The nanoscale friction between an atomic force microscopy tip and graphene is investigated using friction force microscopy (FFM). During the tip movement, friction forces are observed to increase and then saturate in a highly anisotropic manner. As a result, the friction forces in graphene are highly dependent on the scanning direction: under some conditions, the energy dissipated along the armchair direction can be 80% higher than along the zigzag direction. In comparison, for highly-oriented pyrolitic graphite (HOPG), the friction anisotropy between armchair and zigzag directions is only 15%. This giant friction anisotropy in graphene results from anisotropies in the amplitudes of flexural deformations of the graphene sheet driven by the tip movement, not present in HOPG. The effect can be seen as a novel manifestation of the classical phenomenon of Euler buckling at the nanoscale, which provides the non-linear ingredients that amplify friction anisotropy. Simulations based on a novel version of the 2D Tomlinson model , as well as fully atomistic molecular dynamics simulations and first-principles density-functional theory (DFT) calculations, are able to reproduce and explain the experimental observations. The mechanical behavior of materials at the nanoscale may present completely novel and unexpected features as compared to their bulk counterparts. Particularly in the process of understanding and controlling friction at the nanoscale one faces new challenges and paradigm shifts24et al.The advent of single layer graphene opened up new possibilities for studies on the mechanisms of nanoscale friction. Major efforts have been addressed, for instance, to understand why friction increases as the number of atomic layers is reduced789One is then led to the question of friction anisotropy in graphene, which has been observed in exfoliated monolayer graphene, but with a 180° period angular pattern, and not the 60° period expected from the sixfold rotational symmetry of the graphene lattice11121314In this work, we demonstrate that friction anisotropy in graphene is tunable by the normal force and can reach values as high as 80% , which represent a giant enhancement with respect to graphite. This behavior is quite unexpected, since the linear elastic properties of graphene  are isotropic, as expected from symmetry considerations. Using a combination of simulations based on the Prandtl-Tomlinson model16Graphene flakes were prepared in ambient condition by micromechanical cleavage of bulk graphite. A typical flake used in our measurements is shown in L as illustrated in the inset of −1) and armchair (2.3 nm−1) lattice periodicities, respectively (we have considered the graphene lattice constant to be a = 0.246 nm). When the tip scans along the zigzag crystallographic direction (30°), a single prominent peak at 4.0 nm−1 is clearly observed in the FT spectrum. On the other hand, when the tip scans along any other direction, at least two prominent peaks are observed in the FT spectra. These two features have spatial frequencies of 1/λn and 1/λn+1, and the largest separation between them occurs for scanning along the armchair direction (±30°). λn and λn+1 as a function of the scanning angle (with respect to the zigzag direction): full circles and triangles are experimental data, empty squares are results from Prandtl-Tomlinson simulations, and the solid lines represent an analytical approximation , and armchair (c) directions. All profiles present short-period stick and slip jumps superimposed with slow build-up of friction forces, eventually reaching saturation after the tip completes approximately 10 stick-slip cycles. The orientation dependence of the stick-slip patternOnce we have determined the crystallographic orientation of the graphene sheet, friction forces can be measured along specific directions. N). Surprisingly, the energy dissipated along the armchair direction is about 80% larger than the energy dissipated along the zigzag direction, for large normal forces. Interestingly, this giant friction anisotropy can be tuned by the applied normal force: friction enhancement along armchair with respect to zigzag increases from roughly 15% to reaching saturation at ~80% as the normal force increases from 50 nN to 150 nN as shown in Strengthening of tip-surface interaction induced by the tip motion certainly increases energy dissipation, by converting kinetic energy of the tip into lattice vibration energy. The energy dissipated in each scanning cycle is obtained by integrating the friction force over the lateral displacement in the forward and backwards scan loops, as illustrated in 3N4 tip scanning over a single graphene layer. The anisotropy in HOPG observed in our experiments has atomic-scale origin and it is discussed in detail in the Friction anisotropy has been previously observed in bulk graphite, and was associated with the incommensurability of an “effective tip” generated by adhesion of a small flake of graphite to the tip apexAs it is evident from the comparison to the HOPG case, the giant and tunable anisotropy observed in graphene must be related to flexural deformations of the monolayer, which are likely enhanced when the tip moves along the armchair direction, with respect to the zigzag. This is surprising, because linear elastic constants are isotropic in graphene, so one should not expect to be any easier to buckle a graphene sheet by applying forces along any specific direction. Besides, assuming that the graphene sheet is flat when the motion begins, lateral forces exerted by the tip on the sheet are only 15% larger along armchair (the friction anisotropy of HOPG), therefore there must be some sort of nonlinear ingredient responsible to amplify the anisotropy of flexural deformations as the tip moves.m and position rt, interacting with the surface via a potential energy V(rt). For HOPG, we useWe are able to model and understand the giant friction anisotropy in a graphene using a modified version of the well-stablished Prandtl-Tomlinson model16a = 0.246 nm. This potential energy corresponds to placing the zigzag direction along x. The base of the microscope is located at position rM, and the elastic energy of the strained tip is modeled by an isotropic harmonic potential: with Ft of the elastic force along the base trajectory is integrated along the path to give the dissipated energy. Technical details of the simulations are presented in the The stick-and-slip motion of the tip apex gives rise to friction and the component X to describe flexural displacements, which evolves according to the following equation of motion:We then introduce flexural degrees of freedom of graphene by adding an effective dynamical variable M is an effective mass that describes the inert, interacting with the surface via a potential energyrtia of the flexural mode and W(X) is the restoring potential of the flexural mode, in which we include a non-harmonic cubic component on the potential C > 0. The cubic term is important to make the system stable after we introduce the feedback mechanism by which the increase in flexural amplitude enhances the tip-surface interaction, to be discussed below. Damping is introduced by the ΓFL is the lateral force due to the tip, which drives surface buckling only if it becomes larger than a critical force Fc. This is the well-known condition for Euler buckling which occurs not only for macroscopic rods and membranes1921where V0 on the flexural amplitude X: V0→V0 + αX. This is illustrated schematically in 2, it should have no effect on friction anisotropy. The anisotropy amplification effect can be easily understood by considering an extreme situation in which the lateral forces along armchair, initially only 15% larger than zigzag ones when the sheet is planar (as for the HOPG case), become larger than the critical buckling force as the tip moves, whereas zigzag forces do not. In this special case, armchair scanning causes buckling and zigzag scanning does not, leading to giant anisotropy amplification. The effect can be tuned by increasing the normal forces, which in turn increase the lateral friction forces.A feedback mechanism by which the increase in flexural amplitude enhances the tip-surface interaction is introduced simply by adding a linear dependence of the tip-surface potential energy amplitude σvm known as the von Mises stress24σij as:Finally, in order to provide an atomistic visualization of the mechanisms leading to these phenomena, we perform classical molecular dynamics (MD) simulations. Our simulation model is composed of an elastic tip (the AFM tip) and a single layer of graphene, which interacts via van der Waals forces with a horizontal substrate which is held fixed below it. The AFM tip is pushed against the surface using a constant force (~0.3 pN) perpendicular to the graphene plane and forced to move along the horizontal direction, parallel to the membrane, as illustrated in In summary, we demonstrate the existence of a giant and tunable anisotropy in nanoscale friction between an AFM tip and graphene. The microscope tip was observed to move in a discontinuous stick and slip mode allowing the identification of the graphene crystallographic directions. By increasing the normal force, the anisotropy increases from 15% (same as HOPG) to roughly 80% larger for scanning along the armchair direction, with respect to the zigzag one. The nonlinear mechanism responsible for amplifying the anisotropy is related to Euler buckling in membranes, which leads to flexural deformations of the graphene sheet, causing puckering of graphene around the tip and enhancing tip-surface interactions, in a feedback mechanism. The proposed mechanism is supported by theoretical calculations in three levels: density functional theory, molecular dynamics simulations and Tomlinson model. This novel behavior is an important step in understanding and controlling friction in single-atom-thick membrane at the nanoscale.2 layer on top of a Si substrateGraphene flakes were prepared in ambient conditions by micromechanical cleavage of bulk graphite onto a 300 nm SiOAll nanoscale friction measurements showing atomic-scale lattice resolution were acquired using silicon nitride V-shape cantilevers, with tip radii of about 20 nm and calibrated normal and lateral spring constants of 0.40 ± 0.01 N/m and 85.4 ± 4.2 N/m respectively. During quantitative FFM measurements, the scanning direction was kept perpendicular to the cantilever main axis in such a way that the friction force experienced by the tip was measured by the lateral torsion of the cantilever. The microscope was calibrated for each tip used, and the lateral force was obtained from the product between the cantilever’s lateral spring constant and the lateral tangential displacement of the tip measured in the position sensitivity photodetectorHow to cite this article: Almeida, C. M. et al. Giant and Tunable Anisotropy of Nanoscale Friction in Graphene. Sci. Rep.6, 31569; doi: 10.1038/srep31569 (2016).


