The ecological insurance hypothesis predicts a positive effect of species richness on ecosystem functioning in a variable environment. This effect stems from temporal and spatial complementarity among species within metacommunities coupled with optimal levels of dispersal. Despite its importance in the context of global change by human activities, empirical evidence for ecological insurance remains scarce and controversial. Here we use natural aquatic bacterial communities to explore some of the predictions of the spatial and temporal aspects of the ecological insurance hypothesis. Addressing ecological insurance with bacterioplankton is of strong relevance given their central role in fundamental ecosystem processes. Our experimental set up consisted of water and bacterioplankton communities from two contrasting coastal lagoons. In order to mimic environmental fluctuations, the bacterioplankton community from one lagoon was successively transferred between tanks containing water from each of the two lagoons. We manipulated initial bacterial diversity for experimental communities and immigration during the experiment. We found that the abundance and production of bacterioplankton communities was higher and more stable  for treatments with high initial bacterial diversity. Immigration was only marginally beneficial to bacterial communities, probably because microbial communities operate at different time scales compared to the frequency of perturbation selected in this study, and of their intrinsic high physiologic plasticity. Such local “physiological insurance” may have a strong significance for the maintenance of bacterial abundance and production in the face of environmental perturbations. Marine bacterial communities are recognized as major players in the ecology of coastal ecosystems, i.e. nutrient cycling, production and decomposition of organic matter, and the regulation of major biogeochemical cycles Marine bacterioplankton consist of drifting microorganisms that often inhabit large volumes of water. This particularity necessitates the inclusion of their spatial distribution as a central element in understanding the relationship between their diversity and ecosystem functioning. Indeed, while it is intuitive that in connected aquatic systems “everything is everywhere” In a temporally fluctuating environment, diversity may increase the stability of a community through different mechanisms a concentrations, and in their bacterial community structure. Bacterioplankton communities were transferred between lagoons to mimic changes in environmental conditions experienced by natural aquatic bacterial assemblages We investigated how coastal bacterial assemblages with different initial levels of diversity and immigration responded to temporal environmental variation. We used bacterial communities from two distinct Mediterranean lagoons, the Thau and Bagnas lagoons. These two lagoons differ in their salinity, chlorophyll Testing the insurance hypothesis with natural bacterioplankton communities requires the whole community to be subjected to a temporally fluctuating environment. This was achieved by successive transfer of bacterioplankton lagoon communities, with differing levels of diversity and immigration over two contrasting environments.a concentrations (Chl a), over a short geographic distance (<10 km): the Bagnas (43°24′53′′N - 3°41′16′′E) and the Thau (43°19′47′′N - 3°31′13′′E) lagoons. Salinity measured with a Cond probe 197i were 8 and 34 respectively, and Chl a concentrations  and rinsed in deionized water, and were immediately transferred to a field laboratory located at the edge of Thau lagoon. We filled two 900-L polypropylene tanks with either 1 mm filtered water from the Thau or the Bagnas lagoons and allowed a constant water flow (1 day retention time). Our experimental approach consisted of transferring the Thau bacterial community between tanks containing water from the Thau and the Bagnas lagoons to experimentally mimic temporal variation in environmental conditions , most of which are connected to the Mediterranean Sea nditions . In pracnditions . These mPrior to filling with bacterioplankton communities, diffusion chambers were acid-washed (24 h in 10% HCl) and rinsed thrice in deionized water and dried. Chambers were filled with bacterioplankton communities from the Thau Lagoon with three different levels of diversity (see below). −3) and “low” (dilution 10−5) diversity. Measuring band number in a denaturing gradient gel electrophoresis  confirmed that a fraction of the community had been eliminated. As DGGE is effective for detecting dominant bacterial operational taxonomic units (OTU), a decrease in DGGE band number between two dilutions is consistent with a reduction in community diversity. Dilutions of bulk bacterial communities were done with sterile lagoon water (0.22 µm filtrated plus two autoclave cycles at 121°C for 20 min). The dilution treatment could itself influence initial abundances within incubated communities. We thus controlled for bacterial abundances by allowing a 48 h acclimation period in Thau lagoon water before the first environmental transfer.The dilution-to-extinction approach was used to obtain three different starting levels of bacterioplankton diversity. Dilution removes the rare species and thereby different dilutions create communities that differ in their diversity. This approach allowed us to explore the link between community diversity and functioning Identical series of initial diversity levels were prepared for each of the three immigration treatments: no immigration, medium  and high  immigration. Immigration events were performed twice for each chamber, at each transfer  using a FACSCalibur flow cytometer (BectonDickinson) after nucleic acid staining with green fluorescent SYBR®GreenI (Invitrogen). Light scattering was used in conjunction with fluorescence to discriminate bacterial cells -samples .The initial bacterial community diversity was assessed by denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes after touchdown PCR amplification Time series for bacterial abundance and production were first analyzed by a three factor analysis of variance (ANOVA) with time, diversity and immigration included as explanatory variables. No interactions among factors were included. Then, we ran a second fully factorial ANOVA on the residuals (thus taking account of the effect of time) with diversity and immigration as explanatory factors. This reveals the effect of diversity and immigration on the abundance and production of our bacterial communities independently of the effect of time. We performed Tukey-Kramer post-hoc tests  to test for differences between diversity and immigration levels on temporal averaged abundance and production.We also calculated the temporal coefficient of variation (CV) for both bacterial abundance and production, for each replicate population, from 48 h (i.e. after acclimation) to the end of the experiment (240 h). The coefficient of variation is a common and convenient metric for temporal stability The diffusion rate of the solutes through the 0.22 µm chamber membranes must be greater than the incubation time between two transfers (48 h) so that communities experience changes in their environment. When chambers containing Thau water were immersed in Bagnas water, the changes in salinity inside the diffusion chambers were gradual and reached equilibrium after 15 h of incubation . ClogginLagoon bacterial cells did not contaminate the experimental bacterial communities within each chamber during a 10 days submersion period . This coA pre-requisite of the experiment is that bacterioplankton from both lagoons differed in their bacterial community composition. One week before the experiment, triplicate water samples were collected from the Bagnas and Thau lagoons to explore differences in the bacterioplankton composition based on DGGE banding pattern analysis. Both lagoons had a similar number of OTUs, but they differed in their OTUs composition .The DGGE analysis of the diluted Thau bacterial community confirmed that our dilution treatments resulted in a bacterial diversity gradient .Overall, bacterial abundances increased during the first 100 h and then decreased through time . We founBacterial production also varied trough time . Temporaperformance-enhancing effect characterized by an increase in temporal mean bacterioplankton abundance and production, and second, a buffering effect characterized by a reduction in the temporal variance of production The insurance hypothesis predicts a buffering effect of both diversity and immigration on ecosystem functioning based on species asynchrony and compensatory dynamics. Using an experimental approach, we observed two effects of diversity when the bacterial communities are confronted with temporal environmental fluctuations. First, a in situ hybridization with microautoradiography, or nano-scale secondary-ion mass spectrometry, which have been shown as promising tools for correlating microbial identity with specific metabolic functions, for individual cells, within heterogeneous bacterial communities Click here for additional data file.Figure S2We assayed how much our diffusion chambers were resistant to potential contamination from the surrounding waters in a pilot experiment performed before the main experiment. This control of sterility was determined by incubating for 10 days four chambers (2 completely waterproof and 2 with 0.2 µm membranes) containing sterile Thau lagoon water into a flow-through natural Thau lagoon water tank. The sterile Thau lagoon water was obtained after filtration through a 0.22 µm polycarbonate membrane plus two cycles of autoclave at 121°C during 20 min. Using this procedure, bacterial abundance was abated by 98.4% (from 2.3 106 cells ml−1 to 3.6 104 cells ml−1). Most of the persisting cells were considered as dead cell. Two milliliters of water were withdrawn from the chambers at t = 0, t = 1 h, t = 24 h and t = 240 h. Bacterial abundance was determined by flow cytometry as described in the experimental procedures section. Bacterial abundance did not significantly change over time and between the waterproof and 0.22 µm membranes diffusion chambers Click here for additional data file.Figure S3One week before the experiment, 200-ml of water from both Thau and Bagnas lagoons were collected with a 500-ml acid washed glass bottle to determine the DGGE banding pattern of the bacterioplankton communities. The DGGE method is describe in the experimental procedure section. The number of OTUs was similar with 17 and 20 OTUs for Thau and Bagnas bacterioplankton, respectively. However, both communities shared only 5 OTUs (y 5 OTUs . This re(DOC)Click here for additional data file.


