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.


Correspondingly, treatment with exogenous BDNF increased cell proliferation. In in vivo studies in athymic nude mice bearing multidrug-resistant uterine sarcoma cell tumors, we demonstrate suppression of tumor growth by treatment with K252a, but not K252b, as reflected by decreased cell proliferation and increased levels of apoptosis and caspase-3/7 activities without obvious side effects. Our findings indicated that endogenous signaling of the TrkB pathway contributed to uterine sarcoma cell growth, and inhibition of TrkB signaling in these tumors could provide a novel medical therapy for patients with uterine sarcomas.Uterine leiomyosarcoma is an aggressive tumor typically found at advanced stages due to difficulties with early diagnosis. Because uterine leiomyosarcoma is resistant to conventional radiation and chemotherapy, the development of more potent medical therapeutics is anticipated. Using quantitative real-time RT-PCR and immunostaining, we found the expression of brain-derived neurotrophic factor (BDNF) and neurotropin-4/5, together with their receptor, tyrosine kinase B (TrkB), in different uterine sarcoma cell lines and primary tumor samples from uterine leiomyosarcoma patients. We noted that levels of BDNF were more abundant than those of neurotropin-4/5. Moreover, the expression of TrkB and its ligands was elevated in a multidrug-resistant cell line and samples obtained from patients with leiomyosarcoma. In cultured uterine sarcoma cells, inhibition of endogenous TrkB signaling by treatment with either the soluble TrkB ectodomain or the Trk receptor inhibitor, K252a, suppressed cell proliferation and increased apoptosis based on cell viability and proliferation,  Leiomyosarcoma is most common subtype among uterine sarcomas. The most effective treatment for this disease is a complete resection of the primary lesion at an early stage. However, a differential diagnosis between early stage uterine leiomyosarcomas and myomas is difficult. In fact, a uterine leiomyosarcoma diagnosis is often made after surgery for benign uterine myomas Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family, and binds to the receptor tyrosine kinase B (TrkB) and the pan-neurotrophin receptor p75 (p75NTR) with high and low affinities, respectively in vitro cell growth and survival using the soluble ectodomain of TrkB and a pan-Trk receptor inhibitor. We also demonstrated that the levels of TrkB and BDNF transcripts were elevated in samples obtained from patients with leiomyosarcoma as compared with those of uterine myometrium and leiomyoma. Furthermore, we showed that a Trk inhibitor suppressed tumor growth in athymic nude mice bearing uterine sarcoma cell tumors.The placenta is a fast-growing organ that displays some tumor-like properties, e.g., high rates of trophoblast cell proliferation and invasion. We demonstrated that BDNF promotes proliferation and survival of trophectoderm cells before implantation et al. 2.The human uterine sarcoma cell lines, MES-SA and MES-SA/Dx5, and human uterine leiomyosarcoma cell line, SKN were purchased from the American Type Culture Collection  and the Japan Health Science Foundation , respectively. MES-SA/Dx5 is a multidrug-resistant variant of MES-SA developed by Harker To study the expression of tyrosine receptor kinase (Trk) family and thier ligands, conventional RT-PCR was performed. Total RNA was extracted from cells and tissues using an RNeasy Mini kit . Uterine leiomyosarcoma tissue sample was obtained from patients at the Department of Obstetrics and Gynecology, Akita University, during surgical resection with ethical committee approval and informed consent from patients. PCR primers for TrkB, p75NTR, BDNF, NT4/5 and β-actin were used as described Immunofluorescent detection of TrkB and their ligands in MES-SA/Dx5 cells was performed as described Total RNA was extracted from cell lines and frozen surgical specimens of human myometrium  