In the course of decentralization, pastoral communities in Namibia have had to find new ways to share their most salient resource, water, and the costs involved in providing it. Using data from sixty communities, we examine (1) whether and to what extent different sharing rules emerge, (2) how variations can be explained, (3) how rules are perceived and influence success, and (4) what economic consequences they have. Our results reveal that either all members pay the same  or payment is according to usage . We find that although proportional equality provides more success, the rule can only pertain where the state maintains an active role. Simulations show that where it does not prevail, wealth inequality is likely to grow. These findings have political implications and suggest that, in the context of the widespread decentralization policies, the state should not withdraw if it aims to ensure the success of common-pool resource management and to fight poverty. As the impact of humans on communal resources grows, there is an increasing need to better understand how resources can be governed sustainably. Elinor Ostrom has identified eight principles that explain failure and success in shared communal resource management. While the significance of some of the original variables  has been explored to some extent during the last decades Ostrom , comparanumerical and proportional . With proportional equality, the payment received varies according to the time each individual spends actually working, whereas with numerical equality all those who work are paid the same amount. Logically, the latter is a special case of the former. The parable of the workers in the vineyard offers an illustration (Matthew 20:1–16). Here, the winegrower decided to pay all workers the same, independent of when they began working during the day. Thus, he established a relationship between the two variables at stake, ‘work’ and ‘pay’ which is not proportional but numerical in Aristotle’s sense. Not surprisingly, those who had been working since dawn complained.Sharing agreements are based on notions of equality. Equality is a functional relationship between two variables, for example, time worked and payment received. According to Aristotle, there are two kinds of equality: In the case of communal resource governance, cost- and benefit-sharing institutions must resolve the same fundamental issue , and she concluded that institutions are perceived to be fair if there is a ‘congruence between appropriation and provision rules’ Ostrom , p. 90, Currently, Namibia offers a unique opportunity to test these expectations in an investigation of the emergence, success, and consequences of sharing agreements on a large scale. In the course of decentralization, rural communities have had to find new ways to share their most salient resource, water, and the costs involved in providing it. In the arid environment of northwestern Namibia, pastoralism is the dominant livelihood strategy, and almost all households own cattle and small stock. Until some 50 years ago, most African pastoralists obtained water through natural springs, surface water, and hand-dug wells McCabe . Open waIn northwestern Namibia, between 1960 and 1990 the number of boreholes increased almost by a factor of ten Bollig . Until iStarting in the mid 1990s, the implementation of CBNRM has led to a drastic reconfiguration of the organizational and institutional landscape , longitudinal data, or simulations is appropriate. Such data were not available prior to the study reported here. We explore for the first time (1) whether and to what extent different sharing rules emerge, (2) how possible variations can be explained, (3) how different rules are perceived and hence influence success in community water management, and (4) what economic consequences distinct rules are likely to have.The arid Kunene region is sparsely populated. Small communities dot the vast landscape and, on average, they contain 13.1 (SD 3.5) households with 13 (SD 9.3) household members each. In general, dependency on natural resources is high. Pastoralism is the dominant subsistence strategy and the pastoral livelihood is constrained by the environment, most notably the low and unpredictable precipitation Bollig . Annual Livestock possession is unequally distributed in Kunene and other parts of Namibia  as part of a German Research Council (DFG)-funded research project LINGS . The two principle investigators, M. Schnegg and M. Bollig, have been conducting ethnographic fieldwork in the region since 1994 (M. Bollig) and 2002 (M. Schnegg) respectively, and are responsible for the overall design and comparative analysis of the data. In the first phase of the current fieldwork, three anthropologists  stayed for roughly 1 year between 2010 and 2011 in seven communities in the southern (Fransfontein), central (Otwani), and northern (Okangwati) parts of the research area to gain an in-depth understanding of processes entailed in negotiating and crafting new institutions through daily routines. During this time, all 80 households were interviewed about their social and economic livelihoods, including economic possessions and social networks.2 around Fransfontein, Otwani, and Okangwati, all communities were interviewed. We can thus treat the data as a complete sample for those three areas. We decided against a representative sample of the entire Kunene region due to its size, bad road infrastructure, logistical constraints, and the lack of a list of communities that could serve as a sampling frame. In addition, our approach allowed us to make use of the fact that fieldworkers were already known and trusted in the target areas.After an initial analysis of the ethnographic data collected during 2010 and 2011, we returned to the field in late 2012 to conduct the ‘upscaling’ research we had designed to study the distribution of some of the phenomena found in the community ethnographies. Since our study treats communities as cases, it is challenging to collect relatively large numbers of observations that permit meaningful comparisons. To facilitate this requirement, we designed an interview guide to elicit information at the community level. For geographical areas of approximately 250 kmThe research protocol contained three sections. First, we elicited the rules of water management and the composition of community-based organizational structures for water governance in the community. Each interview took place in public and included both female and male informants, some of whom were active in the water point committee. The second and third sections dealt with the success of the water management and the conflicts communities had experienced or observed. Since those questions are more confidential, interviews were done in private, aiming at a sample of males and females of different age groups and from different economic positions. In total, we researched sixty communities. Since information remains incomplete for some of them, the number of communities included in the analysis is sometimes lower than that.All group interviews were taped and two independent interview protocols were written by the main researcher and his/her assistant. Discrepancies were resolved thereafter. The data were entered in MySQL database, designed for the project. The coding was done by the principal investigators in collaboration with the researchers.The measurement of success derives directly from the questions posed and is coded on an ordinal three-point scale. To measure the involvement of the state, we use two indicators. During the interview, we collected two types of information. First, the frequency and purpose of visits by state officials in the community during the last 2 years. If the state officials had visited the community at least once during the last year for consultancies and activities other than urgent repairs of broken infrastructure, we took this as an indication of more than average state involvement. Since CBNRM and the decentralization strategy are highly standardized, we did not observe any variations in the type of rules recommended. Generally, state officials try to push communities in certain directions above all concerning the payment schemes. Second, our ethnographic observations have shown that if employees of the ministry or commissioned NGOs lived in the communities at issue, the impact of the state was significantly stronger, because those people typically wanted their communities to be flagship cases for the state’s mission and ideology (see below). For the analysis, we coded the state involvement to be above average, if either of the two indicators was given.‘‘Recommended is a rate per head of large or small stock, each member paying a certain rate per head of large or small [stock] accordingly, as to raise enough money to sustain the water point”  and/or NGOs contracted by the government  agreed that individuals paid fees according to the number of livestock they owned . Thus, the more water one uses, the more they pay. This fits the notion of proportional equality. In addition, seven communities (12.5 %) used an attenuated form in which the rich paid more, but not exactly in proportion to the number of their livestock.4.6 % agrBefore we offer an explanation why, we want to rule out two common alternative hypotheses: technology and a “majority cost/benefit vote” Ostrom . WhereasThe account we provide instead is formulated on the basis of our ethnographic work and includes four factors. Of those, three apply in all communities and favor a numerical rule. Only the last factor, the involvement of the state, makes a difference, as the statistical analysis reveals.To establish and maintain a proportional rule is more costly than a numerical rule. Most importantly, it requires counting animals. Counting livestock is difficult. Counting individual animals of small stock in large herds from different owners flowing toward a well is nearly impossible. In addition, there are cultural taboos against counting livestock, and people complain that counting livestock brings about bad luck Bollig . In geneNot surprising, wealthy herd owners oppose a proportional rule and opt for numerical equality . Thus, water is only one resource in a larger sharing arrangement and a moral model exists that short-term imbalances in one domain will equal out across all types of transfers and time , the involvement of the state can explain the institutional outcome to a significant degree. In contrast, in communities where the state is only weakly involved the first three dynamics analyzed above are dominant and numerical equality prevails.We have outlined when and why different equality regimes evolve. Since the groundwater in northwestern Namibia comes from aquifers fed in Angola, ecological success cannot be measured locally. We, therefore, base our analysis of the success of the water management regimes on social indicators developed in cooperation with communities. These include (1) satisfaction with the rules, (2) satisfaction with the work of the water point committee (committee), (3) general level of satisfaction expressed concerning cooperation in the community, and (4) the general satisfaction with the water management in the community.Table As we have indicated, livestock is unequally distributed. Thus, with numerical equality in a hierarchical setting, where all individuals pay the same, the institutional regime must have economic consequences that diverge from equality. To explore these in depth, we developed a simulation model. The model starts with the distribution of livestock that we found among 80 households for which we did detailed livestock counts. Small and large stock are aggregated into one measure, livestock units (LSU), for which a head of cattle equals one LSU and small stock  one sixth  has become a guiding principle of many development efforts—the idea that turning ownership and responsibility over to local communities will empower them and help eradicate poverty Ribot . Left al