A total of 19 SNPs on chromosomes (SSC) 3, 5, 8, 10, 14, and 18 were significantly associated with OC lesion scores . The SNPs MARC0098684, MARC00840086, MARC0093124, and ASGA0062794 at SSC14 36.1–38.2 Mb encompass a region of six linkage disequilibrium (LD) blocks. The most significant SNP ASGA0062794 is located in a LD block spanning 465 kb and covering the gene encoding T-box transcription factor 5 (TBX5). A SNP (c.54T > C) identified in TBX5 was significantly associated with OC lesion scores in a single-marker analysis. TBX5 c.54T > C showed highest LD with ASGA00627974 (r2 = 0.96) and superior association with OC lesion scores over other SNPs when included in the genome scan, whereas its treatment as an additional fixed effect in the GWAS statistical model led to a drop of significance of nearby markers. Moreover, real-time PCR showed different transcript abundance of TBX5 in healthy and defect cartilage. The results imply that the association signal obtained on SCC14 is largely attributable to TBX5 c.54T > C likely to be in LD with a regulatory polymorphism of TBX5. The transcription factor TBX5 interacts with GJA5 and MEF2C both being involved in vascularization. This study provides evidence for epistatic interaction of TBX5 and MEF2C, thus supporting deficiency of blood supply to growth cartilage as being fundamental for the initiation of OC.Osteochondrosis (OC) is an orthopedic syndrome of the joints that occurs in children and adolescents and domestic animals, particularly pigs, horses, and dogs. OC is the most frequent cause of leg weakness in rapidly growing pigs causing animal welfare issues and economic losses. In this study, a genome-wide association study (GWAS) was performed using the Porcine 60k SNPChip in animals of the breed Large White ( Osteochondrosis (OC) is a joint disorder characterized by the disturbance of endochondral ossification in the articular cartilage and the epiphyseal growth plates that is common in human beings and several animal species including pigs  gene which represents a functional and according to the GWAS, also a positional candidate gene for OC.In general linkage studies based on genotyping a limited number of highly polymorphic markers, mostly microsatellites, in special family designs have revealed numerous QTL regions for various traits  from SUISAG during 2005–2008. The animals used were selected from the two ends of the distribution of OC lesion scores  recorded from 2,622 animals. In order to avoid bias due to any obvious parameter that might affect OC scores, selection was done accounting for sex, year, body weight, and ancestry; in fact, selected animals represent offspring of 141 boars and 264 dams. The animals were kept in the performance test station of SUISAG and fed ad libitum during the whole testing period from 30 to at average 103 kg of body weight. The animals were examined for osteochondral lesions at seven positions of the carcass after dissection  and the epiphyses was examined at the cutting surface. Scores from 1 to 4 were defined . Osteochondral lesions at the DEU and CMF are scored from 1 to 6, because these lesions were more variable  or OCcat low (n = 162) .Genomic DNA and phenotypic data were obtained from animals of the breed Large White  to 4 (=severe)   and approved standard techniques as recommended by the manufacturer. This array contains 62,163 SNPs distributed throughout the porcine genome. Raw data were normalized and assigned a cluster position for genotyping with the GenomeStudio software . SNPs were filtered with call rates >99%. Markers were excluded if they had low minor allele frequency <5%. Deviation from Hardy-Weinberg equilibrium and the degree of LD were analyzed using HaploView 4.2  and T-box transcription factor 5 (TBX5) both located in close proximity to each other and a number of significant markers, were screened for SNPs in the coding region by comparative sequencing of PCR fragments of eight individuals. The PCR products were purified and comparatively sequenced in both directions using an ABI3130 DNA Analyzer. We detected a single SNP at g.38,175,026T > C or position 54 relative to the start codon (c.54T > C) of TBX5. Then, TBX5 genotyping was performed by PCR-RFLP technique using the restriction enzyme PfeI. The following primers were used: forward 5′-AAACTGCTGGTGGAAGCCTA-3′ and reverse 5′-GCAGAGAAAGGTGGGATTGA-3′. The PCR was performed in 20 μl volume containing 50 μg of genomic DNA, 1 × PCR buffer (with 1.5 mM MgCl2), 0.25 mM of dNTP, 0.2 μM of each primer, and 0.5 U of Taq DNA polymerase . The PCR procedures were performed under the following conditions: initial denaturing at 95°C for 5 min followed by 40 cycles of 30 s at 95°C, 30 s at 62°C, and 1 min at 72°C, and final elongation of 10 min at 72°C. After PCR amplification, PCR products were checked for specific amplification on 1.5% agarose gels  and digested by PfeI enzyme. The digested products were separated using 3% agarose gels.Two genes, namely T-box transcription factor 3 . Although the subset of animals of a total of 2,622 animals was built in order to combat any bias due to population stratification, the success of these measures of the selection procedure in this regard was tested. Accordingly, to determine population stratification, pairwise clustering of identity by state (IBS) distance of the samples was computed using the IBS and multidimensional scaling (MDS) approach within JMP Genomics 5.0. MDS analysis show that all samples cluster together within indication of any subclusters, suggesting that population stratification is not prominent Figure . We obtap-value < 10−5 was used, equaling a false discovery rate (FDR) based q-value ≤ 0.1  was applied in order to define LD blocks across a 3.4 Mb region on chromosome 14. The D′ and 2r coefficient  were used to calculate pairwise LD estimates. Finally, LD measure D′  between pairs of SNPs was estimated to create blocks of consecutive SNPs based on those in strong LD  and control (OCcat low) for testing trait association by a χ2 test with one degree of freedom. Whereas, for the GWA analysis to assess the association between SNPs and the ordinal traits of OC lesion scores, a logistic regression model  was used with the fixed effect of line, sex, and slaughter weight as a covariate. Genotype effects were estimated as odds ratios (ORs) and 95% confidence intervals (95% CIs). ORs were estimated for homozygotes of the minor allele compared with homozygotes for the major allele and heterozygotes (dominant model). OR value less than 1 imply the desired effect of lower OC lesion scores. A significance threshold of  Storey, . The samGene annotations for 1 Mb genomic intervals around the significantly associated SNPs were identified by the Ensemble genome browser (Build9)GAPDH) gene. All RNAs were stored at -80°C for further analysis.Total RNA was isolated from the articular cartilage of CMF of four divergent sib pairs using TRI reagent  according to manufacturer’s protocol. After DNase treatment a column-based purification using the RNeasy Mini Kit  was done. The RNA samples were visualized on 1% agarose gels containing ethidium bromide to check RNA integrity. RNA was quantified by spectrometry with a NanoDrop ND-1000 spectrophotometer . In addition, absence of DNA contamination was checked by using the RNA as a template in standard PCR amplifying fragments of the glyceraldehyde-3-phosphate-dehydrogenase . The 2−ΔCt and 2−ΔΔCt methods were used to calculate relative expression of the target gene TBX5. For the 2−ΔCt method, mean Ct values of TBX5 in each sample was normalized to housekeeping gene values  and affected  tissues, where the ΔCt values in the affected tissues was normalized to the ΔCt values in the normal tissues to obtain the ΔΔCt values. Statistical analysis was performed by mixed model analysis using Proc Mixed of SAS version 9.2  with the fixed effects of sex and OC lesion score; family was used as a random effect and slaughter weight as covariate. Differences were considered significant at p-value ≤ 0.05.RNA was reverse-transcribed into cDNA using random primers and an oligo dT primer in the presence of Superscript III reverse transcriptase . Quantitative real-time RT-PCR (qPCR) was performed on LightCycler480 system using the LightCycler 480 SYBR Green I Master kit . Two common housekeeping genes The phenotype distribution of OC lesion scores at different joints among the animals used is shown in Table p-value ≤ 10−5, q-value ≤ 0.1) with either one or more of the OC lesion score traits. Significant SNPs were distributed along chromosomes (SSC) 3, 5, 8, 10, 14, and 18. The Manhattan plots of −log10 for OC lesion scores are shown in Figure ASGA0060524) was found significantly associated with OC lesion scores at DEU that mapped to position 3.4 Mb. As for CMH, two SNPs (M1GA0019792 and M1GA0019802) were identified and located on 144.9 and 145.2 Mb, respectively. Most interesting, significant associated with OC lesion scores of CMF were found for five SNPs  at 25.4 and between 36.1 and 38.2 Mb. In addition, there were 11 significantly associated SNPs which located on SSC3, 5, 8, 10, and 18  to ASGA0062794. Therefore, the TBX3 and TBX5 gene were selected to screen of sequence variation and to assess LD with the significant SNP-markers and association with OC in the same population used for the GWAS and LD study.We investigated the region between 36.0 and 38.5 Mb on SSC14 where multiple significant SNPs were located, that were associated with OC lesion scores at CMF. LD was calculated between the SNPs in this region across 2.5 Mb. Figure TBX3 revealed no polymorphisms. In the TBX5 gene, a single synonymous SNP was detected at position 54 relative to the start codon (c.54T > C) of TBX5. The SNP TBX5 c.54T > C showed a high LD of 2r = 0.96 and D′ = 1.0 with ASGA0062794. The single-marker association analysis between TBX5 c.54T > C with OC lesion scores is shown in Table p < 0.0001, OR = 0.40, 95% CI = 0.26–0.61), DEU , and OCcat . Moreover, when integrating the SNP TBX5 c.54T > C in the GWAS together with the 48,716 SNPs of the SNPchip, it showed highest statistical significance of association with CMF and DEU among the markers of the haplotype block and even reached the significance threshold for the trait OCcat  of all markers in the region below the significance threshold indicating that the SNP TBX5 c.54T > C explains much of the variation in OC lesion scores depending on this genomic region . This may be simply due to the reduction of residual variance. However, it might also be a hint for epistatic interaction between TBX5 and a gene located in the region on SSC2. Indeed, Ingenuity Pathway Analysis revealed a network of indirect and direct effects between TBX5 and a number of genes all being located between 87.3 and 115 Mb on SSC2 , ELL2 , GLRX [glutaredoxin (thioltransferase)], ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 (endoplasmic reticulum aminopeptidase 2), PAM , TNFAIP8 , FAM170A , FTMT , LOX (lysyl oxidase), and SNCAIP . The logistic regression model complemented with an appropriate interaction term of TBX5 c.54T > C and SNPs on SSC2 revealed pairs of SNPs with significant interaction effects .Comparative sequencing of the coding region of TBX5 did not reveal any SNP leading to amino acid exchange we performed qPCR of CMF cartilage samples of divergent sib pairs with either low or high OC lesion scores in order to test the hypothesis that TBX5 may exhibit a