TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined.TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT.Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general. TARDBP, is well conserved among the multicellular organisms from C. elegans to human [TARDBP gene, the 43 kDa TDP-43 protein is the most abundant one expressed in all tissues [TARDBP gene is indispensible for normal early development of the mouse embryos [The TAR-DNA-binding protein 43 (TDP-43)-encoding gene, to human ,2. Of th tissues . TDP-43  tissues ,6, a spl tissues ,8, and a embryos -12. Late embryos -15. Bioc embryos -15. Loss embryos  and refeTARDBP, have been identified to be associated with 10% of ALS [TARDBP mis-sense substitutions have been identified, almost all of which are mapped in the glycine-rich domain of TDP-43 [A315T is also more toxic to the primary rat cortical neurons than the wild type hTDP-43 [Q331K and TDP-43M337V induce oxidative injury of the motor neuron-like NSC34 cells [The molecular and cellular basis for the pathogenesis of either ALS or FTLD-U is poorly understood yet. Mutations in 11 different genes, including the long studied superoxide dismutase 1 (SOD 1) and % of ALS , which i% of ALS . Further% of ALS . Interesf TDP-43 ,16,22. Af TDP-43 -27. For f TDP-43 . On the f TDP-43 . hTDP-43 hTDP-43 . Further34 cells . One unaTARDBP gene both increase the stability of the TDP-43 polypeptide in motor neuron-like cells as well as in non-motor neuron cells. In addition, the mutant hTDP-43 polypeptides as well as the wild type hTDP-43 induce significant apoptosis of motor neuron-like cells, but much less so in non-motor neuron cells, in a dose-dependent manner. Thus, the major role of the ALS-associated hTDP-43 mutations appears to be the enhancement of the steady-state level of hTDP-43 through stabilization of the polypeptide in the spinal motor neurons, which have a low tolerance to the elevated cellular level of TDP-43 in comparison to the non-motor neuron cells.In the following, we show that two randomly chosen ALS mis-sense mutations of the Wild type (WT) human hTDP-43 with addition of a Myc epitope tag to its 3’-end was generated by PCR of human brain cDNA using the following primers: forward, 5'-CCG CTC GAG CGG ATG TCT GAA TAT ATT CGG GTA AC -3'; reverse, 5'-TCT AGA GCT ACA GAT CCT CTT CCG AGA TGA GTT TTT GTT CCA TTC CCC AGC CAG AAG AC-3'. The A315T and N390D mutations were introduced into the WT cDNA by site-directed mutagenesis using the QuikChange® Site-Directed Mutagenesis Kit (Strategene). The three hTDP-43 cDNAs were first cloned into the pGEM-T vector . Following sequence confirmation, the cDNA inserts were subcloned into the XhoI/ XbaI sites of a pEF vector. Experimental research that is reported in the manuscript was performed with the approval of an appropriate ethics committee.The commercial antibodies used in this study included a rabbit anti-TDP-43 polyclonal antibody (pAb) raised against a.a 1–260 of human TDP-43 and recognizing human as well as mouse TDP-43 (Gene Tex), a human specific mouse monoclonal antibody (mAb) against the same TDP-43 sequence (2E2-D3) (Abnova), anti-Myc mAb (LTK), anti-α-tubulin (Sigma), anti-cleaved caspase 3 (Asp175) (Ac-cap3) , and anti-Hsp70 (Chmicon).6 cells by supplement with the pEF vector DNA. After transfection for different hrs, the cells were harvested and analyzed by Western blotting. In general, under the conditions used by us, the transfection efficiencies of NSC34, Neuro2A, SHSY5Y, and HEK293 were approximately 50%, 70%, 70%, and 90%, respectively.NSC34 cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotics (100 IU/mL penicillin and 100 g/mL streptomycin). Neuro2a cells were maintained in MEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 1% antibiotics, and 1% sodium pyruvate (Invitrogen). SHSY5Y cells were maintained in DMEM/F12 (Invitrogen) with 10% FBS (Invitrogen), 1% antibiotics, and 1% sodium pyruvate (Invitrogen). The cells were transfected with the empty pEF vector and the expression plasmids, respectively, using Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer’s protocol. The amount of the plasmid DNA used in each transfection was kept at 20 μg/103 cells/well and 8 × 103 cells/well, and allowed to grow for 24 hr and 72 hr, respectively. On the average, the cells were at 30% confluency before the assay. For the assay, Caspase-Glo 3/7 reagent (Promega) was added to all the wells in a 1:1 ratio following the manufacturer's instructions. Cells with addition of 5 μM staurosporine  for 6 hr were used as a positive control [t-test was then used to obtain the p values associated with comparisons between the MT and WT.After incubation with the transfectants, the cells were split and seeded with two different densities, 2 × 10 control . After st-test was then used to obtain the p values associated with comparisons between the MT and WT.The cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 5 min, blocked with 10% donkey serum in PBS for 2 hr, and incubated overnight at 4°C with the primary antibodies anti-hTDP-43 (2E2-D3), anti-Myc, and anti-cleaved caspase 3, respectively . The primary antibodies were visualized with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 561 (Molecular Probe), and the nuclei were detected using DAPI . The patterns of immunofluorescence staining were analyzed in a LSM710 confocal microscope (Zeiss). For quantification of cleaved caspase-3 positive cells, several random fields/ sample were analyzed and the percentages of transfected cells displaying anti-cleaved caspase 3 staining signals and apoptotic nuclei were calculated . To determine the percentages of dead cells, four independent experiments were carried out. The data were expressed as means ± SD. The differences in % of the Ac-cap 3-positive cells among the variants were assessed by the ANOVA test. An unpaired two-tailed Student’s t-test was then used to obtain the p values associated with comparisons between the MT and WT.Cells were lysed in RIPA buffer  supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). The protein concentrations of the lysates were measured using the Bio-Rad protein assay reagent on a Beckman Coulter DU-800 machine. The lysates were then resolved by SDS–PAGE and immunoblotted with the indicated antibodies. Quantification of the immunoblot band intensities was performed with use of the Image J software (NIH). There independent experiments on each cell line were carried out for densitometry analysis. The data were expressed as means ± SD. The differences in the relative levels of hTDP-43 among the variants were assessed by the ANOVA test. An unpaired two-tailed Student’s t-test was then used to obtain the p values associated with comparisons between the MT and WT.Cells were transfected with different expression plasmids encoding Myc-tagged versions of WT hTDP-43 and the two MT hTDP-43. Cycloheximide  was added to the media at 40 hr post-transfection. At various time points thereafter, the transfected cells were lysed and the amounts of the Myc tagged TDP-43 proteins were measured by Western blot analysis using the anti-hTDP-43 antibody (2E2D3) or anti-Myc antibody (LTK). Four independent experiments on each cell line were carried out for densitometry analysis. The data were expressed as means ± SD. The differences in relative levels of hTDP-43 among the variants were assessed by the ANOVA test. An unpaired two-tailed Student’s p values associated with comparisons between the MT and WT.The data obtained from independent experiments are expressed as the mean ± S.D.. The differences among the variants were assessed by the ANOVA test. An unpaired two-tailed Student’s t-test was then used to obtain the To examine the relative cytotoxicities of WT and MT hTDP-43 in motor neuronal cells and non-motor neuronal cells, respectively, plasmids encoding the WT hTDP-43 and hTDP-43 carrying two different ALS-associated mutations (A315T and N390D)  of the expression plasmid(s) used for transfection, overexpression of the two MT hTDP-43 forms caused a motor neuronal cell type-specific cytotoxicity significantly higher than that by the WT hTDP-43.To examine the apoptotic cell death induced by overexpression of the WT hTDP-43 in comparison to the MT hTDP-43, we measured activities of the effector apoptotic caspases 3 and 7 of NSC34  have been shown to stabilize hTDP-43 in HeLa cells and in primary fibroblast culture from a human patient . In the Drosophila[In interesting parallel with the dose-dependence of the cytotoxicity of hTDP-43 as derived from this study, previous transgenic mice -44 and trosophila-47 experrosophila and mousrosophila,49 inducrosophila,51. ThusTARDBP gene itself. How the ALS-associated mutations in hTDP-43 stabilize the protein and why the spinal motor neurons have a relatively low tolerance to the elevated level of TDP-43 remain to be investigated.Take together all of the above, we suggest that pathogenesis of ALS could be due to the selective neurotoxicity of the spinal motor neurons caused by elevated level of hTDP-43, which in turn results from mis-regulation of the hTDP-43 metabolism due to different ALS-associated gene mutations including those within the In conclusion, based on our data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general.The authors declare that they have no competing interests.LSW carried out the experiment work and analyzed data. LSW, WCC and CKJS designed the study, coordinated the experiments and analyzed data. LSW and CKJS wrote the manuscript. All authors read and approved the final manuscript.6 cells), as described in the legend of Figure Comparison of apoptotic deaths of cells with exogenous expression of WT hTDP-43 and mTDP-43. Apoptotic cell death was assayed by the activities of caspase 3/ 7 at 24 hr and 72 hr post-transfection of NSC34 and Neuro2a cells with plasmids expressing hTDP-43 and mTDP-43, respectively  of the appropriate expression plasmids. At 72 hr post-transfection, the levels of the exogenous hTDP-43 and mTDP-43 proteins were compared by Western blotting with use of anti-Myc. The mouse Hsp70 and tubulin were analyzed as the internal control. The means of the relative levels obtained from three independent experiments (S.D.) are plotted in the lower 2 panels, with the level of the exogenous hTDP-43 in cells with the transfection dose of 20 μg plasmid DNA/ 106 cells as 100. The differences in the relative levels of the Myc-tagged hTDP-43 or mTDP-43 among the variants were assessed by the ANOVA test. Note the similar levels of hTDP-43-Myc and mTDP-43-Myc at each dose of the expression plasmid(s) used.Expression plasmid dose-dependent increase of hTDP-43 and mTDP-43 in trasnfected NSC34 and Neuro2a cells. NSC34 and Neuro2a cells were transfected with different doses (μg/ 10Click here for file