The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3’ side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. Genes containing introns can produce several transcripts and protein products via alternative use of exons and introns. Alternative splicing (AS) exploits these gene structures to increase transcriptome and proteome complexity and to serve regulatory purposes in higher eukaryotes.Splicing requires a complex molecular machinery, the spliceosome, which is directed by three important splice signals: the 5’ splice site, the branch point and the 3’ splice site with associated polypyrimidine tract. These signals are detected by components of the spliceosome and define the exon-intron boundaries. To enable AS in higher eukaryotes, many auxiliary factors are present such as SR proteins or hnRNPs . These cIn S. cerevisiae, about 4% of all genes contain introns and the organism is able to correctly splice those genes. While complete splicing is well studied in S. cerevisiae, the use of alternative splice sites is only described in individual examples, which we will review below. These examples have been reported in the literature and are not annotated in resources like the Saccharomyces genome database (SGD) or Ensembl. The simple gene structure and certain properties of the S. cerevisiae genome suggest that AS in S. cerevisiae is limited: its genome shows strong 5’ splice sites and a highly conserved branch point sequence (TACTAAC), which facilitate the splicing of introns . FurtherIndividual examples of AS have been described, especially in the context of environmental stress or certain states of the cell cycle. Intron retention and subsequent nonsense mediated decay (NMD) are reported for certain genes during mitosis  or by auMore complex AS has been confirmed on the transcript level, leading to multiple alternative mRNAs for a single gene locus . Intron Finally, changes in the amino acid sequence by AS have been observed. Intron retention generates two different isoforms in PTC7, which determine the protein localization. The intron does not disrupt the reading frame and introduces 93 bases into the mRNA. The resulting additional 31 amino acids contain a transmembrane domain and change the localization and cellular function of the protein . Usage oMoreover, Pelechano et al. have shown that S. cerevisiae is capable of producing a high diversity of transcript isoforms by using different transcription start and end sites. More than 26 transcript isoforms per protein-coding gene have been observed . These rIntron retention, a common mechanism in fungi and plants, is often observed in S. cerevisiae splicing. Most known introns will disrupt the reading frame, leading to a premature stop codon. S. cerevisiae is able to degrade the affected transcripts using the nonsense mediated mRNA decay pathway , and useIn this study we show that S. cerevisiae makes extensive use of its limited alternative splicing capabilities. We analyze a publicly available mRNA sequencing data set of S. cerevisiae and report novel introns, the pervasive use of alternative splice sites and evidence for unspliced transcripts that possibly occur due to intron retention. Results are validated in three different ways. Firstly by using multiple mapping programs, secondly in a complementary mRNA sequencing dataset and thirdly, via PCR, which we performed in independent S. cerevisiae samples. Advances in next generation sequencing technology provide a large number of sufficiently long reads (≥ 100 bp) that can be mapped confidently to unique positions of the genome, even if a read spans a splice junction and two ends of the read need to be mapped individually. We analyze one of the first of these available data sets by Nookaew et al. , observewww.ensembl.org). Read mapping was performed using Bowtie [Sequencing data was obtained from Nookaew et. al. . The S. g Bowtie  and TopHg Bowtie  with defg Bowtie  and Contg Bowtie . TopHat Predicted introns were validated in another, independent next generation sequencing dataset . The rawFinally, several cases of novel introns and AS events are validated via PCR. Details are described in For a given transcriptome sequencing data set, we define a potential intron as chromosomal subsequence which is spanned by a split read mapping, i.e. the first part of the read is mapped to the genome before the potential intron and the other part after the potential intron. Potential introns provide evidence of splicing events and, in case of overlapping potential introns, AS events. They may match or overlap known introns, according to the annotation of the Saccharomyces Genome Database, and they can also constitute novel introns.We focus on potential introns that show similar characteristics to those of annotated S. cerevisiae introns in terms of length and splice signal, and which are supported by sufficient evidence. Potential introns meeting those three criteria are called predicted introns (PIs) as outlined in n) bases upstream or downstream due to repetitive sequence patterns at the junction boundaries, the exact position of a split is unclear. We observe that known introns are spanned by clearly defined split reads, i.e. the intron can be assigned to an exact genomic location in almost all cases. Furthermore, the supporting split reads match well before and after the junction exhibiting a long “anchor” on either side. Combining these two observations into a single score results in a measure for “junction quality”, defined as the anchor length minus n. Hence for each predicted intron we have two junction quality values. For PIs we require similar values as we observe for annotated introns, i.e. the maximum of both values must exceed 85 and the minimum must exceed 25  and two within small nucleolar RNA. Mitochondrial introns are not considered in this study because they are spliced differently from chromosomal genes .With, in general, only one intron per gene, the only possible forms of AS are intron retention, alternative 3’ and alternative 5’ splice site selection. These are summarized in After read mapping we obtain 880,312 mapped split reads, which define 5,093 potential introns in the genome. On average there are 173 reads per intron, but reads are distributed in such a way that a few of the potential introns are supported by a large fraction of all reads: 1,532 potential introns (30%) are supported by only one read, 2,418 by two or less reads. The median read support is 3. An overview of read support is given in Out of 447 predicted introns 277 match known introns. The remaining 170 predicted introns either define an alternative splice site at known introns (71 cases) or constitute novel introns (99 cases). Out of 99 novel introns, 27 are predicted as “single intron” see  and 12 aFour additional novel introns with sufficient read support are disruptive to the reading frame  we also find evidence of the unspliced transcript. Mostly, inclusion of annotated introns will lead to either a disrupted reading frame and/or an introduced stop codon.In our data we find 56 introns located in the 5’ UTR. Among these introns, we identify 20 of 24 known introns and 36 novel introns. In Under the most conservative assumptions we identify a total of 63 annotated introns with variable splice sites. These divide into 44 cases of an alternative 3’ splice site, 16 cases of an alternative 5’ splice site and three cases with combined alternative 3’ & 5’ splice sites. There are 22 (3’) and 8 (5’) cases with 10 or more supporting split reads. Compared to 3’ and 5’ alternative splice sites, combined alternative splice sites exhibit the lowest read support see .Among the alternative 3’ splice sites the median distance to the canonical splice site is 28 bases. There are 11 cases that do not disrupt the reading frame and are able to encode an altered protein product. The AS events are able to remove or introduce between 1 and 53 amino acids compared to the reference sequence. The remaining 33 cases will disrupt the reading frame, if the intron is located in the protein coding region. Examining some of those cases in more detail, we find examples in which the AS event may trigger well known biological functions: Alternative splicing in YDR099W (BMH2) and YBR078W (ECM33) introduces a single additional codon, the splice site is of the NAGNAG form, providing two adjacent acceptor sites. The additional codon introduces a new amino acid into the resulting protein sequence, which has been reported as a mechanism to control protein localization  and as aThe remaining alternative 3’ splice sites are located downstream of the canonical splice site, extending the intron and removing amino acids from the protein product. Intron extension may be explained by a variation in the linear scanning mechanism proposed for detection of the splice acceptor site starting from the branch point .We find evidence of three cases of novel introns overlapping the genes 3’ end . SplicinIn the spliced isoform of YBR101C (FES1) the terminal YVL* is substituted with TFCKMEFIKKVRRGK*. In case the terminal intron in YMR147W is spliced, the alternative protein terminus extends into the reading frame of YMR148W (OSW5), leading to a chimeric protein fusion. This confirms a previously described observation [The alternative splicing events reported in this paper were validated in three different ways: (A) by using different mapping software, (B) in an independent sequencing dataset, and (C) via PCR for selected splicing events.(A) The first validation was performed using two other mapping programs (Star  and Cont(B) Predicted introns were cross checked against read data from another next generation sequencing dataset . We cons(C) We used S. cerevisiae samples to validate our identified splicing and alternative splicing events by PCR. Primers could be designed if the following conditions were met: First, the length difference of the expected PCR products had to be large enough, such that different products would become visible on the gel. Second, the gene and isoform under consideration had to be expressed in our available S. cerevisiae samples. Primers must be unique and close enough to the alternative event and we require them to lie within genomic regions that are transcribed in the used samples. We also designed primers for three additional cases in which we found striking evidence of alternative splicing or splicing in our own S. cerevisiae samples. These cases are also found in our original dataset , but witUnder the abovementioned constraints, we designed primer pairs for 10 predicted novel introns: Five pairs were designed for novel introns in the 5’ UTR, three pairs for novel introns in protein coding regions and two pairs for alternative 3’ splice sites at two known introns. Out of those 10, PCR confirmed alternative splicing in 7 cases: splicing was confirmed for the 5’ UTR introns at genes YLR467W (YRF1-5), YBL111C and YGL063W (PUS2) . Furthermore, AS could be confirmed for the novel introns in YBR101C (FES1), and in YMR147W. In both cases the annotated stop codon is observed as both, included and spliced out in the isoform. The third novel intron in the protein coding region of YGL136C (MRM2) is alternatively spliced according to the sequencing data, and PCR confirms the presence of an intron. Finally, PCR confirmed the alternative 3’ splice site in YBL026W (LSM2), but not in YOR293W (RPS10A). Pictures of the gels and sequences of all primers are listed in the supporting information .We analyzed alternative splicing (AS) in the yeast S. cerevisiae. High throughput mRNA sequencing data with sufficient read length enable us to confidently map split reads to unique positions on the genome. As a result we can identify an unprecedented amount of previously unknown introns and observe several cases of alternative intron usage in S. cerevisiae. Some of these AS events have been described earlier and are referenced in the introduction; in most of these cases the alternative isoform shows high read support in our analyzed data and may be detected more easily in general. In addition to the previously described AS events we find novel cases, indicating a more widespread use of AS in S. cerevisiae than currently known. Strikingly, we validate most of the reported alternative events in a different, independent NGS dataset. We then validate reproducible splicing events via PCR in 7 out of 10 tested cases in a dedicated experiment.In about 20% of annotated introns we find evidence of alternative 3’ or 5’ splice sites, with a strong bias toward 3’ variation. Compared to 5’ splice sites, S. cerevisiae exhibits weaker splice signals at 3’ splice sites, which facilitates alternative splicing [Reads supporting intron retention can be found in every intron containing gene, albeit with low read support in general. The mechanism is common in fungi and plants and serves regulatory purposes, but the retained introns could also be an artifact of the stochastic nature of the splicing process. As such they might be yet