulates that although there are 4 copy number transitions in these patients, there are only two breakpoint junctions . We havePLP1 using long-range PCR  ~50 bp away from the junction that appeared to occur concurrent with the rearrangement, as observed for other CGR mediated by a replicative process  and the quadruplication rearrangement in P113 (insertion of a G) were not seen in the corresponding, contiguous (non-breakpoint containing) section of the X chromosome for these intrachromosomal events, a finding consistent with observations made at the MECP2 locus and de novo mutation with CGR formation /2). Note that RNase P is autosomal and so has two copies per male or female genome . The junctions are located on the X chromosome; there is only one X allele in males, and it is affected in the patients. Female carriers have junctions on one X allele but not the other.(PDF)Click here for additional data file.S6 Tablede novo nucleotide changes, and Inv. PCR primers were used in inverse PCR of sample P1150. Additional primers are available upon request.SNP primers amplify (PDF)Click here for additional data file.


Thermoelectricity is a very important phenomenon, especially its significance in heat-electricity conversion. If thermoelectric devices can be effectively applied to the recovery of the renewable energies, such as waste heat and solar energy, the energy shortage, and global warming issues may be greatly relieved. This review focusses recent developments on the thermoelectric performance of a low-dimensional material, bulk nanostructured materials, conventional bulk materials etc. Particular emphasis is given on, how the nanostructure in nanostructured composites, confinement effects in one-dimensional nanowires and doping effects in conventional bulk composites plays an important role in ZT enhancement. Thermoelectric (TE) materials can directly contribute toward a global joint solution because they are capable of converting a thermal gradient into a voltage, and vice-versa, and thus to recover waste heat Bell, . Many adZT = α2σT/κ, where α, σ, T, and κ are the Seebeck coefficient, the electrical conductivity, the absolute temperature, and the thermal conductivity, respectively. Because α, σ, and the electronic contribution to κ involve band structures , and scattering mechanisms, controlling the parameters independently is difficult  thermoelectric materials.Low-dimensional TE materials such as quantum wells and nanowires are supposed to have excellent thermoelectric properties than their bulk counterparts, because of increase in the density of states (DOS) near Fermi level by quantum confinement which tends to enhance the Seebeck coefficient. And also effectively scatters phonon over a large mean free path (mfp) by high density of interfaces, hence resulting in the lower lattice thermal conductivity. Remarkable enhancement of ZT inside quantum wires is predicted through theoretical studies due to its additional electron confinement. Hicks and Dresselhaus  due to phonon drag effects . This is the first time it has been claimed that phonon-drag can enhance ZT significantly. The argument is that in rough nanowires, the Seebeck coefficient can be increased by the transport of certain phonon modes which have minimal contribution to thermal conductivity. An interesting study published by Hsiung et al.  nanowires synthesized by stress-induced method, representing 10 times higher than compared to its bulk counterpart because of surface-dominated transport and large insulating bulk state in the BSTS nanowires. The thermal conductivity and power factor of the nanowires are shown in Figure Large enhancement of sselhaus  considersselhaus  reportedg et al.  reportedκlat.Nanostructured materials are among the strongest candidates for thermoelectric applications, as they offer a route to suppressing thermal conductivity without hindering electrical properties. Second phase endotaxial nanostructuring , one of the traditional thermoelectric materials, which spontaneously forms nanostructures efficiently scatters phonons, without the need for artificial nanostructuring confirmed from neutron scattering and high resolution transmission electron microscope (HRTEM) investigations  where Ag and Sb randomly occupying the Na site whereas Te is located at the Cl position shown in Figure κlat, dominates largely on the total thermal conductivity, which is related to the propagation of phonons. The κlat in rock salt AgSbTe2 is about three fold lower than that of PbTe at around room temperature. It has been reported that band gap ~0.35 eV at room temperature were obtained by optical diffuse reflectance measurements, whereas strong degenerate nature reflects the electrical conductivity. Recently, the AgSbTe2 compound has attracted considerable attention in constructing so-called bulk nanostructured TE materials, such as (AgSbTe2)x1−(PbTe)x (LAST-m) x1−(GeTe)x (TAGS)  doping; the electrical conductivity was enhanced greatly with an increase in the Se doping concentration. In 2014, Mohanraman et al. (2 compound doped with tin (Sn), the Ag2Te nanoprecipitates with feature size of 100–500 nm were observed in AgSbTe2 matrix are effective in scattering the phonons with mid-to-long mean free paths shown in Figures Recent studies has been reported that element doping or substitution technique has succeeded in enhancing the thermoelectric performance of AgSbTeu et al.  investign et al.  reported2 material demonstrated that the Bi doping has significantly enhanced phonon scattering process through point defects over the entire temperature range, they possessed lower thermal conductivity and achieved a high ZT-value ~ 1.0 at 570 K shown in Figure 2 material, the results showed enhanced power factor over 25–30% because of the increase in Seebeck coefficient related to decreased in carrier concentration and increase of the effective mass caused by In doping whereas lattice thermal conductivities were reduced substantially because of lattice mismatch arise from the dopants and host atoms having different atomic weights and thus resulted in enhanced phonon scattering. The highest ZT = 1.35 is achieved for Ag(Sb0.97In0.03)Te2 sample at 650 K shown in Figure 2 based alloys shows that the thermoelectric performances have been greatly improved by suitable types of dopants. Techniques such as doping or substitution have considerably decreased the lattice thermal conductivity, particularly in the high temperature range. All the results show that doping technique for enhancement of thermoelectric performance for AgSbTe2 based composites is reliable.Moreover, recent study published by Mohanraman et al.  on dopin2Te3 based alloys, the excellent TE materials at room temperature, are extensively used for the commercial thermoelectric devices for thermo-cooling application. Significant enhancement in the ZT-value of Bi2Te3 based bulk materials has been reported recently  method fabricated bulk nanocomposite p-type 2Te3 ingot with a ZT-value of 1.56 at 300 K published by Xie et al. . Lan et al.  demonstr2Te3is very small, or a very thin layer of Bi2Te3 is coated on the surface of Bi grains. Figure The morphology of the samples is studied by SEM. In Figure 2Te3 near the room temperature is at an advantageous position, the small decrease of electrical resistivity and the values of Seebeck coefficient can be explained by the existence of Bi2Te3 thin layer. Furthermore, the thermal conductivity κ was gradually diminished while the grain size was reduced. This also indicates that the quantity of grain boundaries is influenced by the thermoelectric property. The coating process might scatter the phonons but not the electrons. Moreover, the electrical resistivity was also diminished when the Bi2Te3 was coated on. This may be because the coating layer is more flexible than the core material making for better contact of particles than with the single phased material. Hence, the nano-coating process can increase the figure of merit (ZT) because the coating layers provide more boundaries and also prohibit the aggregation of particles within the sintering process. Increasing the number of grain boundaries could efficiently reduce the thermal conductivity without the reduction of electrical conductivity. We found that reducing thermal conductivity leads to a dimensionless figure of merit ZT ~ 0.278 at ~300 K shown in Figure The nano-coating process even slightly decreases electrical resistivity and maintains the values of Seebeck coefficient at the same value shown in Figures ZT-value of 2.2 at 915 K for p type PbTe-SrTe system via grain boundary phonon scattering enabled by nanostructuring to reduce the thermal conductivity.PbTe alloys are one of the premiere TE materials for intermediate range temperature (500–800 K) applications and played a key role in radioisotope thermoelectric generator for deep space exploration program as a power source. Recently, Kanatzidis group has published many reports on significant improvements in the thermoelectric properties of PbTe based alloys by nanostucturing and also modification in density of states through band structures (Zhao et al., 3 based skutterudites are highly promising candidate for medium temperature TE power generation applications because both n and p type materials with high performance can be obtained in the same material system. The most remarkable feature of this compound is that the cage like open structure are found and can be filled with foreign atoms acting as phonon rattlers which scatter phonons strongly thus drastically reduces the thermal conductivity. Nanocomposites, as an efficient way to reduce the thermal conductivity via grain boundaries and nanoinclusions, have also been used in CoSb3 based TE materials. Significant advances have been made in recent years with various kinds of nanoinclusion filling the cages in this compound (Bertini et al., in situ formation of InSb nanoislands in the In0.2Ce0.5Co4Sb12 nanocomposite with enhanced ZT up to 1.43 at 800 K by significant reduction in thermal conductivity. Zhao et al. (0.25Co4Sb12 nanocomposite and well distributed Yb2O3 particles synthesized by in situ reaction. The Yb2O3 nanoinclusions located at the grain boundaries are effective in scattering phonons, there by increases the figure of merit and achieved peak ZT of 1.3 at 850 K. Xiong et al. (0.2-Yb0.26Co4Sb12 nanocomposite exhibiting peak