Initially implicated in the pathogenesis of CFTR and HIV-1 transcription, nuclear factor TDP-43 was subsequently found to be involved in the origin and development of several neurodegenerative diseases. In 2006, in fact, it was reported for the first time the cytoplasmic accumulation of TDP-43 in ubiquitin-positive inclusions of ALS and FTLD patients, suggesting the presence of a shared underlying mechanism for these diseases. Today, different animal models of TDP-43 proteinopathies are available in rodents, nematodes, fishes, and flies. Although these models recapitulate several of the pathological features found in patients, the mechanisms underpinning the progressive neuronal loss observed in TDP-43 proteinopathies remain to be characterized. Compared to other models, Drosophila are appealing because they combine the presence of a sophisticated brain with the possibility to investigate quickly and massively phenotypic genetic modifiers as well as possible therapeutic strategies. At present, the development of TDP-43-related Drosophila models has further strengthened the hypothesis that both TDP-43 “loss-of-function” and “gain-of-function” mechanisms can contribute to disease. The aim of this paper is to describe and compare the results obtained in a series of transgenic and knockout flies, along with the information they have generated, towards a better understanding of the mechanisms underlying TDP-43 proteinopathies. D. melanogaster is the species with the highest degree of homology with humans. Over the years, many mutants of Drosophila melanogaster have been isolated and many transgenic strains have been created as model systems for the study of human disease [Drosophila melanogaster [Drosophila melanogaster is further supported by the fact that it represents an animal model where the efficacy of putative therapeutic agents can be quickly screened and tested [Nowadays, Drosophila is one of the most widely used model organism for studying complex genetic and biological problems. Drosophila was introduced as an experimental model in the early twentieth century. Although there are several different Drosophila species which differ in habitat, morphology, and genetic background,  disease , leadingnogaster . Comparenogaster ; the gennogaster . The impd tested , 5. AltoIn particular, in the last ten years increasing attention has turned towards this animal in order to obtain a better understanding of neurodegenerative disorders , 5. For α-synuclein negative [ Among genes implicated in human disease, TDP-43 is a nuclear factor recently identified as a protein capable of playing a crucial role in the pathogenesis of FTLD, ALS, and other neurodegenerative diseases –24. In Anegative , 26. Thenegative . However, the fact that they may be acting as a cytoplasmic protein “sink” to trap the predominantly nuclear TDP-43 has suggested that the onset of neurodegenerative diseases might be the consequence of the progressive nuclear loss of function of TDP-43 trapped within the cytoplasmic inclusions  .Drosophila melanogaster  has the structure and in vitro functions similar to the human protein, at least for the repression of splicing of specific exons [ In order to clarify these issues it is therefore important to find a suitable animal model that expresses a functional homologue of human TDP-43. In this respect, it is already known that the TDP-43 ortholog of ic exons , 37. TheDrosophila melanogaster. In flies, the main homologue of human TDP-43 is a protein called TBPH . The comparison of the longest protein isoforms shows that the region that includes two RNA recognition motifs  [ Notwithstanding these differences, many experiments have highlighted an extremely high degree of functional similarity and interchangeability between the human and fly proteins . First of all, both proteins share the same preference with regards to RNA binding specificities as recent studies have shown that TBPH is able to preferentially bind the same consensus (UG)n rich sequences tagged by the human ortholog . Importao TDP-43 . Even moo TDP-43 , 38, thehnRNPB1) . Altogether, therefore, these observations indicate that there is an extremely high degree of functional conservation through evolution from flies to human and this observation has represented the starting point for using Drosophila models to address the issue of TDP-43-dependant neurodegeneration.Two complimentary approaches have been utilized for the genetic characterization of ALS-related pathways: loss and gain of function of the Drosophila TDP-43 ortholog and overexpression of TDP-43 variants presenting mutations found in ALS patients. Tables  With regards to loss of function, the general strategy has been to target disruption of the TBPH gene in Drosophila in order to analyze whether ALS-like phenotypes could be generated. The first TBPH-ko model used chromosomal deletions that removed the initial part of the gene regulatory region and the ORF . Homozyg The second loss-of-function model was generated with a point mutation (G>A) that introduced a stop codon at residue 367 (Q367X) [in vivo. In fact, it was shown that overexpression of TDP-43 could efficiently rescue the phenotype caused by TBPH deficiency. Most importantly, both studies demonstrated that the functions of the human TDP-43 overlapped those of TBPH also in vivo the data obtained in vitro with human cell lines and no further molecular characterization was carried out. In particular, this model was used to confirm that the ability of TDP-43 to bind and regulate expression of the HDAC6 mRNA was conserved also by TBPH in Drosophila [ A third null allele model of TBPH was generated by deleting the whole coding sequence of the TBPH gene . Contrarosophila . In conc Finally, a fourth null allele model of TBPH was created by deletion of 932 bp within the 5′ end of TBPH . Similar Taken together, these studies suggest that the genetic background can significantly modulate or modify the effects of the genomic mutations. However, all these initial reports are strongly in agreement that depletion of TDP-43/TBPH can trigger neurodegeneration. As intuitively obvious, a complimentary approach to the creation of TBPH-ko flies consists in the ubiquitous or tissue-specific overexpression and/or silencing of this factor by using the Gal4-UAS system. Consistently, different reports found that the expression of the wild-type form of TDP-43 (both TBPH and TDP-43) in the Drosophila eye causes a severe retinal degeneration with loss of cells –13, 18. In a likewise manner, expression of TDP-43 in mushroom bodies caused neuronal death and axonal loss  and over Another distinguishing feature of TDP-43 proteinopathies is the presence of mutations occurring mainly within the C-terminal region of the TDP-43 protein (that have been identified mostly in both sporadic ALS and FALS patients ). Overex Other types of more artificial mutations, however, were found to be more consistent with regards to their effects. For example, this was the case for the impact of mutations within the nuclear localization and nuclear export sequences. In general, while the expression of the TDP-43 NLS-mutant in the fly eyes resulted in a stronger , 18 or c Finally, and most importantly, it is clear that the RNA-binding activity of TDP-43 is crucial for TDP-43 toxicity , 11, 19, A growing body of evidence indicates that TDP-43 abnormalities are not limited to ALS and FTLD but are present in many neurodegenerative and myodegenerative diseases –46. Of cin vivo; (2) the intracellular redistribution of TDP-43 was sufficient to induce degeneration; (3) enhancement of toxicity was obtained following coexpression of the TDP-M337V mutant with mutant dVCP [ The Valosin-Containing Protein (VCP) is a molecular chaperone VCP that segregates ubiquitinated substrates from multimeric protein complexes. Autosomal dominant mutations in the VCP gene have been identified in the Inclusion Body Myopathy associated with Paget's disease of bone and frontotemporal dementia . Mutations in this gene exhibit variable penetrance and cause progressive muscular bony and neuronal degeneration with lethal exitus . Most imant dVCP . These fant dVCP . Thus, b Another gene found mutated in familial and sporadic ALS is the one coding for fused in sarcoma/translocated-in-liposarcoma (FUS/TLS) protein , 55. Sim Finally, recent studies have found that ubiquilin 1 (UBQLN) is another potential TDP-43 interactor . UBQLN i Taken together, these observations provide evidence that many aspects of neurodegeneration are conserved from Drosophila to vertebrates and strengthen the utilization of the Drosophila models to study systematically modulators of TDP-43-induced neurodegeneration.futsch mRNAs  gene has been implicated in the pathogenesis of Alzheimer's disease and Parkinson's disease by linking together two protein degradation pathways (the ubiquitin proteasome system and autophagy) –69. Recefutsch was observed in the heads of TBPH -/- flies [futsch mRNA without affecting its stability or splicing profile. However, since futsch protein levels were decreased in TBPH -/- animals, this observation suggested that TBPH might be somehow involved in the posttranscriptional regulation of futsch expression [ In parallel, starting from the observation by different research groups that the loss of TBPH in Drosophila alters the morphological organization of the NMJ , 13, 17,/- flies . The Fut/- flies . In thispression . This ob Altogether, the studies with Drosophila models have allowed us to make several general observations.The data gained so far from these models significatively overlap most of the observations made in human patients and in rodent models, so strengthening the usefulness of the fruit fly in investigating the role played by TDP-43 protein abnormalities in ALS and other neurodegenerative diseases.Similarly to what is observed in rodent models, both overexpression and silencing of TDP-43/TBPH in Drosophila produce neurodegeneration. Beyond the particular attention that must be reserved to the expression levels of TDP-43 mutants, whose effect should be tested specifically for any model, these observations also suggest that events able to modify the expression of TDP-43 in humans might elicit neuronal dysfunctions even before the appearance of pathological TDP-43 inclusions.While Drosophila models confirm that toxicity occurs regardless of inclusions, the relevance of the cytoplasmic accumulation for the pathology remains an open question. In flies, expression of mutants accumulating in the cytoplasm showed an increase in toxicity, but a NLS mutant TDP-43 expressed in C. elegans, although cytoplasmic, did not result in alteration of locomotion and this observation led to the suggestion that nucleus is the primary site of the TDP-43 toxicity .Different Drosophila models support the hypothesis that TDP-43 toxicity depends also on retaining the ability to bind RNA targets , 11, 19.The carboxy-terminal domain seems to have a prominent role in the pathogenesis of human diseases. In fact, most mutations found in patients lie within this domain (for a recent review see ). IntrigSeveral studies have addressed the pathogenic role of hyperphosphorylation on the formation of TDP-43 inclusions –34. It sAge-dependent degeneration is an important feature of neurodegenerative diseases. The investigations aimed at understanding the relevance of aging in affecting TDP-43 expression in humans and its possible association with Alzheimer were not conclusive . At pres For all these reasons, fruit flies remain an appealing system in which to continue the characterization of the pathogenesis of TDP-43 proteinopathies through the screening of modifier genes or the search for drugs useful for the prevention and cure of neurodegenerative diseases.