regulatory polymorphism in LD with the SNP TBX5 c.54T > C. Significant differences were found (p < 0.05) with 1.63-fold higher transcript abundance in the normal cartilages compared with OC cartilages . Among these, 12 SNPs were located in previously reported QTL regions for leg weakness and OC related traits in pigs on SSC3, 5, 14, and 18.TBX3 (T-box 3) and TBX5 (T-box 5) as candidate genes for the liability to develop OC lesions. TBX3 and TBX5 belong to the T-box gene family that were demonstrated to function as transcriptional activators and repressors  and the MGMT (O-6-methyguanine-DNA methyltransferase) gene, respectively. PTPRE plays a major role in regulating bone structure and metabolism  gene and 0.13 kb downstream of the PLEKHH2  gene, respectively. There are no obvious functional links between MOB1A, or PLEKHH2 and OC lesions. However, the region (63.3–90.0 Mb) is consistent with the QTL for leg weakness and OC related traits of previous studies  which may play a role in the endothelial inflammatory disease, as atherosclerosis. The mapping site of KRT8 on the porcine SSC5 matches in a QTL region for OC lesions of fissure between cartilage and bone and OC  and the MKLN1 (muskelin 1) gene, respectively. These sites match a QTL for front leg conformation detected by ] which was identified as a target gene of TBX5 and harbors binding site for transcriptional factor of TBX5 in its promoter  was not significant associated with OC lesion (p = 0.18).TBX5 with OC lesion scores. This study provides further positional and genetic-statistical evidence for the candidacy of TBX5. However, beside TBX5 also two other genes (TBX3 and ZSCAN21) reside in the same haplotype block and may potentially be responsible for the association with OC lesions. TBX3 is expressed in the anterior and posterior margins of both fore limb and hind limb buds. The posterior expression of TBX3 is crucial for development of the more distal limb elements  the high LD of TBX5 c.54T > C with the significant SNP-markers at 36.0–38.5 Mb of SSC14, (2) its association with OC lesion scores confirmed by single-marker analysis, (3) the superior association of TBX5 c.54T > C when included in the genome scan, and (4) the LOD-drop when fitting the GWAS model using the TBX5 c.54T > C genotype as an additional fixed. The results imply that the association signal obtained on SCC14 is largely attributable to TBX5 c.54T > C, or other as-yet-unspecified polymorphisms in LD that most likely affect the expression of TBX5. The functional role of TBX in synergy with GJA5 and MEF2C promote deficiency of blood supply to growth cartilage as being fundamental for the initiation of OC.In summary, we identified The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Livestock_Genomics/10.3389/fgene.2013.00078/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


A rough estimation of the density of states is given from temperature measurements.Using high-temperature annealing conditions with a graphite cap covering the C-face of, both, on axis and 8° off-axis 4H-SiC samples, large and homogeneous single epitaxial graphene layers have been grown. Raman spectroscopy shows evidence of the almost free-standing character of these monolayer graphene sheets, which was confirmed by magneto-transport measurements. On the best samples, we find a moderate  It is now widely accepted that graphene-based devices are promising candidates to complement silicon in the future generations of high-frequency microelectronic devices. To this end, the most favourable technique to produce graphene for industrial scale applications seems to be epitaxial graphene (EG) growth. This can be done by chemical vapour deposition on a metal ,2 or by n-type doped, with a low-carrier mobility. On the contrary, on the C-face of the same SiC substrates, there is no need of a C-rich buffer layer at the interface before growing the first graphene layer [2/V s in the work of Ref. [On the Si-face of 6H or 4H SiC substrates, graphitisation at high temperature in an Ar atmosphere close to atmospheric pressure shows promising results for on-axis substrates. In this way, single-layer epitaxial graphene (SLEG) has already been grown at the full wafer scale ,8 but anne layer -12. In t of Ref. .2/Si [For a long time, whatever the growth technique, the uniformity and quality of the EG was not good enough to find evidence of the so-called "half integer" quantum Hall effect (QHE). However, recently, large SLEG areas have been produced on the C-face of on-axis SiC substrates and, on such monolayer graphene, the carriers were holes with mobility close to the one found in mechanically exfoliated graphene films on SiO2/Si . Consequ2/Si . This sh2/Si .In this work, we compare the results of graphene growth on semi-insulating, on axis and and 8° off-axis, 4H-SiC substrates. The quality, uniformity and size of the growth products will be compared using optical microscopy (OM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and micro-Raman spectroscopy (μR). Then, Hall effect measurements will be done at different temperature in order to extract the density of states in the epitaxial monolayers.To produce SLEG, in both cases of on axis and 8° off-axis SiC substrates, we used the recipes of Ref.. On the In Figure 2/Si [-1) which is strong evidence that there is no strain at the layer to substrate interface . Third, this 2D-band can be fitted with a single Lorentzian shape with a FWHM of 30 cm-1 [I2D/IG between the integrated intensities of the 2D-band and the G-band is high, which suggests weak residual doping in the order of 3 to 6 × 1012 cm-2 [Tens of similar monolayer islands grown on, both, on axis and off-axis substrates were probed by Raman spectroscopy. We used the 514 nm laser line of an Ar-ion laser for excitation and got very similar features. At the micrometer size, all spectra reveal that the islands are of the same nature and very homogeneous. First, the D-band, which usually indicates the presence of disorder or edges defects, is very weak and the Raman signature is extremely close to the one found for exfoliated graphene on SiO2/Si . Second, 30 cm-1 . Fourth,012 cm-2 . Altoget012 cm-2 .Gold alignment marks were used to select some SLEG position by OM. Then, they were contacted by e-beam lithography and subsequent deposition of a contact layer made of Cr/Au in Hall bar configuration. A typical example is shown in Figure 12 to 1 × 1013 cm-2 at low temperature, with a weak temperature dependence.Then transport measurements were done at low temperature on the different samples, using a maximum magnetic field of 13.5 T. The contact geometry allowed simultaneous measurement of, both, the longitudinal and transverse voltages with the current flowing between two injection contacts at the flake extremities. In both series of samples, from the sign of the Hall voltage, we found that the carriers were holes (in agreement with other results published on the C-face ). The ho12 cm-2, no QHE could be detected and only Shubnikov-de Haas (SdH) oscillations were found. This is shown in Figure For carrier concentrations larger than 3 × 10RK/4(N + 1/2) in which RK = h/e2 is the Von Klitzing constant [N = 0, 1, 2... As already known, this peculiar sequence of resistance values is the well-known quantum transport signature of the monolayer graphene Landau levels [ns = 1.2 × 1012 cm-2 and mobility μ ~5000 cm2/V s at T = 1.6 K. At B = 12 T, the longitudinal resistance cancels while the transverse resistance tends to 12.9 kΩ which is the expected value for the N = 0 plateau (RK/2).For the low doped layers, the transverse resistance exhibits now quantized Hall plateaus, clearly governed by the sequence constant  and N = u levels . In Figuns = 8 × 1011 cm-2 and a mobility μ ~11,000 cm2/V s. The mobility is high enough and the concentration low enough to make the N = 0 and N = 1 plateaus well resolved and stable up to 13.5 T. The experimental results of Figure xy ~(V(B)+V(-B))/2I, where the voltage V is measured between a lateral probe and the current drain. At high magnetic fields, we identify V(+B)/IG as ρxx, where G~4 is the geometric factor and I is the current.In Figure ρxx(B) is shown in Figure ρxx ~exp(-Ea/kBT) of the N = 0 plateau. This activation energy Ea is the energy separation between the Fermi energy EF and the delocalised states of the N = 1 Landau level. In Figure xx taken at different magnetic fields in the vicinity of the RK/2 plateau. The activation energy Ea varies from 0.7 to 3.3 meV between B = 10 and 13 T, which remains much smaller than the distance between the first and the second Landau level (~120 meV at B = 10 T). This indicates that the Fermi energy is firmly pinned by localised states. Ea has been calculated by taking into account only temperatures above 6 K. At lower temperatures, there is an additional contribution to the conductivity, which is visible in Figure The temperature dependence of E). The filling factor is calculated from B = 10 to 13 T, each filling factor change Δν at a given magnetic field corresponding to a density variation Δns = nsΔν/ν. The Fermi energy shifts by ΔEa to compensate for the density variation and the mean value for the density of states at energy ~Ea is given by ρ(E) = Δns/ΔEa.In principle, from the activation energy, we can reconstruct the density of state ρ increases and becomes one order of magnitude larger than the density of states ρ0(E) without magnetic field at a comparable energy EF ~100 meV: ρ0(E) ~15 × 109 cm-2 meV-1. The shape of ρ(E) gives a rough upper bound of the half-width at half-maximum (HWHM) of the N = 1 Landau Level. We find HWHM ≤ 3 meV. This value is in good agreement with results obtained recently on EG by STM [0 over the whole investigated energy range. This observation, combined with the fact that hopping was neglected, indicates that more detailed investigations are still needed.Following this procedure, already used in the early times after the discovery of the integer QHE , we findG by STM . HoweverB = 13.5 T. This magnetic field is far from the filling factor υ = 2 and; therefore, the breakdown occurs at relatively low current: I = 0.5 μA, which corresponds to a current density j = 0.025 A/m. By comparison, for III-V heterostructures, critical current values of 1 A/m are reported.Finally, since EG has recently been proposed for metrological application, we plot, in Figure To summarize, we have shown the possibility to grow large islands of monolayer graphene on the C-face of on-axis and 8° off-axis commercial 4H-SiC wafers. The graphene layers are continuous, almost free-standing and show quantum transport properties comparable with high-quality, low-doped, exfoliated graphene. We show evidence of half-integer QHE specific of graphene monolayer and give a first estimate of the density of states in the magnetic field.AFM: atomic force microscopy; EG: epitaxial graphene; HWHM: half-width at half-maximum; QHE: quantum Hall effect; SEM: scanning electron microscopy; SdH: Shubnikov-de Haas; SLEG: single-layer epitaxial graphene.The authors declare that they have no competing interests.NC and AC carried out the Graphene growth, the Hall Bars fabrication, the AFM, SEM and Raman characterisation. AT carried out the Raman investigation and interpretation. BJ, BJ and CC carried out the magnetotransport measurements. Finally PG and JC participated in the design and the coordination of this work. All authors read and approved the final manuscript.