ZT-value of 1.45 at 850 K.CoSbo et al.  fabricatg et al.  has repo3 skutterudites, and Bi2Te3 based nanocomposites. Nanostructures such as nanoprecipitates, controlled two-dimensional coated nanostructures, nanoinclusion to atomic defects, and nanoscale inhomogeneities have been found to be potential routes for reducing thermal conductivity to a greater extend without hindering much on electrical conductivity, resulting in an enhanced figure of merit for the bulk nanocomposite material. However, additional approaches such as carrier-energy filtering or quantum confinement effects will likely be key role for enhancing power factor to achieve further significant ZT enhancement. Overall from our practical point of view, bulk nanocomposites shows more exciting than nanowires or nanobelts because the former can reduce the expenses, scale-up, and thermal management issues normally related with the later.This review summarizes the recent progress of nanowires; Ag-Sb based alloys, PbTe, CoSbThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Up to 25% of stroke patients wake up with a neurological deficit, so called wake-up stroke (WUS). Different imaging approaches that may aid in the selection of patients likely to benefit from reperfusion therapy are currently under investigation. The magnetic resonance imaging (MRI) diffusion weighted imaging – fluid attenuated inversion recovery (DWI-FLAIR) mismatch concept is one proposed method for identifying patients presenting within 4.5 hours of the ischemic event.To report our experience with the DWI-FLAIR mismatch concept for selection of wake-up stroke patients to be thrombolysed at our centre.Patients treated with off label intravenous thrombolysis (IVT) for WUS at our centre during a 6.5-month period were included. We performed MRI including DWI and FLAIR in all patients at admission. Each MRI examination was rated as either DWI-FLAIR mismatch or match. National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale were used to measure clinical outcome. Cerebral computed tomography (CT) or MRI was performed within 24 hours after thrombolysis to determine the presence of any intracranial haemorrhage (ICH).Ten patients treated with IVT for WUS were included. Four patients had a DWI-FLAIR mismatch and after IVT treatment the mean reduction in NIHSS in the DWI-FLAIR mismatch group was 4.0. In the DWI-FLAIR match group the mean reduction in NIHSS after IVT therapy was 4.8. None of the ten patients had any signs of ICH on follow-up imaging.In this small series DWI-FLAIR mismatch was not associated with worse outcome or ICH. This suggests that selecting WUS patients using DWI-FLAIR mismatch in clinical trials may exclude a large group of patients who might benefit. Results from large randomized controlled trials have shown a clear benefit for intravenous thrombolysis (IVT) when patients with ischemic stroke are treated within 4.5 hours (hrs) of symptom onset ,2. If IVDifferent imaging modalities that may aid in patient selection for reperfusion therapy are currently under investigation. Besides the traditional non-enhanced computed tomography (CT) and perfusion-CT, two different magnetic resonance imaging (MRI) techniques are discussed . The difWe retrospectively identified WUS patients treated with off label IVT at Stavanger University Hospital between 06.11.2013 and 22.05.2014. All consecutive wake up patients who were eligible for intravenous thrombolysis were included. The WUS patients met the following criteria: (a) asymptomatic <12 but >4.5 hrs from onset; (b) patients could report that they woke with stroke symptoms or were seen to have deficits on awakening; (c) diffusion restriction in less than 1/3 of the middle cerebral artery territory on DWI; (d) fulfilling all other IVT treatment criteria. There were no patients eligible for thrombolysis and considered to have WUS who did not get treatment, neither because of contraindications for MRI nor contraindications for thrombolysis found on MR or CT images; such as haemorrhages, restricted diffusion in more than 1/3 of the middle cerebral artery territory on DWI or intracranial tumours found.All patients underwent an initial cerebral MRI. The MRI protocol comprised transversal DWI (b-factor 0 and 1000), transversal T2-weighted imaging, transversal FLAIR imaging, transversal T2* weighted imaging and transversal time of flight arterial MR angiography; representing a total scan time of 15 minutes. To save time, MR angiography was not performed in one patient (patient 5) who had undergone a CT examination with CT perfusion and CT angiography of precerebral and intracranial arteries prior to the MRI. All MR scans were performed on 1.5 Tesla MRI machines . Follow up imaging with unenhanced CT or MRI was performed within 24 hours after IVT treatment to verify infarction, any eventual haemorrhagic complications and evaluate prognosis.Diffusion restriction (positive DWI) was defined as increased signal on DWI b 1000, and corresponding reduced apparent diffusion coefficient (ADC) values. Increased signal on FLAIR images in the same area as the restricted diffusion was defined as positive FLAIR, whilst a normal signal on the FLAIR images was defined as negative FLAIR. DWI-FLAIR mismatch  on post treatment CT or MRI was recorded. ICH was classified as in the European Cooperative Acute Stroke Study II (ECASS II) .Neurologic deficit was graded on admission and at discharge using the National Institutes of Health Stroke Scale (NIHSS) -20. The Patients were prospectively collected and only included in this study after informed written consent was given. After retrospective analysis of the results, we now include our WUS patients in a nationwide, prospective study (NOR-TEST) to enhance the number of WUS available for analysis. The study was approved by the Regional Ethics Committee.A total of ten patients presenting with WUS were treated with off-label IVT during the study period Table . The meaFollow up imaging with CT or MRI was performed 24 hrs after IVT treatment in nine patients. In one patient (patient 10) the follow up imaging was performed 10 hrs after IVT treatment.Four patients had a clear DWI-FLAIR mismatch . They received IVT treatment and had a reduction in NIHSS of 4.0 on average (range 8 – 2). None of these patients had signs of ICH on follow up imaging with CT or MRI.Four patients had a clear DWI-FLAIR match . In two patients, classification into DWI-FLAIR mismatch or match group was challenging. The first patient patient 1) with extensive confluating hyperintense white matter lesions on FLAIR images presented with an area of positive DWI in the same area. There was a definite DWI-FLAIR match, but it is possible that the increased signal on the FLAIR images was a result of chronic and not acute ischemia in that area. This case was classified as DWI-FLAIR match. Another patient (patient 2) presenting with multiple transitory ischemic attacks (TIAs) within the preceding 24 hrs had multiple small (3–10 mm) lesions with positive DWI. Some of these, mainly the largest, also had a hyperintense signal on the FLAIR images in the corresponding area. However, it was not possible to decide which lesion was responsible for the acute clinical symptoms. This patient was also classified as DWI-FLAIR match. The patients with a DWI-FLAIR match received IVT treatment and had a mean reduction in NIHSS of 4.8 (range 9 – 0)  three patients had normal findings, one patient had a M1 occlusion, one patient an occluded branch of the MCA, two patients showed stenoses in the M1 segment with reduced flow signal distal to the stenosis, one patient showed a P1 occlusion and one patient had a tandem occlusion in the internal carotid artery (ICA) and MCA. Follow up MR examination was not performed in all patients, however, one of the M1 occlusions (patient 4), the MCA branch occlusion (patient 2) and the tandem occlusion (patient 6) all showed restored flow signal on the MRA classified as TICI 3.Three patients (3/6 = 50%) in the DWI-FLAIR match group showed a drop in mRS of one point each at three months compared to their mRS before the stroke. In the mismatch group two patients (2/4 = 50%) showed a drop in mRS of two points each. One patient (patient 10) was severely disabled (mRS 4) before the stroke event. This was a 65 years old male with bilateral blindness and mild cognitive impairment.Although the presented cohort of ten patients is too small to draw general conclusions, we can show that six of the ten patients (60%) would have been excluded from IVT treatment if strict DWI-FLAIR mismatch criteria would have been adhered to . All patThe introduction of the DWI-FLAIR mismatch concept for identification of WUS patients within 4,5 hrs of symptom onset is clearly advantageous ,14. In tBoth the afore mentioned studies have a high PPV, suggesting that WUS patients with a larger ischemic lesion and DWI-FLAIR mismatch are in fact highly likely to be within 4.5 hours of symptom onset. However, the study by Thomalla et al. has a relatively low NPV (0.54) highlighting that many patients presenting within 4.5 hrs already have a DWI-FLAIR match . RigorouOngoing trials selecting patients either with WUS  or beyond the 4.5 time window  based on identification of significant salvageable brain tissue may further contribute to better select patients profiting from IVT ,25.Our study has its limitations: The sample size is too low to draw any firm conclusions and the retrospective nature of the study is a clear disadvantage. We see that the infarct size in our study is on average small. Despite the DWI lesions being small, several patients showed large vessel occlusions on MRA, and one would therefore expect that some of the ischemic lesions seen on DWI would progress if not thrombolysed. We can add to the body of evidence that the DWI-FLAIR mismatch concept is just a stepping stone to other, more reliable imaging techniques. Techniques such as multiparametric CT and MRI may have the advantage of estimating penumbra more precisely and giving us a more reliable indication of salvageable brain tissue . HoweverIn this small series DWI-FLAIR mismatch was not associated with worse outcome or ICH. This suggests that selecting WUS patients using DWI-FLAIR mismatch in clinical trials may exclude a large group of patients who might benefit. Clearly more research is warranted to lead to advances in this field.