We present transport measurements on a strongly coupled graphene quantum dot in a perpendicular magnetic field. The device consists of an etched single-layer graphene flake with two narrow constrictions separating a 140 nm diameter island from source and drain graphene contacts. Lateral graphene gates are used to electrostatically tune the device. Measurements of Coulomb resonances, including constriction resonances and Coulomb diamonds prove the functionality of the graphene quantum dot with a charging energy of approximately 4.5 meV. We show the evolution of Coulomb resonances as a function of perpendicular magnetic field, which provides indications of the formation of the graphene specific 0th Landau level. Finally, we demonstrate that the complex pattern superimposing the quantum dot energy spectra is due to the formation of additional localized states with increasing magnetic field. This makes graphene a promising material for spintronic devices [Graphene ,2, a two devices ,7 and sp devices -11. Addi devices ,5 makes  devices . Advance devices ,15. Alon devices -22 and q devices -30 have  devices -25, quan devices -28 and c devices  have bee devices ,31-36. IHere, we report on tunneling spectroscopy (i.e. transport) measurements on a 140-nm graphene quantum dot with open barriers, which can be tuned by a number of lateral graphene gates . In cont++) bulk material covered with 295 nm of silicon oxide (SiO2), where thickness (and roughness) of the SiO2 top layer is crucial for the Raman [The fabrication process of the presented graphene nano-device is based on the mechanical exfoliation of  graphite by adhesive tapes ,25,28. The Raman  and scanrh) on the island. While there are no visible resist residues on the island of the sample in Ref. [rh ≈ 0.35 nm, there are many dot-like residues on the sample presented here giving rh ≈ 1.1 nm.A scanning force microscope image of the final device studied here is shown in Figure  in Ref.  with rh T = 1.8 K in a variable temperature cryostat. We have measured the two-terminal conductance through the graphene quantum dot device by applying a symmetric DC bias voltage Vb while measuring the source-drain current through the quantum dot with a noise level below 10 fA. For differential conductance measurements a small AC bias, Vb,ac = 100 μV has been superimposed on Vb and the differential conductance has been measured with lock-in techniques at a frequency of 76 Hz.All measurements have been performed at a base temperature of Gqd as a function of back gate voltage at low bias (Vb = 200 μV) highlighting the strong suppression of the conductance around the charge neutrality point (-5 <Vbg < 3 V) due to the so-called transport gap [e2/h inside the gap region may be due to both, (i) resonances in the graphene constrictions acting as tunneling barriers [e2/h is measured at Vbg = 0 V.In Figure port gap -22. Herebarriers  . By sweeping the right side gate (Vrg) we break the left-right symmetry of the transport response  have been performed along the  solid gray line in Figure Ec = 4.5 meV, which is in reasonable agreement with the size of the graphene quantum dot [Corresponding Coulomb diamond measurements , that isntum dot ,25,26. MVbg = -5 to -3.5 V, at Vrg = 0 V  in Figure Vbg) for increasing magnetic field, in good agreement with Refs. [d of the dot. Therefore, the ratio d/ℓB is a relevant parameter for the observation of Landau levels in graphene quantum dot devices. Here, the comparatively large size (d ≈ 140 nm) of the dot promises an increased spectroscopy window for studying the onset and the formation of Landau levels in graphene quantum dots in contrast to earlier work [d ≈ 50 nm). Moreover, we expect that in larger graphene quantum dots, where the surface-to-boundary ratio increases edge effects should be less relevant. In Figure B-field, where (ii) the levels feature a kink, whose B-field onset increases for increasing number of particles, and (iii) we observe that the levels convergence towards higher energies  in Figure B < 2 T and a slight tendency for their bending towards lower energies might be identified  despite the fact that we find some small hysteresis in magnetic field for B < 3 T  typical for two coupled quantum dots [In Figure tum dots . The twoB ≈ 1.5 T corresponding to a magnetic length B is getting smaller than the extension of potential valleys induced by disorder.We interpret the magnetic field dependence in the following way. At low but increasing magnetic field we see in almost all measurements an increase of the conductance through the dot  in the 2: silicon dioxide.BG: back gate; EBL: electron beam lithography; LL: Landau level; LG: left side gate; PG: plunger gate; RG: right side gate; SiOThe authors declare that they have no competing interests.KE, TI, CS and JG designed the experiment. JG fabricated the sample. TF and JG carried out the transport measurements. All authors analyzed the measurements. JG and CS wrote the paper. All authors read and approved the final manuscript.


We report a 52-year-old patient who developed B-cell non-Hodgkin’s lymphoma subsequent to sarcoidosis. Sarcoidosis was diagnosed 16 years ago and remained asymptomatic for 14 years after steroid treatment. She presented with new symptoms of arthralgia, photosensitivity, butterfly erythema, autoimmune antibodies  associated with progression of the known left upper lobe lesion on the chest X-ray suggesting primary autoimmune disease (systemic lupus erythematosus). As steroid treatment was not effective, we started bolus cyclophosphamide therapy after which progression was seen on the chest X-ray. Computed tomography (CT)-guided needle biopsy confirmed malignancy of indefinable origin. Despite of the well-known fluorodeoxyglucose (FDG) avidity in active sarcoidosis, a FDG-positron emission tomography (PET) scan was performed to stage the primary tumour. Intensive FDG uptake was detected in the affected lung segment, with moderate uptake in mediastinal lymph nodes. The patient underwent left upper lobectomy. The histology showed pulmonary mucosa-associated lymphoma (bronchus-associated lymphoid tissue (BALT) lymphoma) in the lung tissue, while only sarcoidosis was present in the mediastinal lymph nodes. Bone marrow biopsy was negative.The association between sarcoidosis and lymphoma is known as sarcoidosis lymphoma syndrome, which is a rare disease. PET-CT was helpful in the differentiation of sarcoidosis and malignancy in this patient. It is important to be aware of the risk of lymphoma in sarcoidosis and FDG-PET, used for adequate purpose, can help the diagnosis. Sarcoidosis is a chronic granulomatous inflammatory multisystem disorder of unknown origin. The association between sarcoidosis and malignancy - particularly lymphoproliferative disease, such as non-Hodgkin’s lymphoma - has been previously described -3. In sa2 monthly) therapy was instituted. Repeated chest X-ray showed rapid and significant worsening of the left upper lobe lesion following the first dose  showed bilateral hilar lymphadenopathy and lung parenchyma involvement. Histology confirmed sarcoidosis, which was treated with systemic steroids for six months, after which persisting diabetes mellitus developed. After 14 asymptomatic years without immunosuppressive therapy, she was referred to our department with symptoms of dry cough, snoring and mild anaemia. On the chest X-ray, progression of the known left upper lobe lesion was noted  lymphoma) in the left upper lobe lung tissue, while only sarcoidosis was present in the mediastinal lymph nodes. Immunophenotyping demonstrated a B cell lineage phenotype: CD20, BCL2 and lambda light chain positivity on the cell surface, with CD10 and CD5 negativity Figure  and 7.Sarcoidosis is a multisystem granulomatous disorder of uncertain origin. The diagnosis of sarcoidosis is based on clinical, radiological, biochemical and histological findings. These findings are erythema nodosum, lymphopenia, hypergammaglobulinemia, anergy, hypercalcemia and elevated angiotensin-converting enzyme. The non-caseating granulomas are B-cell negative . The resBrincker and Wilbek have matched all patients who had been diagnosed with respiratory sarcoidosis during the period 1962 to 1971 against the data of the Cancer Registry. About 1.5 times higher incidence of cancer was seen in sarcoidosis patients. Lung cancer occurred 3 times and malignant lymphoma 11.5 times more frequently than in the control population . In a reet al. described six patients who developed malignancy first, followed by the diagnosis of sarcoidosis. The median interval between the diagnoses of the two diseases was only nine months [et al. reported two cases, where NHL was identified and treated for 3 and 10 months before the diagnosis of sarcoidosis. They proposed that sarcoidosis had been triggered by immunological disturbance induced by chemotherapy or associated with the lymphoma. They supposed an infectious agent, which could have spread due to the immunosuppression (caused by chemotherapy) resulting in granuloma formation. Alternatively antineoplastic therapy might have reduced suppressor T cells leading to lymphocyte activation observed in sarcoidosis [et al. reported a patient with osteosarcoma who developed granulomatous lymphadenopathy and multiple pulmonary nodules confirmed as sarcoidosis following cessation of treatment. They suggested that immunosuppression inhibited the development of sarcoidosis, which became symptomatic when chemotherapy was terminated [There are many theories explaining the possible relationship of this association. The changes in the number and the functions of immune cells in sarcoidosis are well described, suggesting dysfunction in the immunoregulatory pathways leading to granuloma formation. A clone may escape in an environment with defective T suppressor cells regulation resulting in the development of lymphoid malignancy. In this context, chronic sarcoidosis represents a more prolonged and more severe form of the disease. Steroid treatment, by further compromising the immune system, might be an additional predisposing factor . There ae months . There acoidosis . Sybert FDG-PET is a sensitive method for the staging of several malignancies, the underlying biological principle is based on the Warburg effect . FDG is et al. found that BALT lymphomas show heterogeneous but identifiable FDG uptake on PET scans [In a study with 21 patients, Bae ET scans . They anET scans .According to the current literature, our patient had chronic sarcoidosis and had been treated with systemic steroids. The development of autoimmunity (SLE) and its treatment with cyclophosphamide induced changes in the slow growing tumour. This is in line with the theory that abnormal immune function contributes to the development of LD, and that immunosuppressive therapy may enhance it ,17. The According to previous observations, sarcoidosis predisposes for lymphoid malignancies. Sarcoidosis and lymphoma both can cause increased FDG uptake in mediastinal lymph nodes, thus FDG-PET-CT cannot prevent the need for histological verification . In our Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ANA: Antinuclear antibodies; BALT: Bronchus associated lymphoid tissue; CO: Carbon-monoxide; CT: Computed tomography; FDG: Fluorodeoxyglucose; LD: Lymphoproliferative disease; MALT: Mucosa associated lymphoid tissue; NHL: Non-Hodgkin’s lymphoma; PET: Positron emission tomography; SLE: Systemic lupus erythematosus.The authors have no competing interests to declare.AK wrote the paper. NE and LT have been involved in drafting the manuscript. GYL and VM drafted the manuscript and revised it critically for important intellectual content. ACS performed the lobectomy. JCS carried out the histological examination and immunophenotyping. All authors read and approved the final manuscript.


