Tardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-sh TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, indicating that TDP-43 can directly cause neurodegeneration Tar DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) in vitro and in vivoin vivo and observed that mice developed early embryonic lethality, suggesting an important role for TDP-43 in development WT) or mutant hTDP-43, leads to pathological phenotypes consistent with certain TDP-43 proteinopathies. These phenotypes may include some of the following: increased ubiquitination, truncation, aggregation and phosphorylation of TDP-43, cytoplasmic TDP-43 inclusions, neuronal degeneration, motor dysfunction, learning and memory deficits, and mitochondrial abnormalities Tardbp. Additionally, recent studies demonstrate that TDP-43 autoregulates itself to decrease its expression Tardbp levels through this mechanism. However, it remains unclear whether the reduction in mTdp-43 (loss of function) or the overexpression of hTDP-43 (gain of function), or combined events are responsible for the ALS or FTLD-like pathologies observed in hTDP-43 transgenic mice.Previous studies have explored the TDP-43 loss of function hypothesis Tardbp mRNA observed in our hTDP-43 transgenic mice results in significant reduction of mTdp-43 protein. We found that this reduction in mTdp-43 does not increase the levels of splicing variants known to be inhibited by TDP-43. In particular, the levels of mouse Ex17b-containing sortilin 1 (mSort1+Ex17b) and Ex5-deleted translin (mTsn_ΔEx5) mRNAs are significantly upregulated when mTdp-43 protein is reduced in non-transgenic (NT) mice but not in hTDP-43 mice, suggesting that hTDP-43 has similar biological functions to that of mTdp-43 in vivo. Moreover, a further reduction of mTdp-43 in asymptomatic hemizygous hTDP-43 (hTDP-43hemi) mice to the levels equivalent to that of symptomatic homozygous hTDP-43 (hTDP-43homo) mice did not lead to the development of pathological phenotypes indicating that downregulation of mTdp-43 is not sufficient to induce the pathological phenotypes observed in hTDP-43homo mice. Finally, we provide evidence that high expression levels of hTDP-43 beyond a certain threshold lead to alterations in TDP-43 RNA substrates critical for maintaining neuronal structure and function and this, in turn, might cause neuronal dysfunction and the development of pathological phenotypes.To address the aforementioned question, we first generated an antibody that specifically detects mTdp-43 protein, and confirmed that downregulation of mouse hemi and hTDP-43homo transgenic mice. The levels of mTdp-43 protein in hTDP-43hemi and hTDP-43homo mice respectively decreased to ∼55% and 45% of NT mice levels (Tardbp mRNA and mTdp-43 protein levels in both hemizyogous and homozygous hTDP-43M337V mice (data not shown). Of importance, TDP-43M337V and TDP-43WT display similar biological activity in autoregulating the levels of TDP-43 in vivo, suggesting the ALS-associated M337V mutation does not lead to loss of function.Recently, we and others have reported that the mRNA levels of mTdp-43 are significantly decreased in hTDP-43 transgenic mice e levels , suggestin vivosortilin 1 (Sort1) translin (Tsn) mSort1+Ex17b (sortilin 1 including exon 17b) and mTsn_ΔEx5 (translin with exon 5 deletion) , the levels of mSort1+Ex17b and mTsn_ΔEx5 increased 3.7- and 5.2-fold, respectively, when mTdp-43 levels are reduced -shRNA targeting mTdp-43 in neonatal mice expressing only mTdp-43 (NT mice) or mTdp-43 and hTDP-43 (hTDP-43hemi mice). Note that efficient downregulation of mTdp-43 protein and RNA was achieved in both NT and hTDP-43hemi transgenic mice (mSort1+Ex17b and mTsn_ΔEx5 (hemi mice transduced with a control shRNA (shCtrl), the levels of mSort1+Ex17b and mTsn_ΔEx5 were not increased after further reduction of mTdp-43 in shTardbp-treated hTDP-43hemi mice. Similar results were observed in hTDP-43M337V mice . Overall our data suggest that a decrease in mTdp-43 protein in hTDP-43 transgenic mice is not responsible for the observed phenotype in these mice. Rather, it does seem that the human TDP-43 may be driving these events.We have previously reported that hTDP-43n levels . To invemice see . Since homo mice . At 1 mogregates , phosphogregates , and abngregates . These pemi mice  and NT mhomo mice to a certain point, beyond which the overexpression of hTDP-43 induces toxicity, we sought to investigate whether the highly abundant hTDP-43 protein present in these mice would alter the levels of certain TDP-43 mRNA targets and consequently account for the TDP-43 associated pathologies in hTDP-43homo mice. To this end, we measured the levels of several neuronal TDP-43 RNA targets that are critical for neuronal survival and function homo mice compared to NT mice and hTDP-43hemi mice , presenilins (PSEN1 and PSEN2) or microtubule associated protein tau (MAPT) have been overexpressed in animals to make Alzheimer’s disease (AD) mouse models, which are characterized by the production and accumulation of amyloid-β into plaques or of hyperphosphorylated tau into neurofibrillary tangles Generation of transgenic mice is a widely used research technique to model human diseases, given transgenic mice recapitulate key pathological features of disease and also serve as valuable tools to explore the role of disease-causing molecules (e.g. TDP-43). More importantly, the models provide opportunities to evaluate potential therapeutic strategies for translational research. For instance, mutations in genes encoding amyloid precursor protein  and mTsn lacking exon 5 (mTsn_ΔEx5) increase dramatically. These two opposite splicing patterns upon mTdp-43 downregulation (exon inclusion in mSort1 and exon exclusion in mTsn) make our assay more comprehensive. Of note, the levels of mSort1+Ex17b and mTsn_ΔEx5 are not increased in hTDP-43 transgenic mice, despite the fact that these mice present lower levels of mTdp-43 protein than in AAV1-shTardbp injected NT mice, which do show a significant increase in these aberrant splicing isoforms. Moreover, further reduction of mTdp-43 in hTDP-43hemi mice to the levels equivalent to that of hTDP-43homo mice do not lead to an increase in mSort1+Ex17b nor mTsn_ΔEx5 isoforms. As a result, our data indicate that loss of mTdp-43 function in hTDP-43 mice is compensated by the expression of hTDP-43. These results are not surprising since mouse and human TDP-43 proteins are 96% homologous WT and hTDP-43M337V develop phenotypes characteristic of FTLD and ALS diseases. However, hTDP-43hemi transgenic mice are virtually indistinguishable from NT controls homo mice hTDP-43hemi mice have ∼75% hTDP-43 protein or ∼25% less hTDP-43 protein. As such, we hypothesize that expression of hTDP-43 over a certain threshold will disrupt the processing of TDP-43 RNA targets that will consequently cause the neuropathologies observed in hTDP-43homo mice. While we were unable to detect differences in TDP-43 RNA targets known to be altered by mTdp-43 downregulation, we found significant changes in TDP-43 RNA targets that are critical for neuronal function and survival in hTDP-43 mice overexpressing high levels of hTDP-43. In particular, the mRNA levels of synaptic markers Psd-95, Bsn, Gap-43, and Syt7 are significantly decreased in hTDP-43homo mice, but not in hTDP-43hemi mice. Moreover, the number of dendritic spines in the hippocampus of hTDP-43homo mice is also significantly decreased compared to NT. More important, the mRNA levels of the above mentioned targets and the number of dendritic spines are not altered in AAV1-shTardbp treated hTDP-43hemi mice, excluding the possibility that low levels of mTdp-43 are responsible for the synaptic dysfunction in hTDP-43homo mice. Together, our results suggest that expression of total levels of TDP-43 protein (human and mouse) above 2.5 fold of endogenous mouse Tdp-43 in NT mice might disrupt RNA metabolism of several synaptic targets To further determine whether the lower levels of mTdp-43 protein in our hTDP-43 transgenic mice result in loss of mTdp-43 function, we examined the levels of abnormal splicing variants of two of its RNA targets: homo mice. In addition, there is no difference between wild-type and M337V hTDP-43 proteins regarding their ability to regulate RNA splicing. Our findings suggest that the current hTDP-43 transgenic mouse models might not be the most suitable tools to investigate loss of mTdp-43 function. Knock-in transgenic mouse models expressing either wild-type hTDP-43 or ALS-associated TDP-43 mutations at similar levels to mTdp-43 should be generated to understand the roles of TDP-43 in disease pathogenesis.In conclusion, although expression of hTDP-43 reduces the levels of mTdp-43 in hTDP-43 transgenic mice, this reduction does not lead to loss of mTdp-43 function because hTDP-43 compensates for functional losses in mTdp-43. Moreover, expression of hTDP-43 above a certain threshold disrupts RNA metabolism, especially of genes critical for neuronal survival and function, which consequently causes the pathological phenotypes observed in hTDP-43The hTDP-43 transgenic mice expressing wild-type or M337V mutant hTDP-43 have been previously reported The polyclonal mTdp-43 antibody is produced by using synthetic peptide corresponding to residues 203–221 of mTDP-43 (TAEELQQFFCQYGEVVDVF) to immunize rabbits. The resulting sera were affinity purified and the specificity of the antibody was characterized using Western blot.Tardbp, mSort1+Ex17b and mTsn_ΔEx5 mRNA values were normalized to Gapdh values, an endogenous transcript control. The data was analyzed by using Software RQ Manager 1.2 (Applied Biosystems). The primers used were: Gapdh: 5′-CATGGCCTTCCGTGTTCCTA-3′ and 5′-CCTGCTTCACCACCTTCTTGAT-3′; Tardbp: 5′-GGGGCAATCTGGTATATGTTG-3′ and 5′-TGGACTGCTCTTTTCACTTTCA-3′; mSort1+Ex17b: 5′-AAATCCCAGGAGACAAATGC-3′ and 5′-GAGCTGGATTCTGGGACAAG-3′; mTsn_ΔEx5∶5′GGTCTTCCTGGCAGCATTTG-3′ and 5′-TTGACAGACAGCCTCGATGC-3′. To quantify the RNA levels of TDP-43 synaptic targets in human TDP-43 transgenic mice, we also performed q-PCR. There primers used were: Psd-95∶5′-CGCTACCAAGATGAAGACACG-3′ and 5′-CAATCACAGGGGGAGAATTG-3′; Bsn: 5′-TGTGGCTTTAACCCAACACC-3′ and 5′-TTTGGCAGTTCAGACAGAGC-3′; Dlg1∶5′-TCATTCTCATATCTCACCAATAAAGC-3′ and 5′-CAGGGGTCACAGGGACAA-3′; Dlg2∶5′-TCAACTCCCTACCCCCACTA-3′ and 5′-GCAGTACTGTGCTGGGAATG-3′; Dvl1∶5′-CCATGGACCAGGACTTCG-3′ and 5′-GGCAACTTGGCATTGTCAT-3′; Gap-43∶5′-AGAGGATGCTGCCACCAA-3′ and 5′-GGCTTCGTCTACAGCGTCTTT-3′; Syt7∶5′-GAGGCTTGGACATGAAATCC-3′ and 5′-TCCGAAAGCCCTAATACCAG-3′; Snap25∶5′-GCTCCTCCACTCTTGCTACC-3′ and 5′-CAGCAAGTCAGTGGTGCTTC-3′; Syt1∶5′-AAGGAGATTCCAAAAGGAACAA-3′ and 5′-TTTTGGTTCAAGCGGAATG-3′.TRIzol  and Pure Link™ RNA Mini Kit  were used for RNA extraction. A total of 3 µg of RNA were used to synthesize cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT- PCR was performed using an HT7900 analyzer (Applied Biosystems). Each 5 µl reaction contained: 2 µl of cDNA diluted 1∶20, 0.5 µl primer mix (1 µM for each primer), and 2.5 µl SYBR green (Invitrogen). The qRT-PCR program was as follows: 50°C 2min, 95°C 2min, 40 cycles of 95°C 15s and 60°C 1min. For dissociation curves, a dissociation stage of 95°C 15s, 60°C 15s was added at the end of the program. Sort1∶5′-CAAATGCCAAGGTGGGATGAA-3′ and 5′-TTGAATCCAAAGCCTCTACGCC-3′; Tsn: 5′-CCCGAGAGGCTGTTACAGAG-3′ and 5′-CCTCGGATGGAAAGGTCATA-3′.For semi-quantitative PCR, 10 µl of 1∶40 diluted cDNA was used for PCR amplification in a final volume of 20 µl using Taq polymerase (Qiagen). PCR cycling conditions were as follows: 94°C, 3min; 94°C, 30s; 60°C, 30s; 72°C, 30s (10 cycles); 94°C, 30s; 50°C, 30s; 72°C, 30s (20 cycles); 72°C, 10min. PCR products were electrophoresed on 2% agarose gels. The primers used were: Tissues were homogenized at 10 ml/g (volume/weight) in lysis buffer . Following centrifugation, protein concentration of the supernatant was assessed by BCA assay . Following Western blotting, membranes were incubated with rabbit polyclonal mouse specific Tdp-43 antibody that we have generated(1∶1000), mouse monoclonal human specific TDP-43 antibody , rabbit polyclonal GFP antibody , and mouse monoclonal Gapdh antibody  overnight at 4°C. Following incubation with an appropriate secondary antibody, immunoreactivity was visualized by ECL and exposure to film.2O. Antigen retrieval was performed in a dH2O steam bath for 30 min. Tissues were immunostained with cytochrome oxidase subunit IV  and pS403/S404-phosphorylated TDP-43  antibodies, using the DAKO Autostainer (Dako Auto Machine Corporation) and the DAKO EnVision+ HRP system. DAKO Liquid DAB Substrate–Chromogen system was the chromogen. After immunostaining, sections were briefly counterstained with hematoxylin to visualize cell nuclei and coverslipped. Paraffin-embedded sections were also stained with hematoxylin and eosin.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 dHMice were deeply anesthetized with a mixture of ketamine  and xylazine  and perfused with 4% paraformaldehyde (PFA). Brains were removed and put into 4% PFA for additional 20 minutes and then washed overnight in PBS at 4°C. Vibratome sections of 200 µM were prepared and immersed in PBS at 4°C. The Helios Gene Gun System  was used to deliver gold (1.0 µm) and tungsten particles coated with lipophilic dyes into perfusion-fixed brain slices. Preparations of the DiOlistic bullets were performed according the methods described previously Tardbp and AAV1-shControl was prepared by standard methods. Briefly, AAV vectors expressing shTardbp and shControl under the control of the cytomegalovirus enhancer/chicken β-actin promoter, a woodchuck post-transcriptional regulatory element, and the bovine growth hormone, poly (A), were generated by plasmid transfection with helper plasmids in HEK293T cells. All plasmids were sequence verified as described above. Forty-eight hours after transfection, the cells were harvested and lysed in the presence of 0.5% sodium deoxycholate and 50 U/ml Benzonase  by freeze thawing, and the virus was isolated using a discontinuous iodixanol gradient. The genomic titer of each virus was determined by qRT-PCR.AAV1-shOne-way ANOVA with Tukey’s posthoc analysis were used to compare measures among 3 groups. Two-tailed student’s t test was used to compare measures among 2 groups. For data presentation, normalized values were averaged and presented as mean ± standard error of mean (SEM). Values of p<0.05 were considered statistically significant.Figure S1Intraventricular injection of the neonatal mouse brain with AAV1-shTardbpM337V transgenic mice, but cannot cause loss of mTdp-43 function. can further decrease mTdp-43 levels in the brain of hTDP-43 (A–B) Intraventricular injection of AAV1-shTardbp decreases mTdp-43 protein levels (A–B) and RNA levels (C) in the brain of both non-transgenic (NT) and hTDP-43M337V hemizygous mice. There are no significant difference of the protein RNA levels of mTdp-43 between AAV1-shTDP-43 injected hemizygous mice and homozygous mice. (D–E) Injection of AAV1-shTardbp significantly increases the mRNA levels of mSort1+Ex17b (D) and mTsn_ ΔEx5 in the brain of NT mice, but not in hTDP-43M337V hemizygous mice. mSort1+Ex17b and mTsn_ΔEx5 RNA levels between AAV1-shTardbp injected hemizygous mice and homozygous mice are not significantly different. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, N.S. no significance, as assessed by one-way ANOVA with Tukey’s posthoc analysis.(TIFF)Click here for additional data file.Figure S2WT hemizygous mice injected with AAV1-shNo alternations of RNA of synaptic proteins and dendritic spines in hTDP-43Tardbp. (A) Intraventricular injection of AAV1-shTardbp in the brain of hTDP-43WT hemizygous mice does not alter the RNA levels of PSD-95, Bsn, GAP-43 and Syt7. (B–C) Intraventricular injection of AAV1-shTardbp in hTDP-43WT hemizygous mice does not decrease dendritic spine number in the brain. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, as assessed by one-way ANOVA with Tukey’s posthoc analysis.(TIFF)Click here for additional data file.


The estimated median survival of patients with IPF is 2–5 years following diagnosis, and the mortality rate is greater than that associated with numerous malignancies ,2.This has presented significant challenges in patient management and until recently there were no pharmacological treatments approved for patients with IPF in Europe.The timing of this meeting was therefore particularly relevant as there were a number of important advances and new findings during 2011, which included the publication of the updated guidelines from the ATS/ERS/JRS/ALAT Committee and the approval of pirfenidone for the treatment of mild-to-moderate IPF in Europe ,4.In addition, a press release from the National Institutes of Health reported that one arm of an ongoing trial, PANTHER-IPF, had been stopped because patients with IPF receiving a currently used triple-drug therapy consisting of prednisone, azathioprine, and N-acetylcysteine (NAC) had worse outcomes than those who received matching placebo. The interim results from PANTHER-IPF have since been published .Respiratory Research reported the proceedings from this first European IPF meeting, with key articles selected by the AIR Scientific Committee, which were considered essential reading for all clinicians managing patients with IPF in Europe.This supplement to Articles in this supplement included a review of the recently updated ATS/ERS/JRS ALAT diagnostic criteria by Professor Athol Wells, and discussion on how these can facilitate early diagnosis of IPF. It also considered issues that may remain unclear despite the updated guidance. Linked to the diagnosis of IPF, Nicola Sverzellati gave a master class on HRCT imaging and practical requirements for optimising CT scanning techniques.Presentations then focused on the management of IPF. Professor Luca Richeldi discussed the Cochrane Collaboration approach to analysing clinical trial data and the findings of Cochrane meta-analyses that have been performed to determine the effect of treatments used in the management of IPF. This was followed by Professor Vincent Cottin, who summarised the outcomes from prior clinical trials in IPF and the recently published Phase III studies of pirfenidone that led to its approval in Europe. Following these updates, Professor Jürgen Behr and Luca Richeldi discussed how these data can be interpreted in light of the updated recommendations of the ATS/ERS/JRS/ALAT Committee regarding treatment of IPF. Professor Carlo Albera outlined the implications of the press release from the NIH regarding the PANTHER-IPF study.Hopefully the selected proceedings from this meeting will provide IPF clinicians with new insights and support in the management of their patients. The intention is for the AIR meeting to become a fixed date in the calendar for respiratory physicians and researchers dedicated to improving knowledge and bringing new hope for IPF patients.U. Costabel has received consultancy fees from Actelion, Boehringer Ingelheim, Centocor, Gilead and InterMune, and has received lecture fees from InterMune.J. Behr has received fees for speaking from Actelion, Bayer-Schering, Boehringer-Ingelheim, Encysive, GSK, Pfizer, Lilly, Nycomed, InterMune, Novartis, MSD and has served as consultant/advisor for Actelion, Bayer-Schering-Pharma, Lilly, Pari-Pharma, GSK, Pfizer, Optima, Gilead. J. Behr has also received research grants from Actelion, Bayer-Schering, InterMune, Pari-Pharma and attending international and national congresses sponsored by Actelion, Boehringer-Ingelheim, AstraZeneca, Bayer.V. Cottin has received fees for speaking from InterMune, Boehringer Ingelheim, and Actelion, and has participated as a member of steering committees, a member of data safety monitoring boards or as an investigator to clinical trials sponsored by Actelion, Boehringer Ingelheim, Gilead, and InterMune Inc.L. Richeldi has received consultancy fees from Boehringer Ingelheim, InterMune, Celgene and Gilead, and lecture fees from InterMune.The authors thank C. Trenam, I. Mandic and M. Smith of IntraMed Communications for editorial assistance in the preparation of the manuscript. Development of this article was supported by InterMune AG.