and uterine leiomyoma (uterine benign tumor) using RNeasy Mini kit (Qiagen). Due to insufficient number of frozen samples of uterine leiomyosarcoma, total RNA was extracted from formalin-fixed parafine-embeded (FFPE) samples using RNeasy FFPE kit (Qiagen). After reverse transcription of 1 µg tRNA of each samples by Prime Script RT reagent kit , real-time PCR was performed using LightCycler 480 system with SYBR Green I Master mix . Primers for β-actin, TrkB, BDNF, NT-4/5, and galectin-1 were as follows:5′-CCACACTGTGCCCATCTACG-3′; antisense, 5′-AGGATCTTCATGAGGTAGTCAGTCAG-3′; TrkB: sense, 5′-CTGAAGGATGCCAGTGACAA-3′; antisense, 5′-ATGTGCTCATGCTGGAGGTT-3′; BDNF: sense, 5′-GCCAGGACAGAGCTGACAA-3′; antisense, 5′-TCCATGAACAGACAGGATGG-3′; NT4/5: sense, 5′-TGGATTCGAATTGACACTGC-3′; antisense, 5′-CCTGAGGTCTCTCAGCATCC-3′; galectin-1: sense, 5′-TGCAACAGCAAGGACGGC-3′; antisense, 5′-CACCTCTGCAACACTTCCA-5′. In all reactions, HotStartTaq DNA polymerase was activated by an initial denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 10 sec and annealing at/elongation at β-actin, 64°C; TrkB, BDNF and NT4/5, 60°C; Galectin-1, 61°C for 10 sec and elongation at 72°C for 10 sec. Fluorescent signals were monitored at the end of the elongation phase for each cycle. Data were normalized based on β-actin transcript levels. Human tissue samples were obtained from patients at the Department of Obstetrics and Gynecology, Akita University with ethical committee approval and informed consent from patients.β-actin: sense, To examine roles of endogenous Trk ligands in cell proliferation and apoptosis, MES-SA, MES-SA/Dx5 and SKN cells were seeded in 96-well culture plates at a density of 300 cells/well. Cells were incubated overnight before beginning a 48 h culture in serum-free medium with or without different doses of the following compounds: the TrkB soluble ectodomain ; the pan-specific Trk inhibitor, K252a 2 ×0.5. The tumors were resected after 15 days of treatment, weighed, embedded and sectioned. In addition to hematoxylin and eosin (H&E) staining to assess morphology, in vivo cell proliferation and apoptosis were evaluated by Ki-67 immunostaining and the TUNEL assay, respectively, as described 2, 1 mM EDTA, and a protease inhibitor cocktail (Roche). Data were standardized by protein concentration and represented as fold increases relative to controls.To explore the roles of endogenous Trk ligands during t-test. One-way ANOVA, followed by Fisher’s protected least significant difference test, was used to evaluate other differences. Data are expressed as the mean ± SEM.The Mann−Whitney U test was performed to compare the levels of TrkB and its ligands in human uterine sarcoma cell lines. The effects of drug treatment on tumor volume were analyzed using a Students’ The expression of Trk receptors and their ligands in uterine sarcoma cells was examined by PCR and immunostaining. Transcripts encoding TrkB, p75NTR, BDNF and NT4/5 were detected in both MES-SA and MES-SA/Dx5 cells . Other TIn human uterine samples, the expression levels of TrkB and its ligands were compared among uterine myometrium (n = 8), leiomyoma (n = 8) and leiomyosarcoma (n = 7) by quantitative real-time RT-PCR. The levels of TrkB and BDNF transcripts were significantly increased in uterine leiomyosarcoma as compared to those in myometrium and leiomyoma , whereasTo determine the roles of endogenous TrkB ligands in cell proliferation and apoptosis of sarcoma cells, MES-SA and MES-SA/Dx5 cells, were incubated with K252a or the TrkB ectodomain. Cell proliferation was inhibited by treatment with either the K252a or TrkB ectodomain, but not with the K252b . In leioTo examine the effect of BDNF on cell proliferation, cells were cultured with or without different doses of BDNF. As shown in in vitro inhibition of sarcoma cell growth by Trk receptor inhibitors could be translated into in vivo antitumor activity, K252a was administered to athymic nude mice bearing tumor xenografts of MES-SA/Dx5 