Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries.In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 μm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 μm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05).Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK inhibitors accelerate endothelial repair of stented arteries by enhancing EC polarity and migration through regions of bidirectional flow. Given the essential role of EC in suppressing inflammation and thrombosis, the restoration of functional vascular endothelium is an important therapeutic goal to avoid the lethal consequences of in-stent restenosis and thrombosis.,The repair of injured vascular endothelium involves migration of EC from adjacent uninjured sites.The deployment of a stent by balloon angioplasty leads to major changes in vascular mechanics. Of note, the presence of stent struts alters local blood flow patterns thereby modifying wall shear stress (WSS),in vitro and in vivo models to study the influence of stent struts on local hemodynamics and EC migration. Stent struts generated disturbed flow patterns which reduced EC polarization and impeded migration towards cell-free space. Inhibition of ROCK enhanced EC migration over stent struts by promoting migratory polarization of cells via modulating the activity of MLC and cofilin. We conclude that ROCK inhibitors may have beneficial effects in stented arteries by promoting re-endothelialization and thus restoring vascular homeostasis.Here we used For studies of human cells, experiments were approved by University of Sheffield Research Ethics Committee (reference 10/H1308/25) and all subjects gave informed consent. Studies using human cells were used in accordance to the standards set by the Declaration of Helsinki. For animal studies, all procedures were approved by the University of Sheffield ethics committee and performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986 and in accordance with Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.0.6, Ibidi GmbH). Flowing medium was applied using the Ibidi pump system and chamber slides were placed on the stage of an inverted light microscope (Nikon® TE300) enclosed in a Perspex box warmed to 37 °C. Time-lapse imaging was performed for up to 96 h. Individual cells were manually tracked using ImageJ software. 3D stented model vessels were fabricated with PDMS and the internal surface was coated with fibronectin prior to deployment of a Coroflex Blue stent. EC were seeded as a confluent monolayer upstream of the first stent strut. Flowing medium was applied using the Ibidi® pump system in a 5% CO2 humidified atmosphere at 37 °C. EC were identified by light microscopy. Multiple independent experiments were conducted using primary cells isolated from different individuals, and the number of independent experiments carried out is stated in the figure legends.Pharmacological inhibition of ROCK activity was performed using Y27632  or fasudil  isoquinoline; HA-1077; Calbiochem) at 2 µM. Silencing of ROCK1 and ROCK2 was performed using small interfering RNA . Human umbilical vein EC (HUVEC) were isolated using collagenase digestion. Human coronary artery EC (HCAEC) were obtained commercially . EC were seeded into polydimethylsiloxane (PDMS) flow chambers with ridges  or onto flat flow chambers  and Alexafluor568-conjugated secondary antibodies (Invitrogen) to identify the microtubular organizing centre (MTOC), phalloidin-488  to identify actin. Nuclei were identified using DAPI (Sigma). Imaging was carried out using an inverted fluorescence microscope (Olympus IX71) and image analysis was performed using Image J software (1.49p). Polarized cells were defined as those with an elongated morphology with the MTOC positioned upstream from the nucleus as described.® . The protein levels of tissue factor pathway inhibitor (TFPI) or von Willebrand factor (vWF) in cell lysates or cell culture supernatants were quantified by enzyme-linked immunosorbent assay .Total cell lysates were isolated using lysis buffer . Western blotting was carried out using specific antibodies against phosphorylated cofilin , phosphorylated MLC , and pyruvate dehydrogenase complex component X  with horse radish peroxidase-conjugated secondary antibodies (Dako). Chemiluminescent detection was carried out using ECL Primeen face staining using anti-PECAM-1 antibodies followed by confocal microscopy.Male Yorkshire White pigs (22–27 kg) were studied. EC denudation of carotid arteries was achieved by inflating a balloon 5 times for 30 s at a balloon/artery diameter ratio of 