In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copperII (CuII(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copperII (CuII(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the CuII(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The CuII(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either CuII(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. CuII(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, CuII(atsm) reduced accumulation of C-terminal (219–414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that CuII(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable  Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease in which death of upper and lower motor neurons leads to progressive brain and spinal cord deterioration. Patients with ALS rarely survive more than a few years after diagnosis with respiratory failure the most common cause of death In 2006, TAR DNA binding protein 43 (TDP-43) was identified as the major protein constituent of ubiquitinated neuronal inclusions in non-SOD1 ALS cases TDP-43 is a 414 amino acid protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and consists of two RNA recognition motifs and a C-terminal glycine rich region The hallmark neuropathological features of primary TDP-43 proteinopathies include clearing of TDP-43 from the nucleus and accumulation of TDP-43 in cytoplasmic inclusions. These inclusions are enriched in ubiquitinated and hyperphosphorylated (phospho-Ser409/410) CTF-TDP-43 The early disease processes associated with abnormal TDP-43 metabolism are not well understood. Recent studies have shown that an early change to TDP-43 processing and metabolism in ALS may involve its localization to RNA stress granules (SGs) in vivo hallmarks of disease While these studies have begun to delineate some of the initial processes in TDP-43 accumulation, it is not known whether this early stage of TDP-43 aggregation can be developed into a therapeutic target. Recently, we developed a cell model involving paraquat-induced mitochondrial inhibition in neurons and demonstrated that this stress induced changes to TDP-43 that re-capitulated II complexes (CuII(btsc)s) on TDP-43 SG formation. These complexes have traditionally been investigated for their potential as imaging agents for hypoxic tumors II(btsc)s have a potent neuroprotective effect in several different mouse models of neurodegenerative disorders, including models of Alzheimer’s disease, Parkinson’s disease and ALS II(btsc)s is likely related to their ability to modulate kinase signaling mechanisms including Akt, ERK and JNK in both cell culture and animal models bis(methylthiosemicarbazonato)-copperII (CuII(atsm)) has a potent protective effect in an animal model of ALS (SOD1G93A) II(atsm) also modulated TDP-43 processing in the SOD1G93A murine model. Treatment of SOD1G93A mice with CuII(atsm) inhibited accumulation of phosphorylated TDP-43 in the spinal cord of these mice II(btsc)s and other Cu-complexes in animal and cell models II(btsc)s could modify the processing and localization of TDP-43 to SGs. We found that CuII(atsm) and glyoxalbis(methylthiosemicarbazonato)-copperII (CuII(gtsm)) were potent inhibitors of SG formation, blocking accumulation of TDP-43 and HuR into SGs caused by treatment with paraquat. Further studies showed that the protective action was associated with inhibition of stress-induced ERK phosphorylation by CuII(atsm). The protective effect of CuII(atsm) on TDP-43 aggregation was also associated with an inhibitory effect on accumulation of ubiquitinated proteins and decreased paraquat neurotoxicity. Moreover, CuII(atsm) reduced accumulation of C-terminal (219–414) TDP-43 in transfected SH-SY5Y cells. These studies show for the first time that treatment of neuronal-like cells with a copper-complex can inhibit stress-induced cell toxicity and prevent early aggregation of TDP-43 into SGs. This may offer a potential insight into novel targets and therapeutic treatments for TDP-43 proteinopathies.In the present study, we have used this cell model to examine the effect of bis(thiosemicarbazonato)-copper4′,6′ Diamino-2-phenylindole dihydrochloride (DAPI) was obtained from Invitrogen . N,N′-Dimethyl-4,4′-bipyridinium dichloride (paraquat), rotenone, 1-methyl-4-phenylpyridinium (MPP+) and sodium arsenite, were from Sigma Aldrich  and LDH assay kit was purchased from Roche Diagnostics . SP600125 and PD98095 were purchased from Merck Biosciences .Polyclonal TDP-43 antisera were purchased from Proteintech Group . Antisera to ubiquitin were from Santa Cruz Biotechnology . Monoclonal antisera to HuR were obtained from Invitrogen . Antisera to total and phosphorylated forms of ERK and JNK, as well as antibodies to actin and GAPDH were purchased from Cell Signalling Technologies .2 at 37°C.The cell lines used in this study were the human neuroblastoma SH-SY5Y cell line and human epithelial HeLa cell line. Cells were passaged and maintained in DMEM plus 5% FBS (HeLa cells) or DMEM/F12 plus 10% FBS (SH-SY5Y cells) as previously reported An assay for cell death (LDH) was performed as previously described II(btsc)s were synthesized as previously described II(btsc)s were prepared as 10 mM stock solutions in DMSO and added at indicated concentrations. CuCl2 and FeCl2 10 mM stock solutions were prepared in dH2O and added at indicated concentrations.Cu2O and added at indicated concentrations and the medium was briefly mixed by aspiration. Incubations were performed for periods stated in individual experiments. Where indicated, cells were co-treated with kinase inhibitors (SP600125 (JNK) or PD98095 (ERK)) from stock solutions prepared at 10 mM in DMSO. Control cultures were treated with vehicle alone. For immunoblotting, cells were harvested into Phosphosafe Extraction Buffer  containing protease inhibitor cocktail (Roche Diagnostics) and stored at −80°C until use. For immunofluorescence studies, cells were grown on glass coverslips and fixed after treatment in 4% w/v paraformaldehyde for 30 min.Cells were grown in 24 or 6-well plates or on 15 mm coverslips (for immunofluorescence) for 2–3 days before experiments (∼80% confluent). Paraquat, rotenone, MPP+ or sodium arsenite were prepared in dHCell lysates were analyzed for ATP content using an ATP Assay kit (Sigma-Aldrich) as previously reported 2) multiwell plates . After treatment with paraquat and or CuII(btsc)s or CuCl2, cell samples were collected and centrifuged at 720 g for 10 min. Supernatant was removed and the cell suspension was re-suspended in 60 µl Phosphosafe Extraction Buffer with 5% (v/v) DNAse Sigma Aldrich  and 1% phenylmethylsulfonyl fluoride (v/v). The suspension was then centrifuged at 15,000 g and the supernatant was collected and normalized to 8 µg/µl using the BCA assay. The cell suspension was then diluted 1∶2 in sample buffer  bromophenol blue in dH2O). 10 µl was loaded per well of non-denaturing polyacrylamide gels without SDS. Gels were run in Tris-glycine running buffer at 150 V for 1 hr. Gels were incubated in the dark in nitrotetrazolium blue chloride (Sigma Aldrich) solution (2 mg per ml dH2O) for 20 min on a rocking platform in the dark, at room temperature. Nitrotetrazolium blue chloride solution was removed from gels and replaced with developer solution  for 1 hr. Gels were subsequently imaged using a Fujifilm LAS3000 Imager . Quantification of SOD activity was done using Image J analysis, comparing levels of SOD activity based on nitrotetrazolium blue reduction.Cells were grown and treated in 6-well  against TDP-43. For detection of total and phospho-forms of JNK and ERK, membranes were probed with anti-JNK or anti-ERK  and antisera to phospho-forms of each protein . Secondary antiserum was rabbit-HRP at 1∶5,000 dilution. Blots were developed using GE Healthcare ECL Advance Chemiluminescence and imaged on a Fujifilm LAS3000 imager. Expression of GAPDH or actin was determined using antisera at 1∶5,000 and 1∶3,000 respectively for protein loading controls where necessary.SH-SY5Y cells were grown on 15 mm diameter coverslips and treated as indicated. Cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After blocking for 1 hr with 10% normal goat serum, cells were incubated with primary antibody , ubiquitin (1∶150) or HuR (1∶50), for 2 hr at room temperature or overnight at 4°C. This was followed by labeling with secondary AlexaFluor or FITC goat anti-mouse or anti-rabbit antisera at 1∶500 for 2 hr at room temperature or overnight at 4°C. After washing, the coverslips were incubated with DAPI at 0.5 ng/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Images shown are representative of multiple fields and triplicate coverslips per experiment. TDP-43 and HuR-positive stress granules (SGs) were counted in cultures where indicated. A minimum of 500 cells was counted across multiple fields of view (and multiple coverslips) for each treatment. The number of TDP-43 and HuR-positive SGs were counted in these cells. The total number of cells was divided by the total number of SGs to provide a measure of mean SGs per cell. SGs were not observed in untreated cells.Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer’s instructions. DNA was quantified and TDP-43 inserts were identified positively by digestion with BamHI and XhoI.Plasmid DNA corresponding to GFP-tagged full-length wild-type (WT) TDP-43 (pEGFP-TDP WT) and the C-terminal fragment of TDP-43, (pEGFP-TDP 219–414) or empty expression vector pEGFP-C1 were prepared as described by Nonaka et. al. 51 cells per well in 24 well–plates on coverslips. Cells were transfected 24 hr after seeding with the pEGFP-C1 empty vector, pEGFP-TDP WT and pEGFP-TDP 219–414 using Dreamfect (Qiagen) according to manufacturer’s instructions. After 48 hr incubation, cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After washing, the coverslips were incubated with DAPI at 0.5 ng/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Expression of TDP-43 was determined by the EGFP-tagged construct. Efficiency of transfection with pEGFP-C1 vector was approximately 20–25% SH-SY5Y cells were seeded at 2×10t-test and ANOVA where appropriate.All data described in graphical representations are mean ± standard error of the mean (SEM) from a minimum of three experiments. Densitometric analysis of immunoblots was performed using NIH ImageJ 1.43 software on two-three separate blots and adjusted for loading control (GAPDH or actin). Results were analysed using a two-tailed Student’s II(atsm) (II(gtsm) (II(gtsm) has a higher potential to induce toxicity than CuII(atsm) due to the fact that CuI is readily released in the intracellular reducing environment from CuII(gtsm) II(atsm) is more resistant to reduction of CuII to CuI under normoxic conditions and therefore is thought to release less CuIII(gtsm) was used at a concentration 20-fold less than CuII(atsm) due to its higher potential to release bioavailable Cu.To induce TDP-43-positive SGs, SH-SY5Y cells were exposed overnight to 1 mM paraquat . As reported previously, this exposure to paraquat induces only mild loss of cell viability (∼15% as determined by MTT assay) and no cell death (as determined by LDH assay) II(atsm)  or 50 nMII(gtsm) . These cII(atsm) or CuII(gtsm) substantially inhibited formation of TDP-43 and HuR-positive SGs induced by paraquat (II(atsm) and CuII(gtsm) with CuCl2 at 1 µM and 100 µM and FeCl2 at 100 µM. ZnCl2 was not examined as we have reported