cells, which were chosen for these studies due to their high BDNF and TrkB expression levels and multidrug-resistant character that could be more severe model of uterine sarcoma for in vivo analyses. When the tumor volume reached >80 mm3, animals were given vehicle alone, K252a, or K252b at 1 mg/kg every 3 days for 15 days. Tumors in animals treated with K252a were significantly smaller than those that received vehicle or K252b  and uterine leiomyosarcoma cell lines (SKN) . Due to In uterine leiomyosarcoma samples obtained from patients, the levels of BDNF and TrkB were significantly high as compared with uterine myometrium and leiomyoma. Interestingly, the leiomyosarcoma patients with high BDNF expression also showed significantly high levels of TrkB, but not NT4/5. Due to poor outcomes exhibiting overexpression of BDNF and TrkB in neuroblastomas The expression of galectin-1 is shown to regulate by the activation of TrkB in neuroblastoma cells Although NT4/5 is another TrkB ligand, its expression levels in uterine sarcoma cells are low, suggesting that BDNF acts as the predominant ligand in the context of uterine sarcoma cell growth. Similar expression patterns were demonstrated in choriocarcinoma cells in vitro, K252a is likely to be specific for TrkB, although other members of the Trk family, including TrkA and TrkC, were expressed in these sarcoma cells , easily delivered to tumor xenografts via i.p. administration with no overt side effects. By contrast, the TrkB ectodomain is a large glycosylated protein (MW = 75–100 kDa), which is unstable in the body. Thus, we used K252a for in vivo studies. Although uterine sarcoma is a highly invasive and metastatic cancer, MES-SA tumor cells xenografted into mouse uterine muscle failed to metastasize after 42 days The expression of both TrkB and TrkB ligands in uterine sarcoma cells suggested the presence of autocrine loop of TrkB signaling. However, Several molecular targeted agents have been developed and introduced into clinical trials for the treatment of uterine leiomyosarcoma in vitro and in vivo studies. Moreover, we demonstrated the expression level of TrkB and BDNF in uterine leiomyosarcoma associated with clinical malignancy in patients. Further elucidation of the mechanisms of BDNF/TrkB-induced leiomyosarcoma growth could lead to novel therapeutic approaches for the treatment of patients with this disease.In conclusion, our findings demonstrated important roles for TrkB signaling in uterine leiomyosarcomas based on Figure S1Expression of TrkA, TrkC, NGF and NT3 in uterine sarcoma cell lines. Expression of TrkA, TrkC, NGF and NT3 mRNA was detected by RT-PCR in the uterine sarcoma cell lines, MES-SA and MES-SA/Dx5. As loading controls, β-actin mRNA levels were assessed. The negative controls lacked template DNA.(TIF)Click here for additional data file.Figure S2Expression of TrkB, p75NTR and their ligands in uterine leiomyosarcoma cell line, SKN and roles of endogenous TrkB signaling in in vitro cell proliferation and survival (A) Expression of TrkB, p75NTR and their ligands mRNA was detected by RT-PCR. The levels of β-actin mRNA were assessed as loading internal controls. The negative controls lacked template DNA. nc, negative control. (B) Effects of suppression of endogenous TrkB signaling on cell proliferation. SKN cells were cultured in medium alone , or with different doses of the TrkB ectodomain (TrkB EC), the pan-Trk inhibitor, K252a, or its inactive analogue, K252b. Cell proliferation activity was determined using the cell proliferation reagent WST1 (n = 6). Columns, mean; bars, SE. *, P<0.05 vs. control. (C) Effects of suppression of endogenous TrkB signaling on cell survival. SKN cells were treated without or with K252a (1 µM). Apoptosis was determined using the caspase-3/7 assay (n = 6). Data were represented as fold increases relative to controls at individual culture times. Columns, mean; bars, SE. *, P<0.05 vs. control.(TIF)Click here for additional data file.