unspliced transcripts or defective splicing, which will be targeted by NMD. However, we find evidence of eight novel introns that are not disruptive to the reading frame. The relatively weak evidence for the spliced, novel isoforms could explain why these introns have not been identified earlier. Intron retention has been shown to cause alternative proteins in at least one S. cerevisiae gene (PTC7) ), where There are 10 annotated cases of multi intron genes, and we predict a second, novel intron in the gene YFR031C-A (RPL2A). The respective genes contain two introns each and allow the possibility of AS induced exon skipping or cassette exons. Exon skipping has been reported for the SUS1 gene , but in We conservatively analyzed a single dataset with the hypothesis of limited AS in S. cerevisiae. We used a small dataset that comprises two conditions and systematically screened for variation among annotated introns. Nevertheless, we detect novel alternative splicing events that are subsequently validated using independent data. In general the predicted novel introns are supported by less reads than annotated introns. We conclude that the respective transcripts are minor isoforms, possibly triggered by specific conditions. They may coexist with the major isoform and are therefore rather hard to detect. PCR of alternative events resulted in the successful validation of most of the tested isoforms. While isoforms with weak evidence should be considered with care or examined under different experimental conditions, we suggest that highly supported alternative isoforms are more likely functional. This observation is supported by recent work of Kawashima et al. , in partKawashima et al. also publish a list of 728 splicing events, of which 522 suggest alternative splicing. However, the authors do not discuss them in detail. The overlap between our study and Kawashima et al. is not expected to be high, since our analysis pipeline is different  and AS is likely to be context specific. Consequently, differences in yeast strain and growth conditions result in different AS. Furthermore, technical factors like NGS library preparation method and sequencing depth and length will influence the detection of isoforms in each study. The overlap between 170 potential introns in our study and the 522 AS events is 13. Refer to In conclusion, AS in S. cerevisiae is possible and observed on the transcript level. Using split reads in mRNA-sequencing data we identify several cases of alternative transcripts, which can be explained by an alternative splicing mechanism in S. cerevisiae. Systematic analysis of AS events will help to investigate the impact on the proteome and to identify the regulatory mechanisms leading to isoform production. AS in S. cerevisiae is triggered by conditions like elevated heat  or restrS1 TableList of all predicted novel introns, sorted by evidence (read support). For completeness we also report potential introns with a read support lower than three. Columns include Chromosome, Strand, Start, End, Length specifying the position of the novel predicted intron. Additional columns areRead Support: evidence for the predicted intron.Anchor—Slack: junction quality scores.Gene: affected gene.PCR: if PCR has been performed, capital letters identify the lane in (PDF)Click here for additional data file.S1 FigPCR gels of 10 tested novel introns and alternative splicing events. The expected position of PCR products confirming novel events are marked with arrows. Numbers describe the adjacent PCR product size (in bases) of the two marker bands. Chromosomal locations of the events are listed below.μg of total RNA was transcribed in a 25 μl reaction using the M-MLV reverse transcriptase  according to manufacturer instructions. 1 μl of the resulting first strand cDNA was used as template in 50 μl PCR reactions  applying splice variant specific primer pairs .The S. cerevisiae strain BY4741   was cultivated in YPD at 30°C. Exponentially growing S. cerevisiae cells at an OD595 of 0.8 were collected and used for preparation of RNA applying the SV Total RNA Isolation System . For first strand cDNA preparation 1 er pairs  and GoTaThe above experiment was repeated three times. The first time without any sequencing attempt. The second time sequencing failed. The third time sub-clonig and subsequent sequencing was done:For sub-cloning, 50 µl PCR reactions were completely separated on 2% agarose gels and the bands corresponding to the splice product were purified using the MiniElute PCR Purification Kit . The purified splice product was sub-cloned using the TOPO TA Cloning Kit for Sequencing  according to the manufacturer’s protocol. Positive clones were selected after DNA-preparation by control PCRs with the respective primer set and further analyzed by sequencing .In Experiment J the “expected novel fragment length” could be sequenced and is matching the expected sequence. This coincides with the best visible PCR band out of any of the “expected novel fragment length” bands.(PDF)Click here for additional data file.S2 TableLocation and sequences of the PCR primers used.(PDF)Click here for additional data file.S2 FigWe compare our results with the recently published results of Kawashima et al. Venn diagrams compare potential intron assignments at two different levels of confidence in our analysis to introns reported by Kawashima et al. In each plot the set “All” contains all introns fulfilling our length requirements and presenting a valid splice signal. The set “Only accepted” only contains introns that additionally fulfill our support and junction quality criteria.A) Overlap limited to annotated introns. We observe a large overlap of about 90% introns reported in Kawashima et al.  and abouB) Overlap without annotated introns. In these diagrams we compare true alternative splicing events reported here and by Kawashima et al. The overlap among proposed isoforms is not expected to be high, since our analysis pipeline is different  and AS is likely to be context specific. Some isoforms are exactly found in both studies, so those results are reproducible under different experimental and technical conditions.c) Out of the highly confident AS events reported in the tables in the main text, five are also reported by Kawashima et al. Strikingly, all of them are at the 3’ splice site, and four preserve the reading frame. The set “Reported” only contains the predicted introns that are listed in the tables in this manuscript excluding any novel introns (n = 20), because those are not considered by Kawashima et al.The potential introns in the sets “All” and “Only accepted” are derived following our workflow, depicted in (PDF)Click here for additional data file.S3 FigThe data published by Kawashima et al.  enables In summary, we confirm the findings of Kawashima et al. Additionally, we detect some unknown AS events that seem to be unaffected by NMD:Panel A shows how the split read support for annotated introns changes between WT and the three NMD defective strains . Note that annotated introns have a higher median and mean read support in WT, than in any knockout strain. This changes among the unannotated introns in Panel B.Panel B shows the read support for predicted, unannotated introns. By requiring read support in at least two knockout strains, we gain confidence for each predicted intron. The negative fold change from knockout to WT is a result of efficient NMD in WT. This confirms the findings of Kawashima et al.In panel C we show all the predicted introns that are supported by at least two reads in WT, and by at least one read in one of the knockout strains. As a result, we are confident about the predicted introns in WT and able to compare them to the knockout strains. The boxplot clearly shows that the lack of NMD (in the knockout strains) has little or no effect on isoforms that are confidently expressed in WT. Consequently, such isoforms are unlikely targets of NMD in WT and, therefore, more likely functional. Also, there are other nuclear RNA decay pathways than NMD that might target those transcripts.Scatter plot D shows the same data as box plot B (including outliers): for each predicted intron, the WT read support is plotted against the mean read support of the knockout strains. In particular, the predicted introns with high support in WT do not show a significant fold change, so those transcript isoforms are not depleted in wildtype S. cerevisiae and, therefore, unlikely NMD targets.Unfortunately, a large scale comparison of the predicted isoforms based on our original dataset is not feasible, because the overlap of isoforms between the different datasets is not sufficiently large see .(PDF)Click here for additional data file.S4 FigSome additional evidence for a predicted intron would be its validation in a different, independent dataset. We successfully validated the most confident intron predictions by detecting their split reads in data from Waern and Snyder . In thisρ = 0.71). Among the unknown splice junctions (red circle), the correlation drops (ρ = 0.43); finally, those with a non-canonical splice signal (blue x) tend toward lower read support in general and the read support appears not to be correlated (ρ = −0.01).Figure on top: Evidently, annotated introns show high read support in either dataset and their read support seems to be correlated among datasets (Pearson’s ρ = 0.16), qualifying them as possible read or mapping errors. However, there are some potential introns covering long stretches of the S. cerevisiae genome. Some of these can also be validated in the independent dataset (green crosses around the center of the plot). These read mappings can either be explained by genomic rearrangements in the used strains or point toward post-transcriptional mechanisms in S. cerevisiae. Since there is no known mechanism explaining such transgenic splicings, the presence of these transcriptional products needs to be validated by other means.In the figure at the bottom we additionally show potential introns that do not pass our length filter (green x). The majority of those long introns shows a low read support and no correlation among datasets ((PDF)Click here for additional data file.S1 FileSome predicted isoforms might be translated into functional protein isoforms. In this file we list an in silico translation of all S. cerevisiae genes for which we predict alternative isoforms. The sequences might aid further research on the respective genes. In each record, the first line reports the gene along with the genomic location. The next line reports the predicted intron, followed by all annotated introns of the gene. Next, the length difference of the annotated and the predicted alternative transcript is given in bases: negative values indicate deletion and positive values indicate insertion events. Finally, the transcript and translated protein sequence are reported for the gene as annotated (ORI) and for the predicted isoform (VAR).(TXT)Click here for additional data file.S5 Fig(A) Split reads defining an intron. Reads r1 through r4 are mapped to the genome in a spliced form; the potential intron is supported by 4 reads. Reads r1 and r2 represent the same fragment, because they have exactly the same sequence and hence the same start and end position on the genome. As a result, the potential intron is supported by three distinct fragments. (B) Intron retention. The total read coverage (black) drops in the area of the intron and the intronic area is spanned by split reads (green). Ungapped (red) reads containing the 3’ or 5’ splice site are evidence of intron retention. (C) Alternative 3’ splice site. The total read coverage is lower in the intron, but shows an increase towards the 3’ site. There are two different types of overlapping split reads (green and blue), using the same 5’ splice site, but different 3’ splice sites. (D) Alternative 5’ splice site. Similar to alternative 3’ splice sites, but mirrored. (E) 5’ UTR intron. The intron is located before the start codon and the coding sequence (yellow) starts downstream. (F) Novel terminal intron. The intron overlaps the annotated stop codon and splices it out, extending the coding sequence to the next downstream stop codon.Alternative transcript models together with their idealized read coverage in next generation sequencing data. The black line represents the total read coverage of all mapped reads. The green, red and blue lines represent characteristic reads for certain isoforms. (PDF)Click here for additional data file.