The thermal conductivity of monolayer graphene nanoribbons (GNRs) with different tensile strain is investigated by using a nonequilibrium molecular dynamics method. Significant increasing amplitude of the molecular thermal vibration, molecular potential energy vibration and thermal conductivity vibration of stretching GNRs were detected. Some 20%∼30% thermal conductivity decay is found in 9%∼15% tensile strain of GNR cases. It is explained by the fact that GNR structural ridges scatter some low-frequency phonons which pass in the direction perpendicular to the direction of GNR stretching which was indicated by a phonon density of state investigation. GNRs have high in-plane electron mobility and high out-plane electron resistance, making them the most promising candidates for the next generation of logic devices and sensors [Graphene nanoribbons (GNRs) are several layers of two-dimensional honeycomb lattice of sp sensors . Potenti sensors .The thermal conductivity of GNRs plays an important role on the design and application of high quality GNR electronic devices. Investigations by experiments and molecular dynamic (MD) simulations have demonstrated that the thermal conductivity of graphene is ultrahigh ,8,9. ResStretching of GNRs would be a critical issue in the manufacture and application of GNR electronic devices, such as the graphene resonators for showing the images of scanning electron microscopy (SEM) ,16. ActuTherefore, an in depth investigation concerning the relationship between the tensile strain and thermal conductivity of GNRs is quite necessary. The measurement of GNR's thermal conductivity from experiments is quite difficult due to their limited size. In the present work, in order to correlate the thermal conductivity with the tensile strain of mono-layered GNRs, a non-equilibrium molecular dynamic method (NEMD) with quantum correction was applied to construct the MD models, and then the effect of tensile strain on the thermal conductivity of GNRs was investigated .2.For MD simulations, firstly, the classical MD method based on the COMPASS potential function is applied to describe the valence-bond and non-bond energy of graphene. The COMPASS force field calculation fits well with that of experiment work, and has been verified in simulating the mechanical properties of carbon nanotubes (CNTs) .ab initio forcefield because most parameters were derived based on ab initio data. Compared with other force fields, COMPASS valence terms include high-order (cubic and quartic) bond, angle, torsion angle, out-of-plane angle terms, and the cross-coupling terms between them [The COMPASS force field is first a high quality forcefield and focuses on high accuracy in prediction. It is an een them . The COMeen them .sink) was applied on the left. On the right, a heat source with a temperature of 500 K (Tresource) was applied. Then the response as the resulting heat flux was measured.To ensure a reasonable molecular configuration, before NEMD calculations, atoms are assumed to achieve equilibrium in a NVT Nosé–Hoover thermostat (300 K) for 10,000 time steps with a fixed time step of 1.0 fs . GNR str5 time steps giving a total molecular dynamics time is 0.3 ns which is a sufficiently time for a COMPASS force field model [J is the heat flux, ÄT is the temperature gradient along the length direction, S is the cross-section area of graphene layers.After the equilibration, NEMD approach run for 3 × 10ld model . After tThe cross-section area of GNR layers is a product of the width of GNF and the thickness of graphene layer. In order to verify the feasibility of our simulation approach, the thickness of single graphene layer was used as 0.335 nm referred to a previous simulation .−1·K−1, which is in agreement well with that of the previous study [im is the atom's mass, and Bk is the Boltzmann constant.It was found the thermal conductivity in the strain-free GNR is 525 Wm3.For the train-free GNR, MD simulations show that there are some structural distortions on the graphene surface that look like ripples in , that isTo understand the effect of GNR tensile strain, MD simulations were performed for the same size and temperature gradient GNRs with different tensile strains, namely, 2.5%, 5%, 7.5%, 8%, 9%, 10%, 11%, 12%, 12.5%, 15%, separately. We find GNR under a larger tensile strain yields necking due to Poisson's contraction in the length direction, and this phenomenon is evident in close-up SEM images of suspended epitaxial graphene resonators ,16.−1·K−1. Then the thermal conductivity begins to rapidly decrease to 371 Wm−1·K−1 while the tensile strain changes from 7.5% to 10.0%, which is almost 30% smaller than that of a strain-free GNR.MD simulation results show GNRs with different tensile strains have different thermal conductivities. In versus time curve also exhibits an outrageous oscillation caused by the increasing potential energy vibration. The final values of thermal conductivity were determined by averaging 400 fore-and-aft time step points in calculated thermal conductivity versus time step curve.The change of thermal conductivity relates to some molecular configuration changes of GNRs. Graphenes under different tensile strains exhibit different atomic configuration characteristics. In graphene under tensile strain bigger than 7.5% cases, graphene's structural ripples are clearly elongated and changed into some structural ridges which have larger sizes in the stretching direction. These structural ridges of stretching GNR shown in  have als−1·K−1.This thermal conductivity decrease coincides with the generation of GNR structural ridges. It implies a stretching graphene's thermal conduction is influenced by the structural ridges on the GNR. When the tensile strain increases from 12.5% to 15%, the thermal conductivity remains steady at about 425 Wm−1) compared with that of strain-free GNR. These phonons scattered by structural ridges result in a decrease in stretching GNR thermal conductivity.Theoretical and experimental studies have demonstrated two-dimensional phonon transport in nano-ribbons ,23,25,26The thermal conductivity reduction is attributed to the increasing lattice anharmonicity of stretching GNR . Thermal4.In summary, molecular dynamic simulations have shown that GNR surface ripples will be changed into structural ridges by tensile strain. The tensile stretching of a GNR results in a significant increase of amplitude of the molecular thermal vibration and thermal conductivity vibration of the GNR. The thermal conductivity of GNR can be decrease 20%–30% by stretching. It is explained as that phonon waves propagating across graphene structural ridges will be scattered by these graphene structural ridges and cause a decrease in thermal conductivity. Stretching should be an effective way to control the thermal conductivity of monolayer nanostructures.


Frontotemporal lobar degeneration (FTLD) has been subdivided based on the main pathology found in the brains of affected individuals. When the primary pathology is aggregated, hyperphosphorylated tau, the pathological diagnosis is FTLD-tau. When the primary pathology is cytoplasmic and/or nuclear aggregates of phosphorylated TAR-DNA-binding protein (TDP-43), the pathological diagnosis is FTLD-TDP. Notably, TDP-43 pathology can also occur in conjunction with a number of neurodegenerative disorders; however, unknown environmental and genetic factors may regulate this TDP-43 pathology. Using transgenic mouse models of several diseases of the central nervous system, we explored whether a primary proteinopathy might secondarily drive TDP-43 proteinopathy. We found abnormal, cytoplasmic accumulation of phosphorylated TDP-43 specifically in two tau transgenic models, but TDP-43 pathology was absent in mouse models of Aβ deposition, α-synucleinopathy or Huntington’s disease. Though tau pathology showed considerable overlap with cytoplasmic, phosphorylated TDP-43, tau pathology generally preceded TDP-43 pathology. Biochemical analysis confirmed the presence of TDP-43 abnormalities in the tau mice, which showed increased levels of high molecular weight, soluble TDP-43 and insoluble full-length and ~35 kD TDP-43. These data demonstrate that the neurodegenerative cascade associated with a primary tauopathy in tau transgenic mice can also promote TDP-43 abnormalities. These findings provide the first in vivo models to understand how TDP-43 pathology may arise as a secondary consequence of a primary proteinopathy.The online version of this article (doi:10.1007/s00401-013-1123-8) contains supplementary material, which is available to authorized users. MAPT gene that encodes the tau protein, whilst mutations in Granulin (GRN), Valosin Containing Protein (VCP), or C9ORF72 genes can cause FTLD-TDP or amyotrophic lateral sclerosis [The major classes of frontotemporal lobar degeneration (FTLD) are those characterized by the presence of neuronal and glial inclusions composed of either tau protein (FTLD-tau) or TAR DNA-binding protein of 43 kDa  , 40. Famclerosis , 22, 54.clerosis , 47, 51.clerosis  and cortclerosis . The mecOne common feature in most human TDP-43 proteinopathies is the presence of cytoplasmic phosphorylated TDP-43 (pTDP-43), while normally TDP-43 is readily detected in the nucleus. Several studies have shown that antibodies specific for phosphorylated S403/404 and S409/410 TDP-43 recognize TDP-43 proteinopathies in humans , 38 and To study TDP-43 in the context of amyloidosis, we utilized TgCRND8 mice that overexpress a double mutant (K670N/M671L and V717F) form of human amyloid precursor protein (695 amino acid isoform—APP695) leading to age-dependent cognitive deficits and Aβ amyloid pathology  and Tg25Anti-TAR-DNA-binding protein 43 (TDP-43) rabbit polyclonal antibody was purchased from ProteinTech Group . Rabbit anti-phospho Ser409/410 TDP-43 antibody is from CosmoBioUSA . Rabbit anti-phospho Ser410 TDP43 antibody is from Sigma-Aldrich . pSer129 is a mouse monoclonal antibody that specifically recognizes phosphorylation of α-synuclein at S129 . AT8 (Th202 solution for 10 min. Sections were incubated with primary antibodies in 0.1 M Tris pH 7.6/2 % FBS overnight at 4 °C and subsequently incubated with biotinylated anti-rabbit or anti-mouse (Vector) for 1 h. To detect signal, a standard peroxidase ABC system (Vector) was used with a DAB reagent kit (KPL). Sections were counterstained with hematoxylin, rehydrated by an ascending series of ethanols and xylene, and cover slipped with Cytoseal (Thermo Scientific).Mice were humanely euthanized. Fixed brains were paraffin embedded and sectioned. Intact spinal columns were immersion fixed, followed by fine dissection to remove vertebrae and subsequent post fixation. Sections were deparaffinized in xylene and rehydrated by immersion in a descending series of ethanols and steamed for 30 min. Peroxidase activity was quenched by incubation in an 80 % methanol/2 % HPost-embedding immunogold electron microscopy (IEM) is essentially