TARDBP gene that encode TDP-43 as causative for cases of sporadic and familial ALS http://www.molgen.ua.ac.be/FTDMutations/. Certain mutant forms of TDP-43, such as M337V, are associated with increased low molecular weight (LMW) fragments compared to wild-type (WT) TDP-43 and cause neuronal apoptosis and developmental delay in chick embryos. Such findings support a direct link between altered TDP-43 function and neurodegeneration.Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Researchers have identified 44 mutations in the M337V) carrying this mutation. hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, and intranuclear and cytoplasmic phosphorylated TDP-43 aggregates were frequently detected. The levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased in the transgenic mice. Moreover, overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP-43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice.To explore the pathogenic properties of the M337V mutation, we generated and characterized two mouse lines expressing human TDP-43 (hTDP-43M337V mouse model indicates that overexpression of hTDP-43M337V alone is toxic in vivo. Because overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, the mechanisms causing pathogenesis in the mutant model remain unknown. However, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity. The mutant model will serve as a valuable tool in the development of future studies designed to uncover pathways associated with TDP-43 neurotoxicity and the precise roles TDP-43 RNA targets play in neurodegeneration.Our novel TDP-43 TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS [n motif and is involved in gene transcription, pre-mRNA splicing, mRNA stability, and mRNA transport [M337V under control of the mouse prion (PrP) promoter [TDP-43 is the major component of ubiquitinated inclusions in most cases of ALS and FTLD-U ,2, and tlial ALS . TDP-43 ransport ,9. Underransport -16. Thatpromoter .M337V mice, hTDP-43M337V is mainly expressed in the brain and spinal cord, a finding that is consistent with transgenic mice overexpressing wild-type TDP-43 that we previously reported [M337V mice exhibited certain features similar to those seen in ALS, such as TDP-43 cleavage, phosphorylation, aggregation, increased ubiquitination, gliosis, gait disturbances, and early lethality; however, the mice also exhibited other features not yet reported in humans, which may be due to the effect of hTDP-43M337V overexpression. Such features include: down regulation of mTDP-43, abnormal mitochondrial aggregation and abnormal tau phosphorylation.In the TDP-43reported . TDP-43MWT line 3c hereafter termed TDP-43WT), which we previously generated with the same promoter [M337V showed that protein expression was highest in the brain and spinal cord, with low levels in other tissues  using real time-PCR analyses of hemizygous and homozygous brains  TDP-43, we generated transgenic mice using the mouse prion promoter to constitutively drive expression of full-length hTDP-43 carrying the M337V mutation. Three  of eight independent founder lines showed germline transmission. The expression level of human TDP-43 was low in line 1 (~20% of endogenous levels), while lines 4 and line 6 had similar hTDP-43 expression levels to that of wild-type TDP-43 mice . They failed to show proper escape extension by splaying their hindlimbs upon elevation, as shown by their NT counterparts  was similar to that described for line 4.At approximately post-natal day 21, homozygous TDP-43s Figure . They dis Figure . By ~ 1 s Figure . Due in s Figure . We had M337V and NT mice. The results showed that TDP-43 was mostly in nuclei in the brain and spinal cord of TDP-43M337V and NT mice and that the TDP-43M337V mice specifically expressed high levels of hTDP-43 that were not seen in the controls ; however, increased ubiquitination and reactive gliosis were observed in TDP-43M337V mice and not in NT mice  showed similar pathological changes.A striking histological feature in TDP-43s Figure  located s Figure . Neuronae Figure . Ubiquite Figure . Ubiquite Figure , and co-e Figure . Reactive Figure ; Table 1WT model . HomozygM337V mice were immunoreactive for the mitochondrial marker, COX-IV  results confirmed that the majority of neuronal cytoplasmic inclusions in the spinal motor neurons of TDP-43M337V mice contained aggregates of mitochondria .The eosinophilic aggregates in spinal motor neurons of TDP-43V Figure , which iV Figure  did not a Figure  that wera Figure . The agga Figure . The dege Figure  were smae Figure . In NT me Figure ; howevere Figure . The mitns Table . HomozygWT mice is accompanied by changes in protein levels and/or phosphorylation of proteins that regulate mitochondrial fission and fusion [M337V mice and our previously described TDPWT mice, we sought to determine if mitochondrial