Massive chromosomal aberrations are a signature of advanced cancer, although the factors promoting the pervasive incidence of these copy number alterations (CNAs) are poorly understood. Gatekeeper mutations, such as p53, contribute to aneuploidy, yet p53 mutant tumors do not always display CNAs. Uterine Corpus Endometrial Carcinoma (UCEC) offers a unique system to begin to evaluate why some cancers acquire high CNAs while others evolve another route to oncogenesis, since about half of p53 mutant UCEC tumors have a relatively flat CNA landscape and half have 20–90% of their genome altered in copy number.We extracted copy number information from 68 UCEC genomes mutant in p53 by the GISTIC2 algorithm. GO term pathway analysis, via GOrilla, was used to identify suppressed pathways. Genes within these pathways were mapped for focal or wide distribution. Deletion hotspots were evaluated for temporal incidence.Multiple pathways contributed to the development of pervasive CNAs, including developmental, metabolic, immunological, cell adhesion and cadmium response pathways. Surprisingly, cadmium response pathway genes are predicted as the earliest loss events within these tumors: in particular, the metallothionein genes involved in heavy metal sequestration. Loss of cadmium response genes were associated with copy number changes and poorer prognosis, contrasting with 'copy number flat' tumors which instead exhibited substantive mutation.Metallothioneins are lost early in the development of high CNA endometrial cancer, providing a potential mechanism and biological rationale for increased incidence of endometrial cancer with cadmium exposure. Developmental and metabolic pathways are altered later in tumor progression. Cancer genomes can evolve through acquisition of favorable mutations, copy number variations, or both. Determination of those mutations which recur on individual genes or even codons of individual amino acids within a gene has aided the development of targeted therapeutics, such as Vemurafenib to target BRAF V600E mutant tumors. The efficacy of targeting of mutations is improved for those mutations which arise early in tumor development (“trunk” mutations) and thus occupy the vast majority of tumor cells . HoweverHER2 [BRCA1/2 [BRCA mutant tumors do not always display a largely altered copy number landscape. There must be other genetic changes which enable certain tumors to acquire a larger burden of CNAs.Copy number alterations (CNAs) are another route to oncogenesis. Some dramatic alterations, such as massive amplification of HER2 , have beHER2 . MutatioHER2 , 6 or BR[BRCA1/2  can contOne cancer which allows an identification of these genetic changes which create a varied CNA profile is endometrial cancer. Endometrial cancer has been extensively sequenced by The Cancer Genome Atlas (TCGA) research consortium . The majHere we investigated the genetic pathways altered by copy number between TP53 mutant uterine endometrial carcinoma tumors with high overall copy number diversity and those tumors with low copy number diversity. The enriched gene sets identified which pathways are involved in the creation of an endometrial tumor’s genetic diversity.TP53 mutations , B cell , and T cell response  were significantly enriched for deletion/losses among high CNA tumors. Statistical enrichment was q< 3.4x10-11.The somatic alteration patterns analyzed in this study are specific for the tumor and are not alterations in the peripheral blood. Nonetheless, the most significantly enriched pathway for deletions in endometrial tumor cells was that of the immune response. Genes involved in the natural killer cell (PCDH genes), FAT2 and DLL4 , tyrosine kinases BLK and PEAK1, ECM2 , EGFL7 , FERMT2 (an ECM protein), B2M (a MHC I protein often overexpressed in cancer and which can promote metastasis), NEDD4 (an E3 ubiquitin ligase which negatively regulates PTEN levels), NEO1 (a FAK interaction protein), NPTN (neuroplastin), CD276 (an immune checkpoint protein), ITGAE (an E-cadherin receptor involved in autoimmunity), and PSTPIP1 (an autoinflammatory response protein). Statistical enrichment was q< 8.4x10-11The second most enriched pathway for deletions in high CNA TP53 mutant tumors was that of cell-cell adhesion. Suppressed genes included those in the protocadherin family of integrins , mitotic cytokinesis , and general development genes  were enriched for deletions. Statistical enrichment was q< 7.5x10-6.The broad class of developmental genes were also prevalent among the high CNA mutant TP53 tumors. Nervous system development , dsRNA metabolism , oxoacid metabolism , serine phosphorylation , nitrogen response (many genes), organic cyclic compound response (many genes), S-methylmethionine metabolism (BHMT and BHMT2), and hormone level regulation  including serotonin . Statistical enrichment was q< 2.0x10-5.While less statistically significant, metabolism genes had the greatest overall incidence of losses of any pathway in this analysis. Specifically, metabolic GO terms enriched for deletion included Coenzyme A metabolism (SPARC deletion), 9q (GSN deletion), 15q , and a region on 16q containing seven metallothionein genes . SPARC, also known as osteonectin, may be an adhesion suppressing gene and has been linked to cadmium by its specific downregulation as noted following oncogenic exposures of cadmium in bladder cancer [-3.The gene losses in the GO term “cadmium response” (CR) include a swath of genes regulated by heavy metal ions and distributed across the genome; some are in hot spots with other pathway deletions . These rr cancer . It is er cancer . Gelsolir cancer , and trar cancer . Sorbitor cancer , possiblr cancer . The cytr cancer , includir cancer . The losIn vivo studies have validated that knockdown of genes within gene clusters independently contribute to oncogenesis[TP53, may be commonly deleted due to neighboring tumor suppressors EIF5A and ALOX15B. Since some biological pathways utilize many genes within a short region of the genome in arrayed gene sets, it is possible some of our GO term enrichments were found due to local clustering. To address this possibility, we mapped the genes enriched for loss in PMCH tumors to their chromosomal locations and graphed their distribution   . Taken tum test) . Such daTP53 mutant tumors: those which evolved primarily by acquiring mutations (PMCL cohort) and those which evolved in part by acquiring copy number changes (PMCH cohort). Our gene pathway analysis of regions enriched for deletions in PMCH tumors revealed that immunological, metabolic, developmental, adhesion and matrix signaling, and cadmium response pathways are enriched for deletions. The CR pathway loss, in particular the loss of metallothionein genes, occurred earlier in tumor evolution than any other regional loss. Tumors with genetically suppressed CR largely did not alter their mutational spectrum or increase mutational load, but rather increased the amount of copy number changes by 186% , NOTCH1egulator ), and GRppressor ). But deegulated , as wellOylation . Howeverppressor , 48, yetppressor , 56. ExaChallenges surrounding genomic cancer experiments are progressing from the initial simple difficulty of obtaining genetic information to those difficulties associated with interpretation and applying genomic information . This stTP53 mutation. These patients were defined as having a diagnosis of endometrioid adenocarcinoma or endometrioid serous