Graphene oxide composites with photocatalysts may exhibit better properties than pure photocatalysts via improvement of their textural and electronic properties.2-Graphene Oxide (TiO2 - GO) nanocomposite was prepared by thermal hydrolysis of suspension with graphene oxide (GO) nanosheets and titania peroxo-complex. The characterization of graphene oxide nanosheets was provided by using an atomic force microscope and Raman spectroscopy. The prepared nanocomposites samples were characterized by Brunauer–Emmett–Teller surface area and Barrett–Joiner–Halenda porosity, X-ray Diffraction, Infrared Spectroscopy, Raman Spectroscopy and Transmission Electron Microscopy. UV/VIS diffuse reflectance spectroscopy was employed to estimate band-gap energies. From the TiO2 - GO samples, a 300 μm thin layer on a piece of glass 10×15 cm was created. The photocatalytic activity of the prepared layers was assessed from the kinetics of the photocatalytic degradation of butane in the gas phase.TiO-1), while sample labeled TiGO_075 (k = 0.00774 h-1) demonstrated the best activity under visible light.The best photocatalytic activity under UV was observed for sample denoted TiGO_100 (k = 0.03012 h It has been well documented that GO is heavily oxygenated graphene that is readily exfoliated in water to yield stable dispersions consisting mostly of single-layer sheets. The use of graphene oxide as the nanoscale substrates for the formation of nonocomposites with metal oxides is widely explored due to an idea to obtain a hybrid which could be combined both properties of GO as fascinating paper-shape material and the features of single nano-sized metal oxide particles.Traditionally, TiO2-GO) and titanium oxide nanoparticle-reduced graphene oxide (TiO2-RGO) composites were early provided and emphasized on the different synthetic strategies for the preparation of graphene-TiO2 materials. For example, TiO2-GO materials have been prepared via the hydrolysis of TiF4 at 60°C for 24 h in the presence of an aqueous dispersion of graphene oxide (GO). The reaction proceeded to yield an insoluble material that is composed of TiO2 and GO 2+ induced by the hydrolysis of Ti(SO4)2 diffused into graphene oxide interlayer by electrostatic attraction [TiOfate TiSO2 as initRecently, Min et al.  presente2 nanocomposites was published [2 to CH4. Another novel method [2 composites as a highly efficient photocatalyst by in situ depositing TiO2 nanoparticles on graphene oxide nano-sheets by a liquid phase deposition, followed by a calcination treatment at 200°C.A new technique for producing of novel solvent-exfoliated graphene-TiOublished , which al method  was deve2-graphene oxide nanocomposite, which was prepared by thermal hydrolysis of suspension with graphene oxide nanosheets and titania peroxo-complex. It should be mentioned that we are able to produce pure graphene nanosheets in large quantity from natural graphite by using high intensity cavitation field in the high-pressure ultrasonic reactor. Graphene oxide sheets, prepared by such safe and friendly method, can be used as a good support for TiO2 to enhance its photocatalytic activity. In this paper, we reported on an "one-pot" thermal hydrolysis of titanium peroxo-complexes in the presence of graphene oxide in aqueous solution. Our "hydrogen peroxide route" is based on hydrolysis of Ti(IV) sulfate, next treatment of the resulting gel by hydrogen peroxide, and thermal decomposition of so formed peroxo-titanate under reflux at ambient pressure producing directly the TiO2-GO photocatalyst. The relative photocatalytic activity of the as-prepared thin layers of titania/graphene nanocomposite in poly(hydroxyethyl methacrylate) was assessed by the photocatalytic decomposition of butane under UV and visible light.In our previous work , we repo4, hydrogen peroxide H2O2, ethylene glycol OHCH2CH2OH and ammonium hydroxide NH4OH were supplied by Sigma-Aldrich. Graphene was produced in large quantity from natural graphite (Koh-i-noor Grafite Ltd. Czech republic) using high intensity cavitation field in a pressure batch-ultrasonic reactor  [All chemical reagents used in the present experiments were obtained from commercial sources and used without further purification. Titanium oxo-sulfate TiOSOcs GmbH) .2SO4 and 10 ml of H3PO4, 1 g of graphene and 3 g of KMnO4 were mixed in round bottom flask. The reaction was then heated to 40°C and stirred for 6 hours and pink squash suspension was obtained. Subsequently was poured onto ice with 200 ml of 30% H2O2. The pink squash suspension quickly changed to lemon-like yellow suspension. The whole reaction product was purged by dialysis , washed with ethanol and dried at 105°C.Graphene oxide was prepared by our safety method, a 60 ml of H2 - GO nanocomposite preparation, 100 ml of 1.6 M titanium oxo-sulfate (TiOSO4) was hydrolyzed by slow addition of ammonium hydroxide solution (10%) under constant stirring at temperature of 0°C in ice bath. The stirring last until the reaction mixture reaches pH 8.0. The obtained white precipitate was separated by filtration. The consequent depuration of sulfate ions from precipitate with distilled water was confirmed by the BaCl2. The wet precipitate is mixed with 100 ml of 15% hydrogen peroxide solution; thereby a yellow solution of titania peroxo-complex is obtained.In the typical procedure of TiO2 - GO nanocomposite was filtered off and dried at 105°C. Ten samples of TiO2-GO nanocomposites doped as TiGO_XXX, where XXX are grams of GO, were prepared.Well defined quantity (see Table 2 - GO nanocomposite powder (2 g) was dispersed in a mixture of 5 ml poly(hydroxyethyl methacrylate) and 10 ml of ethanol. From this suspension, a 300 μm thin layer on a piece of glass, 100 × 150 mm was created.The prepared TiODiffraction patterns were collected with diffractometer PANalytical X´Pert PRO equipped with conventional X-ray tube  and a linear position sensitive detector PIXcel with an anti-scatter shield. A programmable divergence slit set to a fixed value of 0.5 deg, Soller slits of 0.02 rad and mask of 15 mm were used in the primary beam. A programmable anti-scatter slit set to fixed value of 0.5 deg., Soller slit of 0.02 rad and Ni beta-filter were used in the diffracted beam. Qualitative analysis was performed with the DiffracPlus Eva software package  using the JCPDS PDF-2 database . For quaAFM images were obtained using an NTEGRA Aura (NT-MTD) microscope. A sample of the diluted dispersion was placed on synthetic mica as an atomically smooth support and evaporated at room temperature. The measurements were performed in air at room temperature in non contact mode, with Si tips of the 1650–00 type at resonance frequencies ranging from 180 to 240 kHz.The morphology of sample powders was inspected by transmission electron microscopy (TEM) and the crystal structure was analyzed by electron diffraction (ED) using a 200 kV TEM microscope JEOL 2010 F. As specimen support for TEM investigations a microscopic copper grid covered by a thin transparent carbon film was used. The samples were studied in both bright field and by electron diffraction with a selecting aperture (SAED) mode at an acceleration voltage of 200 kV.The surface areas of samples were determined from nitrogen adsorption–desorption isotherms at liquid nitrogen temperature using a Coulter SA3100 instrument with outgas 15 min at 150°C. The Brunauer–Emmett–Teller (BET) method was used for surface area calculation , the porThe Raman spectra were acquired with DXR Raman microscope (Thermo Scientific) with 532 nm (6 mW) laser, 32 two-second scans were accumulated with laser 532 nm (6 mW) under 10× objective of Olympus microscope.