previously that ZnCl2 induces nuclear aggregates of TDP-43 2 had no effect on TDP-43 or HuR SGs (2 induced an effect indistinguishable to 1 µM CuII(atsm) and 50 nM CuII(gtsm) (II(btsc)s are highly efficient at delivering intracellular Cu when compared to CuCl2 and therefore a higher level of CuCl2 is required to obtain the same increase in cellular bioavailable Cu 2 at 100 µM (2 ligand had no effect on SG formation (data not shown). These findings demonstrated that co-treatment of SH-SY5Y neurons with low concentrations of CuII(btsc)s inhibited paraquat-mediated SG formation, including TDP-43-positive SGs and this was likely to be related to the ability of the CuII(btsc)s to efficiently deliver bioavailable Cu into the cell. Subsequent studies were performed using CuII(atsm) due to the significant potential of this compound as a therapeutic agent for ALS based on efficacy in our preliminary in vivo studies Co-treatment with either Cuparaquat . To dete HuR SGs , howeverII(gtsm) . These ft 100 µM . SimilarII(atsm) and 1 mM paraquat overnight blocked formation of SGs as observed in SH-SY5Y cells (data not shown). To determine if CuII(atsm) could inhibit against TDP-43-positive SGs generated by another common inhibitor of mitochondrial function, we treated HeLa cells with 500 µM sodium arsenite for 1 hr. This is the most common method used for SG induction and we have shown previously that sodium arsenite induces robust TDP-43 and HuR SGs in HeLa cells. However, co-treatment with 1 µM CuII(atsm) for 1 hr, 4 hr, or overnight prior to the 1 hr sodium arsenite treatment had no effect on the formation of TDP-43 and HuR-positive SGs (Next we examined if paraquat-mediated TDP-43-positive SGs could be inhibited in HeLa cells. Co-exposure of HeLa cells with 1 µM Cutive SGs . The reaII(atsm) inhibited TDP-43 and HuR-positive SGs, we first determined if there was any effect on TDP-43 or HuR expression. Cells treated with 1 µM CuII(atsm) alone overnight revealed no substantial alterations in full length TDP-43 or HuR expression (data not shown). Interestingly whilst paraquat induced a slight increase in 35 kDa TDP-43 (II(atsm) further increased this expression (II(atsm) did not affect 35 kDa CTF-TDP-43 levels in the absence of paraquat treatment (data not shown). The effect on 35 kDa CTF-TDP-43 was not observed with 1 µM CuCl2 (2 did slightly increase the expression of the 35 kDa band (II(atsm) lead to elevated 35 kDa CTF-TDP-43 levels. HuR expression was not substantially altered in any condition tested (II(atsm).To examine how Cua TDP-43 , co-expopression . Using NµM CuCl2 , althougkDa band . We repon tested . These rII(atsm) on kinases. As reported previously II(atsm) inhibited ERK activation (II(atsm) did not inhibit levels of phosphorylated ERK when examined after 2 hr of incubation (II(atsm) having no effect on JNK phosphorylation after 2 hr incubation with paraquat (II(atsm), both ERK and JNK phosphorylation were significantly inhibited compared to paraquat alone (II(atsm) alone had no effect on basal ERK or JNK phosphorylation over the same time period  on sodium arsenite-treated HeLa cells. Exposure of HeLa cells to 500 µM sodium arsenite for 1 hr induced significant activation of JNK and ERK (II(atsm) did not substantially affect activation of either kinase (II(atsm) in sodium arsenite-treated cells  was found to inhibit ERK phosphorylation in SH-SY5Y cells treated with paraquat, we compared the effect of CuII(atsm) and an ERK inhibitor on SG formation. We found that both CuII(atsm) and PD98059 (ERK inhibitor) induced the same inhibition of TDP-43 and HuR-positive SGs when co-treated with paraquat (compare II(atsm) with II(atsm) has an analogous effect to inhibition of ERK, the results contrast with the more specific action of JNK inhibition on TDP-43 alone (II(atsm) could inhibit TDP-43 and HuR-positive SG formation through inhibition of prolonged ERK activation. This is consistent with our observations that paraquat does not induce TDP-43/HuR-positive SGs until after 8 hr of exposure II(atsm) did not block SGs induced by 1 hr treatment with sodium arsenite, as prolonged exposure to CuII(atsm) e.g. overnight in the presence of the mitochondrial inhibitor, is needed to obtain a protective response from the copper complex.As Cu43 alone . These fII(atsm) only inhibited ERK (and JNK) activation after a prolonged exposure with paraquat, we further investigated the potential neuroprotective mechanism of CuII(atsm) that might account for this. We examined the effect of CuII(atsm) on paraquat-induced changes to ubiquitin. Previous reports have demonstrated that paraquat treatment leads to altered proteasomal function and can result in accumulation of ubiquitinated proteins II(atsm) resulted in complete abrogation of the ubiquitin accumulation in paraquat-treated cultures  may have a broad neuroprotective effect leading to decreased cell stress and therefore lack of SG formation.Due to the observation that Cucultures . Moreovecultures . As expebiquitin . This waII(atsm) to protect against paraquat-induced cell death. As 1 mM paraquat did not induce substantial cell death, we exposed cultures to 10 mM paraquat overnight (II(atsm) significantly reduced this neurotoxic action to 35% (II(gtsm) (50 nM) also significantly decreased paraquat-mediated toxicity -like activity, we examined the SOD activity using an in-gel assay. Exposure of SH-SY5Y cells to 1 µM CuII(atsm), 50 nM CuII(gtsm) or 1 µM CuCl2 in the presence or absence of 1 mM paraquat resulted in no significant increase in SOD-like activity in treated cells as compared to untreated controls (II(btsc)s mediated through inhibition of protein accumulation and kinase activation rather than through enhanced SOD or SOD-like activity.To confirm this protective action, we measured the ability of Cuvernight . This trvernight . Co-trean to 35% . In additoxicity  (28%). Acontrols . These fII(atsm) could inhibit endogenous TDP-43-containing SGs induced by mitochondrial inhibition, we were interested to know if this action could be extended to prevention of pathological aggregation of TDP-43 associated with ALS. To investigate this, we transfected SH-SY5Y cells with wild-type (WT) full length TDP-43-GFP and a C-terminal fragment known to aggregate in cultured neurons (CTF-TDP-43 219–414-GFP) II(atsm) either at the time of CTF-TDP-43 219–414 transfection or 24 hr later. At 48 hr after transfection, cells were assessed for the level of CTF-TDP-43 219–414 aggregation. CuII(atsm) significantly reduced the number of TDP-43 aggregates in transfected cells when added at time of transfection (0 hr) or at 24 hr post-transfection  had a greater inhibitory effect when added after 24 hr, reducing the number of cells with aggregates by ∼50% (II(atsm) from the cell before accumulation of CTF-TDP-43 219–414 when added at time of transfection. Most aggregates form in the second 24 hr after transfection. As we have shown previously that JNK inhibition, but not ERK inhibition, significantly inhibits accumulation of CTF-TDP-43 219–414 II(atsm) inhibition of CTF-TDP-43 aggregation here is mediated through its inhibitory effect on JNK (associated with cell stress induced by CTF-TDP-43 aggregation). However, these studies show that CuII(atsm) can significantly reduce cellular accumulation of both endogenous and transfected TDP-43.Finally, although we had shown that Cusfection . CuCl2 aormation . Interes by ∼50% . This maII(btsc) Cu-complexes to inhibit stress-mediated TDP-43-positive SGs in neuronal cell culture. We found that CuII(atsm) and CuII(gtsm) inhibited paraquat-mediated sustained ERK phosphorylation resulting in abrogation of SG formation and improved survival of cells treated with paraquat. In addition, both CuII(atsm) and the ERK inhibitor, PD98059, inhibited accumulation of ubiquitin in paraquat-treated cells indicating that this may be a protective action of CuII(atsm) mediated through inhibition of prolonged ERK activation. CuII(atsm) also reduced aggregation of CTF-TDP-43 in transfected cells.In this study, we investigated the ability of CuII(btsc)s have been investigated extensively as hypoxia imaging agents and as potential anti-cancer compounds for delivery of radionuclides to tumors II(atsm) has a relatively negative CuII/I reduction potential (0’E = −0.59 V vs SCE) and this limits reduction of the metal ion to CuI and the subsequent release of bioavailable CuI into cells under normal cell conditions but CuI release is enhanced under hypoxia, cellular energy impairment and probably within select sub-cellular compartments within the cell II(gtsm), it is easier to reduce the CuII to CuI (0’E = −0.43 V vs SCE) so the complex is able to release considerable CuI under basal cellular reducing conditions. In previous studies, we have harnessed these properties to develop CuII(btsc)s as potential therapeutic agents to deliver small levels of bioavailable Cu to the brain. This is achieved through the ability of these compounds to cross the blood brain barrier II(btsc)s can modulate cell signaling pathways including phosphorylation and activity of phosphoinositol-3-kinase (PI3K), ERK, glycogen synthase kinase 3 (GSK3) and JNK II(btsc)s II(atsm) significantly delayed disease onset and extended life span in a low transgene copy SOD1G93A murine model of ALS. We observed that CuII(atsm) delayed disease onset, extended survival and inhibited oxidative damage and glial activation in treated mice II(atsm), the therapeutic action of this complex in the SOD1G93A mice remains unknown II(atsm) did inhibit accumulation of phosphorylated full length and C-terminal TDP-43 in the treated SOD1G93A mice. Our data here and previously II(atsm) has also protected against neurodegeneration and improved motor performance in models of Parkinson’s disease CuII(btsc)s could inhibit formation of TDP-43-positive SGs induced by the mitochondrial inhibitor, paraquat.We previously reported that treatment of neuronal and non-neuronal cell cultures with paraquat overnight induced a robust induction of TDP-43-positive SGs and that this was mediated through ERK and JNK activation II(atsm) to inhibit TDP-43-and HuR-positive SG formation during paraquat-mediated stress was likely due to its ability to inhibit ERK phosphorylation. Although CuII(atsm) also inhibited prolonged JNK phosphorylation, we have shown that inhibition of JNK in this model stops TDP-43 association with SGs but not HuR SG formation. In this study, CuII(atsm) and the ERK inhibitor, PD98059, both prevented formation of HuR-and TDP-43-positive SGs making the inhibition of ERK potentially more important to these protective effects than JNK inhibition in this cell model. However, the JNK inhibitory action of CuII(atsm) was perhaps more likely to be involved in the reduced aggregation of CTF-TDP-43 219–414 in transfected cells. This is because we reported previously that JNK, but not ERK inhibition, reduced accumulation of CTF-TDP-43 219–414 and 162–414 in the same cell type II(atsm) could mediate inhibition of CTF-219–414 TDP-43 aggregation through an unrelated mechanism.The ability of CuII(atsm) did not inhibit ERK or JNK phosphorylation after 2–4 hr exposure to the Cu-complex but did inhibit both kinases after an overnight treatment. These findings suggested that CuII(atsm) had a delayed effect and prevented prolonged activation. This may be due to inhibition of complex upstream pathways and could be associated with the