carcinomas. All patients must have required surgery which extracted the tumor, and no patients at the time of surgery had detectable metastatic lesions. No prior chemotherapy or radiotherapy was allowed for incorporation into this dataset. Somatic alteration determination was made possible by paired sequencing of normal tissue from the patient, either from blood or from tissue >2cm from the tumor. We defined tumors with p53 mutation and CNAs at higher than median levels as “PMCH” and the tumors with p53 mutation and CNAs at lower than median levels as “PMCL”. Median age of patients with PMCH and PMCL tumors were both 68 years.The NCI Cancer Genome Atlas cohort was utilized for this study, due to its consistent data structure and large sample size . Tumors TP53 mutant tumors were identified as those annotated with a p53 mutation in cBioPortal [All data were extracted and analyzed from The Cancer Genome Atlas (TCGA) consortium’s sequencing of Uterine Corpus Endometrial Carcinoma (UCEC) patient-tumor pairs, as maintained in the UCSC Cancer Genome Browser. The provisional dataset of 548 tumors, 242 of which had both CNA and mutation data, were downloaded and analyzed for this study. ioPortal , which wioPortal  to deterioPortal . GISTIC2ioPortal . SNP datioPortal  to generα:GISTIC scores are gene specific GISTIC2 [LOSS score is a gene deleted in many more PMCH tumors than PMCL tumors, and a gene with a very low αLOSS score is a gene deleted more often in PMCL tumors or a gene amplified in PMCH tumors.We used the following equation to determine loss enrichment scores for a given gene  GISTIC2  scores wLOSS scores were then input directly in GOrilla [Ranked α GOrilla  and outp GOrilla . For anyWhole genome gene IDs were input into bioDBnet  and convRaw reference normalized SNP signal was downloaded from the UCSC Cancer Genome browser, sliced for those tumors and genes under evaluation, and compared to GISTIC2 copy number calls. All GISTIC2 calls of -1 in any tumor were included in the analysis. SNP values were used to determine timing of region loss using the following assumptions: only monoallelic, not biallelic, losses occurred (chromosome 8p did not fit this assumption and was excluded), the stronger the frequency of the variant the earlier the loss (as it is present in more cells within the sample), and variants of a log2 ratio of <0.2 were potential noise events and excluded from analysis. All genes resulting from GO term enrichment analysis, as depicted in Figs TP53 mutation and/or Cadmium Response Loss cohorts, as described above. Tumors with a loss in any of the 12 genes enriched of loss in the Cadmium Response pathway were counted in the “CR” bar of For determination of transitions and transversions detected in whole-genome sequencing, we downloaded the COSMIC database  of canceS1 Fig2 genomic signal for the indicated genes. Each pathway panel is sorted left to right from highest to lowest median deletion depth. *Indicates genes on chromosome 8p, which may be confounded by an artificially high signal due to populations of cells with homozygous deletions. Some clusters of highly related and physically adjacent genes are collapsed into a single stacked column for clarity: MT , PCDHB , PCDHA , IFNA .Stacked histograms of copy number alteration magnitudes are generated from tumors with a -0.2 or greater decrease in log(PDF)Click here for additional data file.S1 TableThe table lists the sample identifier, p53 mutation status, as well as the tumor type classification, number of annotated mutations and the other diagnostic criteria for the clinical data used in the study.(XLSX)Click here for additional data file.S2 TableThe table lists the GO term, with their brief description, followed by the both q and p vales for significance of the observed losses and a listing of the genes included. Note that some genes are listed to impact more than one pathway, and appear more than once in the table.(XLSX)Click here for additional data file.


Caenorhabditis briggsae has been suggested to result in selection against certain combinations of nuclear and mitochondrial alleles, raising the possibility that mitochondrial–nuclear (mitonuclear) epistasis reduces hybrid fitness. To test this hypothesis, cytoplasmic–nuclear hybrids (cybrids) were created to purposefully disrupt any epistatic interactions. Experimental analysis of the cybrids suggests that mitonuclear discord can result in decreased fecundity, increased lipid content, and increased mitochondrial reactive oxygen species levels. Many of these effects were asymmetric with respect to cross direction, as expected if cytoplasmic–nuclear Dobzhansky-Muller incompatibilities exist. One such effect is consistent with the interpretation that disrupting coevolved mitochondrial and nuclear loci impacts mitochondrial function and organismal fitness. These findings enhance efforts to study the genesis, identity, and maintenance of genetic incompatibilities that precipitate the speciation process.In order to identify the earliest genetic changes that precipitate species formation, it is useful to study genetic incompatibilities that cause only mild dysfunction when incompatible alleles are combined in an interpopulation hybrid. Such hybridization within the nematode species  The formation of species is generally thought to depend on a period of reduced mating and gene flow between two populations . NeutralC. elegans  complexes, and are essential for oxidative phosphorylation , many re.g.,  specific to one cross direction when working with hybrid crosses, y 163 bp . Thus, te.g., The first step toward identifying the genetic basis of AF16-HK104 hybrid dysfunction is to determine whether mitonuclear epistasis exists. Deleterious effects of hybridization on fitness resulting from mitonuclear epistatic interactions have been recently described in a number of species. Together, these studies have established requirements for demonstrating that mitonuclear epistasis causes interpopulation hybrid dysfunction  is essential. A cybrid combines the nuclear genome of one population with the mitochondrial genome of the other. If any individual genotype will elicit dysfunction because of the separation of coevolved mitochondrial and nuclear alleles, this is the one. In the presence of negative mitonuclear epistasis that elicits hybrid dysfunction, cybrids are expected to exhibit reduced mitochondrial function and reduced fitness compared to the parental strains. Any phenotypic differences between a cybrid and its paternal parental population (nuclear genome donor) can be attributed to having replaced one mitotype with the other. This comparison rules out the possibility of confounding nuclear–nuclear epistatic interactions that might exist between the parental strains.C. briggsae and promote the use of C. briggsae to study the genetic basis of hybrid dysfunction and speciation.In the present study, we experimentally tested the hypothesis that the AF16 and HK104 populations contain mitochondrial and nuclear alleles that negatively interact to produce dysfunctional hybrids. The presence of asymmetric effects of cybridization suggests the accumulation of mitochondrial–nuclear DMIs, while an observed symmetrical effect could be due either to the presence of multiple DMIs or to separation of coevolved mitonuclear loci . DistingC. briggsae AF16 and HK104 strains were initially obtained from the Caenorhabditis Genetics Center. Strain maintenance and storage