the same as previously described . Rabbit Sections were deparaffinized in xylene, rehydrated by immersion in a descending series of ethanols, steamed for 30 min, incubated with blocking solution (1 % fish skin gelatin/1 % BSA/2 % FBS/0.1 M Tris pH 7.5) for 2 h, incubated with primary antibodies in 0.1 % fish skin gelatin/5 % BSA/0.2 % FBS/0.1 M Tris pH 7.6 with azide overnight, and subsequently incubated with Alexa Fluor 594 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit (Invitrogen) for 1 h. Sections were post-fixed with 10 % phosphate buffered formalin, immersed in amino-black to quench lipofuscin auto-fluorescence, and counterstained with DAPI (Pierce). Sections were mounted and cover slipped with Fluoromount G (Southern Biotech). Pictures were obtained using an Olympus BX51 fluorescent microscope with FITC, Texas Red, and DAPI filters. To visualize co-localization, images from each filter were layered in Photoshop.Sagittal half forebrains of rTg4510 and NT mice were frozen on dry ice and then fractionated as previously described [Immunohistochemistry to visualize pTDP-43 (S409/410) was performed on mouse models of Aβ amyloidosis , tauopathy (rTg4510 and JNPL3), α-synucleinopathy (Lines M47 and M83), HD (HD586-82Q), and non-transgenic (NT) controls at time points when each model has robust aggregates of their primary pathologic protein . For rTg4510 mice, mice expressing the tTA activator in the absence of the tau responder were also used as a control.In control mice, low levels of pTDP-43 S409/410) were observed in the nucleus of some neurons  . Given tGRN, VCP, and C9ORF72 genes [MAPT gene that encodes tau protein [C9ORF72 expansion mutation, but similar elevation of tauopathy were not observed in FTLD associated with GRN mutations suggesting that an overlap of FTLD-TDP and FTLD-Tau may occur in the context of C9ORF72. Interestingly, King et al. [MAPT gene as well as the C9ORF72 expansion. This individual presented with dominant Pick-like tau pathology as well as the TDP-43 and p62 pathology that characterizes C9ORF72 carriers; however, her siblings lacked this tau variant and developed typical pathology associated with the C9ORF72 hexanucleotide repeat. Surprisingly, we were unable to find publications in which the authors clearly screened FTLD-tau cases with known pathogenic MAPT mutations for the level and/or distribution of phosphorylated TDP-43. TDP-43 pathology has been identified in a subset of different proteinopathies including tauopathies that occur in the absence of MAPT mutations [TDP-43 aggregation, cytoplasmic redistribution, phosphorylation and misprocessing characterize the pathology found in FTLD-TDP, associated with mutations in the 72 genes , 54. In  protein . Recentlg et al.  reportedutations , 37. TheIn healthy neurons, TDP-43 is primarily localized within the nucleus, and the redistribution and aggregation of TDP-43 within the cytoplasm are thought to be critical events in TDP-43-proteinopathies . Much ofIn order to determine if the accumulation of pTDP-43 is directly related to the aggregation of hyperphosphorylated tau or simply the expression of high levels of human mutant tau protein, we examined brain and spinal cord from young rTg4510 and JNPL3 mice, respectively, with high levels of transgenic tau expression and minimal levels of hyperphosphorylated, aggregated tau protein. No change in TDP-43 phosphorylation or cytoplasmic distribution was observed in young tau mice. In contrast, cytoplasmic pTDP-43 was observed in both rTg4510 and JNPL3 mice after they developed overt tau pathology and, in the case of JNPL3, motor dysfunction. These results strongly support the idea that mutant tau expression alone does not induce pTDP-43 accumulation within the cytoplasm and that aggregation of tau is also critical.DCTN1, the large p150glued subunit of the dynactin complex [The distribution of neurofibrillary tau pathology and neuronal loss in rTg4510 and JNPL3 mouse models of tauopathy closely correlated with the distribution of the neurons containing cytoplasmic accumulations of pTDP-43 protein. Hyperphosphorylated tau and pTDP-43 protein co-localized within many of the affected neurons; however, the overlap was incomplete since hyperphosphorylated tau aggregates could be found in some neurons in the absence of cytoplasmic pTDP-43. Rarely, the converse was observed. This data suggests that tau pathology precedes the redistribution of TDP-43 into the neuronal cytoplasm. Interestingly, pTDP-43 was present within the neurites of rTg4510 mice in the absence of AT8 immunopositive neuritic tau, suggesting that TDP-43 cytoplasmic accumulation may develop in other cellular domains after cytoplasmic redistribution is initiated by tau aggregation or other factors in the perikarya. Another interpretation is that neuritic pTDP-43 may co-localize with tau that is not phosphorylated at S202 or S202/T205, the phospho-tau epitopes recognized by CP13 and AT8 in the current study, respectively. Since most remaining cortical and hippocampal (CA1) neurons in the rTg4510 mice examined had tau pathology within the neuronal cell body, it seems likely that the pTDP-43 positive neurites originate from these affected cells; however, the methods utilized in this paper cannot exclude other origins of pTDP-43 localization within the neurites. Nevertheless, tau is a microtubule binding and stabilizing protein and it is possible that its aggregation perturbs normal microtubule function leading to the cytoplasmic accumulation of TDP-43 that might normally be transported to the nucleus or the presynaptic domains . The not complex  and cell complex .To determine if the association between pTDP-43 and hyperphosphorylated tau altered the biochemical profile of TDP-43, we performed protein fractionation from brains of rTg4510 tau transgenic and NT mice. There was no change in total levels of TDP-43 in the soluble tau fraction across genotypes; however, rTg4510 mice did have a significant increase in a higher molecular weight smear that was immunopositive for TDP-43. The nature of these high molecular weight species is unclear; however, similar high molecular weight species have been identified in affected human brains although these tend to be found in insoluble, not soluble, fractions , 39, 40.The association between cytoplasmic pTDP-43 and tau appears specific since we saw no evidence of cytoplasmic relocalization of pTDP-43 in mouse models of Aβ amyloidosis, α-synucleinopathy or polyglutamine disease (HD), regardless of the broad spectrum of ages and stages of primary proteinopathy examined. A number of papers report TDP-43 pathology in AD with estimates ranging from 23 to 56 % , 30, 51.The amyloid models that we utilized in the current study do not develop tauopathy similar to that observed in AD; therefore, it is possible that amyloidosis and tauopathy act in concert in AD to produce TDP-43 pathology. Caccamo et al.  reportedTDP-43 pathology is frequently observed in the brains (18–60 %) of patients with DLB , 19, 37,Our group has also shown that constitutive overexpression of wild-type and less so mutant TDP-43 can cause aggregation of hyperphosphorylated tau protein at S202, one of the two epitopes that is recognized by the AT8 antibody used in the current study . This daMAPT mutation carriers should assess their autopsy tissue for overlapping tau and TDP-43 pathology; however, such screens are generally precluded by the availability of tissue from known MAPT carriers. Functional studies which exploit the capabilities of the in vivo model systems utilized in this report could compliment these human studies. For example, the rTg4510 model of tauopathy can now be crossbred with conditional TDP-43 models created by our group and others [Our results show a clear link between tau pathology and cytoplasmic accumulation of phosphorylated TDP-43 in the controlled in vivo systems of tau transgenic mice. It is currently unknown if this association between tau and TDP-43 can affect the disease course in either the mouse models or in human tauopathies. Our studies lay the groundwork for such investigations. Certainly, our data would suggest that groups with large cohorts of d others , 24 to dd others . In addid others  that areSupplemental Figure 1. Neither extracellular amyloid plaques nor intracellular aggregates of α-synuclein and huntingtin cause accumulation of pTDP-43 within the cytoplasm of associated cells. Brains from 13 month old TgCRND8 amyloid precursor protein mice were immunostained with (a) anti-Aβ antibody 33.1.1 to visualize extracellular plaques and (b) anti-pTDP-43 (S409/410) antibody within the hippocampus. Hippocampus from 6 month old HD586-82Q huntingtin transgenic mice (labeled HD82) were immunostained with (c) anti-huntingtin antibody 2B4 and (d) anti-pTDP-43 (S409/410) antibody. Brain stem from 15 month old M47 α–synuclein transgenic mice were immunostained with (e) anti-pSer129 α-synuclein antibody to visualize α-synuclein pathology and (f) anti-pTDP-43 (S409/410) antibody. Brain stem from 15 month old M83 α–synuclein transgenic mice were immunostained with (g) anti-pSer129 α-synuclein antibody to visualize α-synuclein pathology and (h) anti-pTDP-43 (S409/410) antibody. Regions where the primary proteinopathy was robust were chosen for each model. The bars indicated 100 μm. Additional ages (Electronic Supplementary Table 1) for each model were examined and also found negative for cytoplasmic pTDP43 (409/410). Supplemental Figure 2: Cytoplasmic, phosphorylated TDP-43 (S410) co-localizes with tau pathology in cell bodies of the cortex of rTg4510 mice. Immunofluorescence shows pre-tangles and neurofibrillary tangles composed of hyperphosphorylated tau recognized by the antibody AT8 (a), AT100 (d), and PHF-1 (g) which co-localizes with cytoplasmic aggregation of pTDP-43, recognized by the S410 antibody . Co-localization between pTDP-43 (S410) and AT8 (c), AT100 (f), and PHF-1 (i) is shown in yellow and is similar to that observed between the dual phosphorylation TDP-43 epitope examined in Figure 4. Nuclei were stained with DAPI (blue). Neurons shown are from the frontal cortex of an 8 month rTg4510 mouse at 20X magnification. White bar indicates 50 μm. Supplemental Figure 3. rTg4510 show robust biochemical signatures of hyperphosphorylated, aggregated tau at 10 months of age. Soluble and sarkosyl-insoluble fractions from the saggital half forebrains of 10 month old rTg4510 (Tg) and NT mice were analyzed by Western blot using the CP13 antibody for phosphorylated tau (S202). These samples were the same fractions utilized for the biochemical analysis of soluble and insoluble TDP-43 levels . Human transgenic tau from rTg4510 mice shows a mobility shift from its normal MW at ~50kD into a 64kD species (arrow), representing the high degree of aggregation and hyperphosphorylation in these sample. Loading for soluble fractions was normalized to GAPDH (PDF 3853 kb).Below is the link to the electronic supplementary material.