prevention of ubiquitin accumulation observed in cultures treated with paraquat. The fact that both ERK inhibition and CuII(atsm) blocked the ubiquitin aggregation suggested that ERK inhibition is upstream of changes to ubiquitin in paraquat-treated cells. Interestingly, other mitochondrial inhibitors did not induce effects on ubiquitin or induce SGs supporting a close connection between ubiquitin changes and SG formation, possibly controlled by ERK. It has been reported elsewhere that ERK (and JNK) have important roles in the toxic effects of paraquat Interestingly, CuII(atsm) is inhibiting ERK is not known but is likely to involve release of Cu as we observed the same effect with CuII(gtsm), which is known to release intracellular Cu. We have also found CuII(gtsm) to potently inhibit ERK at low concentrations in other cell lines including PC12 neurons and primary cortical neurons (data not shown). Interestingly, we have shown that under certain conditions, CuII(atsm) can also activate ERK rather than inhibit its activity II(atsm)-mediated inhibition of ERK may initially appear contradictory, it is likely that the different outcomes of CuII(atsm) treatment are related to basal ERK activity in each model system. Our unpublished studies have shown that if ERK phosphorylation is low, CuII(atsm) (under conditions of energy inhibition) can increase phosphorylation, whereas if ERK phosphorylation is already high (as in paraquat-treated cultures in this study) CuII(atsm) can inhibit ERK. The reason for this dynamic effect is not known but may be related to changes in subcellular Cu pools.How CuII(atsm) and CuII(gtsm) in this system were supported by our finding that both compounds inhibited cell death induced by paraquat.It has been reported elsewhere that Cu can enhance function of ubiquitin-associated activities, although excess Cu can also induce abnormal ubiquitination II(atsm) inhibited paraquat-induced TDP-43 and HuR SGs, there was no effect on SGs formed by sodium arsenite treatment. The reason for this may be the time frame required for the Cu-complex to act. We tested CuII(atsm) against sodium arsenite by using pre-treatment regimes of overnight or 4 hr but these had no effect, indicating that the CuII(atsm) should be present at the same time as the stress and that the effect is then mediated through a relatively slow action. Whether this is related to Cu release from the complex under the paraquat-induced stress or the need for Cu to induce protein synthesis is unknown. However, as ALS is a slowly progressing disease, these complexes may have therapeutic potential. We have shown that CuII(atsm) inhibits disease progression in a SOD1 model of ALS II(atsm) inhibited formation of phosphorylated cleavage fragments of TDP-43 in the treated SOD1 mice II(atsm) protects mice from disease induced by overexpression or mutation of TDP-43 While CuII(bstc) complexes can inhibit formation of TDP-43-positive SGs and prevent paraquat-mediated cell death. This neuroprotection appeared to be associated with inhibition of ERK phosphorylation and may be mediated through modulation of cellular ubiquitination by Cu. Further studies are warranted to determine if CuII(atsm) or related CuII(btsc)s are potential therapeutics for ALS and other neurodegenerative diseases through kinase modulation and maintenance of ubiquitin homeostasis.In summary, we have found that co-treatment of cells with CuFigure S1ATP levels in paraquat-treated cells. SH-SY5Y cells were treated overnight with 1 mM paraquat and total cellular ATP levels were determined in cell lysates. *p<0.05 compared to control cells.(TIF)Click here for additional data file.Figure S22 on TDP-43 expression.Effect of CuClA: Cells were treated with 1 mM paraquat overnight in the presence or absence of 1 µM CuII(atsm) or 100 µM CuCl2. Cells were examined for expression of full length TDP-43 and 35 kDa CTF-TDP-43 by western blot. B: Effect of CuII(atsm) on ERK and JNK phosphorylation. Cells were treated overnight with 1 µM CuII(atsm) and examined for expression of phosphorylated ERK and JNK.(TIF)Click here for additional data file.


Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has been retained in the mtDNA of various angiosperms: the matR gene encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4). Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4), which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory splicing cofactors.Group II introns are large catalytic RNAs (ribozymes) which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA) in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated  Group II introns have not yet been identified in the nuclear genomes of eukaryotes, but their predicted descendants , are highly abundant in eukaryotes, comprising together more than half of the genome in mammals.Interestingly, many plant organellar group II introns lack an ORF capable of specifying a maturase, or they encode a degenerate maturase protein which is probably unable to promote intron mobility  and a site-specific endonuclease (D/En) motif at the C-terminus. Genetic and biochemical data show that the processing of group II introns in bacteria is facilitated by a single maturase-ORF encoded within DIV of the intron itself  for nMAT4  , and five new nuclear maturase loci in Physcomitrella . Building upon this new information a comprehensive phylogeny was constructed to determine the evolutionary relationships among the different maturase sequences; a simplified version of this phylogeny, displaying only the relationships among the seed plant (six maturase genes), Marchantia (eight related genes) and Selaginella maturases, is shown in Figure A recent study by Guo and Mower  has provmat genes fall into two main categories . It is likely, therefore, that the angiosperm type I nMat1 and nMat2 genes resulted from a nuclear gene duplication, following the translocation to the nucleus of a single mitochondrial maturase related to Marchantia maturase mat-Mpo1   Figure . It is f) Figure ; this ev) Figure , this po) Figure .Different RNA-binding proteins were shown to function in the splicing and processing of introns in plant organellar genomes (see below). Some, such as a DEAD RNA-helicase (PMH2) and a CRM-domain protein (mCSF1) are required for optimal splicing of a large set of introns, whereas others  rather appear to be specific for a single, or few, individual introns. Although this review focuses mainly on maturases, this section summarizes the information available about other types of mitochondrial group II intron splicing factors.E.coli, YhbY is mainly associated with the maturation of the pre-50S ribosomal subunit  proteins are characterized by a conserved RNA-binding domain of ~10 kDa (Pfam-PF01985), which is related to a conserved bacterial ORF (YhbY)   domain which is represented in a small family of proteins in angiosperms (15 in Arabidopsis and 17 in rice)   which binds to chromatin and interacts with the nuclear Ran GTPase Dasso, . SimilarNicotiana sylvestris (nms1) was shown to disrupt the splicing of the first intron in the nad4 transcript  often results in severe developmental defects.Plants possess some of the most complex organelle compositions of all known eukaryotic cells. As sessile organisms, land-plants must cope with multiple environmental stresses. During their evolution, plants have acquired complex regulatory mechanisms to coordinate cellular functions with environmental stresses. The participation of nuclear-encoded factors in organelle RNA metabolism may therefore provide means to control the biogenesis of the respiratory (and photosynthetic) machineries, and thus to link cellular metabolism and environmental and developmental signals. Following this idea, the splicing of both plastidial Barkan,  and mitoccmFc, cox2 and the ribosomal rpl2 and rps3 genes, the splicing mutants are strongly affected in respiratory associated functions and cellular metabolism  (reviewed in Millar et al., The importance of these stages is further reflected in plant mutants affected in genes required for the splicing of mitochondrial introns. Such mutants exhibit reduced germination and seedling establishment, and are often affected in growth and development (see Keren et al., Chlamydomonas reinhardtii demonstrated that the trans-splicing of the two group II introns in its chloroplast genome, is also facilitated by a large set of nuclear-encoded RNA binding cofactors (Goldschmidt-Clermont et al., Clostridium tetani, was shown to undergo alternative splicing reactions resulting with five alternative mRNAs products, each encoding a distinct protein isoform (McNeil et al., trans” in a primitive spliceosome, and the group II structural features of the target primordial introns degenerated. Interestingly, features of some plant mitochondrial introns reflect possible stages that may have occurred in such a process. Several undergo trans-splicing: different portions of the intron are assembled from two and in one case, three different transcripts. At least one trans-splicing intron undergoes a significant degree of mis-splicing, and this is lost in plants in which the intron is further fragmented, a process that may have played a role in the formation of the primordial spliceosome. Also, several introns lack key structural features, such as the bulged adenosine in the DVI stem, which serves as the nucleophile in the first splicing step (Figure Based on the structural features, the similarity of exon-intron boundaries and a common splicing-mechanism, group II introns are proposed to be ancestors of the eukaryotic spliceosomal introns Sharp, . While bp Figure  and may trans.” The homology of group II maturases to the core spliceosomal splicing factor (Dlakic and Mushegian, Maturases and their accessory splicing factors in plant mitochondria, thus represent a highly versatile set of proteins which differ from their presumptive monospecific ancestors. These features may correspond to a further evolutionary link between mitochondrial group II introns and the spliceosomal machinery in nuclear genomes of most eukaryotic cells. Likewise, the recruitment of different protein cofactors to assist with the splicing of mitochondrial introns in plants (see Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