were performed as described  derived from populations that had been maintained for at least two generations with food ad libitum.escribed . Worms wC. briggsae wild isolates AF16 and HK104 were obtained from GenBank (accessions AC186293 and EU40779). Comparison of translated mitochondrial coding regions was performed using ClustalW and the PAM250 matrix  that distinguish AF16 and HK104 alleles were amplified by polymerase chain reaction (PCR) using published primer sequences  per nuclear chromosome and one SNP in the mitochondrial genome, an approach used in the evaluation of SexAI restriction site. PCR amplification from the AF16 and HK104 mitochondrial genomes (mtDNA) is predicted to produce a 798-bp amplicon at cb18178. SexAI should not digest the amplicon from AF16; digest of the HK104 allele is expected to result in 164 and 633 bp products. Forward and reverse PCR primers CACTTACAATGATAGGGTTTAAAATTC and CCATTTCTGCGAAAAAGAAAC were designed to amplify cb18178 from the mitochondrial genomes of both strains. All amplifications occurred in a Bio-Rad T100 thermal cycler using the following cycle sequence: 95° 2 min, 30 cycles of 95° 1 min, 45° 1 min, 72° 2 min, followed by a final 72° 5 min extension, and subsequently stored at 4°. After amplification, cb18178 amplicons were digested using restriction endonuclease SexAI (New England Biolabs) using the manufacturer’s recommended protocol. All amplicons were separated electrophoretically on 1% agarose gels in 1x TRIS-acetate EDTA buffer containing 2 µg/mL ethidium bromide. Micrographs of gels were obtained using an AlphaImager HP system (Alpha Innotech).The AF16-HK104 SNP cb18178 was previously determined to be located in the mitochondrial genome (ND5 gene (ND5∆) was assessed in purified genomic DNA extracts by PCR as above with the following change: the primers used to amplify the ND5 locus were CbMt_1F and 58R  levels in individual nematodes was performed as described MitoSOX Red fluorescence was then quantitated using ImageJ . The terIn vivo measurement of lipid content was performed by staining using the lipophilic fluorescent dye Nile red (Sigma) following Strains CP129–CP133 are available upon request.To assess the potential for mitonuclear coevolution in AF16 and HK104, their mitochondrial genome sequences were aligned. Comparison of mitochondria-encoded electron transport genes from AF16 and HK104  reveals Figure S1). The two cybrids (CP129 and CP130) whose F1 hybrid ancestor was repeatedly backcrossed to HK104 males were homozygous for HK104 alleles at each nuclear locus tested. The three cybrids (CP131–CP133) whose F1 hybrid ancestor was repeatedly backcrossed to AF16 males were homozygous for AF16 alleles at each nuclear locus tested.The two cybrid replicate strains with AF16 as the male (nuclear donor) parent and HK104 as the hermaphrodite  parent (CP129 and CP130), and three strains with HK104 as the male parent and AF16 as the hermaphrodite parent (CP131–CP133), were genotyped to confirm the success of the cybrid cross scheme  (Table S1). There was no significant difference between HK104(nuc); AF16(mt) cybrids and HK104.To identify the effect of genotype on organismal phenotype, the number of hermaphrodite self-progeny surviving to adulthood (self-brood size) was determined for the parental strains AF16 and HK104, and for the cybrid strains CP129 and CP130 , and CP131 and CP133 . Comparison of AF16(nuc); HK104(mt) to the fecundity of wild-type AF16 revealed a significant decrease in cybrid fecundity in this hybrid cross direction . AF16(nuc); HK104(mt) cybrid ROS levels are increased compared to AF16, but HK104(nuc); AF16(mt) cybrid ROS levels do not differ from HK104. As a positive control, the addition of paraquat to the nematode medium significantly increased detected ROS levels in every strain. Even in the presence of paraquat, the AF16(nuc); HK104(mt) cybrids maintained a significant increase of in vivo ROS compared to AF16, and the HK104(nuc); AF16(mt) cybrids still demonstrated no significant difference from HK104.in vivo Nile Red fluorescence in parental and cybrid strains revealed a statistically significant increase in whole-body lipid content in both cybrid cross directions compared to the paternal parental strains (Table S3).Quantification of C. briggsae was first suggested by asymmetric patterns of MTRD in reciprocal hybrid crosses . This result is remarkable, considering that no HK104 hermaphrodites  were employed in the ten generations of crossing used to produce CP132.For all but one of the strains, the mitochondrial genotype was consistent with the cybrid cross design. All CP132 amplicons are digested in the presence of Two notable implications of this finding are i) that paternal mitochondrial transmission occurred, and ii) that the resulting heteroplasmy for AF16 and HK104 mitochondria that must initially have existed following paternal transmission was rapidly and completely resolved in favor of transmission of HK104 mitochondria, producing a cybrid line apparently homoplasmous for HK104 mitochondria within 10 generations.C. briggsae mitochondria suggests that a conservative estimate of the limit of detection of heteroplasmy by PCR is at least as low as 5%  and/or increased production of ROS . This explanation is related to the idea that the existence of multiple DMIs is also predicted to produce more symmetrical effects . Thus, te.g., , or by selection for compensatory mutation in epistatic pairs (coevolution). It is important to distinguish between these in order to understand the forces that act to generate hybrid dysfunction and ultimately speciation.Mitochondrial–nuclear epistasis provides a valuable opportunity to more readily distinguish the two possibilities than with nuclear–nuclear epistatic pairs. Under the neutral process by which DMIs accumulate in separate lineages, only one hybrid cross direction should result in the production of dysfunctional hybrids if one of the epistatic loci is in the maternally inherited mitochondrial genome . Thus, wOn the other hand, when hybrid dysfunction is due to the separation of coevolved mitochondrial and nuclear loci, one would expect to observe symmetrical effects, because coevolved alleles at two (or more) loci should exist in both parental populations. Thus, any hybrid, regardless of cross direction, should experience the deleterious effects of separating the coevolved alleles of epistatic gene pairs. However, it is also possible that the neutral accumulation of multiple mitonuclear DMIs produces a symmetrical effect .Our data reveal asymmetric phenotypic effects in cybrids on fecundity and ROS levels in AF16 (nuclear); HK104 (mt) cybrids. We attribute these effects to the neutral accumulation of mitonuclear DMIs, with the incompatibilities being located in the HK104 mitochondrial and AF16 nuclear genomes. This conclusion can be reconciled with evidence of marker transmission ratio distortion patterns in AF16-HK104 hybrids. We previously found evidence for selection against the combination of AF16 nuclear chromosome V alleles, and HK104 mitotypes, in AI-RIL , it has C. elegans and C. briggsae are true biological species  and Hawaii (CB4856) strains  and a temperate clade strain (HK104); most reported incompatibilities do not completely eliminate hybrid fitness. As has been noted, the Dobzhansky-Muller model does not require that the epistatic interaction cause absolute loss of hybrid fitness   . This isbilities , and C.