Sporadic inclusion body myositis (sIBM) is the commonest idiopathic inflammatory muscle disease in people over 50 years old. It is characterized by slowly progressive muscle weakness and atrophy, with typical pathological changes of inflammation, degeneration and mitochondrial abnormality in affected muscle fibres. The cause(s) of sIBM are still unknown, but are considered complex, with the contribution of multiple factors such as environmental triggers, ageing and genetic susceptibility. This review summarizes the current understanding of the genetic contributions to sIBM and provides some insights for future research in this mysterious disease with the advantage of the rapid development of advanced genetic technology. An international sIBM genetic study is ongoing and whole-exome sequencing will be applied in a large cohort of sIBM patients with the aim of unravelling important genetic risk factors for sIBM. Sporadic inclusion body myositis (sIBM) is the commonest form of idiopathic inflammatory myopathy among individuals aged over 50. It has The clinical diagnosis of sIBM is confirmed by muscle biopsy assisted by the clinical phenotype, electromyography (EMG), determination of serum muscle-enzyme levels and muscle imaging with MRI. CurrentsIBM is a complex disease. The primary pathogenic mechanism is still hotly debated and it is believed that many factors including environmental factors contribute to the disease process. There are many clinically and/or histologically sIBM-like muscle diseases with known genetic defects, which may provide clues to understand the pathogenesis of sIBM. In this review we will comprehensively summarize known and candidate genetic susceptibility risk factors for sIBM.. Human leukocyte antigen (HLA) class II genotypes were reported in a strong association with some families, particularly the DR3 allele (DRB*0301/0302) in four western families[DR15(2)/DR4 (DRB1*1502/0405) in two Japanese sisters[There are several IBM case reports of two or more affected siblings in the same family-26 and ofamilies,25 and tHereditary inclusion body myopathies (hIBM) include several autosomal-recessive and autosomal-dominant muscle disorders with various clinical presentations, but with a number of pathological features similar to those of sIBM, including rimmed vacuoles, protein accumulations and tubulofilaments on electron microscopy. Patients with hIBM can be distinguished from sIBM by the earlier age of onset, rarity of inflammatory features and negative MHC class I expression, hence the term ‘myopathy’ instead of ‘myositis’. The hIBBeyond the above conditions, there are various other neuromuscular disorders showing rimmed vacuoles in the muscle tissue, which make the differential diagnosis of sIBM even wider. Mutated genes have also been found in these called ‘rimmed vacuolar myopathies’, such as oculopharyngeal muscular dystrophy (OMIM#164300), X-linked recessive Emery-Dreifuss muscular dystrophy OMIM#310300), limb girdle muscular dystrophy (LGMD) type 2G (OMIM#601954), LGMD type 1A (OMIM#159000), LGMD type 1G (OMIM#609115), and rigid spine syndrome (OMIM#602771). Some in0300, limMYH2 (gene associated with hIBM3) significantly increased the risk of developing sIBM (RR = 12.2). However, none of the patients with that variant reported joint contractures or external ophthalmoplegia, a family history of IBM or consanguineous marriage[MYH2 gene mutation described in non-familial cases. Immunohistochemical studies of myosin heavy chain isoforms showed atrophy or loss of muscle fibres expressing myosin heavy chain IIa or IIx, which may provide important clues for establishing the pathogenicity of this variant. In the same study, a novel missense mutation c.1719G > A (p.V566M) in the LIM domain binding 3 gene , coding for Z-band alternatively spliced PDZ-motif-containing protein, was reported in one of the patients[LDB3 mutations might cause abnormalities in the Z-band in skeletal muscle resulting in the disease observed in this patient. Further studies with a larger number of cases will be needed to clarify the contribution of these genes to the pathogenesis of sIBM. In addition, investigations should be carried out on more hereditary myopathies candidate genes, including α-B crystalline (CRYAB), dystrophin (DMD) and myotilin (MYOT) in the future.Overall, these common features raise the possibility that sIBM and these diseases share certain etiologic factors. Indeed, a recent study, reportemarriage. This wapatients. This fiCurrently there are two most popular theories for the pathogenesis of sIBM – an autoimmune pathway and a degeneration pathway. In addition, ageing is also considered an important factor contributing to mitochondrial abnormalities Figure .+ T cells and T cell-mediated cytotoxicity, which ultimately result in damage or death of muscle fibres[The theory of an inflammation pathway stands for viral or inflammatory triggering factors leading to the clonal expansion of CD8e fibres. This the fibres. Degenere fibres. The dege fibres.However, sIBM is poorly responsive to even vigorous immunosuppression. Even where there is histopathological evidence that inflammation was reduced, this was not accompanied by clinical improvement. Therefore, some investigators are supporting a degenerative hypothesis over inflammation as the primary pathogenesis of sIBM. The identification of aberrant protein aggregates in sIBM vacuolated muscle fibres has shown the remarkable parallels of those features in brain tissue of Alzheimer’s disease (AD) and Parkinson’s disease with Lewy bodies. This theory suggests that inflammation is secondary to the degeneration-associated processes in sIBM muscle fibres includinDespite sIBM not being an inherited Mendelian disease, multiple genetic risk factors are being shown likely to play important roles in the development and progression of sIBM. A list of possible susceptibility genes that could be important candidate genes for understanding the pathogenesis of sIBM is shown in Table HLA-DR3 and the extended 8.1 ancestral haplotype (AH) with sIBM[HLA-A*01, -B*0801, -DRB1*0301, -DRB1*0101, -DRB1*1301, -DQB1*0201, -DQA1*05[HLA-DRB1*0301 and *1301 also influence on the age at onset and the severity of muscle weakness[HLA-DRB1 locus may contribute to both disease susceptibility and clinical phenotype. Other alleles and haplotypes have also been associated with increased risk of sIBM, including the 35.2AH  in Caucasians[HLA-B*5201, DRB1*1502) in Japanese[HLA-DR53 and HLA-DRB1*0401, *0701, *0901, *1101, *1501, HLA-DRB1*0301/*0401 and HLA-DRB1*0301/*0701 diplotypes, and HLA-DRB1*1501, were found protective for sIBM[Garlepp and colleagues first reported the strong association of ith sIBM. This as -DQA1*05-65. Furtucasians and the Japanese. Interesfor sIBM,65,67.HLA-DR and C4[HLA-DRB1 and pre-B-cell leukemia homeobox2 [BTNL2) to telomeric of HLA-DRB1 including three protein-coding genes – part of BTNL2, HLA-DRA and HLA-DRB3[NOTCH4; a transmembrane receptor which regulates cell fate decisions), advanced glycosylation end product-specific receptor gene , testis-specific basic protein gene , and G-protein signalling modulator 3 gene [A potential region of MHC-associated susceptibility genes was first defined between DR and C4, and thes HOX12). A furth HLA-DRB3. Other g as G18),63.NOTCH4 gene polymorphisms (rs422951 and rs72555375) have shown a strong association (OR > 2) with sIBM in two independent Caucasian cohorts[Two  cohorts. These v cohorts.The MHC contains important genetic contents which have been reported as being associated with sIBM and among them 8.1AH which is predominant in Caucasians, supporting an immune-mediated mechanism underlying sIBM. Identification of the actual genes within the MHC will be crucial to understand the role of MHC in the pathogenesis of sIBM.NT5C1A) was recently identified in sIBM by two independent groups[NT5C1A and NT5C1B genes[An autoantibody against the cytoplasmic 5′-nucleotidase 1A , and AβPP-mRNA in both sIBM and hIBM muscle fibres[APP gene in sIBM have not been fully understood. The fact that mutations in exons 16 and 17 of APP gene have been found to be pathogenic in AD, resulting in early-onset familial AD, suggests that mutations in this gene might also play a role in sIBM. In line with a previous small study[APP mutations were found in our 58 sIBM cases.It has been widely recognized that there are abnormal accumulations of beta-amyloid (Aβ) protein, N-terminal and C-terminal of beta-amyloid precursor protein  and presenilin 2 (PSEN2) genes are also found to be responsible for early onset familial AD, with mutations in PSEN1 gene as the most common cause[PSEN1 and PSEN2 have not been investigated in either sIBM or hIBM cases.Abnormal accumulation of presenilin proteins occurs in the brains of both sporadic and familial AD patients. Autosomal dominant inheritance of mutations in presenilin 1 [DYSF gene might link to the pathogenesis of sIBM though the possible mechanisms is unknown[Dysferlin is a modular type II transmembrane protein newly identified as a binding partner of AβPP that co-aggregates with Aβ42 in sIBM muscle fibres. In norm(LGMD2B). Similar(LGMD2B). In addi unknown.APOE ϵ4 allele is recognized to be an important risk factor for developing late-onset AD. Several studies have been carried out to investigate the roles of APOE alleles in sIBM. Only one study of a cohort of 14 Australian sIBM patients suggested that ϵ4 allele may be a susceptibility factor for the development of sIBM[APOE ϵ4 allele as a susceptibility factor for developing sIBM. However, the authors demonstrated a non-significant trend towards an earlier age at onset in patients with the APOE ϵ2 allele[ApoE protein deposits but not mRNA have been identified in both sIBM and hIBM muscle fibres,83. The  of sIBM. However of sIBM,51,85-87 of sIBM, includi2 allele.APOE ϵ4 allele. Interestingly, we found a statistically significant decreased frequency of APOE genotype ϵ2/ϵ3  in the patient group compared with our British control individuals (unpublished data), and thus we found an association between ϵ2 allele and a reduced risk of developing sIBM  is consistent with the above meta-analysis with respect to the association with MAPT) gene mutations have been identified in many different tauopathies including AD, frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The majority of them affect exons 9–13[MAPT mutations, a common extended haplotype (H1) in MAPT appears to be a risk factor for sporadic PSP[MAPT intron 9 is inherited as a part of the less common H2 sub-haplotype[del-In9) in 81 sIBM DNA cases and 159 British healthy controls, but no significant differences were found between the two groups.Accumulation of phosphorylated-tau is widely known to be another major protein present in sIBM muscle aggregates, which is strikingly similar with those found in taupathies which share hyperphosphorylated deposited tau protein in the brain. Differeons 9–13. In addiadic PSP. A 238 baplotype. We analSERPINA3, previously known as ACT) is considered a major acute-phase protein and belongs to the serpin superfamily specifically inhibiting serine proteases. Similarly to AD brains, abnormal accumulations of α1ACT, located closely with β-amyloid protein containing ubiquitinated amyloid plaques, have been described in sIBM muscle[SERPINA3/AA (rs4934) was found to alter the AD risk associated with APOE ϵ4/ϵ4 genotype[APOE ϵ4/ϵ4 genotype.Alpha-1-antichymotyrpsin  gene was more prevalent in sIBM , when compared with controls[Prion protein and mRNA  have been described as being abnormally accumulated in vacuolated muscle fibres in both sIBM and hIBM,93. Lampcontrols. But thicontrols and our TARDBP have been detected in both sporadic and familial