FOXC2. However, FOXC2 mutation analysis has been performed in only 1 family, and no mutation analysis has been performed on sporadic  SEDACs. We recruited 17 SEDAC subjects consisting of 2 familial and 7 sporadic cases and examined FOXC2 mutations by Sanger sequencing and structural abnormalities by TaqMan copy number assay. We identified 2 novel FOXC2 mutations in 2 familial cases. Incomplete LDS penetrance was noted in both families. Four subjects presented with SEDACs only. Thus, SEDAC caused by the heterozygous FOXC2 loss-of-function mutation should be considered a feature of LDS, although it often manifests as the sole symptom. Seven sporadic SEDAC subjects had no FOXC2 mutations, no symptoms of LDS, and showed differing clinical characteristics from those who had FOXC2 mutations, suggesting that other gene(s) besides FOXC2 are likely to be involved in SEDAC.Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space from a defect in the dura mater. Most cases are sporadic; however, three familial SEDAC cases have been reported, suggesting genetic etiological factors. All familial cases are associated with lymphedema-distichiasis syndrome (LDS), whose causal gene is  Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space via defects in the dura mater Fig. 1. The etiological factors of SEDAC remain unclear. Its origin has been attributed to congenital dural defects, arachnoid proliferation and inflammation, previous surgery, and closed spinal trauma.FOXC2, a forkhead family transcription factor (OMIM 153400); in fact, molecular screening of 81 probands resulted in the detection of FOXC2 mutations in 100% of LDS patients.FOXC2 is a good candidate gene for SEDAC; however, FOXC2 mutation analysis has been performed in only 1 SEDAC family associated with LDS, and no mutation analysis has been performed on sporadic SEDACs or SEDACs unrelated to LDS (solitary SEDACs). The relationship between SEDAC and FOXC2 mutations remains unclear.LDS is an autosomal dominant disorder with variable expressivity. Its major features are lymphedema and distichiasis. The penetrance of lymphedema or distichiasis is 70% to 80%.FOXC2 mutations in 2 familial and 7 sporadic cases of SEDAC.To gain insight into the genetic etiology of SEDAC, we examined The study was approved by the institutional review boards of RIKEN Center for Integrative Medical Sciences, Keio University and Fukushima Medical University. A written informed consent was obtained from all participants and/or guardians on the behalf of the minors/children participants.We recruited a total of 17 Japanese SEDAC subjects. Seven of them were from a previously reported familyFOXC2 (NM_005251.2) was PCR-amplified using KOD FX  and the primers 5′-TCTCCCCCTCTGGCTCTCT-3′ (forward) and 5′-TCTGCAGCCCCTTAATTGTC-3′ (reverse). PCR products were sequenced by the dideoxy termination method, using an ABI 3730 automated sequencer , and screened for mutations.DNA was extracted from saliva using the Oragene DNA self-collection kit  according to the manufacturer's protocol. The single coding exon of FOXC2 variant on its ability to bind DNA, we used FoldX as previously described.To evaluate the effect of the N118K FOXC2. The primers and probes for the qPCR were designed using Primer Express software v3.0 (Applied Biosystems). The RNase P gene was used as a reference gene. All assays were performed with the TaqMan Universal PCR Master Mix according to the manufacturer's protocol (Applied Biosystems).The TaqMan real-time quantitative PCR (qPCR) method was used to examine the copy number of FOXC2 genes were PCR-amplified using the primers 5′-TATGAATTCAATGCAGGCGCGCTACTC-3′ (forward) and 5′- TATAGATCTTCAGTATTTCGTGCAGTCGTAG-3′ (reverse). The PCR products were cloned into the EcoRI and BglII sites of the pFLAG-CMV-4 expression vector . The nucleotide sequences of the clones were confirmed by Sanger sequencing.The wild-type, c.354C>G, and c.733delG 2 at 37°C.COS-7 and HeLa cells were grown in Dulbecco's modified Eagle's medium and 10% foetal bovine serum. Transfection was performed using FuGENE6 transfection reagent  according to the manufacturer's protocol. For protein expression, COS-7 cells were transfected in a 12-well plate using 500 ng of the FOXC2-pFLAG construct. For luciferase assays, HeLa cells were transfected in a 24-well plate using 500 ng of the FOXC2- pFLAG construct, 50 ng of the FOXC2 luciferase reporter , and 1 ng of the pRLTK control vector (Promega).Transfected COS-7 cells were washed with PBS and harvested by scraping after 48 h of incubation. The cell lysates were resolved by SDS-PAGE. The N-terminal FLAG epitope was detected by immunoblot analysis, using a mouse anti-FLAG monoclonal antibody (Sigma Aldrich).Transfected HeLa cells were washed with PBS and dissolved in 150 ml of passive lysis buffer after 48 h of incubation. The dual-luciferase assays were performed in triplicate, using the Promega Dual Luciferase Assay kit (Promega), according to the manufacturer's protocol.FOXC2 mutations in 2 familial cases. In Familiy 1 .No mutation was found in the other 7 subjects with sporadic SEDACs. Structural abnormalities such as deletion or duplication of FOXC2 mutations were also diagnosed as LDS, since the family members had lymphedema and/or distichiasis  and 48.3 (range: 36–64) years, respectively. Male/female ratios of SEDAC with and without FOXC2 mutations were 4/6 and 4/3, respectively. Furthermore, the number and location of the cysts were different. All SEDAC subjects without FOXC2 mutations had a single cyst, which occurred in the thoracolumbar junction, whereas 7 of 10 SEDAC subjects with FOXC2 mutations had multiple cysts that occurred in various areas, ranging from the upper thoracic to the sacral regions.The age of diagnosis of the SEDAC patients with utations Table 1:FOXC2. Whole cells were resolved by SDS-PAGE and subjected to immunoblot analysis. Detection of the N-terminal vector-encoded FLAG epitope demonstrated a stable product, 56 kDa in size, for both wild type and c.354C>G FOXC2. It also demonstrated a product, 31 kDa in size, for the c.733delG FOXC2 protein  mutation in 12 of them. This mutation co-segregated with LDS: distichiasis, lymphedema and SEDAC were observed in 12, 11, and 7 of the 12 subjects, respectively. Taken together, these data suggest that familial SEDAC occurs as a feature of LDS caused by FOXC2 mutation, although symptomatic SEDAC is a rare complication of LDS.We identified 2 novel ema, 20% Fig. 2. FOXC2 mutations presented with SEDAC alone and only 3 subjects with SEDAC had both of the 2 major features of LDS (lymphedema and distichiasis). Consequently, the mode of inheritance of the disease in the family was sometimes unclear . The identification of new SEDAC associated genes will help not only in early detection but also in clarifying the pathology of SEDAC.Our study suggests that additional etiological factors of SEDAC, other than the features Table 1.FOXC2. As a result of our study, SEDAC patients and their family members will be able to use genetic screening to evaluate their risk and undergo close examination and surgery before developing irreversible neurological defects. It is easy to screen for the FOXC2 mutation since FOXC2 is a small 1-exon gene that can be covered by a single PCR reaction. To date, we have observed that SEDAC caused by FOXC2 mutations is also associated with LDS. Hence, examination of family history and clinical investigation for the features of LDS are important for the early detection of the disease. However, most cases of SEDAC are not caused by the FOXC2 mutation and show different clinical features from those with FOXC2 mutations. Further studies aimed at identifying new SEDAC causal gene(s) are necessary to clarify their etiological factors and pathogenesis.In summary, we showed that SEDAC is caused by heterozygous loss-of-function mutations in


Programmed cell death-1 (PD-1) is a co-inhibitory receptor expressed on lymphoid and non-lymphoid-derived cells that negatively regulates peripheral T-cell responses. PD-L1, the major PD-1 ligand, is expressed on various tumors and is being investigated as a possible predictive marker for anti-PD-1 therapy. Few studies have determined PD-L1 expression across tumor types using a consistent process. We evaluated PD-L1 expression across human tumor samples using a novel, automated, sensitive, and specific PD-L1 IHC assay (developed by Dako) using the 28-8 antibody.PD-L1 expression in 654 commercially available tumor samples was explored. Percentage of tumor plasma membrane staining was determined at ≥1+ intensity. Macrophage and lymphocyte PD-L1 status was determined by compartment . Immune cell density and PD-L1+ immune cell frequency were assessed on a scale of 0 (none), 1 (mild), 2 (moderate), and 3 (heavy). PD-L1 positivity was evaluated using 5% surface expression values for tumor cell membrane staining.Of 654 samples examined, spanning 19 tumors from different sites, 89 (14%) were PD-L1+ (≥5% frequency). Highest PD-L1+ frequencies were seen in head and neck , cervical , cancer of unknown primary origin , glioblastoma multiforme , bladder , esophageal , triple negative (TN) breast , and hepatocarcinoma . Across a subset of head and neck tumor samples, high PD-L1 expression was seen: lip , tongue , larynx , and oral cavity . In breast tumors, TN tumors showed a higher frequency PD-L1+ status  compared with estrogen receptor, progesterone receptor, or HER2+ positive tumors (0/45). PD-L1+ was higher in squamous (SQ) compared with other histologies within esophageal , cervical , and bladder  tumors. PD-L1+ tumors were associated with immune cell density and PD-L1+ immune cells, especially in TN breast and laryngeal tumors.PD-L1 is expressed in various human tumors and is associated with tumor grade, squamous histology, immune cell density, and co-localization of PD-L1+ immune cells. Highest PD-L1+ frequencies were seen in head and neck, cervical, CUP, GBM, bladder, esophageal, TN breast cancer, and hepatocarcinoma.