The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection. There is currently a great effort world-wide to develop interventions such as vaccines and microbicides to decrease, or hopefully block, HIV transmission. To model the infection of women, the field utilizes the rhesus macaque vaginal transmission model. Understanding the initial events leading to infection after viral challenge of the female reproductive tract (FRT) is crucial for the development of functional prevention strategies. To this end, we developed a novel method for detecting infection in the rhesus macaque FRT after vaginal inoculation. This method utilizes single round replication defective vector that expresses dual reporter proteins, Luciferase and mCherry. Monitoring Luciferase expression allows us to identify the sites of infection within the intact FRT, while fluorescent protein mCherry allows us to visualize the single infected cells. Our studies revealed that virus can access the entire upper and lower reproductive tract. Infection occurs primarily in vaginal and ectocervical tissue, but can spread as far as the ovary and local draining lymph nodes. All classically defined susceptible cell types can be infected with the broadly tropic HIV envelope utilized in this study. Prevention strategies aimed at protecting from HIV infection should consider the entire FRT architecture as potentially susceptible and design interventions accordingly. Macaca mulatta; RM) SIV vaginal transmission model.The majority of new female human immunodeficiency virus (HIV) infections are the direct result of vaginal intercourse with an infected male partner Current paradigms imply that a small number of initial transmission events occur, which subsequently rely on the host immune response to infection to fuel viral spread To date, the only clinically demonstrated efficient prevention mechanism is based on oral pre-exposure prophylaxis (PrEP) that results in systemic distribution of antiviral drugs in vivo imaging system (IVIS). Luminescence is used as macroscopic guide for detection of tissue sites where individual infected cells can be subsequently identified with fluorescence microscopy of cryosections.The reporter system we use in these studies is designed to examine the localization and phenotype of cells that are susceptible to infection by the initial viral inoculum. We use a non-replicating vector that allows determination of the initial targets of infection, because any transduction events detected must be a derived from the challenge inoculum. Gene delivery through such a vector is commonly described as transduction, rather than infection because it only encompasses the replication steps up to the integration of the reporter genome. The reporter genome is delivered by a SIV-based lentiviral delivery system  which isThe vector strategy allows different envelopes to be utilized to determine vector tropism. Here we compare HIV-1 envelope . This pilot study revealed that we could detect as few as 10 cells in close proximity to an injection site . Four of the animals were pretreated with Depo-Provera (DP) to increase the likelihood of finding transmission events To initiate our development of a transmission model in rhesus macaques, we began with a VSV-G pseudotyped version of the vector, anticipating that the broad tropism would more readily generate a detectable luciferase signal. To increase the possibility of detecting a luciferase signal we utilized a vaginal biopsy, which we anticipated would provide a site of focused transduction and allow detection of signal because of the potential limitation of detection described above. The biopsy site would simulate the effects of microtrauma and generate a place where the vector could bypass mucosal barriers. Six RMs were atraumatically inoculated, vaginally and rectally, with high titer VSV-G pseudotyped reporter virions (TCID2) showing luciferase activity facilitated identification of single cells in thin tissue sections.The initial analysis for luciferase activity delivered by the VSV-G pseudotyped vector revealed that virions could access viable cells throughout the entire FRT . The bacExpression of mCherry fluorescence revealed that epithelial cells in the ectocervix  and vagiThrough pilot studies with VSV-G pseudotyped dual reporter vector we were able to demonstrate that we can detect vector expressed luciferase signals after vaginal exposure to virus. Importantly, we were able to optimize techniques to unequivocally identify mCherry expression of transduced cells by spectral imaging and luciferase staining in cryosections of small pieces of tissue exhibiting luciferase activity. Through this process we developed criteria to identify the bulk of transduced cells on a fluorescence microscope by 3 criteria: 1) mCherry positive, 2) luciferase positive after antibody staining, and 3) minimal TRITC fluorescence consistent with a signal from mCherry fluorescence rather than broad-spectrum tissue autofluorescence.50 range: 104.8–106.2) were inoculated into eight RMs with vaginal biopsies; four animals were DP treated. During luciferase detection of these animals, dissection into subsequently smaller regions enabled focusing of the luminescent signal into foci , CD3 (T cell specific) or CD68 (macrophage specific). We find that both CD4 T cells and macrophages are capable of being infected within the same vaginal domain . The JRFThroughout the vaginal vault, where viral challenge was administered, transduced cells were consistently located in the lamina propria below the basal epithelium . The ectFurther into the FRT, we find infection was also possible within the endocervix, consistent with previous studies that have focused on this site. Only 1 out of the 8 animals had evidence of infection by luminescence analysis of endocervical tissue. However, in animal CT82, a focus containing many transduced cells was identified . The lumcorpus albicans structures. The high density of blood and lymphatic vessels in the helus does not have a corresponding increase in the density of transduction. As seen in Although no luminescent signal was detected in the uterus or oviduct, we did find that virus was able to localize to and transduce cells in the ovary. A luminescent signal was identified in ovarian tissue of 6 out of 8 animals. When ovarian tissue was stained for CD3, 79% of transduced cells found within the ovary are found to be T cells, a further bias towards infection of T cells than is seen on the whole of the FRT. Transduced cells were primarily present in stromal tissue of the cortical zones of the ovary, proximal to developing follicles although not seen in Within local draining lymph nodes, only infrequent evidence of infection is found. Two animals were identified with modest luminescent signal in inguinal lymph nodes. Further examination revealed a number of CD4 cells expressing mCherry and luciferase reporters , but hig4 cells, was added per PCR reaction and at least 24 reactions were performed per tissue extract. All final products were separated on agarose gels and visualized by ethidium bromide staining. Amplification of the mCherry gene was confirmed by extracting DNA from the gel and sequencing with the inner set of primers.To validate our findings of mCherry and luciferase reporter expression we utilized nested PCR designed to detect rare transduction events within the tissue of exposed animals. Such PCR based approaches have been previously utilized to find evidence of rare infections events within days of vaginal or oral exposure of SIV Tissues that had a detectable luciferase signal corresponded to those in which there was a positive PCR signal . The ovaTo summarize, the regions of the FRT where luciferase and mCherry expressing cells were detected in the JRFL pseudotyped vector exposed RMs, varied between animals and could be identified throughout the FRT and could extend to local draining LNs . The majThe anatomical site and phenotype of the initial cells infected by immunodeficiency viruses within the FRT is heavily debated. Previous studies using similar high virus challenges have suggested that the endocervix is a primary site of infection in a RM vaginal challenge model There are disparate models of the initial cell type of infection during male-to-female transmission, with some studies implicating CD4 T cells While some have suggested that target cells in the upper FRT (uterus) might be sites of transmission trans-infection of resident T cells by migrating DCs carrying virions to lymph nodes is unlikely, though a possibility at later time points The mechanism by which infected cells are present in the lymph node is not apparent; vaginal or rectal inoculation could lead to virions accessing draining lymph nodes. Because the kinetics of expression require a day post-entry for reporters to be detectable, The distribution of sites transduced by either the VSV-G or JRFL pseudotyped vectors was quite similar. Both were able to reach the ovaries, and typically were observed to breach mucosal barriers in discrete foci. Yet the two different vectors reflected very different and anticipated tropism. This indicates that the distribution of viral particles in the FRT after vaginal exposure is not strongly influenced by envelope tropism. Throughout most of the reproductive tract, the mucosal barriers functioned to exclude virions and prevent infection from occurring, but where the barriers were overcome, there were multiple cells being infected. It is also notable that T cells are abundant where infection occurs, irrespective of the envelope protein . Where mThe phase of the menstrual cycle was not controlled as a part of this study, but could play a significant role in viral acquisition These findings on the wide range of locations and cell types susceptible to SIV have fundamental implications for development of prevention strategies. The sites of initial infection are not restricted to specific domains within the FRT tract and are not consistent between RMs. The observed abundant infection within the macaque vaginal vault is consistent with the failure of the diaphragm to protect women from HIV acquisition All animal studies were conducted in accordance with protocols approved by Northwestern University and Tulane National Primate Research Center local Institutional Animal Care and Use Committees (IACUC), protocol P0153. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and with the recommendations of the Weatherall report; “The use of non-human primates in research”. All procedures were performed under anesthesia using ketamine hydrochloride, and all efforts were made to minimize stress, improve housing conditions, and to provide enrichment opportunities . Animals were euthanized by ketamine hydrochloride injection in accordance with the recommendations of the panel on Euthanasia of the American Veterinary Medical Association.−1 penicillin, 100 µg mL−1 streptomycin and 292 µg mL−1 l-glutamine (Gibco). Rhesus macaque peripheral blood mononuclear cells (PBMC) were separated over Ficoll gradient and cultured at 2.5×106 cells mL−1 in RPMI containing 10% fetal bovine serum, 100 U mL−1 penicillin, 100 µg mL−1 streptomycin and 292 µg mL−1 l-glutamine. 24 hours prior to infection with vector, cells were activated with 100 IU ml−1 IL-2 and 5 µg mL−1 Phytohaemagglutinin (PHA). 1000 TCID50 JRFL pseudotyped vector was added to activated PBMC. Infected cells were sorted for mCherry expression on a Beckman Coulter MoFlo system.293T cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (HyClone) containing 10% fetal bovine serum, 100 Um L50) on TZM-bl cells as previously described and stored at −80°C until use 50 ranged from 104.8–106.2 for JRFL pseudotyped virions and 105.4–106.3 for VSV-G pseudotyped virions.A SIV-based pseudovirus vector system was generated by modification of the SIV3 vector system Macaca mulatta; RM) were used in this study, 5 of which have had one offspring and 8 of which are multiparous. Animals treated with Depo-Provera (DP) were given a single 30 mg dose one month prior to viral inoculation. All animals received a vaginal biopsy in the upper vagina/fornix, and were subsequently atraumatically inoculated vaginally and rectally with virus supernatant. Groups of 6 (2 DP Treated) and 8 RMs (4 DP Treated) were inoculated with VSV-G or JRFL Env expressing virions, respectively. Animals were sacrificed 48 hours after inoculation and their genital tracts were removed, stored in RPMI and shipped on ice overnight for further processing. This timeframe was chosen because it was the shortest time interval in which we could reliably detect reporter expression. Whole reproductive tracts, inguinal, and iliac lymph nodes were washed in PBS, then soaked in 100 mM d-Luciferin (Biosynth) and placed in the IVIS device to examine luciferase expression. Reproductive tract tissue was next divided in 5 regions: labia and lower vagina, upper vagina/fornix, cervix, uterus and ovaries and reimaged. Foci of luminescence detected in the second screen were excised and examined again for luciferase expression. Domains with luciferase detected in the third screen were cut to 2×2 mm2 pieces and frozen in optimal cutting temperature (OCT) compound. All analysis of luminescent signals was performed on Living Image Software.In total 14 female rhesus macaques (5′-ATAACATGGCCATCATCAAGGAGT-3′ (forward) and 5′-GTACTGTTCCACGATGGTGTAGTC-3′ (reverse). In the second round, 2 µl of the first round reaction products were amplified with 5′-CCGACTACTTGAAGCTGTCCTT-3′ (forward) and 5′-GTCTTGACCTCAGCGTCGTAGT-3′ (reverse). Amplification was performed using a Bio-Rad iCycler Thermal Cycler system . Each DNA sample was tested in ≥24 replicates. Negative controls were rhesus macaque ovary tissue from animals not exposed to the mCherry containing reporter vector. Second round PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. Sequences were confirmed by extracting DNA with Qaigen QIAquick Gel Extraction Kit analysis with the second round primers.Genomic DNA was isolated from 3 to 5 mg of frozen tissue using the Qaigen DNeasy Blood & Tissue Kit . In each nested PCR reaction, 250 ng of genomic DNA was utilized to detect a 268-bp DNA fragment of the mCherry reporter gene using an amplification procedure based on previously described methodology Cryosections of tissue (20 micron thickness) were fixed in 1% formaldehyde in PIPES buffer. To decrease background, tissue was treated with 100 mM L-Lysine. Tissue was stained for luciferase expression with polyclonal rabbit anti-firefly (Abcam), pre-labeled with Xenon AlexaFluor-647 (Invitrogen). Phenotyping target cells was achieved by blocking with donkey serum and subsequently staining with either rabbit anti-human CD3  followed by AlexaFluor488 conjugated donkey anti-rabbit Ig (Jackson Immuno), mouse anti-human CD4  followed by AlexaFluor488 conjugated donkey anti-mouse Ig (Invitrogen), or mouse anti-human CD68  followed by AlexaFluor488 conjugated donkey anti-mouse Ig. Spectral imaging of mCherry expression was performed on a Nikon A1R laser scanning confocal microscope equipped with a 60× objective and Nikon Elements Software. 5 color  image stacks containing 20–40 sections in the Z plane in 0.5 µm steps were acquired and deconvolved using softWoRx software (Applied Precision) on a DeltaVision inverted microscope.Figure S1Quantification of luminescent signal in transduced cells transferred to nude mice. (a) Rhesus macaque PBMCs are infected with JRFL pseudotyped virions, and mCherry expressing cells are selected by cell sorting. mCherry positive cells were defined by fluorescence signal relative to uninfected cells. The gating strategy isolates leukocytes by cell size and ensures cells are singlets and have bright mCherry expression, relative to uninfected controls. (b) Increasing numbers of cells are transferred via subcutaneous injection into nude mice (strain: SKH1), followed by intraperitoneal delivery of 100 mM d-Luciferin. Luminescent flux across the area of injection is measured by in vivo imaging system; a representative image of one mouse is shown. (c) Luminescent signal at the site of injection is related to the number of mCherry expressing cells transferred by a power law. Horizontal line indicates limit of detection (LOD), defined by 2.5× background signal. n≥3 for each measurement. Error bars indicate standard error.(TIF)Click here for additional data file.Figure S2Background luminescence levels from unchallenged macaque. In vivo imaging analysis of an unchallenged macaque treated with Luciferin was used to define background signal and threshold the luminescence from all LICh inoculated macaques. .(TIF)Click here for additional data file.Figure S3Infection of PBMC from two Rhesus Macaques with JRFL pseudotyped virions. 48 hours after infection with vector, infected cells are identified by fluorescence microscopy. Cells express mCherry, and stain positive for luciferase expression and HIV-1 receptor CD4, while remaining dim for TRITC. mCherry, luciferase, or TRITC signal is shown in red. CD4 is shown in green. Nuclear counterstain (DAPI) is shown in blue.  EH99, (b) FM27) Scale bars, 10 µm.(TIF)Click here for additional data file.Figure S4Secondary-only antibody controls are used to set thresholds for specific fluorescent signal. Tissue sections from the same vaginal vault tissues as shown in (TIF)Click here for additional data file.Figure S5The inguinal lymph node of a JRFL inoculated Rhesus Macaque harbors infected CD4 expressing cells. Although a high degree of auto-fluorescence limits the scope of tissue examination, some reporter signal distinguishable from background is observed in susceptible cells. mCherry, luciferase, or TRITC signal is shown in red. CD4 is shown in green. Nuclear counterstain (DAPI) is shown in blue. Scale bars, 10.7 µm. .(TIF)Click here for additional data file.


FTO gene, robustly related to body mass index (BMI) and obesity risk. We investigated the association between binge eating and the individual and combined effect of 32 SNPs robustly associated with BMI in a population-based sample. We hypothesized that higher BMI and binge eating might share a common genetic etiology.Cognitions and behaviors characteristic of binge eating are associated with a polymorphism in the n = 5,958) and 16 years . We tested associations between 32 BMI-related SNPs and binge eating in crude and BMI-, age-, and gender-adjusted regression models.Binge eating was assessed in adolescents from the Avon Longitudinal Study of Parents and Children at age 14 (FTO) that persisted after adjustment for BMI . A weighted allelic score consisting of all 32 BMI-related SNPs was associated with binge eating (P = 8 × 10−4); this association attenuated (P = 0.08) when rs1558902 was removed from the weighted allelic score.Crude analyses showed an association between binge eating and rs1558902 (FTO polymorphism. Although replication is needed, our findings have biological plausibility and are consistent with a postulated effect of FTO on appetite and food intake. Future studies should aim to understand the mechanisms underlying the relationship between FTO, binge eating, and obesity.BMI-related genes are associated with adolescent binge eating, in particular an  Binge-eating disorder (BED) is the most common eating disorder (ED) in the general population, with a lifetime prevalence of about 1.5% in adolescents and adults It is widely accepted that ED are partly explained by genetic factors MC4R (melanocortin 4 receptor) variants and BED FTO locus and loss-of-control eating, which is one of the main characteristics of BED/binge eating behavior Genetic association studies have investigated risk-conferring genes particularly in the dopamine, serotonin, and appetitive systems. Two studies found an association between Based on these studies and evidence from our studies that BED predicts overweight st December 1992 were invited to take part in the study. All women gave informed and written consent. The study website contains details of all the data that are available through a fully searchable data dictionary .The Avon Longitudinal Study of Parents and Children (ALSPAC) is a longitudinal, population-based, prospective study of women and their children The children from 14,541 pregnancies were enrolled; 13,988 children were alive at 1 year. At age 14 years 10,303 adolescents (singletons) were eligible for follow-up Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees.Binge eating was assessed using a two-part question (21). Participants were first