-1, respectively, with single-reflection horizontal accessory on Si crystal. The samples were mixed with KBr and pressed to conventional pellets at ambient conditions and measured in the transmission mode.Infrared spectra were recorded by using Thermo-Nicolet Nexus 670 FT-IR spectrometer approximately in 4000–500 and 500–50 cm-8 mbar. XPS survey-scan spectra were made at pass energy of 40 eV; the energy resolution was set to 0.5 eV. While individual high-resolution spectra were taken at pass energy of 10 eV with 0.05 eV energy steps. A software tool CasaXPS was used to fit high-resolution multi components peaks. The proper surface charge compensation was done by fitting C-C, C-H component of C 1 s peak to reference binding energy 284.5 eV. The atomic concentration of compounds was evaluated with relative sensitivity factors (RSF) defined in standard table of CasaXPS software.XPS apparatus was equipped with SPECS X-Ray XR50 (Al cathode 1486.6 eV) and SPECS PHOIBOS 100 Hemispheric Analyzer with 5-channels detector. A background pressure in XPS during the measurements was under 2×104 as a standard was used. The reflectance data were obtained as relative percentage reflectance to a non absorbing material (BaSO4) which can optically diffuse light.Diffuse reflectance UV/VIS spectra for evaluation of photo-physical properties were recorded in the diffuse reflectance mode (R) and transformed to absorption spectra through the Kubelka-Munk function . A Perki-2). The gas concentration was measured with the use of Quadrupole Mass Spectrometer JEOL JMS-Q100GC and gas chromatograph Agilent 6890 N. A high-resolution gas chromatography column  was used. Samples were taken from the reactor automatically through the sampling valve  in a time interval of 2 hours.Kinetics of the photocatalytic degradation of butane (0.87%) was measured by using a home-made stainless steel batch photo-reactor  with a N2, and consequently neither butane absorbed on the poly(hydroxyethyl methacrylate) matrix. The injection volume of butane into the photoreactor was 30 ml.Blank tests (a layer of poly(hydroxyethyl methacrylate) without titania) were performed in order to establish the effect of photolysis and catalysis on the conversion of butane. The UV irradiation detects that there was none or immeasurable conversion of butane, as a testing gas, into CO and/or CO001 = 6.33 Å [001 = 6.718 Å which is in good agreement with published results [Early published experimental XRD pattern of graphene oxide demonstrated arising of strong 001 reflection peak at 2θ ~10° with a basal spacing of d= 6.33 Å . We calc results ,17.2-GO nanocomposites. All samples show only the anatase phase (PDF 21–1272). Higher concentrations of GO (samples TiGO_300 and TiGO_400) probably lead to partial reduction of GO to graphene and a weak peak at 2Θ = 26.50° appears (see arrow G). Nevertheless, the intensity of graphene X-ray lines is under possibility for quality Rietveld refinement. Furthermore, in the position at 2Θ ~ 10-12° (see arrow GO), there is an obvious hint of GO peak, which is due to the small particle size and its concentration featureless.The Figure 4+ are transformed to Ti3+, because of the larger ionic radius of Ti3+ (0.670 Å) compared to Ti4+ (0.605 Å) [Crystallite size, a and c cell parameters of anatase, calculated by the Rietveld refinement procedure using Topas v.4.2 programme, based on the full width at half-maximum of the peak at 25.4° are presented in Table 0.605 Å) .2-GO nanocomposite.Atomic force microscopy and electron transmission microscopy were used to determine quality of delamination of graphene oxide (GO) used for preparation TiO2 nanoparticles and graphene oxide sheet prevents the re-aggregation of the sheets of graphene oxide. The TEM images of GO prepared by modified oxidation method is presented in Figure 2-GO nanocomposite and demonstrates that TiO2 nanoparticles were dispersed uniformly on the graphene plane.In our previously work we have been demonstrated that the thermal hydrolysis of titania peroxo-complex leads to spindle-like particles . Our expFigure 2-GO nanocomposites calculated by the multi-point Brunauer-Emmett-Teller (BET) method and total pore volume are listed in Table 2-GO nanocomposites have mesoporous texture. With increasing content of GO in the composite decreasing trend of specific surface area is obvious  was found compared to the theoretical value of the graphene and graphene oxide, respectively. Nevertheless mesoporous TiO2-graphene oxide nanocomposites have specific surface area in interval ~ 80–200 m2g-1 being considerably larger than those of P25 and similarly prepared neat TiO2 particles without using graphene oxide.The specific surface area of the prepared TiO4+ to Ti3+[4+, somewhere an oxidation must occur, and it is probably on the graphene skeleton in free positions of Π bonds. Following oxidation of graphene due to reduction of Ti4+ corresponds to an increase of intensity of Raman bands GO in the nano-composite TiO2-GO.Raman spectroscopy is a powerful tool to characterize the crystalline quality of graphene or GO. Additional file + to Ti3+. However-1, whereas the intensity of the D band at 1350 cm-1 increases substantially. Increased background of the spectrum is caused by the sample fluorescence. G band is common to all sp2 carbon forms and provides information on the in-plane vibration of sp2 bonded carbon atoms and the D band suggests the presence of sp3 defects [In our Raman spectrum, the G band is broadened and shifted slightly to 1605 cm2-GO is presented in Figure -1 (Eg), 396 cm-1 (B1g), 512 cm-1 (B1g + A1g) and 631 cm-1 (Eg) indicating the presence of the anatase phase in all of these samples. The D bands of GO are located at 1349 cm-1 and G band at 1601 cm-1. With increasing concentrations of GO the intensity of both bands also increases. The background of the sample labeled TiGO_075 is increased by the sample fluorescence.The Raman spectra of series nanocomposites TiO2-GO nanocomposites was evaluated from the high resolution spectra of O 1 s, C 1 s and Ti 2p. The compositions are summarized in the Additional file High resolution XPS spectra were accumulated 10 times to enhance the signal and to noise ratio, and are presented in Figure 4)2 with GO, plenty of oxygen-containing groups, such as C = O located in the interlayer of graphene oxide were consumed during the nucleation and growth of TiO2 crystallites, which means graphene oxide has been partly reduced [2 nanoparticles, resulting in hydrogen bonds between them [The binding energy calibration was made on C-C component of C 1 s peak after a deconvolution process. The intensity of C 1 s peak is rather small and the signal is noisy, but the C 1 s peak was fitted by two main components to represent C-C/C-H bond at binding energies about 284.5 eV and C = O bond at higher binding energies about 288 eV. The C = O bond was expected because it is a part of the graphene oxide precursor. Furthermore,during reaction of Ti and had C-C/C-H character; therefore it can be expected that the carbon comes from the precursor rather then from additional contaminations. The other expected bonds as O = C-OH and C-OH were neglected because of the weak signal .3/2 and Ti 2p1/2 were identified at binding energies of 458.5 and 464.1 eV, respectively. The position of Ti 2p peaks corresponds to the literature reported values and also to the FWHM values about 1.6 eV for Ti 2p3/2 and 2.4 eV for Ti 2p1/2, which are reasonable for single peak character. The Ti is probably bonded in the TiO2. The equal area/RFS ratios were found on both spin states within precision about 10%. The limited precision is due to the background removal process when the Shirley background was used. The peak positions and the FWHM were found to be practically identical for all investigated samples.The Ti 2p2-GO nanocomposite prepared by thermal hydrolysis of titania peroxo-complexes. The broad absorption peaks about 3427 cm-1, and the band at 1631 cm-1 correspond to the surface absorbed water and the hydroxyl groups [-1 can be assigned to hydroxyl C-OH and that at 1258 cm-1 to epoxide C-O-C [-1 is most probably related to the Ti-O vibration modes of the Ti3+-O-Ti4+ framework [The Additional file l groups . The bande C-O-C . The ban2-GO nanocomposite. The presence of different amounts of GO influences the optical properties of light absorption significantly. With increasing GO content in the nanocomposite light absorption intensity in the UV region grows and a red shift to higher wavelength in the absorption edge at about 400 nm also observed [2[3+.The Additional file observed . Increasserved [2 and part2/2R, R is the reflectance of the "infinitely thick" layer of the solid [1/2 versus photon energy (hν = 1239/λ) [2 is 3.20 eV, [2-GO nanocomposite has been reduced from value 3.15 eV (sample TiGO_001) to value <2.50 eV for sample TiGO_100. The band-gaps of samples TiGO_200 to TiGO_500 are impossible to be identified because the absorption edges for TiO2 entirely overlapped with those for GO.According to the equation for band gap:he solid , B is ab 1239/λ) ,31, whic3.20 eV,  whereas The effect of oxidation time variation on the electronic, physical and chemical properties of GO was investigated and it was found that the bandgap of GO increased with oxygen content of the GO material . GO has 2 to form a Ti–O–C structure, which then shifted up the valence band edge and reduce d the band gap [Graphene oxide is non-stoichiometric material, which retains the lamellar structure of graphite. After treatment in the presence of strong acids and oxidation agents, many oxygen-containing groups such as carboxyl (C − OOH), hydroxyl (C − OH), epoxide (C − O − C) become covalently attached to its layers surfaces. In our experiment GO reacted in the presence of Ti-peroxo-complex, and some functional group such as  on the GO surface were removed. We can can suppose that some unpaired ∏ − electrons bonded with the free electrons on the surface TiOband gap .