fission and fusion proteins were altered in the TDP-43M337V mice. Surprisingly, the alterations to pDLP1 (Ser616), Fis1, and MFN1 that were observed in the TDP-43WT mice, regardless of line, were not present in the TDP-43M337V mice, despite the comparable mitochondrial aggregation in each transgenic model . Abnormally phosphorylated tau was rarely found in the spinal cord (data not shown). The regional distribution of hyperphosphorylated tau is summarized in Table Abnormal, phosphorylated tau was found in the brains of both TDP-43V Figure  and TDP-e Figure  showed se Figure . Additioe Figure . Westerne Figure , while te Figure . To expln Figure . The levM337V mice) develop phenotypic and pathologic features including gait disturbances, gliosis, increased ubiquitination, TDP-43 truncation, phosphorylation, and both nuclear and cytoplasmic phosphorylated inclusions.In the current study, we generated and characterized a novel transgenic mouse model that overexpresses hTDP-43 carrying the M337V mutation under the mouse prion promoter. Homozygous TDP-43 M337V mice  were also noted in a second independent line of TDP-43M337V mice (line 6), suggesting that these features were not simply due to transgene insertional effects.One limitation of our model is that although many of the features are consistent with those observed in ALS, there are several features of this model that are incongruous with the human disease. Phenotypically, our mice show retarded growth from early life; whereas, humans with ALS typically show progressive disease after initially normal development into adulthood. While our TDP-43se model ; howevergression . TDP-43Mpairment ,31, whicWe observed some regional differences in our TDP-43 transgenic models, despite the relatively uniform expression of the TDP-43 proteins throughout the gray matter of the brain and spinal cord. For example, nuclear pTDP-43 inclusions, abnormal mitochondrial aggregation and chromatolysis were mainly observed in spinal cord, while cytoplasm pTDP-43 inclusions and tau phosphorylation were primarily seen in the brain. This indicates that the mechanisms regulating TDP-43 and being regulated by TDP-43 are likely to be different in the brain and spinal cord and warrant further investigation.M337V mouse model indicates that overexpression of hTDP-43M337V alone is toxic in vivo. TDP-43M337V mice recapitulate certain pathologic features seen in neurodegenerative diseases, including TDP-43 fragmentation, phosphorylation, aggregation, increased ubiquitination and gliosis. While these features could also indicate general neuronal dysfunction, the mice also exhibit down regulation of mouse TDP-43, abnormal mitochondria aggregation and abnormal tau phosphorylation. Because overexpression of mutant hTDP-43 produces phenotypes similar to the wild-type TDP-43 model, the mechanisms causing pathogenesis in the mutant model remain unknown. However, these results should not be surprising, given the mutant TDP-43 and wild-type TDP-43 biochemically are similar in human disease. As such, the TDP-43M337V mice may serve as a valuable tool for future studies of yet-to-be-examined disease pathways and the precise roles TDP-43 RNA targets play in neurodegeneration.In summary, this novel TDP-43XhoI site of the pcDNA 3.1. To generate the M337V mutation, site directed mutagenesis was performed using Quikchange kit (Strategene). The primers used for the mutation were: 5'-CAGTTGGGGTATGGTGGGCATGTTAGC-3' and 5'-GCTAACATGCCCACCATACCCCAACTG-3'. After confirming the mutation by sequencing, the M337V TDP-43 cDNA was inserted into the XhoI site of MoPrP vector [NotI, gel purified and digested with β-agarose. DNA was filtered, concentrated and diluted to 3 ng/μl in microinjection buffer. The transgene was microinjected into fertilized C57BL/6 (B6) mouse eggs and re-implanted into pseudopregnant females. Eight founders were mated with B6 mice to determine germline transmission and establish expression levels. TDP-43 founder lines (line 4 and 6) that have similar transgene levels to the TDP-43WT mice we previously described [The human wild-type TDP-43 cDNA was generated as previously described  and was P vector . Followiescribed , were usTransgenic mice were identified by PCR using hTDP-43-specific primers: 5'- TGGAGAAGTTCTTATGGTGCAGGTC-3' and 5'-GGTATTAGCCTATGGGGGACAC-3' against control actin-specific primers (5'-CGGAACCGCTCATTGCC-3' and 5'-ACCCACACTGTGCCCATCTA-3'). Homozygous mice were identified by Quantitative real-time PCR .® Gene Expression Assays . Total RNA was isolated either from tail samples for identification of homozygous mice or from brain or spinal cord tissue to determine the levels of human and mouse TDP-43 transcripts. TRIzol  and Pure Link™ RNA Mini Kit  were used for RNA extraction. 