V 1.2/KV 3.1 channels and blocked K+ currents which lie downstream of ERK activation. Taken together, these findings underpin the role of AT2R as a novel target that regulates cell proliferation mediated by Ang II with implications for therapeutic intervention for regulation of neurogenesis.Angiotensin II (Ang II), known a potent vasoactive substance in the renin-angiotensin system in the brain, plays a critical role in systemic blood pressure control. However, increasing evidence indicated that the physiological role of Ang II go beyond its vasoactive effect. In the present study, we demonstrated that Ang II type-1 receptor (AT1R) and type-2 receptor (AT2R) were expressed in primary rat hippocampal neuronal stem cells (NSCs). Treatment of rat hippocampal NSCs with Ang II increased cell proliferation. Pretreatment of NSCs with specific AT2R, but not AT1R, antagonist significantly suppressed Ang II-induced cell proliferation. Furthermore, Ang II stimulated ERK and Akt phosphorylation in NSCs. Pretreatment of MEK inhibitor, but not PI3K inhibitor, inhibited Ang II-induced ERK phosphorylation as well as cell proliferation. In addition, stimulation of NSCs with Ang II decreased expression of K It is well known that Angiotensin II (Ang II) is the principal vasoactive substance of the renin-angiotensin-system (RAS) with a variety of physiological actions, including vasoconstriction, aldosterone release, and cell growth It is now well-known that neurogenesis is persistent in mammalian brain until adult Ang II has been clearly demonstrated to induce cell proliferation V) affect cell mitogenesis, which was initially reported in human T lymphocytes V play an important role in controlling proliferation in numerous types of cell, including glial cells, lymphocyte, endothelial cells, bereast and prostate cancer cells, and stem cell + current have been found in NSCs, such as IKDR , IA (encoded by KV 1.4), TEA-sensitive IKDR (encoded by KV 3.1) V 1.3 and KV 3.1 channels has been reported to increase NSC proliferation + current and K+ channel expression in different types of cell, including rostral ventrolateral medulla neurons V and A-type K+ currents + currents in cultured neurons V channels modulate Ang II-mediated proliferation in NSCs and, if so, AT1R or AT2R is involved in regulation of K+ currents.It has been demonstrated that voltage-gated potassium channels , α-Dendrotoxin (α-DTX), 4-Aminopyridine (4-AP) and tetrodotoxin (TTX) were purchased from Sigma Chemicals . The specific MEK1/2 inhibitor U0126 and PI3K inhibitor LY294002 was purchased from Calbiochem .Pregnant female Sprague Dawley rats were purchased from Charles River Laboratories, Inc. . All of animals were housed under conditions of constant temperature and humidity on a 12-h light, 12-h dark cycle, with lights on at 0700 h. Food and water were available ad libitum. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center.tal5 cells/well for 24-well plate, 1×104 cells/well for 96-well plate) and were used if found more than 90% nestin+ (a marker for progenitor cells).NSCs derived from the hippocampus of embryonic day18 (E18) fetus were cultured in substrate-free tissue culture T75 flasks as reported by Tian eNSCs were lysed using the Mammalian Cell Lysis kit . Cell lysis containing 20 µg of protein were subjected to electrophoresis on a 12% sodium dodecyl sulfate-ployacrylaminde (SDS) gel and blotted onto a polyvinylidene difluoride membrane with a semi-dry blotting apparatus. After blocking, membranes were then probed with antibodies recognizing the AT1R , AT2R , p-ERK , p-Akt  and β-actin . Secondary antibodies were alkaline phosphatase conjugated to goat anti mouse/rabbit IgG (1∶5000). Signals were detected by chemiluminescence . All of the Western blot experiments were repeated three times individually and representative blots are presented in the figures.Dissociated NSCs were plated on the coverslip. The cells at different time were fixed with 4% paraformaldehyde for 20 minutes at room temperature according to the experiment protocol. The cells were permeabilized and blocked with solution containing 10% normal goat serum (NGS), 0.3%Triton X-100 in PBS at room temperature for 2 hours. Next, the cells were incubated with primary antibody  in 10% NGS, 0.3%Triton X-100 in PBS at 4°C overnight. Following 3 washes with PBS, the cells were exposed to secondary fluorescence antibody for 2 hr. After washing with PBS for 3 times, the cells were mounted on the slide with anti-fade reagent with DAPI at room temperature. The slides were examined with a laser confocal microscope (Leica TSC STED).V channel after Ang II treatment was first normalized against GAPDH RNA as ΔCT, and then the relative expression was compared to untreated group using the ΔΔCT method for quantification in Opticon Monitor software . Relative fold changes were determined as RQ values. The primers were designed using online software at https://www.genscript.com/ssl-bin/app/primer and synthesized in the Eppley Cancer Institute Molecular Biology Core Laboratory on the campus of the University of Nebraska Medical Center.RNA was extracted using TRIZOL Reagent  according to the manufacturer’s instructions. The RNA quality and quantity was verified using NanoDrop 2000 . Total RNA was reverse-transcribed using the iScript cDNA synthesis kits  according to manufacturer’s protocols. Quantitative RT-PCR assays were carried out using SsoFast Eva Green Supermix RT-PCR kit . Relative quantification was analyzed using PTC-200  and Chromo 4 continuous fluorescence detector . In each experiment, GAPDH RNA was amplified as a reference standard. Expression data for different K+ currents in NSCs. This experiment was carried out using an Axopatch 200 A amplifier . Measurements of the K+ current were performed in episodic-stimulation mode. For the K+ current measurement, patch pipettes were filled with (in mM) 135 KCl, 5 EGTA, 10 HEPES, 2 MgCl2, 0.25 CaCl2, 1 ATP, 0.1 GTP and 15 glucose, pH 7.2. The extracellular solution consisted of the following composition (in mM): 134 NaCl, 5.4 KCl, 2 MgCl2, 10 HEPES, 10 glucose, 1.35 CaCl2, 0.3 NaH2PO4 and 0.3 CdCl2, pH 7.4. Na+ channels were blocked by TTX (3 µM). Cell membrane capacitance was determined by integrating the capacitive current evoked by a voltage step from 0 to 5 mV and dividing the resulting charge by the voltage step. Currents were not leak subtracted. Current traces were sampled at 10 kHz and filtered at 5 kHz. Holding potential was −80 mV. Current-voltage relations were elicited by test potentials over the range of −80 mV to +80 mV, with duration of 400 ms in 20 mV increments (5 s between steps). Peak currents were measured for each test potential.The whole cell patch-clamp technique was used to determine the effect of Ang II on K4 cells/well for 2 days and were pre-treated with AT1R antagonist-Losartan, AT2R antagonists-PD123319, MEK inhibitor-U0126 or PI3K inhibitor-LY294002 for 1 hr followed by subsequent treatment with Ang II for 48 hrs. Then, 200 µl of the CyQUANT® GR dye/cell-lysis buffer was added into each well and incubated in the CO2 incubator for 15 min. Without washing, fluorescence intensity of each well was obtained using a Dynatech MR5000 plate counter at excitation and emission wavelengths of 480 and 520 nm, respectively.Cell proliferation was measured by CyQUANT® NF Cell Proliferation Assay Kit . Briefly, NSCs dissociated from neurosphere were seeded in 96-well plates at a density of 105 cells/well. At this time, Ad5-SYN-AT2R-IRES-EGFP or Ad5-SYN-EGFP were added to the culture medium (5×107 infectious units per well), three days later followed by monitoring for AT2R expression using western-blot analyses based upon previous report NSCs dissociated from neurosphere were seeded in 24-well plates at a density of 10Data were expressed as mean ± SEM. Significance of differences between control and samples treated with various drugs was determined by one-way ANOVA followed by post hoc least significant difference (LSD) test. Values of P<0.05 were considered as statistically significant.To assess the effects of Ang II on the cell proliferation of NSCs, rat NSCs were exposed to varying concentrations of Ang II for 48 h and cell proliferation assessed by CyQuant assay. As shown in Since Ang II mediates signaling via binding to its cognate receptor, AT1R and AT2R, we next examined the expression pattern of AT1R and AT2R in rat NSCs. Interestingly, we observed increased expression of both AT1R  and AT2RERK/mitogen-activated protein kinase (MAPK) and PI3K/Akt pathway has been demonstrated to play a crucial role in cell proliferation + channel activity, Ang II has been shown to regulate both K+ current and K+ channel expression in different types of cell + channel expression and K+ current in NSCs. As shown in + channel, in which KV1.2 and KV 3.1 decreased more than 10 fold. Functional implication of K+ channel in NSC proliferation induced by Ang II was further corroborated using proliferation assays, wherein 4-AP , a specific KV channel blocker, increased NSC proliferation in a concentration-dependent manner  respectively, as shown in + channel in regulation of NSC proliferation.Since cell proliferation can be regulated by modulation of Kt manner . These fV channels mediate cell proliferation and that Ang II regulated K+ currents in other cell system, it was rationalized that K+ currents through AT2R is a pre-requisite for increased NSC proliferation mediated by Ang II. NSCs were treated with Ang II and CGP42112A for 48 hours, then subsequently assessed for K+ currents using whole-cell patch clamp. As shown in + currents in NSCs. The effect of Ang II on K+ currents was restored by pretreatment of PD123319, but not by losartan. These findings underpinned the role of AT2R in Ang II-mediated blockage of K+ currents. To further unravel the role of ERK pathway in Ang II-mediated blockage of K+ currents, NSCs were pretreated with the MEK inhibitor-U0126 and assessed for K+ currents. As shown in + currents.Based on the premise that KIt is well-recognized that new dentate granule cells are continuously generated from NSCs and are integrated into the existing hippocampal circuitry in the adult mammalian brain through an orchestrated process termed adult neurogenesis It is well-known that the cardiovascular and other actions of Ang II are mediated by AT1R and AT2R, which are seven transmembrane glycoproteins with 30% sequence similarity In this study, we observed that Ang II induced ERK and Akt phosphorylation. These signaling pathways have been shown to regulate NSC proliferation Another interesting finding herein was the observation that inhibition of PI3K/Akt failed to reverse Ang II-mediated NSC proliferation thereby ruling out the potential role of this pathway. It is well known that activation of PI3K/Akt and its downstream pathway-NF-κB plays a key role in enhancing NSC proliferation following exposure to a wide array of stimuli + channel in Ang II-mediated increased proliferation, thereby lending credence to previous reports indicating the involvement of K+ channels and K+ currents in cell proliferation + channel with the neural progenitor cells isolated from subventricular zone V channels were expressed in NSCs. Ang II is known to exert its action by regulation of K+ currents in neuronal cell system + currents in NSCs remains less clear. Herein we report that Ang II induced inhibition of K+ currents through activation of AT2R. Both AT1R and AT2R belong to the large family of G protein-coupled receptors with seven transmembrane domains + channels can be activated by G-protein-coupled receptors + current via the activation of AT1R + current via AT2R + current through AT2R + currents via AT2R, but not AT1R. A key finding here is that Ang II-induced inhibition of K+ currents via ERK activation. It was evident from our findings that Ang II-mediated inhibition of K+ currents is ERK-dependent activation, and ensuing NSC proliferation. To further address the relevance of KV channel in NSC proliferation, we test the effect of pan KV channel blocker and specific KV 1.2/KV 3.1 blocker in NSC proliferation. 4-AP, α-DTX and low dosage of TEA significantly increased NSC proliferation, while high dosage of TEA inhibited NSC proliferation. It is reported that high concentration of TEA induced mammalian neuroblastoma cell swelling and decreased cell proliferation V channel in NSC proliferation Another novel finding of this study is the role of KV channels, which in turn, resulted in increased NSC proliferation. Taken together, our findings suggest that although the two pathways were upregulated by Ang II, only ERK was involved in Ang II-mediated increased proliferation of NSCs. A better understanding of these molecular pathways could be critical for the understanding how Ang II play important role in the NSC proliferation in the central nervous system.In summary, activation of the AngII/AT2R axis resulted in stimulation of ERK pathways leading to inhibition of K