asked about the frequency during the past year of eating a very large amount of food. Those who answered yes were directed to a follow-up question that asked whether they felt out of control during these episodes, i.e., whether they could not stop eating even if they wanted to stop. Adolescents who answered yes to both questions were classified as engaging in binge eating.For the purpose of the study we used binge eating either at 14 or 16 years of age as an outcome. We also created an ordinal variable for frequency of bingeing reported in the year prior to assessment , in order to determine whether increasing frequency of binge eating was associated with genetic risk.P < 5 × 10−7) were also excluded from analyses In total, 9,912 participants were genotyped using the Illumina HumanHap550 quad genome-wide SNP genotyping platform by the Wellcome Trust Sanger Institute, Cambridge, UK, and the Laboratory Corporation of America, Burlington, NC. Individuals were excluded from analyses on the basis of excessive or minimal heterozygosity, gender mismatch, individual missingness (>3%), cryptic relatedness as measured by identity by descent (genome-wide IBD >10%) and sample duplication. Individuals were assessed for population stratification using multi-dimensional scaling modeling seeded with HapMap Phase II release 22 reference populations. Individuals of non-European ancestry were removed from further analyses. SNPs with a final call rate of <95%, minor allele frequency (MAF) <1% and evidence of departure from Hardy-Weinberg equilibrium (HWE) . Data on BMI were available on 4,048 adolescents (82.3% of those with available data on binge eating and genotype).BMI, weight (kg)/height (m)Data on binge eating and genotype were available on 4,360 adolescents  at 14 years, and 3,663  at 16 years. Overall 4916 adolescents had genotype data and information on binge eating at either age 14 or 16 (2725 girls and 2191 boys).FTO variant to the allelic score, two models were tested, one including all 32 SNPs and one comprising 31 SNPs and excluding the FTO variant.We tested the effect of BMI associated SNPs in unadjusted and adjusted logistic regression models where binge eating was the outcome. Adjusted models included age, BMI and gender as covariates. Unadjusted logistic regression models were also conducted stratifying by gender to investigate whether the pattern of association differed between males and females. In order to increase the power of our analyses, we examined the relationship between a weighted allelic risk score and binge eating at either age in an unadjusted logistic regression model. We used effect size coefficients as reported in Speliotes et al. Finally, we investigated the relationship between BMI associated SNPs and an ordinal indicator of binge eating (frequency-based) in age stratified adjusted (for gender and BMI) ordinal logistic regression models.Analyses were run using STATA 12 and the R software package 2.15.2.The prevalence of binge eating in adolescence was 5.6% at age 14 years and 11.2% at age 16 years amongst adolescents included in the study (See TableFTO) and increased risk of binge eating . This association showed little attenuation after adjusting for BMI (OR = 1.20 (95% CI: 1.05-1.38), P = 8.0 × 10−3) (TableNRXN3) and binge eating in unadjusted (OR = 0.85 (95% CI: 0.72-0.99), P = 0.038), but not adjusted analyses .Minimally adjusted analyses (adjusted for gender and age) revealed an association in the hypothesised direction between the A allele of rs1558902 (P = 3.3 × 10−4) displayed slightly stronger evidence for association at the FTO locus than males , but the difference was not significant (P = 0.26)  and binge eating in the expected direction (OR=1.21 (95%CI: 1.04-1.40), P = 0.012) (see TableNEGR1) and binge eating in the expected direction  and rs10150332 (NRXN3) and bingeing in the direction opposite to expected (OR = 0.70 (95%CI: 0.49-1.01), P = 0.047).Amongst girls there was nominal association between rs2112347 (FTO) and higher binge eating frequency at age 14 years  and at 16 years . In other words, the presence of the A allele increased the odds of higher binge eating frequency  by 28% for each increase in level at age 16 : OR = 1.21 (95%CI: 1.01-1.46), P = 0.03] or 16 years  in the expected direction; but these did not replicate across ages.Two SNPs showed nominal association with frequency of binge eating at either age 14  has been examined in the literature and resulted in conflicting findings. In particular a recent study found an association between the obesity predisposing allele of the FTO variant rs9939609 and AN The association between SNPs at the FTO SNP (r2 = 0.93 in CEU HapMap 2) and a recent meta-analysis has shown similarly strong associations between each of these SNPs and overweight/obesity in children and adolescents FTO locus SNPs may be related to binge eating. Both rs1558902 and rs9939609 have previously been associated with eating behavior, such as loss of control in adolescents FTO) and the cognitive restraint subscale from the three factor eating questionnaire (a measure of restrained eating) FTO locus might increase the risk for both binge eating and overweight/obesity via higher palatable food and energy intake and decreased satiety; or that binge eating might be a mediator on the pathway between genotype and increased BMI/obesity.The rs1558902 polymorphism is in high linkage disequilibrium with the rs9939609 FTO locus SNP and binge eating, that showed little reduction in strength after conditioning on BMI, (b) the lack of association with other BMI-increasing SNPS, and (c) an association between the allelic risk score and binge eating when FTO was included that attenuated markedly after excluding FTO from the allelic risk score suggests that the effect of FTO on binge eating is not mediated by BMI. Therefore our findings suggest either pleiotropic effects of variants in or near the FTO locus, or that binge eating might be on the causal pathway between genetic variants and obesity. We have shown in two large population-based cohorts (including the one under study) that BED significantly increases the odds of overweight/obesity even when taking into account baseline weight Our findings of: (a) an association between an FTO polymorphisms, with lower activity (hence lower inhibitory control of eating) in this brain region post-prandially in subjects carrying the risk allele FTO rs9939609 AA carriers have a different brain response to food and ghrelin expression compared to TT carriers Recent findings have highlighted differences in prefrontal cortex activation in the processing of food stimuli associated with FTO locus might confer risk of binge eating. In our study, the strongest associations between the FTO polymorphism and binge eating were most evident in girls; although this might be due to a lower prevalence of binge eating in boys and hence lower power to detect differences, or a true differential gender effect .Thus, evidence from behavioral genetics and neuroimaging studies supports our findings that genetic variation at the FLJ35779/HMGCR in girls, and a polymorphism in NEGR1 with binge eating in boys). A variant at MTCH2 was associated with frequency of binge eating at age 14 years, and a polymorphism in TNNI3K with frequency of binge eating at age 16 years. These findings might reflect type I error given the large number of statistical comparisons performed in this paper, and the rather weak P values. Future studies should aim to replicate these findings. FLJ35779/HMGCR has not, to our knowledge, been previously implicated in appetite regulation. In contrast the TNNI3K and MTCH2 obesity risk-conferring SNPs have both been shown to be positively associated with emotional eating and uncontrolled eating In gender-stratified analyses some polymorphisms were nominally associated with binge eating:  and the Pro Opio Melanocortin (POMC) genes might be implicated in appetite control BDNF gene have been shown to be associated with ED phenotypes Amongst the SNPs studied we did not find an association between binge eating and variants within FTO) which has a risk allele frequency of 0.4, assuming 586 cases and 4331 controls , a heterozygote relative risk of 1.1, a multiplicative disease model , we only have ∼40% power to detect an association at α = 0.05 using an allelic test of association. This calculation illustrates that our study has low to moderate power to detect association at individual variants if the effect size of these variants is small (as is likely to be the case). However, assuming that one or more variants jointly contribute to binge eating, combining the variants into an allelic score should increase our power to detect association. Whilst an allelic score comprised of BMI associated variants did show some association with binge eating, our results suggest that most of this association was due to the FTO variant, rather than the other BMI related SNPs. We were unable to carry out a replication of our findings in an independent sample within the current study; therefore our findings need replication. However, it should be noted that Tanofsky-Kraff et al. FTO locus and loss-of-control eating; thus, our finding could be considered a replication.Strengths of our study include a relatively large sample size in comparison to previous studies on genetic risk for binge eating, an investigation of binge eating as a dichotomous outcome and of as an ordinal outcome , and a candidate gene approach based on 32 SNPs robustly associated with BMI. We also investigated a possible relationship between a weighted allelic risk score and binge eating. Limitations include the fact that data on binge eating were collected using self-report, however our questions were validated in a population-based sample and showed high reliability and validity FTO gene and adolescent binge eating, particularly in girls. Taken together with previous research, it appears that variants within or near the FTO locus are associated with a preference for energy-dense foods, greater food intake, less sensitivity to satiety cues, and loss-of-control eating episodes, all of which characterize binge eating. A recent GWAS of 339,224 individuals This study suggests a positive association between a polymorphism in the