- and h+ is strongly reduced, which increases the process yield.On another hand after the photoactivation of titanium dioxide the electrons can easily transfer to the graphene-nanosheets and photoinduced holes migrate into titania; recombination of e2-GO nanocomposites were assessed from the kinetics of the photocatalytic degradation of butane in the oxygen atmosphere. Photocatalytic oxidation of butane are based on the following overall reactions [The photocatalytic activity of the as-prepared layered of TiOeactions :(2)2C4H12 and the measurement is run. If the measured gas chromatogram shows only the oxygen peak at retention time 1.52 min then a gas for the photocatalytic decomposition (in our case the butane) can be injected. After measuring the subsequent chromatogram, peaks for the oxygen and the butane are marked each by value of 1. The value of gases, which are expected to be formed due to photo-activity of the TiO2-GO nanocomposite  are assigned each by value 0. By this normalization of input data is performed and the measurement with time period of 2 hours can be started.Before each measurement, the photocatalytic reactor is evacuated, then rinsed with the O2 and H2O after time reaction 60 hours is presented in Table In a typical chromatograph of the effluent obtained from butane photocatalytic degradation only oxygen, carbon monoxide, carbon dioxide, water and butane were detected at retention times of 1.52, 1.54, 2.41, 6.42 and 9.53 min . The nor0 is the initial concentration, c is the concentration of butane after time (t), and k is the first-order rate constant.The typic corresponding experimental dependencies of butane, oxygen, carbon monoxide and carbon dioxide in time are plotted in Figure 2 at ambient temperature. In general, they observed that alkanes (CnH2n+2) formed ketones (CnH2nO) and other aldehydes CmH2mO with 2 < m < n. If the alkane was branched, the ketone was CmH2mO with 3 < m < n. The reactivity of different types of carbon atoms followed the sequence: Ctertiary > Cquaternary > Csecondary > Cprimary. Paper [2 and H2O.The photocatalytic degradation of butane was fitted by curves of the first-order kinetics; the corresponding rate constants are given in Table y. Paper  shows, t-1) and TiGO_075 (k = 0.00774 h-1), respectively. As for the TiGO_075 sample, there is also the largest increase in CO and CO2 content. It is evident from Table 2 at the expense of the primary decomposition of butane and the value for rate constant decreases. This suggests that in these samples a much deeper mineralization occurs, which leads to the preferential decomposition of intermediates to end products of photocatalytic decomposition, i.e. CO2 and H2O. This increase in photoactivity for some samples is probably due to reduction of Ti4+ to Ti3+ and oxidation of free ∏ − bonds during preparation. The interaction of CO2 molecules with the excited states of Ti3+-O- leads to the formation of CO2•¯ anion radicals [2•¯ anion radicals readily react with poly(hydroxyethyl metacrylate), which leads to the formation of carbon monoxide due to its strong adsorption on Ti sites of the TiO2 surface [2 nanoparticles, ii) graphene oxide sheets can enhance adsorption ability of the TiO2 - graphene oxide nanocomposite, iii) Graphene oxide works as electron acceptor and photosensitizer to efficiently enhance the butane photodecomposition.The results presented in Table radicals . The CO2 surface . The fun2, we developed a simple ("one-pot") method to prepare nanocomposite by depositing TiO2 on the graphene oxide through liquid phase deposition.Using graphene oxide sheets as a substrate for TiO2-GO nanocomposite systems irrespective of light sources could be attributed to synergy effects including the increase in specific surface area with graphene oxide amount as well as to the formation of both π–π conjugations between butane molecules and aromatic rings and the ionic interactions between butane and oxygen-containing functional groups at the edges or on the surfaces of carbon-based nanosheets. Graphene oxide works as the adsorbent, electron acceptor and photosensitizer and efficiently enhances the butane photodecomposition. The best photocatalytic activity under UV and visible light was observed for samples denoted TiGO_100 (k = 0.03012 h-1) and TiGO_075 (k = 0.00774 h-1), respectively. Both the used synthesis route and the GO modification are promising ways to cheap and efficient photocatalysts for the Vis light activation.The better photocatalytic properties on the TiOThe authors declare that they have no competing interests.VŠ was the main author of the work, performed syntheses, and coordinated all characterization and catalytic studies. SB was responsible for electron microscopy, TMG for Raman spectroscopy, MK for XPS measurement, JB assisted with manuscript writing. All authors read and approved the final manuscript.Figure S2. IR spectrum of the TiO2-GO nanocomposite. Figure S3. UV–vis absorption spectra of the TiO2-GO. Table S1. C Composition of nanocompsite base on XPS.The Raman spectrum of prepared graphene oxide. Click here for file


D/IG down to < 0.02 in Si-face samples and < 0.05 in C-faces over the 1 cm2 wafer surface grown at 1,450°C. AFM examination shows a better morphology in face-down surfaces. This study suggests that the optimum configuration slows the thermal decomposition and allows the graphene to form near the equilibrium. The Si-face-down orientation (in opposition to the temperature gradient) results in a better combination of low disorder ratio, ID/IG, and smooth surface morphology. Mobility of Si-face-down orientation has been measured as high as approximately 1,500 cm2/Vs at room temperature. Additionally, the field effect transistors have been fabricated on both Si-face-down and C-face-down showing an ambipolar behavior with more favorable electron conduction.We have explored the properties of SiC-based epitaxial graphene grown in a cold wall UHV chamber. The effects of the SiC surface orientation and silicon loss rate were investigated by comparing the characteristics of each formed graphene. Graphene was grown by thermal decomposition on both the silicon (0001) and carbon (000-1) faces of on-axis semi-insulating 6H-SiC with a "face-down" and "face-up" orientations. The thermal gradient, in relation to the silicon flux from the surface, was towards the surface and away from the surface, respectively, in the two configurations. Raman results indicate the disorder characteristics represented by I Then, devices were fabricated on these graphene films. Figure -1 and 2,710 cm-1, respectively. The Si-face-down surface showed the narrowest G-peak with the full width at half maximum of 19 cm-1. Defect related D-peaks were also found at around 1,350 cm-1, and the relative intensity of D-peak and G-peak in the Raman spectra was used to estimate the quality of graphene and the degree of disorders. This ID/IG ratio has been known to be inversely proportional to the grain size and provides a good estimation for the quality of graphitic materials  with a magnetic field of 0.2 T. RT mobility as high as approximately 1,500 cm2/Vs was measured from the graphene on Si-face-down sample with a sheet carrier density of 2 to approximately 3 × 1013 cm-2. As reported previously on epitaxial graphene, mobility increased as carrier density decreased [2/Vs. The degradation of the field effect mobility is likely caused by the dielectric layer on top of the graphene as well as the effects of device fabrication.The signature of graphene as measured by Raman spectroscopy was observed in all the samples. Figure aterials . The ID/ecreased . Figure ecreased . Specialecreased ) or fixeD/IG ratio below 0.02 with smooth and uniform surface morphology. Electrical properties were characterized by test structure-fabricated and Hall-effect measurements. In transistor characteristics, it shows an ambipolar behavior, and the electron conduction is favored over the hole conduction, which is determined by the polarity of initial majority carriers in graphene layer during the formation of graphene. The Hall mobility at RT is as high as 1,500 cm2/Vs, and the value of field effect mobility is close to that of silicon devices. This approach to form a graphene film could be used to grow high quality epitaxial graphene in case a relatively lower-annealing temperature is required.By comparing the face-down (low Si evaporation rate) and face-up (high Si evaporation rate) mounting method, the optimum growth condition producing a higher quality graphene in a cold wall UHV chamber was found. The use of graphite enclosure and the face-down scheme provide a condition that can reduce Si loss rate and allow higher temperature growth. The best quality film was obtained with the Si-face-down configuration with the IThe authors declare that they have no competing interests.MK and JH contributed equally to this work. They carried out the graphene growth and device fabrication/characterizations. VS participated in the growth of graphene and material characterizations. ST participated in the design of devices and its coordination. MS and J-WL conceived of the study and design most of this project. All authors read and approved the final manuscript.