In conclusion, this study is the first to demonstrate that the infiltration of PD1 positive lymphocytes and PD-L1 expression in STS cells could be used as novel prognostic indicators for STS. Moreover, the evaluation of PD1- and PD-L1-positivity in STS is also available as possible criteria for selection of patients suitable for PD1-based immunotherapy. Recently, the possibility of PD1 pathway-targeted therapy has been extensively studied in various human malignant tumors. However, no previous study has investigated their potential application for soft-tissue sarcomas (STS). In this study, we evaluated the clinical impact of intra-tumoral infiltration of PD1-positive lymphocytes and PD-L1 expression in tumor cells in 105 cases of STS. Intra-tumoral infiltration of PD1-positive lymphocytes and PD-L1 expression were seen in 65% and 58% of STS, respectively. Both PD1-positivity and PD-L1 expression were significantly associated with advanced clinicopathological parameters such as higher clinical stage, presence of distant metastasis, higher histological grade, poor differentiation of tumor, and tumor necrosis. Moreover, both PD1-positivity and PD-L1 positivity were independent prognostic indicators of overall survival (OS) and event-free survival (EFS) of STS by multivariate analysis. In addition, the combined pattern of PD1- and PD-L1-positivity was also an independent prognostic indicator for OS and EFS by multivariate analysis. The patents with a PD1 Programmed death 1 (PD1) is a member of the CD28 receptor family and attenuates immune responses by negatively regulating T-cell proliferation and function . The inhSoft-tissue sarcomas (STS) account for less than 1% of human malignant tumors  and apprP = 0.063). When we performed additional analysis by combining PD1- and PD-L1-positivity (the PD1/PD-L1 pattern), it was significantly associated with the age of patients, tumor stage, depth of sarcoma, distant metastasis, histologic grade, tumor differentiation, mitotic count, and tumor necrosis  and EFS   and EFS  (P < 0.001) and EFS  , and PD1+/PD-L1+ groups were 90%, 74%, and 13%, respectively. The factors significantly correlated with OS and EFS by univariate analyses were the age of patients, tumor stage, depth of tumor, lymph node metastasis, distant metastasis, histological grade, tumor differentiation, mitotic count, tumor necrosis, intra-tumoral infiltration of PD1-positive lymphocytes, PD-L1 expression, and the PD1/PD-L1 pattern  Table 3Table 3. 7-6.803) . The exp6-6.036) . The PD1< 0.001) . The PD1P < 0.001) and EFS  (P < 0.001) and EFS   and high stage (stage III and IV) subgroups. In the low stage subgroup, the PD1/PD-L1 pattern was significantly associated with OS  . In the = 0.003) . The infiltration of PD1-positive lymphocytes, the expression of PD-L1, and the combined expression pattern of PD1 and PD-L1 in soft-tissue sarcomas are independent prognostic factors of shorter event-free survival and poorer overall survival P < 0.001) and a 3.174-fold  greater risk of shorter EFS (P < 0.001). In addition, the patients with PD-L1 expression had a 4.827-fold  greater risk of death (P < 0.001) and a 2.629-fold  greater risk of shorter EFS (P = 0.002). When we included the PD1/PD-L1 pattern and excluded PD1- and PD-L1 positivity from the multivariate analysis (Model 2), the PD1/PD-L1 pattern was also an independent prognostic indicator of OS and EFS of STS patients  greater risk of death , 1(weak staining), 2 (intermediate staining), and 3 (strong staining). The area of staining was scored as 0 (0-10% of the cells stained), 1 (11-33% of the cells stained), 2 (34-66% of the cells stained), and 3 (67-100% of the cells stained). Thereafter, the sum of intensity score and staining proportion score was used for further analysis. Subsequently, the immunohistochemical expression of PD-L1 was scored by adding the sum score of two different TMA cores. The maximum combined score was twelve and the minimum combined score was zero.The tissue microarray (TMA) established after review of original H&E slides. Two 3.0 mm cores were taken from the most intact solid area of the highest histological grade which did not showing necrosis or degenerative change. Immunohistochemical staining for PD1  and PD-L1  was performed on 4 µm thick sections of TMA slides after microwave antigen retrieval. For negative controls, tissue sections were incubated with antibody diluent  without primary antibody. Immunohistochemical scoring was performed by two pathologists (Jang KY and Kim KM) without the information of clinicopathological factors. To minimize inter-observer variability, two authors simultaneously counted PD1-positive cells and evaluated staining area and staining intensity for PD-L1 immunostaining with the consensus under the multi-viewing microscope . The consensus for immunostaining scoring was reached after discussion by two authors. To quantify tumor infiltrating PD1-positive lymphocytes, the number of PD1-positive lymphocytes were counted at the highest numbered five high-power fields (HPF) in each TMA core. Thereafter, the number of PD1-positive lymphocytes was scored by adding the number of PD1-positive lymphocytes of the two different TMA cores. The field diameter of HPF was 0.55 mm, and the area of one HPF was 0.238 mmversus the false positive rate (1 - specificity) for the determination of the death of patients, the cut-off level for the ideal test is presented as the sensitivity 1 and specificity 1 (AUC 1.000). Therefore, we choose the cut-off point at the highest AUC value. The cut-off point of the PD1 immunostaining was one PD1-positive lymphocyte in 10 HPF from two TMA cores. The area under the curve was 0.719 for PD1  of the receiver operating characteristic (ROC) curve at the highest positive likelihood ratio point for overall survival (OS) . Because for PD1 . When th for PD1 . The immFigure S1Immunohistochemical expression of PD-L1 in intra-tumoral non-neoplastic cells. A) In contrast to tumor cells, intra-tumoral non-neoplastic cells negative for PD-L1. B) In a PD-L1-negative case, tumor cells did not express PD-L1 but intra-tumoral endothelial cells are positive for PD-L1. C) Intra-tumoral endothelial cells (arrow) and inflammatory cells (arrow head) express PD-L1. Original magnification, x400.(TIF)Click here for additional data file.Figure S2Analysis of sensitivity and specificity of PD1 and PD-L1 score for the event of overall survival (death of the patient) by receiver operator characteristic curves. Arrow indicates a cut-off point for the number of intra-tumor PD1-positive lymphocytes and arrow head indicates a cut-off point for the PD-L1 immunostaining.(TIF)Click here for additional data file.Table S1The association between histologic type of soft-tissue sarcoma and tumor stage.(DOC)Click here for additional data file.Table S2Statistical data on receiver operating characteristic curves.(DOC)Click here for additional data file.


In this article, PtAg alloy nanoislands/graphene hybrid composites were prepared based on the self-organization of Au@PtAg nanorods on graphene sheets. Graphite oxides (GO) were prepared and separated to individual sheets using Hummer's method. Graphene nano-sheets were prepared by chemical reduction with hydrazine. The prepared PtAg alloy nanomaterial and the hybrid composites with graphene were characterized by SEM, TEM, and zeta potential measurements. It is confirmed that the prepared Au@PtAg alloy nanorods/graphene hybrid composites own good catalytic function for methanol electro-oxidation by cyclic voltammograms measurements, and exhibited higher catalytic activity and more stability than pure Au@Pt nanorods and Au@AgPt alloy nanorods. In conclusion, the prepared PtAg alloy nanoislands/graphene hybrid composites own high stability and catalytic activity in methanol electro-oxidation, so that it is one kind of high-performance catalyst, and has great potential in applications such as methanol fuel cells in near future. The unint years -10, fabrnt years -13, dye-nt years , lithiumnt years ,16, and nt years . Up to dnt years , epitaxint years ,20, chemnt years , and rednt years -26 have 3O4 nanoparticles (NPs) on graphene sheets. Zhang et al. [2 NPs on both sides of single layer graphene sheets as anode materials in Li-ion batteries. They found much higher retention of SnO2-graphene composite than commercial SnO2 powder after 50 cycles. Apart from these studies, a lot of efforts had been paid on metal oxide/graphene hybrid composites [In the field of electrochemistry, graphene is an excellent substrate to load active nanomaterials for energy applications due to its high conductivity, large surface area, flexibility, and chemical stability. For example, Dai and colleagues  made higg et al.  preparedmposites . Howevermposites -30. TherIn the field of catalysis, Pt (and Pd) is intensively applied in direct methanol fuel cells (DMFCs) ,32, becamAgn to represent the samples, and m and n are percentage determined by EDX.) The self-assembly technology enables loading a lot of Au NRs and Au@AgPt alloy nanorods on individual graphene sheets with uniform morphology. It was investigated that the prepared Au@AgPt alloy nanorods/graphene hybrid composites were used as a fuel cell electrocatalyst for methanol electro-oxidation. The utilization ratio of Pt was 23.4%, but its catalytic activity was 124 mA mg Pt-1, which was close to 162.5 mA mg Pt-1 (99.2% utilization ratio of Pt) reported previously [In this study, we reported to prepare PtAg alloy nanoislands/graphene hybrid composites based on the self-assembly of positively charged gold nanorods and Au@AgPt alloy nanorods on negatively charged graphene sheets. , chlorauric acid (HAuCl4·3H2O), silver nitrate (AgNO3), and potassium tetrachloroplatinate(II) (K2PtCl4), L-ascorbic acid (AA), methanol, sulfuric acid, potassium permanganate (KMnO4), hydrogenperoxide (H2O2), sodium nitrate (NaNO3), were purchased from Shanghai Sigma Company and used as-received. Milli-Q water (18 MΩ cm) was used for all solution preparations. All glassware used in the following procedures were cleaned in a bath of a piranha solution (H2SO4/30%H2O2 = 7:3 v/v) and boiling for 30 min.10000 mesh (dimension: 1.5µm) graphite, etyltrimethylammonium bromide (CTAB), PVP  were obtained from Alfa Company and used as-received. Sodium borohydride (NaBH-1) was obtained. PVP was used to prevent flocculation when reduced graphene oxide to graphene sheets. In a typical procedure for chemical conversion of graphene oxide to graphene (GN), 100 mL 8 mg mL-1 PVP solution was added to 50 mL 0.5 mg mL-1 GO solution, then stirred vigorously for more than 12 h. Afterward, 1.75 mL 0.5% hydrazine solution and 2 mL 2.5% ammonia solution were added. The mixture was stirred for 1 h at 95°C. After that, graphene was cooled at room temperature. The whole reduction process was repeated once more to reduce GO further. The stable black dispersion of GN was filtered under the condition of vacuum with 200 nm membrane as filter paper to collect