forms of ALS[TARDBP gene have not yet been examined in a sIBM.A predominantly nuclear heterogeneous nuclear ribonucleoprotein (hnRNP) TAR DNA-binding protein-43 (TDP-43) is found as a major component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD-U). Mutations in s of ALS. Severals of ALS,96-98 has of ALS. Similars of ALS. The simhnRNPA1 and hnRNPA2B1 have been identified in familial multisystem proteinopathy with similar myonuclei depletion and sarcoplasmic aggregates[Abnormalities in the distribution of two other hnRNPs, hnRNPA1 and hnRNPA2B1, were identified in sIBM muscle fibres. Similargregates. These gC9orf72 has been identified as the most common genetic cause of familial ALS, FTLD or a combination of both phenotypes and TDP-43-based pathology (FTLD-TDP)[C9orf72 may involve disrupted protein degradation[C9orf72 might also play a role in the pathogenesis of sIBM. Fifty-eight sIBM cases were analysed for C9orf72 repeat by our group, but no expanded repeats were found.An expanded GGGGCC hexanucleotide repeat in the noncoding region of TLD-TDP). In a reradation. TherefoTYMP, previously known as ECGF1), solute carrier family 25  member 4 gene , chromosome 10 open reading frame 2 (C10orf2) and polymerase gamma 1 (POLG1), which are important for mtDNA maintenance and replication[Mitochondrial abnormalities are another important pathological feature in sIBM muscle biopsies, consisting of ragged-red fibres and mostly showing enzyme histochemical deficiency of COX activity. These changes are more prevalent in sIBM than in polymyositis, dermatomyositis and normal ageing muscle fibres. It is tlication. Putativlication,104. ButHLA-DR3 positive[HLA-DR3, which is in a genomic region strongly associated with sIBM. Further studies are required to investigate whether this variant plays a pathogenic role in sIBM and/or its possible interaction with other genetic factors.In addition to mtDNA deletions, mutations at mtDNA nucleotide positions 3192, 3196, 3397 and 4336, which are associated with late-onset AD, are possible risk factors for sIBM. In sIBM cases only the frequency of the common 16311C variant has been found more frequent than in AD and controls, but these differences were not statistically significant. Interespositive, suggestTOMM40) which is adjacent to and in linkage disequilibrium with the APOE locus on chromosome 19, has been implicated in AD[TOMM40 encodes an outer mitochondrial membrane translocase facilitating the transport of unfolded proteins such as amyloid-β from the cytosol into the mitochondrial intermembrane space[TOMM40 gene together with the APOE genotypes has been shown to influence disease susceptibility of AD[APOE ϵ3 allele with a very long (VL) polyT repeat alleles in TOMM40 had reduced risk of sIBM compared to controls, and this was also associated with a later age at onset of symptoms[TOMM40 and/or APOE to influence disease susceptibility. This also comes to a hypothesis that genetic variants of TOMM40 could be associated with altered mitochondrial pore function and transport of proteins into mitochondria. This could result in energy metabolism changes and increased reactive oxygen species formation, further contributing to the impairment in mitochondria and degeneration in muscle fibres[Recently a gene called ‘Translocase of Outer Mitochondrial Membrane 40’ (ed in AD. TOMM40 ne space. A polyTty of AD. It has symptoms. The rs1e fibres.TREM2 were recently found to be associated with increased risk of Alzheimer’s disease by using whole-genome and whole-exome sequencing (WES)[HLA gene, fragment analysis for repeat variants, and comparative genomic hybridization (CGH) analysis for copy number variants which cannot be detected by WES.Over the past five years, next-generation sequencing technologies have already shown considerable promises in the identification of rare variants associated not only with Mendelian but also with complex diseases. For example, rare variants in ng (WES). Up to rng (WES). Therefong (WES), but no In the future, besides genetic defects identified in sIBM, RNA sequencing in muscle tissue and lymphoblast lines will identify transcriptomic profiles and expression quantitative trait loci (eQTL) in sIBM and other inflammatory myopathies. The application of RNA sequencing will also allow us to study and dissect splicing abnormalities which may not be detected by exon arrays. Investigating the transcriptomic and genomic factors will be important in revealing dysregulated molecular pathways in these disorders, identifying a diagnostic signature in blood and revealing biomarkers for future therapeutic strategies. A searchable database to the research community is also planned to build up to integrate WES, RNA sequencing and eQTL data, further to aligned with the biobank database with disease progression, response to treatment and muscle imaging.In conclusion, sIBM remains an enigmatic muscle disorder even though it has been about half a century since the first case was described. The primary pathogenesis of sIBM is still controversial, but it is more likely to due to multifactorial mechanisms, including immune changes, degenerative changes, mitochondrial abnormalities and the ageing milieu in the muscle fibres, which involve a complex interaction between environmental triggers  and genetic susceptibility. Despite the strikingly phenotypic similarities between sIBM muscle fibres and the brains of AD, research on the candidate genes encoding the deposited proteins have not shown any positive result so far. Genes located within the MHC region are still the most strongly associated candidates with sIBM. Genes linked to hIBM and pathologically sIBM-like myopathies may provide important clues for sIBM genetic research. Effective WES and RNA sequencing analysis of a large sIBM cohort will significantly contribute to our knowledge in this under-explored area.References for this Review were identified by searches in PubMed between 1969 and May 2014. The search terms “inclusion body myositis”, “inclusion body myopathy”, “IBM”, “rimmed vacuolar myopathy”, and “inflammatory myopathy” were used. References were also identified from the bibliography of the identified articles. Only papers published in English or with available English translations of relevant data were reviewed. The final reference list was generated on the basis of relevance to the topics covered in this Review.The authors declare that they have no competing interests.QG contributed to the literature search and to drafting the manuscript. All authors contributed to the critical revision process and approved the final manuscript. MGH, HH, and PM are principal investigators of the International IBM Consortium Genetic Study (IIBMCGS).


With rising energy concerns, efficient energy conversion and storage devices are required to provide a sustainable, green energy supply. Solar cells hold promise as energy conversion devices due to their utilization of readily accessible solar energy; however, the output of solar cells can be non-continuous and unstable. Therefore, it is necessary to combine solar cells with compatible energy storage devices to realize a stable power supply. To this end, supercapacitors, highly efficient energy storage devices, can be integrated with solar cells to mitigate the power fluctuations. Here, we report on the development of a solar cell-supercapacitor hybrid device as a solution to this energy requirement. A high-performance, cotton-textile-enabled asymmetric supercapacitor is integrated with a flexible solar cell via a scalable roll-to-roll manufacturing approach to fabricate a self-sustaining power pack, demonstrating its potential to continuously power future electronic devices. Activated cotton textiles are promising for wearable energy storage due to their flexibility and high conductivity, but scalability is an issue. Here, the authors combine supercapacitors and solar cells on activated cotton textiles via scalable roll-to-roll assembly to make self-sustaining power packs. With ever-increasing global energy consumption and the depletion of fossil fuels, finding a sustainable and clean energy supply has become one of the most important scientific and technological challenges facing humanity todayCompared with lithium-ion batteries, the relatively low energy density of supercapacitors limits their wide application. Developing new electrode materials with well-defined nano-architecture and high active surface area is key to improving the electrochemical performance of supercapacitors. To date, according to the different mechanisms of charge storage, typical supercapacitor electrode materials can be categorized into electric double-layer capacitive materials including various carbon materials3567910IIx1-MIIIx(OH)2]x+[An−]x/n·mH2O, where MII and MIII are divalent and trivalent metal cations and An− are the charge-balancing anions1921et al. reported that nickel-aluminium LDH (Ni-Al LDH) deposited on nickel foam exhibits an ultrahigh specific capacitance of 2,123 F g−1 at 1 A g−1  film had a significantly enhanced specific capacitance of 2,682 F g−1 at 3 A g−1  and a capacitor-type electrode as power source 13g−1 ref. . In our Consumer demand for portable/wearable electronics has triggered a technological race to drive innovation in flexible mobile phones, bendable displays, electronic skin and distributed sensors34353738394344in situ on flexible ACTs to realize ACT/Co-Al LDH hybrid by a facile hydrothermal method. A highly conductive graphene coating is wrapped around ACT fibres to form ACT/graphene composite by a simple dipping, drying and reducing process. A flexible, all-solid-state asymmetric supercapacitor (ACT/Co-Al LDH//ACT/graphene) is assembled using the nanostructured ACT/Co-Al LDH as the positive electrode, ACT/graphene as the negative electrode, and PVA-KOH gel as both the solid-state electrolyte and separator. The asymmetric cell works synergically to achieve excellent electrochemical performance in terms of its working potential (1.6 V), energy density (55.04 Wh kg−1), power density (5.44 kW kg−1) and cycling stability . Moreover, we demonstrate a practical roll-to-roll manufacturing approach to combine the flexible asymmetric supercapacitor with a flexible solar cell to fabricate an integrated self-sustaining power pack, which is scalable for industrial manufacturing. Importantly, the assembled power pack can power a commercial light-emitting-diode (LED) continuously, with or without sunlight, demonstrating its potential for flexible, self-powered energy devices.Cotton textile, a source of flexible, ‘green', renewable, breathable clothing, has been shown to be an excellent wearable platform for constructing flexible energy storage devices as activated cotton textiles (ACTs) exhibit eminent flexibility and excellent conductivity46in situ on ACT fibres using a facile hydrothermal method (step ii), which resulted in a pink coating on the surface of ACT fibres. in situ anchored on ACT fibres not only enhances the contact between the active material and ACT substrate but also serves as a reservoir for electrolyte ions, shortening ion diffusion path and facilitating charge transfer, jointly enhancing the electrochemical performance.2 g−1. A lattice spacing of 0.26 nm (The structure and morphology of the Co-Al LDH nanosheets were further investigated by transmission electron microscopy (TEM) and X-ray diffraction (XRD). TEM inspection  reveals  0.26 nm  can be a 0.26 nm . The cor 0.26 nm  shows he 0.26 nm  shows a −1) were first prepared by direct conversion of a cotton T-shirt. As shown in π–π stacking or hydrogen bonding49d-spacing of ∼0.37 nm in the HRTEM image suggest the existence of defects in the graphene nanosheets. The crystalline nature of the graphene nanosheets was validated by the well-defined diffraction spots in the SAED pattern. More importantly, the ACT fibres showed a porous structure as revealed by the TEM image  compared with the ACT/graphite oxide with the ID/IG ratio of ∼1.03. The increased ID/IG ratio is ascribed to the re-establishment of graphene network during the reduction process, indicating smaller sizes, more defects and disordered structures of the graphene1453The as-prepared ACT/Co-Al LDH was used as the positive electrode, ACT/graphene as the negative electrode and the PVA-KOH gel as both the solid electrolyte and separator to construct a flexible asymmetric supercapacitor. The energy storage performances of the assembled cells and individual electrodes were studied by cyclic voltammetry (CV), galvanostatic (GV) charge/discharge and electrochemical impedance spectroscopy (EIS) in both two-electrode and three-electrode testing systems.