We report on a novel electrically tunable hybrid graphene-gold Fano resonator. The proposed metamaterial consists of a square graphene patch and a square gold frame. The destructive interference between the narrow- and broadband dipolar surface plasmons, which are induced respectively on the surfaces of the graphene patch and the gold frame, leads to the plasmonic equivalent of electromagnetically induced transparency (EIT). The response of the metamaterial is polarization independent due to the symmetry of the structure and its spectral features are shown to be highly controllable by changing a gate voltage applied to the graphene patch. Additionally, effective group index of the device is retrieved and is found to be very high within the EIT window suggesting its potential use in slow light applications. Potential outcomes such as high sensing ability and switching at terahertz frequencies are demonstrated through numerical simulations with realistic parameters. The idea of electromagnetically induced transparency (EIT) and the concept of Fano resonance were originally discovered in the context of quantum mechanics2346Since its first practical isolation by Novoselov and Geim in 20048121415166 times the diffraction limit), are easier to tune (obtained via applying a gate voltage to the graphene), and propagate longer distances and have narrower spectral support (due to the lower intrinsic losses in graphene)192123242629In this work, graphene's ability to support SPPs at THz frequencies is exploited to design a Fano resonator that hybridizes SPPs generated on graphene and gold surfaces. Graphene surface plasmon polaritons (GSPPs) have several advantages when compared to SPPs generated on metallic surfaces: GSPPs have higher volume confinement . Variation in cμ can be achieved by applying a gate voltage to the graphene patch using nearly transparent electrodes without perturbing the response of the resonator as suggested recently in referenceObviously, in this design, SPP of the graphene patch acts like the “dark mode” of the traditional Fano resonator designs made of only metalsS1 = 5.5 μm, the dimension of the graphene patch S2 = 1.6 μm, the relative permittivity of the substrate dε = 3.5, and graphene's electron mobility μ = 10,000 cm2/Vs and chemical potential cμ = 1500 meV. It should be noted here that the value assigned to μ is a rather conservative choice considering the latest experimental resultsD1) and the graphene patch (marked as D2) have broad (continuum-like) and very narrow spectral supports, respectively. D1 and D2.The hybridization of the dipolar SPPs induced on the gold frame and the graphene patch is demonstrated by an example. For this simulation, the dimension of the gold frame D1 are clearly more dominant at point I, which is far away from the resonance frequency of D2. Surface charge distributions at the frequency points II, III, and IV (around the Fano resonance), clearly demonstrate that D2 interferes with D1. Additionally, comparison of charge distributions (and magnetic field norms) at points II and III reveals that the sharp roll-off of the transmittance between the EIT point at 8.35 THz and the graphene's dipolar SPP at 9 THz is due to the phase change of the field distribution at points II and III.The dipolar nature of the SPPs is exhibited in S1 = 5.5 μm, dε = 3.5, μ = 10,000 cm2/Vs, and cμ = 1500 meV, while S2 is varied between 1 μm and 2 μm. Transmittance of the resonator is computed for each value of S2  and this quantity should be equivalent to the extinction coefficient 1 − T of the Fano resonator. By minimizing the difference between P and 1 − T numerically, iω, iγ, iφ, and Ωij can be found. Once these coefficients are known, one can easily obtain the physical parameters, iR, iL, iC, i = 1,2,3 and C12, C13, and C23, which describe the RLC circuit.The optical response of the proposed Fano resonator can be mathematically modeled using coupled oscillator equations. In this work, an RLC circuit model is used to replicate the optical response of the Fano resonatoriφ, i = 1,2, represent the amount of relative power each resonant mode receives from the incident electromagnetic field. Non-zero values of iφ indicate that both modes have dipole moments along the polarization vector of the incident field and they can be directly excited. Variables Ω12, Ω13, and Ω23 represent the amount of (energy) coupling between the modes. For example a high Ω12 means that the coupling between D1 and D2 is strong, which indicates high transmittance T within the EIT region. This is demonstrated by an example as described next.The mathematical model described by the system of P and 1 − T of the Fano resonator with S1 = 5.5 μm, S2 = 1.6 μm, dε = 3.5, and μ = 10,000 cm2/Vs for various values of cμ between 500 meV and 1500 meV. The values of the extracted parameters are provided for each value of cμ in cμ can be seen with a blue shift in the resonance frequency ω2 that corresponds to D2. Additionally, a consistent increase in Ω12, which represents the strength of coupling between D1 and D2, is observed. As mentioned above, the increase in coupling strength, i.e., increase in Ω12 translates as increased T within the EIT region [see cμ does not effect characteristics of D1. As shown in ω1 and γ1, corresponding to D1 remain unchanged.Parameters of the circuit with three loops is obtained by minimizing the difference between gion see . Note thM.A. and M.F. carried out the simulations and prepared the figures. HB supervised the research. All authors contributed equally to writing the main manuscript.


We investigated the neural bases of navigation based on spatial or sequential egocentric representation during the completion of the starmaze, a complex goal-directed navigation task. In this maze, mice had to swim along a path composed of three choice points to find a hidden platform. As reported previously, this task can be solved by using two hippocampal-dependent strategies encoded in parallel i) the allocentric strategy requiring encoding of the contextual information, and ii) the sequential egocentric strategy requiring temporal encoding of a sequence of successive body movements associated to specific choice points. Mice were trained during one day and tested the following day in a single probe trial to reveal which of the two strategies was spontaneously preferred by each animal. Imaging of the activity-dependent gene c-fos revealed that both strategies are supported by an overlapping network involving the dorsal hippocampus, the dorsomedial striatum (DMS) and the medial prefrontal cortex. A significant higher activation of the ventral CA1 subregion was observed when mice used the sequential egocentric strategy. To investigate the potential different roles of the dorsal hippocampus and the DMS in both types of navigation, we performed region-specific excitotoxic lesions of each of these two structures. Dorsal hippocampus lesioned mice were unable to optimally learn the sequence but improved their performances by developing a serial strategy instead. DMS lesioned mice were severely impaired, failing to learn the task. Our data support the view that the hippocampus organizes information into a spatio-temporal representation, which can then be used by the DMS to perform goal-directed navigation. Extensive research in rodents using navigation tasks has made a distinction between simple egocentric (response learning based on stimulus response-like associations) and allocentric (or place-learning) navigation sequential egocentric is encoded concomitantly with the allocentric strategy This parallel functioning of the dorsal striatum and the hippocampus is however not always clear-cut as revealed by recent findings. On one hand, the dorsal striatum has been shown to be anatomically One fundamental characteristic of hippocampal function is to enable the relational organization of declarative memory Concerning the DMS, the first demonstration of its implication in goal-directed learning comes from instrumental conditioning Functional connectivity between the DMS and the hippocampus was demonstrated in rodents during the choice period in a T-maze task We first examined the possible co-activation of the DMS with the hippocampus in the allocentric as well as the sequential egocentric strategies in the starmaze task. The use of either strategy can be detected accurately by analyzing the response made in a subsequent probe trial for which the start point is changed All behavioral experiments were performed in accordance with the official European guidelines for the care and use of laboratory animals (86/609/EEC) and in accordance with the Policies of the French Committee of Ethics (Decrees n° 87–848 and n° 2001–464). Animal housing facility of the laboratory is fully accredited by the French Direction of Veterinary Services . Animal surgery and experimentation are authorized by the French Direction of Veterinary Services for LRR  and for AW .All efforts were made to minimize suffering and animal discomfort. Prior to surgery, mice were anesthetized by intraperitoneal injection of ketamine/xylazine. After surgery, mice were allowed 15-day rest and were observed regularly during this period to check if there was no infection and no weight loss.Brain extraction for Fos imaging and histological verification of lesions section). Mice were deeply anesthetized by intraperitoneal injection of ketamine/xylazine and perfused transcardially to extract the brain.At the end of the experiment, mice were sacrificed for Fos imaging and lesion analyses  were used throughout the experiment (n = 24 in the Fos imaging study and n = 47 in the lesion study). Mice were housed in groups of 5 in standard conditions: 12 h light/dark cycle, with Prior to training in the starmaze task, motor coordination and balance were evaluated in an accelerating rotarod see .The starmaze is a complex goal-directed navigation task which consists of five alleys forming a central pentagonal ring and five alleys radiating from the vertices of this pentagonal ring see . White nThe starmaze task relied on a massed training phase given over one day (day 1), composed of 10 sessions of 4 trials in the presence of all visual cues (see The day before the training phase (day 0), all mice were given a pretraining session of 4 trials in the starmaze to limit stress and habituate them to swim and climb on the platform. The pretraining session was a cued version of the starmaze in which a proximal cue (a flag) was placed on the top of the submerged platform in the absence of any other visual cues (the starmaze was surrounded by a circular black curtain). On day 0, an additional training session was run on all mice included in the lesion study to insure optimal learning during the training phase.The day following training (day 2), a probe trial was run to identify the strategy spontaneously used by each animal , namely The departure point was changed . In addition to the habitual platform located in alley 7, another hidden platform was placed in alley 1 so that both strategies were equally rewarded. If a mouse used the configuration of distal visual cues, it performed the [5–6–7] trajectory revealing the use of an allocentric strategy. Executing the temporal sequence of body movements (left–right–left) led to the [5–4–2–1] trajectory indicating a sequential egocentric strategy. Any other path was classified as 'other strategy'.To evaluate the mouse’s ability to learn the correct sequence of turns and thus to elaborate a direct path towards the platform , we compThe mice were classified according to their most used path during the last five training sessions, i.e. the second part of training. Different categories were used: “Direct path” corresponding to the most direct path to the platform  blocked with Plexiglas walls in presence of all visual cues  and alloMice were randomly assigned to one of four groups: hippocampus sham [Hipp PBS] (n = 15), hippocampus lesion [Hipp IBO] (n = 14), dorsomedial striatum sham [DMS PBS] (n = 10), dorsomedial striatum lesion [DMS IBO] (n = 8). Excitotoxic lesions were performed by means of bilateral microinjections of ibotenic