it, at the same time it was washed with Milli-Q water (18 MΩ cm). Finally, the prepared GNs were dissolved in 50 mL water (0.5 mg mL-1).Graphene oxides (GO) were synthesized from flake graphite (1.5 µm graphite) using modified Hummer's method ,42. ThenAu@AgPt nanorods were prepared using an etching method described by Wu . The spe-1 GNs was added to 1 mL of the gold nanorods solution (0.5 mmol L-1) or Au@AgPt nanorods solution. The mixture solution was then shaken vigorously and sonicated for 30 s. Afterward, the mixture was left undisturbed and aged at room temperature for more than 24 h. The color of the solution changed from red (Au nanorods) or dark gray (Au@AgPt nanorods) to colorless, and the hybrid composites precipitated at the bottom of the vessel. Afterward, the precipitate was collected by centrifugation (12000 rpm for 5 min). Finally, the precipitate was redispersed in 100 µL water for electrochemical testing.A certain volume of 0.5 mg mL-1 H2SO4 and 2 mol L-1 CH3OH in the potential range of -0.25 to 1.0 V at a sweep rate of 50 mV s-1.UV-Vis-NIR absorption spectra were obtained from a Varian Cary 50 spectrophotometer. Scanning electron microscopy (SEM) images and energy dispersive X-ray (EDX) analysis were taken on a field emission scanning electron microscope . Transmission electron microscopy (TEM) images were captured on a JEM-2010/INCA OXFORD at an accelerating voltage of 200 kV. Zeta potential results were carried out on zeta potential/particle sizer (Nicom 380ZLS). CHI660C electrochemical workstation  was carried out for the electrochemical measurement. Cyclic voltammetry was performed in a three-electrode glass cell at room temperature. Glassy carbon (GC) electrode was used as working electrode. Before testing, the electrode was rejuvenated by polished with 0.3 and 0.05 µm alumina powders, respectively, then sonicated sequentially in alcohol, pure water in each for about 20 min. 5 μL as-prepared samples were drop-casted onto GC electrodes, and dried overnight in vacuum conditions. A platinum wire and an Ag/AgCl (saturated KCl) electrode were used as counter electrode and reference electrode, respectively. The electrolyte solution was purged with high-purity nitrogen for 30 min and protected under nitrogen during the measurements. Methanol was electro-oxidized in an electrolyte containing 0.5 mol L6H2O3 proposed by Boehm [Figure by Boehm . After r2+ ions were used, the nanodots of Ag-Pt alloy particles deposited almost on the two ends of Au NRs as shown in Figure 2+ ion increased, the nanodots of Ag-Pt alloy particles distributed uniformly on the surface of Au NRs. The amount of Ag and Pt in the shell layer was determined by EDX spectra. To mention the samples relatively easily, we used Au@PtmAgn to represent the samples. Here, m and n were percentage determined by EDX spectra.Figure -1) was mixed with Au NRs solution with different weight ratios  and sonicated for 15 min to form a homogeneous mixture. Self-assembly of positively charged gold nanorods and Au@AgPt alloy nanorods with negatively charged graphene sheets resulted in formation of heavier entities; therefore, after 24 h, precipitation could be found at the bottom of the vessel. For the front four samples , the corresponding supernatants were colorless. By contrast, the corresponding supernatants of the last two samples were still red color which suggested extensive Au NRs used. As shown in Figure Characterization of Au@PtAg alloy NRs/graphene hybrid composites was carried out by zeta potential test, SEM, and TEM. The zeta potential data were shown in Table TEM was also carried out for the sample of weight ratio 2:1 and 5:1 . Among the five test samples shown in Figure 0.64Ag0.36 NRs had the highest catalytic activity.In recent years, DMFCs have intensely been studied because of their numerous advantages, which include high-energy density, the ease of handling a liquid, low operating temperature, and their possible applications to micro-fuel cells. Electrocatalytic materials restricted the performance and application of DMFCs. Herein, cyclic voltammetry (CV) was carried out to investigate the electrocatalytic activity of various graphene/Au@PtAg NRs hybrids materials for the oxidation of methanol. Three samples of Au@PtAg alloy nanorods and one sample of Au@Pt nanorods were used to prepare graphene hybrids materials and measured. In the blank control test, cyclic voltammetry was carried out in 0.5 mol LIf) to the reverse current peak (Ib), If/Ib [QH is the charge consumed for the electrooxidation of adsorbed hydrogen; Qe is the elementary charge or charge of an electron; APt is the averaged atomic area of surface Pt atoms, which is 7.69 × 10-2 nm2 according to the atomic density of a Pt surface which is 1.3 × 1019 m-2; and WPt is the Pt loading at the working electrode. This equation is based on the well-established hydrogen-adsorption stoichiometry at a Pt surface (H: Pt = 1:1). Utilization of Pt was determined by Equation 2. Nt is Pt atom loading on the working electrode; Ns is utilizated Pt atom for electrooxidation [If/Ib value could be used to evaluate the catalyst tolerance to the poisoning species. Low If/Ib value indicates poor oxidation of methanol to carbon dioxide during the anodic sweep and excessive accumulation of carbonaceous residues on the catalyst surface. High If/Ib value shows the converse case.To gain more insights into the three catalysts, some electrochemical parameters such as electrochemically active surfaces (EAS) ,45, util), If/Ib -49 were xidation . If/Ib vIf/Ib) of the three graphene/Au@PtAg NRs hybrids materials  had high catalytic activity reached 124 mA mg Pt-1, which was just a bit lower than the sample of 99% Pt utilization in the sixth row. This result suggested graphene could enhance catalytic activity of Pt material. As Pt utilization was not high for our three samples tested in the experiment, if Pt utilization even enhanced, catalytic activity might even reach a new high platform. Furthermore, the ratio of If/Ib was all higher than the commercial E-TEK catalyst (0.74) [0.64Ag0.36 NRs (sample e) was tested by long-term repeated sweep by cyclic voltammetry in 0.5 mol L-1 H2SO4 with 2 mol L-1 CH3OH at 298 K , graphene/Au@Pt0.64Ag0.36 NRs (sample e) in this study is good electrode catalyst for methanol electro-oxidation.Electrochemical parameters  . It indi0.57Ag0.43 NRs (sample a) and the NRs hybrid compositions of graphene and Au@Pt0.57Ag0.43 NRs (sample b), whose results were shown in Figure 0.57Ag0.43 NRs without graphene), it was hard to find an oxidation peak in the first cycle . With cycles went on, oxidation peak current gradually appeared and increased. The 25th cycle of sample a was shown in Figure 0.57Ag0.43 NRs with graphene), obvious methanol-oxidation peaks were observed at 0.69 V in the forward sweep and at 0.49 V in the backward sweep , which were similar to that in the 25th cycle of sample a. For this reason, sample b had good oxidation current of methanol oxidation, and electrical excitation process was not needed.As mentioned above, graphene/Au@PtAg alloy NRs hybrid compositions were excellent materials for methanol electro-oxidation. To make out what role graphene played in the course, we done controlled experiment using pure Au@Ptad stripping voltammograms to explain the role graphene played in this reaction. The different state of CO adsorption on Pt/graphene was inferred to traditional Pt catalysts supported on carbon black [If/Ib were 1.46 and 1.24, respectively, for graphene/Au@PtAg alloy NRs hybrid compositions (the first sweep) and Au@PtAg alloy NRs (the 25th sweep) without graphene. The different onset potential and If/Ib value in backward sweep could be attributed to different CO adsorption state. The different CO adsorption state on graphene/Au@PtAg alloy NRs hybrid compositions and ordinary PtAg alloy NRs materials influenced the catalytic activity for methanol electrooxidation. Graphene in hybrid compositions could enhance anti-poisoning effect in the backward sweep. Graphene in the hybrid composition could change adsorption state of reactant, so the electrochemical process was affected. The higher oxidation peak in the first cycle of graphene/Au@PtAg alloy NRs hybrid compositions might result from the different interaction between graphene and methanol. Therefore, graphene in the hybrid compositions could improve the catalytic activity for methanol electrooxidation.Another important parameter to value catalytic activity of the samples is onset potential in electrical oxidation process. In forward sweep, all the samples had the same onset potentials (0.216 V). Otherwise, in backward sweep, sample b had frontier onset potentials (up to 124 mV) than sample a (without graphene). As mentioned above, the oxidation current of methanol oxidation in backward sweep represented the removal activity of the incompletely oxidized carbonaceous species  generated in the forward sweep. The frontier onset potentials of graphene/Au@PtAg alloy NRs hybrid compositions indicated easier remove of the incompletely oxidized carbonaceous species. This phenomenon was very similar to that discovered by Yoo et al. before. In their research, Yoo et al. had done COon black . In our In addition, graphene had the advantages of good dispersion, high conductivity, large surface area, flexibility, and chemical stability. The higher catalytic activity of graphene architecture was attributed to the larger surface area which led to large currents and good dispersion of Au@PtAg NRs on the surface. The good dispersion of Au@PtAg NRs on graphene would give reactants easy access to the catalytic active sites, which would help to improve proton diffusion and mass transport.In this study, PtAg alloy nanoislands/graphene hybrid composites based on self-assembling of Au@PtAg NRs on graphene sheets were successfully prepared. The high-loading Au@PtAg NRs distributed uniformly on the surface of graphene sheets. It is confirmed that PtAg alloy nanoislands/graphene hybrid composites own better catalytic activity and longer stabilization for methanol oxidation compared with traditional method. Because large-scale graphene can be prepared by chemical reduction of graphene oxide; therefore, the PtAg alloy nanoislands/graphene hybrid composites can be obtained by large scale with low cost; therefore, as-prepared PtAg alloy nanoislands/graphene hybrid composite has great potential in applications such as electro-catalyst for DMFCs in near future.The authors declare that they have no competing interests.LF carried out the whole study. GG participated in the taking of SEM images. PH participated in the taking of TEM images. XW, CZ, JZ participated in the discussion of this research. DC and SG participated in the design of the study and gave instruction of the study. All authors read and approved the final manuscript.Figure S1. UV-Vis-NIR absorption spectra of the Au NRs. Figure S2. TEM images of Au NRs (A) and Au NRs/graphene hybrid composites with weight ratios: 2:1 (B), 5:1 (C). Scale bar: 200 nm. Figure S3. Cyclic voltammetric curves of the following electrocatalysts: (a) graphene; (b) graphene/Au@Pt NRs; (c) graphene/Au@Pt0.34Ag0.66 NRs; (d) graphene/Au@Pt0.57Ag0.43 NRs; (e) graphene/Au@Pt0.64Ag0.36 NRs in 0.5 mol L-1 H2SO4 solution at 298 K. 0.64Ag0.36 NRs electrocatalyst over 200 cycles of methanol electrooxidationFigure S4. Stability of the graphene/Au@Pt.Click here for file