−1 with the potential windows ranging from −0.2 to 0.6 V are shown in The three-electrode testing system was used to study the electrochemical reaction mechanisms of the as-prepared ACT, ACT/graphene and ACT/Co-Al LDH electrodes. The CV curves of the aforementioned electrodes at the scan rate of 25 mV s−1. Compared with the triangular-shaped charge/discharge curves of the ACT and ACT/graphene electrodes, the ACT/Co-Al LDH electrode exhibited two charge/discharge voltage plateaus, which are the pseudocapacitive plateaus from the Co-Al LDH. The specific capacitance values of the ACT, ACT/graphene and ACT/Co-Al LDH electrodes were measured to be 90.1, 136 and 977.3 F g−1, respectively. The CV curves of the ACT/Co-Al LDH electrode at the scan rates of 25, 50, 75 and 100 mV s−1 in 6 M KOH electrolyte solution are shown in −1 are 977.3, 863, 727 and 545 F g−1, respectively, with a capacitance retention of ∼55.8% even when the current density increased from 2.5 to 20 A g−1.−1. The rectangular-shaped CV curves of the ACT/graphene symmetric cell suggest that the ACT/graphene mainly worked as an electrochemical double-layered capacitor. The broad peak at 0.25 V of the ACT/Co-Al LDH symmetric cell indicates the Faradic reaction from the battery-type Co-Al LDH. The CV area of the ACT/Co-Al LDH//ACT/graphene asymmetric cell was much larger than that of the ACT/graphene//ACT/graphene and ACT/Co-Al LDH//ACT/Co-Al LDH symmetric cells, suggesting the larger specific capacitance of ACT/Co-Al LDH//ACT/graphene asymmetric cell resulting from the high accessibility of electrolyte ions. −1 for the ACT/Co-Al LDH//ACT/graphene asymmetric cell, ACT/Co-Al LDH symmetric cell and ACT/graphene symmetric cell at the current density of 12.5 mA cm−2, respectively. −1. The working voltage of the ACT/Co-Al LDH//ACT/graphene asymmetric cell can be extended to 1.6 V, which is essential for practical application. −1 at an operation window of 1.6 V. The current density increases with increasing scan rate. All the CV curves at different scan rates in −1 was achieved even when the current density increased from 7.5 to 50 mA cm−2, indicating the excellent rate performance of the ACT/Co-Al LDH//ACT/graphene asymmetric cell. The rate performance and corresponding coulombic efficiency of ACT/graphene//ACT/graphene and ACT/Co-Al LDH//ACT/Co-Al LDH symmetric cells as well as ACT/Co-Al LDH//ACT/graphene asymmetric cell are comprehensively compared in −2) in a wide range of current densities (7.5–50 mA cm−2). Whereas the ACT/Co-Al LDH symmetric cell showed poor rate performance (35% capacitance retention) and low coulombic efficiency (91.8% at 50 mA cm−2). Compared with the ACT/Co-Al LDH symmetric cell, the ACT/Co-Al LDH//ACT/graphene asymmetric cell exhibited not only improved capacitance retention (52.3% capacitance retention) but also enhanced coulombic efficiency (98.5% at 50 mA cm−2). At high charge/discharge rates the ions on electrode decreased rapidly with increasing rate, and the ions in the electrolyte diffused too slowly to satisfy the need of ions near the solid–liquid interface, leading to the decrease of capacitanceRate performance and coulombic efficiency are important factors for the real power application of supercapacitors. −1 at the power density of 387.9 W kg−1 and maintained 28.72 Wh kg−1 at the power density of 5.44 kW kg−1. For an in-depth understanding of the electrochemical behaviour of the assembled symmetric and asymmetric cells, the EIS tests were carried out on the aforementioned cells (Cdl) and the charge transfer resistance (Rct) at the electrode and electrolyte interface, corresponding to the charge transfer-limiting processRct was directly measured from the diameter of the semicircle arc in the Niquist plot. Clearly, compared with the ACT/graphene (1.1 Ω) and ACT/Co-Al LDH (1.89 Ω) symmetric cells, the relatively smaller Rct of the asymmetric cell resulted from the synergistic effect of the ACT/graphene and hierarchical ACT/Co-Al LDH architectures that facilitated the access of electrolyte ions to the active surface and shortened the ion diffusion path.Energy density and power density are important factors for evaluating the practical application of supercapacitors. ed cells . A simil−2 using a GV charge–discharge technique. An obvious capacity decay (∼35%) was observed for the ACT/Co-Al LDH symmetric cell, whereas there was a capacity increase (∼8%) for the ACT/graphene symmetric cell after 2,000 cycles. For the ACT/Co-Al LDH//ACT/graphene asymmetric cell, ∼16% decay of its original capacitance was noted after 2,000 cycles. The asymmetric cell exhibited linear and symmetrical characteristics in its charge/discharge curves, and no obvious ‘IR drop' was observed, indicating its low internal resistance . A flexible, all-solid-state asymmetric supercapacitor (ACT/Co-Al LDH//ACT/graphene) was assembled using the nanostructured ACT/Co-Al LDH as the positive electrode, ACT/graphene as the negative electrode and PVA-KOH gel as both the solid-state electrolyte and separator. The hierarchical flower-like Co-Al LDH nanoarrays with interconnected nano-petals and pompon-like nano-stamens provided a highly open and porous scaffold-like structure, facilitating the transportation of electrolyte ions. In addition, the wrapped graphene on porous ACTs rendered a highly accessible surface area and good electrical conductivity, endowing the flexible asymmetric supercapacitor with higher rate performance. The assembled ACT/Co-Al LDH//ACT/graphene asymmetric supercapacitor exhibited an excellent combination of electrochemical and mechanical performance. Moreover, we demonstrated a low-cost, roll-to-roll manufacturing approach to combine the flexible asymmetric supercapacitor with a flexible solar cell to build an integrated, self-sustaining power pack, which is scalable for industrial manufacturing. Importantly, such hybrid energy storage devices could continuously power a commercial LED, demonstrating a great potential for the future of self-powered nanotechnology.Two-dimensional battery-type Co-Al LDH nanosheets were anchored in situ on ACT fibres via a simple hydrothermal process. Briefly, 0.582 g of Co(NO3)2·6H2O, 0.518 g of Al(NO3)3·6H2O, 0.296 g of NH4F and 0.6 g of urea were dissolved in 36 ml distilled water. The resulting solution was transferred into a 50-ml Teflon-lined stainless autoclave with a piece of vertically suspended ACT (1 × 2 cm2) in the solution. Then, the autoclave was placed in an electric oven at 100 °C for 24 h. Finally, the as-prepared products were washed thoroughly with ethanol and distilled water and dried at 80 °C overnight to produce ACT/Co-Al LDH composite.All of the chemicals were used after purchasing without further purification. A piece of commercial cotton T-shirt was first cleaned using distilled water in an ultrasonic bath before activation. Activation of the cotton T-shirt was performed as described in detail in our previous report−1. A piece of ACT (1 × 2 cm2) was then soaked with the graphene oxide aqueous colloid. After drying at 80 °C for 6 h, the as-prepared ACT with graphene oxide coating was heated at 450 °C for 1 h with argon/hydrogen mixture gas  to reduce flexible ACT/graphene oxide to ACT/graphene-negative electrode.Graphite oxide was synthesized by exfoliating natural graphite flakes using a modified Hummers methodKα radiation . The Raman spectra of the ACT/graphene were recorded by a Renishaw InVia Raman microscope at 785 nm. Surface areas of the active materials were measured by physical adsorption of N2 at 77 K  and calculated by the BET method.The morphology and microstructure of the as-prepared products were characterized by SEM (FEI Quanta 650), TEM (JEOL 2000FX), HRTEM (FEI Titan), and atomic force microscopy (Nanoscope IIIa). The crystallographic structure of the synthesized materials was determined by a PANalytical X'Pert Pro Multi-Purpose Diffractometer equipped with Cu 2) and a saturated calomel electrode were used as the counter and reference electrodes, respectively. The mass of the ACT, ACT/graphene and ACT/Co-Al LDH was measured to be 2, 3.45 and 4.1 mg cm−2, respectively. The asymmetric supercapacitor used in this paper was assembled with two pieces of flexible, face-to-face electrodes (ACT/Co-Al LDH-positive and ACT/graphene-negative) separated by the solid-state, polymer PVA/KOH gel electrolyte (ACT/Co-Al LDH//ACT/graphene). The polymer gel electrolyte was prepared by mixing 3 g KOH and 6 g PVA in 60 ml deionized water by magnetically stirring the solution at 80 °C until the solution became clear. Then, the above solution was transferred into a flat Petri-dish to naturally solidify to PVA/KOH gel film electrolyte. Two pieces of flexible electrodes separated by the PVA/KOH gel film were used to assemble the flexible asymmetric cell. The as-obtained solid-state, polymer PVA/KOH gel film severed as both the separator and electrolyte. The electrochemical properties of the assembled flexible asymmetric cell were measured using a CHI 660E electrochemical workstation. CV, GV charge/discharge curves and EIS in the frequency range from 100 kHz to 0.05 Hz with an AC perturbation of 5 mV were used to evaluate the electrochemical performance of the flexible solid-state asymmetric supercapacitors. The integrated flexible energy conversion and storage unit was assembled by combining a piece of commercial flexible solar panel  with an energy storage unit consisting of two pieces of asymmetric cell connected in series using an electrical rolling machine . Two pieces of assembled asymmetric flexible supercapacitors were connected in series and then coated with double-side adhesive tapes. At the same time, the bottom surface of the flexible solar cell was also coated with double-side adhesive tapes. As illustrated in The electrochemical performances of the prepared ACT, ACT/graphene and ACT/Co-Al LDH electrodes were characterized by both three-electrode and two-electrode systems. The three-electrode tests were carried out in 6 M KOH aqueous electrolyte at room temperature. The prepared ACT, ACT/graphene and ACT/Co-Al LDH were used as the working electrodes, platinum foil , I (A), t (s), m (g) and ΔV (V) are the specific capacitance, the discharge current, the discharge time, the mass of the active material and the total potential window, respectively.where E) and power (P) density derived from GV tests were calculated from the following equations:Energy density (E (Wh/kg), C (F/g), V (V), P (W/kg) and t (s) are the energy density, specific capacitance, potential window, power density and discharge time, respectively.where How to cite this article: Gao, Z. et al. Cotton-textile-enabled flexible self-sustaining power packs via roll-to-roll fabrication. Nat. Commun. 7:11586 doi: 10.1038/ncomms11586 (2016).Supplementary Figures 1-3.