Foliar fungal communities of plants are diverse and ubiquitous. In grasses endophytes may increase host fitness; in trees, their ecological roles are poorly understood. We investigated whether the genotype of the host tree influences community structure of foliar fungi. We sampled leaves from genotyped balsam poplars from across the species' range, and applied 454 amplicon sequencing to characterize foliar fungal communities. At the time of the sampling the poplars had been growing in a common garden for two years. We found diverse fungal communities associated with the poplar leaves. Linear discriminant analysis and generalized linear models showed that host genotypes had a structuring effect on the composition of foliar fungal communities. The observed patterns may be explained by a filtering mechanism which allows the trees to selectively recruit fungal strains from the environment. Alternatively, host genotype-specific fungal communities may be present in the tree systemically, and persist in the host even after two clonal reproductions. Both scenarios are consistent with host tree adaptation to specific foliar fungal communities and suggest that there is a functional basis for the strong biotic interaction. Endophytic fungi live in the tissues of leaves and other plant organs without causing symptoms of disease in planta diversity; class 3: broad host range, mostly above-ground tissues of plants, horizontal infection of hosts, high in planta diversity; class 4: broad host range, present in roots). Vertically transmitted endophytes typically infect their hosts during reproduction, while horizontally transmitted endophytes randomly infect their hosts from environmental sources. The group typically associated with tree leaves is the "class 3", nonclavicipitaceous endophytes. Class 3 endophytes are highly diverse, occur in the above-ground tissues, and are generally considered to be horizontally and stochastically distributed Host plant characteristics are known to influence fungal community assembly. It has been shown that plant genotype, taxonomic identity, as well as specific plant traits such as chemical properties can affect microbial community composition and diversity Populus balsamifera L.). This is a well-studied North-American tree species, with a vast range covering most of Canada, Alaska, and the northern U.S.A. The current distribution is the result of a northward range expansion after the last glacial maximum In this study we investigate potential effects of the host genotype in shaping the composition of the foliar fungal communities of balsam poplar  since 2007. In 2009 cuttings from these trees were sent to Fairbanks, Alaska, rooted in the greenhouse, and planted into a new common garden. The newly rooted trees originating from these cuttings grew for 12 months in the Fairbanks common garden until our sampling in 2010. The Fairbanks garden is located on a cleared area of the University of Alaska campus . The opening is surrounded by coniferous forest. All trees were genotyped using single nucleotide polymorphisms (SNP) from 590 gene fragments To ensure that the fungal communities did not change during transportation to the lab we rapidly dried the leaves by placing them immediately in silica gel. Leaves dry very rapidly under these conditions (within a few hours from the time of the sampling). We assumed that the rapid drying also prevents preferential fungal growth in the collected leaves . Once dried, the sampled leaves were carried to the lab in plastic bags filled with silica gel. Within 2 months of sampling, DNA was extracted from dried leaves using Qiagen DNeasy Plant Mini Kit (Qiagen Inc. USA), without surface-sterilization. We are aware that avoiding surface-sterilization may cause further complexity in the data. We consider that our dataset represents both endophytic communities and fungi found on the surfaces of the leaves.DNA extraction, PCR conditions, and primers can strongly influence community composition recovered by amplicon sequencing CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) fungal primers Using ITS1F . We quantified the tagged PCR product concentration with Quant-iT™ PicoGreen®dsDNA assays . MID-tagged community amplicons from five additional, non-genotyped poplar specimens were also multiplexed in the same sequencing run, along with the PCR products for the current study s = 60, PyroNoise initial clustering cutoff c = 0.01; SeqNoise cluster size s = 30, SeqNoise initial clustering cutoff c = 0.08). Trimmed reads shorter than 300 bp were discarded, and all reads were truncated to 450 bp to remove noisy ends. Chimera checking was performed with PerseusD After removing MID tags, binary SFF files were converted into text flowgrams with sffinfo v2.5.3, a program from the 454 Sequencing System software (454 Life Sciences). Trimming information provided by the sequencer in the raw data files was used for a preliminary trimming of the 3′ ends of poor quality fragments of the flowgrams with sffinfo. The trimmed flowgrams were processed with the AmpliconNoise v1.23 pipeline We downloaded all annotated fungal ITS1, 5.8S, and ITS2 sequences from NCBI  and used these to build a BLAST v2.2.21 We removed all sequences not identified as being of fungal origin by the previous BLAST/MEGAN assignment. For the rarefaction analysis we clustered the remaining forward reads  with a grammar-based approach in GramCluster v1.3 We analyzed fungal community structures by selecting all BLAST hits assignable to the lowest taxonomic level . We completed this matrix with all hits assigned to the genus level. This resulted in a taxonomic matrix consisting almost exclusively of species and genera, containing also six families . We omitWe used linear discriminant analysis (LDA) as implemented in the R package MASS The 454 Titanium runs on two 1/4th plate, and two 1/16th plate fractions produced 204,052 reads. Of these reads 203,459 featured correct MID tags (corresponding to the 28 samples multiplexed for sequencing). After quality trimming of read ends, filtering for overall sequence quality and chimeras, and discarding short (less than 300 bp) reads, 126,402 reads were retained . The final number of reads after filtering for 5′-3′ oriented forward sequences was 51,596. Individual samples contained 1,388–3,057 forward-oriented reads.The combined BLAST/MEGAN analysis assigned the reads to 125 taxa. The communities were dominated by a few very abundant taxa. However, most of the taxa were rare, 80% of them being represented by less than 43 reads combined in the 23 host clone leaves . Individgof = 0.475).We plotted different similarity thresholds and showed that sequence cluster discovery approaches, but does not reach, a saturation plateau . The spea priori groups defined on the basis of their host genotypes .Linear discriminant analyses revealed that the genotype of the poplar trees and the composition of their foliar fungal community are tightly linked . Most foenotypes . Of the enotypes . The strOur survey of the leaf-associated fungal communities found in and on poplar leaves sampled from a common garden revealed that the host genotype is a significant determinant of the composition of leaf-associated fungal communities. This finding is in conflict with the expectation that foliar fungal communities represent a random sample of fungi present in the environment. The results imply the existence of specific relationships between the host tree and its foliar fungal microbiome. Intriguingly, the geographic divergence of the balsam poplar subpopulations happened relatively recently, after the last glacial maximum , relatives of known plant parasites (Mycosphaerella sp.). Some reads were assigned to unexpected taxa, e.g. Blumeria graminis and Pyrenophora tritici-repentis (both known to occur only in Poaceae), Leucosporidium golubevii (known from freshwaters). The unexpected assignments may also result from epiphyllous diaspores. Alternatively, the unexpected hits may come from presently unknown taxa, which are happen to be closely related to species present in GenBank. The list of BLAST hits should also be interpreted cautiously, as it strongly relies on the quality of our reference database  are known as ballistosporic yeasts without host specificity. The genus Fusicladium  includes almost 100 species, some of them known to be highly host specific, as shown for example on Populus spp. by Newcombe sensu Pompanon et al. Each of the 25 most common taxa had some discriminating value in the LDA, but only two of them had above-average discriminating values . SporoboSimilar to previous reports on tree leaf endophytes characterized by 454 pyrosequencing Two basic mechanisms possibly explain the patterns observed in the present study. First, host traits may promote infections with specific strains from the environment. Host morphology, physiology Our results show that foliar fungal communities of trees growing in a common garden show host genotype-specific structures. This raises the possibility that at least some members of the leaf fungal community adapt to plant genotypes. Given that many endophytes alter plant fitness, this could be a form of coevolution. We hypothesize that host-foliar fungal relationships in trees might have been obscured by the low resolution of cloning and culturing methods available before next-generation metabarcoding. Most of the strongly discriminating taxa were relatively rare in the foliar fungal communities sequenced in this study . TechnolTable S1a priori LDA groups and LDA results of foliar fungal communities from balsam poplar.Collection data, 454 MID tags of the samples during sequencing, (XLS)Click here for additional data file.Table S2List and functions of foliar fungal taxa discriminating among host balsam poplar specimens on the basis of genotype group and regional translocation events.(XLS)Click here for additional data file.Material S1Pruned sequences produced by the 454 runs for the foliar fungal communities of each balsam poplar host specimen in FASTA format.(ZIP)Click here for additional data file.Material S2The number of pruned 454 reads assignable by MEGAN to low-level taxa (125), on the basis of BLAST hits against annotated fungal sequences present in GenBank.(ZIP)Click here for additional data file.Material S3Clusters delimited at sequence-divergence based equivalents of 6 grammar thresholds with GramCluster.(ZIP)Click here for additional data file.