acid  dissolved in phosphate-buffered saline  and sham lesions consisted in PBS 1X  solution injections. Mice were anesthetized with ketamine  and xylazine  and placed in a stereotaxic frame (Stoelting). The skull was exposed and holes were drilled above injection sites. Targeting coordinates were determined from the atlas of Franklin and Paxinos Ninety minutes after the completion of the last probe trial (or free swimming session) in the starmaze, mice were deeply anesthetized with an intraperitoneal injection of a mixture of Ketamine (150 mg/kg) and Xylazine (12 mg/kg) and perfused transcardially with saline (0.9%), followed by an ice-cold solution of 4% paraformaldehyde in phosphate buffer . After postfixation overnight in the same fixative at 4°C, brains were cryoprotected for 48 hours in a sucrose solution  at 4°C. 50 µm-thick coronal sections were cut on a freezing microtome and stored in PB 0.1 M solution containing 0.02% of sodium azide.2O2 hydrogen peroxide (0.3% in PB). After four 10 min PB-rinses, sections were incubated overnight with a Fos-specific primary rabbit polyclonal antibody  diluted in blocking solution . A biotinylated goat anti-rabbit antibody  was used as secondary. After washing, staining was revealed using the avidin-biotin peroxidase method  coupled to diaminobenzidine as chromogen. Sections were mounted on gelatin-coated slides. Quantitative analyses of Fos-positive nuclei were performed by using a computerized image processing system  coupled to an optical microscope. The quantification of Fos positive nuclei was carried out at 10x magnification. Structures were defined according to the Franklin and Paxinos atlas 2) was divided by the mean count in that region of the respective control group to generate a normalized count for each animal. Results expressed as a percentage were averaged to give the final means of each group.Free floating sections were rinsed in 0.1 M PB and then incubated for 30 min with HFree floating sections were incubated overnight at room temperature with a mouse primary antibody against neuronal-specific nuclear protein NeuN  in blocking solution . Sections were mounted on gelatin-coated slides and coverslipped with Vectashield mounting medium . Analyses of lesion extent were performed using a computerized image processing system  coupled to a fluorescence microscope. One mouse injected with ibotenic acid in the dorsal hippocampus and five mice injected with ibotenic acid in the dorsomedial striatum (DMS) were excluded of the study due to absence of lesion.Results were expressed as means ± standard error mean (S.E.M.). Statistical analyses were run using the Statview 5.0 software. Learning performances were compared using analyses of variance with repeated measures (two-way repeated ANOVA and one-way ANOVA). Differences in normalized Fos density were assessed using Kruskal-Wallis non parametric tests followed by Mann-Whitney comparisons when indicated .1,14 = 0.01, p = 0.91; F1,14 = 0.7, p = 0.41; t14 = 0.23, p = 0.82; Upon completion of training, learning using either the allocentric or the sequential egocentric strategy led to the same direct path  p<0.01, Mann-Whitney U, p<0.01 for all comparisons to the swimming controls, allocentric versus sequential egocentric Mann-Whitney U, p = 0.79 for dCA1 and p = 0.96 for dCA3; p<0.01, sequential egocentric versus swimming controls Mann-Whitney U, p<0.01; p<0.01, Mann-Whitney U, p<0.01 for all comparisons to the swimming controls for the DMS). We did not observe any Fos activation in the dorsolateral striatum compared to the control group. As for Fos counts in the dorsal CA1 and CA3, Fos counts in the DMS were similar in allocentric and sequential egocentric group . Thus, the possibility that the difference in direct path score was due to the difference in swimming speed was unlikely. When analyzing the evolution of the direct path score across sessions, control and lesioned groups both improved their scores during training  [Hipp IBO] and the sham control mice (n = 15) [Hipp PBS] . Control mice with PBS injection essentially relied on the direct path (60%). 27% of them used a serial strategy and 13% no clear strategy. By contrast, only two mice  used the direct path. However during the probe test, one of these two mice used a serial strategy while the other did not use any clear strategy. Thus in the hippocampal group, no mice were clearly identified as using the allocentric or the sequential egocentric strategy. 57% of them relied on the serial strategy  and 29% even expressed no clear strategy  [DMS IBO] was significantly lower than that of sham control mice (n = 10) [DMS PBS] . 70% of the control mice used the direct path while no DMS lesioned mice did. Instead, half of them demonstrated no clear strategy (compared to 10% for the PBS mice), a quarter used the serial strategy (compared to 20% for the PBS mice), and the last quarter adopted a different path, the long path to the goal   .Although both dorsal hippocampus and DMS lesions impaired learning in the starmaze, the behavior exhibited by the lesioned animals was clearly different depending on the lesioned structure, as revealed by the distribution of the paths used , 4D. HipWe investigated the structures involved in using the allocentric and sequential egocentric strategies in the starmaze task. Both strategies are encoded in parallel Using Fos imaging, we first identified the structures involved in the use of the strategies during the probe test. We then focused on the role of the hippocampus and the DMS in learning the task by lesioning each of these structures.The profile of Fos expression revealed a common neural network supporting both allocentric and sequential egocentric strategies. This network included the dorsal hippocampus, the DMS and the medial prefrontal cortex .The higher levels of Fos density in infralimbic and prelimbic cortices of mice achieving the starmaze task compared to swimming controls is consistent with the role of these areas in a variety of executive processes such as working memory for spatial location information, decision-making, temporal order memory and planning  required to ensure successful navigation to the goal. Indeed the hippocampal mice adopted a serial strategy consisting in visiting all radiating alleys successively and systematically reaching the goal. Notice that the serial strategy does not require any learning of the sequential organization of the body movements, an animal using this strategy just explores all encountered alleys by turning toward a defined direction independently of specific choice points. The use of this non-optimal strategy in the starmaze task nevertheless allows reaching the goal doing a small number of errors thus explaining the improvement of the direct path score.These results, indicative of a navigation deficit affecting both allocentric and sequential egocentric strategies, highlight the role of the dorsal hippocampus in the spatio-temporal organization of information Our Fos imaging data also revealed the implication of the DMS in the allocentric and sequential egocentric strategies used in the starmaze task. Whatever strategy spontaneously chosen by mice to reach the goal, Fos levels were significantly higher in DMS compared to swimming controls. On the contrary we did not observe any significant DLS activation during the probe test whatever the strategy chosen. To investigate that Fos activation in the DMS highlights its functional involvement in learning the starmaze task, we disrupted the DMS’s function and examined the effects on learning performance in the starmaze. Mice with selective lesions of the DMS were unable to learn the starmaze task. Unlike hippocampal mice, there was no improvement of the direct path score. More specifically, the examination of the paths used during training showed that the majority of DMS mice exhibited no clear strategy. These results bring to light the role of the DMS in learning and using allocentric and sequential egocentric strategies.The DMS has been shown to play a crucial role in processes mediating goal-directed behavior and decision-making such as action-outcome associations, behavioral flexibility and action selection The failure of DMS lesioned mice to learn the starmaze task may be explained by the inability to create a link between a goal localized far from the animal and the spatio-temporal sequence needed to reach that goal. Indeed our data suggest that a functional DMS is required to learn the correct actions leading to the goal, irrespective of the representation used, i.e. spatial or sequential. While most hippocampal mice developed the serial strategy and therefore improved their performances, most DMS mice were unable to adopt any clear strategies. Our result corroborates previous studies arguing that the DMS is engaged in the cognitive control of behavior Goal-related information has been shown to influence hippocampal place fields Early studies on the DMS by Devan et al. Our data show on the one hand that the goal-related information coding in the hippocampus is not sufficient to allow optimal goal-directed behavior as no mice learned the direct path when their DMS was lesioned, and on the other hand that the DMS does not process spatial information sufficiently to allow learning the direct path to the goal.Taken together, our results demonstrate that the hippocampus and the DMS are involved in learning a complex goal-directed navigation task: the spatio-temporal organization of information performed by the hippocampus can be processed by the DMS to perform optimal goal-directed navigation and both structures cooperate to perform either allocentric or sequential egocentric strategies successfully.Figure S1The control group used to evaluate the nonspecific aspects of the training procedure on expression of the c-fos activity-dependent gene. Mice were allowed to swim in only one alley, corresponding to the departure alley of the experimental group, namely alley 1 during training trials and alley 5 during probe trials. Swimming occurred in the presence of all visual cues for a duration matching the mean amount of swimming time of the experimental group.(PDF)Click here for additional data file.Figure S2Significant correlations were found between normalized Fos counts measured in the CA1 (A) or the CA3 (B) fields of the hippocampus and the DMS for all mice groups. (PDF)Click here for additional data file.Figure S3Accelerating Rotarod performances. Performances on the accelerating rotarod were not significantly different for all mice groups. In particular, lesions did not affect motor performances on the rotarod test.(PDF)Click here for additional data file.Table S1Examples of direct path score calculation for different trajectories. The direct path to the goal is represented by the sequence of three successive turns following the 1, 10, 8, 7 alleys. A turn score is given for each of the three intersections encountered along the direct path: intersection I , intersection II  and intersection III . The intersection turn score is equal to 100 for a correct turn and 0 for a wrong turn. Each intersection turn score is normalized by the number of alleys visited between the evaluated intersection and the first encounter of the previous one. The normalized turn scores for the three intersections are averaged to obtain the direct path score. This direct path score enables to evaluate the ability of the mouse to navigate directly toward the platform. This actually corresponds to making the correct turn as from the first encountered intersection (intersection I). Not turning to the alley number 10 at the first intersection also reveals an inability of the mouse to orient itself towards the platform. Indeed, the configuration of the starmaze is such that when a mouse turns right at the first intersection, it goes towards the opposite direction from the location of the platform. Thus when considering two serial behaviors, namely left and right serial paths, the former corresponds to a turn towards the platform’s location whereas the later indicates the mouse is swimming away from it.(PDF)Click here for additional data file.