Activation of the HSR clears insoluble TDP-43 and increases cell survival.Insoluble TDP-43 inclusions are the pathological hallmark of ALS and tau-negative frontotemporal lobar degeneration. Chen  TARDBP) neuronal cytoplasmic inclusions are the pathological hallmark in ∼95% of amyotrophic lateral sclerosis and ∼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a  as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with sporadic amyotrophic lateral sclerosis. This implies that the HSF1-mediated DNAJB2a/HSP70 heat shock response pathway is compromised in amyotrophic lateral sclerosis. Defective refolding of TDP-43 is predicted to aggravate the TDP-43 proteinopathy. The finding that the pathological accumulation of insoluble TDP-43 can be reduced by the activation of HSF1/HSP pathways presents an exciting opportunity for the development of novel therapeutics.Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron degenerative disease, characterized by progressive motor neuron loss from the spinal cord, brainstem and motor cortex, leading to muscle weakness and eventual respiratory failure. The average age of disease onset is 55 years and affected individuals typically die within 3 to 5 years of diagnosis. No effective treatments are available and consequently there is a high unmet need for novel therapeutic development  and this process is facilitated by molecular chaperones. When cells are acutely stressed by insults that affect proteostasis, such as heat shock, they induce a transcriptional programme orchestrated by the transcription factor heat shock factor 1 (HSF1) to upregulate selected chaperones, known as heat shock proteins (HSPs), to protect them from being overwhelmed by the accumulation of aberrant proteins. HSPs are categorized into several families according to their function and size -independent, HSP70-dependent mechanism. This suggests that the HSF1-DNAJB2a pathway refolds TDP-43 and returns it to its natural physiological state rather than facilitating its degradation. We also demonstrated that the levels of HSF1 and HSPs are markedly reduced in both a transgenic TDP-43 mouse model and in spinal cord tissues from sporadic ALS patients. These results highlight the potential of HSF1 and HSP activation as an important therapeutic strategy for the TDP-43 proteinopathies.The green fluorescent protein (GFP)-TDP-43 constructs were generated and used in a previous study A were gPrimary antibodies used in this study included: rabbit anti-ubiquitin, K48-specific ; mouse anti-phospho TDP-43 ; rabbit anti-mouse TDP-43 . Cells were left for 48 h after transfection to be harvested for analysis unless otherwise stated.HEK293T and SH-SY5Y cells were cultured using Dulbecco’s modified Eagle medium (DMEM) and DMEM/F12 (Life Technologies) supplemented with 10% foetal bovine serum (Life Technologies), and maintained at 37°C, 5% COWTand TDP-43Q331Kmice as previously described  Act 1986, and were approved by the Kings College, London ethics review panel. Compound transgenic mice were generated by crossing TDP-43WTxQ331Kanimals and their single and non-transgenic littermates (n = 3–4) or 50 mg of spinal cord post-mortem tissues from controls or patients with sporadic ALS were homogenized in the appropriate amount of RIPA buffer , sonicated and stored at −80 °C. Protein concentration was determined with DC™ Protein Assay Kit (Bio-Rad). Western blotting was run with 10 μg of protein lysate.Whole spinal cords from 8-week-old TDP-43WTxQ331Kmice and age-matched littermates were anaesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in phosphate buffer. Spinal cord was postfixed in 4% PFA in 15% sucrose for 5 h, cryoprotected in 30% sucrose for 24 h and cut into 30 µm sections on a cryostat. Sections were incubated in rabbit anti-HSF1 antibody , washed and incubated with a biotinylated anti-rabbit antibody  and then incubated in an ABC kit (Vector). Sections were imaged using a Zeiss light microscope and Axiovision software.Eight-week-old, end-stage TDP-43Cells were harvested in immunopurification buffer . After centrifuging (14 000 rpm for 30 s at 4°C), the supernatant was collected and incubated with immunopurification antibody and Dynabead protein A (Life Technologies) overnight at 4°C. The Dynabead® protein A-antibody-protein complex was purified using magnetic separation and washed with immunopurification buffer before eluted in loading buffer.gfor 20 min at 4 °C. After centrifugation, the supernatant was collected as the RIPA solubility fraction. The pellet, after being washed once with RIPA buffer, was then suspended in 20% of the original lysis volume with urea buffer  and collected as the insoluble, detergent-resistant fraction.The fractionation for protein solubility was performed using a protocol described byhttp://imagej.nih.gov/ij/). Integrated band intensities were normalized to that of loading control or the RIPA fraction.Protein quantification and western blotting were performed as described before  for 20 min and washed with PBS three times for 5 min. Cells were permeablized by incubation in PBS containing 0.5% Triton™ X-100 (Sigma) for 15 min at room temperature, followed by blocking in PBS containing 1% donkey serum for 1 h at room temperature. Cells were incubated with primary antibody diluted in blocking solution overnight at 4°C. After washing in PBS, cells were subsequently incubated with fluorescent secondary antibodies diluted in blocking solution for 1 h at room temperature. DAPI (Sigma) was then used to stain for nuclei before being mounted on coverslips using FluroSave .For cell survival assay, SH-SY5Y cells were trypsinized and stained with Calcein Violet 450 AM Viability Dye  for 30 min at room temperature. After washing twice with PBS, cells were resuspended in PBS and analysed with the BD FACSCanto II (BD Biosciences).This study used dominant active and negative forms of HSF1 to investigate the effect of the HSR and HSP upregulation on TDP-43 proteinopathy in cellular models. HSF1 is a transcription factor that upregulates HSPs during the stress response. In the unstressed condition, HSF1 is bound to HSP90 via its regulatory domain and is kept in an inactive form. During cellular stress, HSF1 dissociates from HSP90, trimerizes, translocates into the nucleus, and binds to the heat shock element (HSE) through its DNA binding domain (DBD), where it promotes the transcription of downstream genes via its transactive domain (TAD) A. This P < 0.001). Conversely, HSF1(−) significantly increased the level of insoluble TDP-43 by ∼250% (P < 0.001). The mutation status of TDP-43 when co-transfected with HSF1 did not significantly change TDP-43 protein solubility, nor did the presence of a mutation in TDP-43 when transfected alone (P < 0.01 for dNLS-TDP-43), suggesting the expression of HSF1(−) results in an increased tendency for overexpressed cytosolic TDP-43 to shift from soluble monomer to insoluble aggregates.The expression level of endogenous TDP-43 in our cells was not high enough to cause any proteinopathy phenotype similar to that seen in disease tissues, and was not found in the insoluble fraction A and B.d alone C. HSF1 substantially enhances the clearance of insoluble TDP-43, whereas inhibition of the HSR via HSF1(−) expression results in an increase in insoluble TDP-43, likely resulting from an inhibition of the endogenous, basal HSR.P < 0.01) and enhanced by HSF1(−) (P < 0.001). No phospho-TDP-43 staining was identified in GFP-vector transfected cells alone (P = 0.012). The genotype of the overexpressed TDP-43 had no significant impact on TDP-43 phosphorylationper se reduction in cell death. Overexpression of TDP-43 in HEK293 cells did not show cytotoxicity (data not shown), supporting the observation in ALS studies that neuronal cells are particularly sensitive to TDP-43 cytotoxicity.As reported in other cellular and transgenic animal studies, overexpression of TDP-43 can be cytotoxic to some cells, especially neurons , indicaIn summary, we have demonstrated that overexpressing TDP-43 in cells recapitulates pathological features of the TDP-43 proteinopathies and that the presence of these features results in an increase in cell death. Manipulating the heat shock response (HSR) by expression of HSF1(+) reduces this pathological TDP-43, and improves cell survival. In contrast, when cells are deprived of their basal heat shock response activity by the expression of HSF1(−), the degree of TDP-43 proteinopathy is intensified.HSF1 is a transcription factor that is responsible for the induction of a wide range of HSPs and other stress-responsive proteins upon its activation B. To deDNAJB2and also known as HSJ1a, is a target of HSF1  suffering from the most severe motor deficits and TDP-43 pathology, and a drastically reduced life span compared to both single and non-transgenic mice (To study the role of TDP-43 in the development and progression of ALS, lines of transgenic mice expressing either wild-type-TDP-43, Q331K-TDP-43 or both were established (Q331Ksingle transgenic mice that displayed a mild, non-lethal motor defect at later stage (WTxQ331Kmice (WTxQ331Kmice, whereas HSP90 remained unaffected. Immunohistochemical staining also showed reduced levels of HSF1 in the remaining motor neurons in the end-stage diseased mice spinal cord sections, (Interestingly, HSF1 was decreased in the TDP-431Kmice A. As a ctions, B indicaExamination of HSF1 and the HSPs in human tissue identified a significant decrease in HSP70 D and HSTaken together, our results suggest that the HSR is compromised in both the TDP-43 transgenic ALS mouse model and in patient tissues, which could be initiated either directly or indirectly by long term TDP-43 protein overexpression. Additionally, these results demonstrate that a reduction in the HSR leads to decreased TDP-43 refolding efficiency, promoting the accumulation of phosphorylated insoluble TDP-43.In this study, we generated a cellular model of the human TDP-43 proteinopathies that developed detergent-resistant, hyperphosphorylated cytoplasmic insoluble TDP-43 aggregates that are cytotoxic. Co-expression of a dominant positive version of the master regulator of the heat shock response HSF1 induced a dramatic clearance of insoluble TDP-43 aggregates without affecting the levels of soluble TDP-43. Conversely the dominant negative version of HSF1 lead to a massive accumulation of insoluble and hyperphosphorylated TDP-43 aggregates confirming a vital role of the HSF1-HSP axis in physiological TDP-43 proteostasis. We have shown that HSF1-induced TDP-43 clearance is partly mediated by HSP70 and its co-chaperone DNAJB2a in a proteasome- and autophagy-independent manner. We propose that DNAJB2a recognizes and binds to insoluble TDP-43 taking it to the chaperone protein HSP70, which then refolds the protein. Once it has been returned to its soluble, properly folded status, TDP-43 is then released to perform its biological functions . BecausWe have also shown that levels of several HSR proteins are reduced in transgenic mice and sporadic ALS tissues implying that the HSF1–HSP axis may be compromised due to the disease process. Q331K TDP-43 transgenic mice develop a slowly progressive motor phenotype and have moderately reduced levels of HSF1 and HSP-40, whereas the severely-affected double transgenic mice have a dramatic loss of HSF1, HSP40 and HSP70. In human spinal cord tissues the levels of HSP70 and HSP40 were significantly reduced but not HSF1.The data from our cellular, animal and human tissue studies indicate that the HSF1/HSP70/DNAJB2a pathway plays a vital role in the physiological maintenance of TDP-43 proteostasis. In the disease state, however, the HSR appears to be compromised and may contribute to the accumulation of insoluble TDP-43 protein aggregates and exacerbate defective proteostasis .Molecular chaperones are essential for maintaining proteostasis B. As thInteractions between HSPs and TDP-43 have been documented previously.TDP-43 is an aggregation-prone protein that is found to form detergent-resistant aggregates or stress granules during conditions of cellular stress such as heat shock B; howevHSP90 binds to and inactivates HSF1 by preventing it from entering the nucleus and inducing the HSR. HSP90 inhibitors such as 17-AAG have therefore been tested in cellular and animal models of neurodegeneration with some success (Supplementary DataClick here for additional data file.