3 μg RNA were used to synthesize cDNA using the High Capacity cDNA Reverse Transcription Kit . The qPCR assay used the following: hTDP-43 Hs00606522_m1, mTDP-43 Mm00523866_m and 18S rRNA Hs99999901_s1. The PCR was run on the ABI 7900 and data analyzed using Software RQ Manager 1.2 .Levels of human and mouse TDP-43 transcripts were determined via TaqManSagittal half brain and spinal column were immersion fixed in 10% formalin for immunohistochemistry, and the other half brain was frozen on dry ice for biochemistry. After 24 hours, the spinal cord was removed from the vertebral column and fixed overnight.Tissues were homogenized at 10 ml/g (volume/weight) in lysis buffer . Following centrifugation, supernatant was assessed by BCA assay . Following western blotting, membranes were incubated with mouse monoclonal TDP-43 antibody, which was generated using amino acids 1 to 261 of hTDP-43 as the immunogen and was found to recognize amino acids 205-222 of hTDP-43 by epitope mapping ; rabbit 20. Antigen retrieval was performed in a dH2O steam bath for 30 min. Tissues were immunostained with monoclonal TDP-43 antibody or antibodies toward pS403/S404-phosphorylated TDP-43, ubiquitin, glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (IBA-1), cytochrome oxidase subunit IV (COX-IV), or phospho-tau  using the DAKO Autostainer (Dako Auto Machine Corporation) and the DAKO EnVision+ HRP system. DAKO Liquid DAB Substrate-Chromogen system was the chromogen. See Additional file Tissues were embedded in paraffin, sectioned (5 μm thick) and mounted on glass slides. Sections were deparaffinized in xylene and rehydrated in a graded series of alcohol, followed by dH4, dehydrated in alcohol and propylene oxide, and finally infiltrated and embedded in Epon 812. Ultrathin sections mounted on copper grids were stained with uranyl acetate and lead citrate. Images were obtained with a Gatan CCD camera using a Philips 208S electron microscope.Spinal cords from 4%-paraformaldehyde-perfused mice were immersed in 2.5% glutaraldehyde-0.1 M cacodylate buffer, post-fixed in 1% OsOFront paws were painted red, and hind feet were painted blue with nontoxic paint. The mice were placed in a plastic tunnel with a strip of white paper covering the floor. At the end of the tunnel was an enclosed black box to encourage mice to cross the paper and into the box. The test was repeated until mice left at least five pairs of adjacent footprints.p < 0.05 were considered statistically significant.One-way ANOVA with Tukey's posthoc analysis were used to compare measures among 3 groups. Student's t-test analysis was used to compare measures between 2 groups. Kaplan-Meier methods were used for survival analysis. For data presentation, normalized values were averaged and presented as mean ± standard error of means (SEM). Values of ALS: amyotrophic lateral sclerosis; FTLD-U: frontotemporal lobar degeneration with ubiquitin-positive inclusions; TDP-43: TAR DNA binding protein-43; hTDP-43: human TDP-43; mTDP-43: mouse TDP-43. pTDP-43: phospho-TDP-43.YX, YZ, JL, and LP are inventors of this and related mouse models; however, no royalties have been generated from this invention.YX and YZ performed experiments, data analysis and co-wrote the manuscript. XC performed experiments, WL performed Electron Microscopy study. CS edited the manuscript. JL, DWD and LP conceived of the study, participated in its design and coordination and edited the manuscript. All authors read and approved the final manuscript.M337V mice but hTDP-43 itself is not ubiquitinatedFigure A1: Increased ubiquitin levels in TDP-43. Human TDP-43 was immunoprecipitated from brain homogenates derived from nontransgenic and homozygous TDP-43m337v mice. Briefly, brain homogenates containing 500 μg protein were incubated with 1.5 μg mouse monoclonal TDP-43 antibody overnight at 4°C with gentle shaking. Protein G agarose was added for 4 h at 4°C then pelleted by centrifugation. Protein thus captured was eluted using sample loading buffer and resolved by SDS/PAGE for Western blot analysis. Shown are immunoblots of the inputs and the immunoprecipitated proteins, probed using an antibody to ubiquitin or to total TDP-43. Note that a marked increase in ubiquitin levels is observed in the homogenates derived from homozygous mice prior to immunoprecipitation. Nonetheless, the immunoprecipitated human TDP-43 is not immunopositive for ubiquitin. Arrow = IgG Heavy Chain.Click here for fileM337V miceFigure A2: No mitochondrial fission and fusion protein changes in TDP-43. Immunoblot analysis of Ser616-phosphorylated DLP1, DLP1, Fis1, and mitofusin 1 (MFN1) expression level in brain lysates of nontransgenic(NT), hemizygous(Hemi), and homozygous(Homo) TDP-43M337V mice of both line 4 and line 6. There are no significant protein changes among different mice groups.Click here for fileAdditional Table: Primary Antibody List. Full list of the primary antibodies used in this study.Click here for file