More specifically, we quantify the effect of either pharmacological upregulation of the heat shock response or specific genetic upregulation of a small heat shock protein, CG14207, on the neurotoxicity of both TDP-43 and of its disease associated 25 kDa fragment (TDP-25) in a Drosophila model. Inhibition of the aggregation of TDP-43 by either method results in a partial reduction of its neurotoxic effects on both photoreceptor and motor neurons, whereas inhibition of the aggregation of TDP-25 results not only in a complete suppression of its toxicity but also its clearance from the brain in both neuronal subtypes studied. The results demonstrate, therefore, that aggregation plays a crucial role in mediating the neurotoxic effects of both full length and truncated TDP-43, and furthermore reveal that the in vivo propensity of these two proteins to aggregate and their susceptibility to molecular chaperone mediated clearance are quite distinct.Almost all cases of sporadic amyotrophic lateral sclerosis (ALS), and some cases of the familial form, are characterised by the deposition of TDP-43, a member of a family of heteronuclear ribonucleoproteins (hnRNP). Although protein misfolding and deposition is thought to be a causative feature of many of the most prevalent neurodegenerative diseases, a link between TDP-43 aggregation and the dysfunction of motor neurons has yet to be established, despite many correlative neuropathological studies. We have investigated this relationship in the present study by probing the effect of altering TDP-43 aggregation behaviour  Although the ability to form intractable aggregates is an intrinsic property of all polypeptide chains, the formation of such species under physiological conditions is largely restricted to a subset of proteins associated with progressive pathological conditions In order to investigate the molecular origins of ALS and related disorders, a number of cellular and animal models of TDP-43 expression have now been established. Whilst some of these models have indicated a toxic gain of function role for TDP-43 in protein aggregation and neurodegeneration, others have suggested that the cause of neuronal dysfunction is the loss of normal TDP-43 function, or indeed that the retention of the RNA binding activity of TDP-43 resulting in aberrant binding is a cause of its toxicity in vivo, clearly indicate that these molecules play an important role in the regulation of pathological protein aggregation In the present study we set out to investigate specifically the role of aggregation of full length TDP-43 and of an N-terminally truncated and disease-associated fragment, TDP-25, in causing neurodegeneration by upregulation of molecular chaperones, the inherent machinery of all biological systems that combats misfolding and aggregation; it is well established that molecular chaperones are able to inhibit the aggregation of proteins associated with neurodegenerative diseases Drosophila melanogaster, and these model systems have been formed to capture many of the key molecular features of these diseases Drosophila model of TDP-43 proteinopathy and demonstrate that both pharmacological and genetic upregulation of chaperones enable a dissection of the distinct roles played by aggregation in neurodegeneration induced both by TDP-43 and its N-terminally truncated fragment TDP-25.Many neurodegenerative disorders, including AD, PD and Huntington's disease (HD) have been modeled in Drosophila were generated, each expressing one of these proteins. In the first line, the human TARDBP gene was introduced into the Drosophila genome using a modified UAS-GAL4 vector Drosophila genome in the same way , two lines of transgenic same way  [17]. BoDrosophila typically cause a characteristic ‘rough eye’ phenotype in which the normal linear array of ommatidia and sensory bristles become disordered, and in addition the eye loses its natural pigmentation to varying degrees that depend on the toxicity of the proteins expressed gmr-GAL4 driver was found to result in a rough eye phenotype in both cases, although its severity was observed to vary dramatically between the two genotypes. Thus, flies expressing TDP-43 have a strong rough eye phenotype, characterised by an almost complete absence of structured ommatidia when examined by either optical or scanning electron microscopy, whereas TDP-25 flies show only a weak phenotype that is only clearly evident on high magnification analysis using scanning electron microscopy , a disease that exhibits a similar cytoplasmic aggregation phenotype to the TDP-43 proteinopathies Drosophila, we decided that CG14207 was an excellent candidate to establish whether chaperones can affect the aggregation and the subsequent toxicity of TDP-43 and TDP-25.17-AAG is known to have pleiotropic effects on the regulation of multiple cellular processes, including autophagy and apoptosis, as well as inducing chaperone expression Flies overexpressing CG14207 were crossed with those expressing TDP-43 and TDP-25, and the effects on their neurotoxic phenotypes were monitored. A very similar pattern of results was obtained to those observed with 17-AAG treatment, as CG14207 expression is only partially able to rescue the neurotoxic effect of TDP-43 on developing photoreceptors , and redgmr-GAL4 driver was used, which was able to drive expression of TDP-25 to a much higher level than that achieved by the driver used in In a similar series of experiments with flies expressing TDP-25, where the TDP-25 neurotoxic rough eye phenotype is mild in comparison to that elicited by TDP-43 expression, complete rescue upon CG14207 co-expression was observed. It was important to determine whether the difference in the extent of the rescue by CG14207 of TDP-43 and TDP-25 toxicity was indeed due to the two proteins having different intrinsic susceptibilities to CG14207 activity or is attributable to the apparently lower steady state concentration of TDP-25 present compared to TDP-43. To address this question a stronger D42-GAL4 driver). The median lifespans of the flies in which TDP-43 or TDP-25 expression was driven in these motor neurons were reduced by 57±2% and 17.7±3%, respectively, compared to control flies , such as α-crystallin and αB-crystallin may prevent the formation of insoluble, amyloid fibrils of other proteins but, depending on their stoichiometric ratios with respect to their client proteins, may also stabilise or even enhance the formation of relatively soluble but potentially neurotoxic non-fibrillar aggregates We also find that chaperone upregulation, and subsequent reductions in the microscopic and biochemical detection of aggregates of TDP-43, is associated with a reduction in its pathogenic effects demonstrated by photoreceptor degeneration or motor neuron dysfunction. In the case of flies expressing TDP-43, unlike those expressing TDP-25, this reduction in toxicity is incomplete, a finding consistent with the continued presence of aggregated TDP-43 that we have demonstrated biochemically by the detection of urea insoluble protein, albeit at much lower levels, in the brains of in vivo aggregation behaviour of TDP-43 and TDP-25, and its relationship with neurotoxicity. Our results demonstrate that whilst both TDP-43 and TDP-25 form aggregates in vivo, the abundance of these aggregates, the ease with which they can be prevented from forming by the small heat shock protein CG14207 and their neurotoxic effects are very substantially different. The important role of aggregation in the TDP-43 proteinopathies is therefore consistent with our understanding of other protein misfolding diseases such as AD, PD and HD.In conclusion we have exploited the upregulation of chaperones to probe the D. melanogaster and synthesised by Genscript . Each construct was cloned into the multiple cloning site of pUAST-AttB and then subcloned into a modified pCasper vector . Differences in the expression of the constructs that could arise from their integration at different genomic loci were therefore eliminated as the pCa4B vector contains sites for exploiting the PhiC31 system for site-specific integration of transgenes. All injected constructs were incorporated at the same genomic locus (51D) on Chr. II  HA-tagged TDP-43 constructs were codon optimised for expression in gmr-GAL4 flies (Bloomington Stock ID: 1104 & 8121). All flies were kept in a temperature and humidity controlled incubator maintained at 25°C, 70% humidity. Optical microscope pictures were taken of 1-day-old TDP/gmr-GAL4 offspring at a magnification of 7.5×. SEM images were collected as described previously TDP-43 and TDP-25 flies were crossed with Third-instar larval imaginal eye discs and adult brains were dissected, fixed in 4% PFA and blocked in 5% BSA in 0.05% PBS-Triton. This procedure was followed by incubation overnight with rat anti-HA-biotin high affinity antibody  and mouse anti-ubiquitin antibody . Discs or brains were then incubated overnight in Streptavidin Alexa Fluor 594 conjugate (Invitrogen) and anti-mouse FITC, and counterstained with TOTO-3 (Invitrogen) to detect nucleic acids.Five flies per genotype were decapitated, homogenised in RIPA buffer and centrifuged to pellet the insoluble forms of proteins. The pellets were then resuspended in 9 M urea and centrifuged again to collect urea soluble protein. Each of the protein samples was separated on 4–12% Bis-Tris gel (Invitrogen) under reducing or non-reducing conditions and transferred to PVDF membranes (Millipore). Blots were blocked with 5% non-fat milk and then incubated with primary antibodies: anti-HA-biotin, high affinity (3F10) antibody (Roche) followed by incubation with anti-rat secondary antibody conjugated to HRP. Bands were detected using a SuperSignal® West Femto Maximum Sensitivity Substrate kit (Thermo Scientific). Loading controls using a mouse anti-β-actin antibody (Abcam) and anti-mouse HRP (Dako) were performed on all western blots.gmr-GAL4 flies and maintained on either standard cornmeal-agar food treated either with distilled water or 1.5 µM 17-AAG to allow the larvae to ingest the drug. Microscopy, immunohistochemistry and western blot analyses were performed on drug treated and control offspring as described above.The geldanamycin analogue 17-allylamino geldanamycin  was resuspended in ethanol and diluted in distilled water to a concentration of 1.5 µM. 300 µL aliquots of drug were added to the surface of pre-made fly food and allowed to dry for 2 days. TDP-43 and TDP-25 flies were crossed with D42-GAL4 is activated by heat shock and so simply moving flies from 25°C to 29°C induces expression in motor neurons. Median survivals for each vial were calculated using Kaplan-Meier survival statistics (n = 100 per experiment) and differences between genotypes were analysed using a Mann-Whitney U test (n = 10 tubes of 10 flies each).Crosses were set up at 25°C. Non-virgin females were kept 10 per vial and moved to 29°C within 24 hours of eclosion and were transferred to fresh food and counted every 2–3 days. The experiments were performed in a temperature-controlled incubator at 29°C. Temperature sensitive Gal80 Motor function was assessed by a climbing assay. Flies expressing TDP-43 and TDP-25 in motor neurons from the day of hatching, with and without CG14207, were generated in separate experiments as described above. 10 flies were placed into a single tube and analysed every second day after the initial heat shock; this procedure was repeated three times on the same tube of flies for each day. A climbing index score was calculated as described previously Figure S1Light microscope images demonstrating the effects of TDP-43 when expression is driven by gmr-GAL4, in the presence and absence of a non-toxic control protein (UAS-GFP).(TIF)Click here for additional data file.