1.2:1. A Coroflex™ stent was deployed over the denuded region. Animals were then treated with fasudil (30 mg/day) or with vehicle alone using osmotic minipumps. Aspirin (150 mg/day) and clopidogrel (75 mg/day) were given orally for 3 days following stent implantation. After 3 days, EC coverage of the stented segment was determined by ,η = 10−3 Pa.s, density, ρ = 103 kg/m3) and steady flow simulations were carried out at Reynolds numbers, 2 respectively . Studies of fluid flow in stented PDMS tubes or arteries required import of cross-sectional lumen slices from μCT analysis to CFD analysis using the marker-cell method with matched voxel resolution . For stented PDMS tubes, WSS measurements were extrapolated as for the 2D system and taken from steady state flow Re = 410 with the Newtonian viscosity taken as that of culture medium . For stented carotid arteries, Re = 150 with the Newtonian viscosity taken as blood .Fluid flow in the 2D (3D) chamber was studied using the two-dimensional [D2Q9 (D3Q19)] two-relaxation-time (TRT) incompressible lattice Boltzmann (LB) methodt-test or one-way ANOVA followed by a Bonferroni multiple comparison post hoc test when appropriate using the Prism6 software. A P value of less than 0.05 was considered to be statistically significant.All experiments were performed at least three times and data are presented as mean ± standard error mean. Statistical significance was determined using either Student’s in vitro flow chamber that contained three ridges that were 100 µm high and 100 µm wide and separated by 400 µm (approximating the geometry of second generation stent struts). The chamber was linked to a continuous-pump flow system with ridges positioned perpendicular to the flow direction  and coupled to a live cell imaging platform. CFD demonstrated that cell culture medium introduced at a flow rate of 21.6 ml/min would generate flow velocities that were highest through the centre (0.25 m/s) and decreased in magnitude when approaching the top and bottom of the chamber . The pressure along the length of the parallel plates was mostly homogeneous with a gradual 5% decrease, while the presence of ridges generated variation in the pressure gradient around these features . Mapping the WSS revealed that it was relatively high (13 dyn/cm2) and unidirectional at the inlet, whereas areas immediately upstream and downstream from the ridges were exposed to a low time-averaged WSS values, correlating with recirculating flow patterns observed in the flow path analysis . In detail, WSS was reduced to 0 dyn/cm2 just upstream of the ridge, followed by a sharp increase in WSS along the first vertical plane of the ridge (blue) that peaked at > 100 dyn/cm2 at the interface between vertical and horizontal planes of the ridge . The WSS remained elevated (30 dyn/cm2) on top of the ridge (red), decreased sharply along the second vertical plane of the ridge (blue) and remained low in the flow recirculation zone downstream from the ridge. The pattern of WSS variation repeated for each of the ridges. The disruption in flow was confirmed by particle imaging velocimetry which revealed that the accumulation of polystyrene beads was greater downstream from the ridge (407 ± 35 beads per mm2) where a large recirculating zone was present, compared to a site immediately upstream  or further upstream  from the ridge . Overall, these data indicate that the ridges alter high unidirectional flow (at the inlet) to create local flow disturbances including a region of very high unidirectional WSS at the top of the ridge and recirculating bidirectional flow with low-average WSS downstream from the ridge .To study the influence of stent strut geometries on flow patterns and consequent effects on EC migration, we developed an Figure  and Supplementary material online, Movie 1). Cells on the ridged slide negotiated the first ridge (despite the elevated WSS at the upper surface) and reached the region downstream of the ridge effectively . However, EC did not migrate beyond the region of bidirectional flow located downstream from the ridges .To determine the effect of ridges on EC migration, HUVEC were seeded onto the ridged flow chamber (between the inlet and the first ridge) or onto a flat surface as a control. Flow was applied to the chamber and EC migration monitored by live cell imaging. Cells migrated over the flat surface in the direction of flow and covered a distance of > 500 µm in 24 h , but less uniform direction in cells under bidirectional flow . This was quantified by measuring the angle between the radial axis  and the flow axis (nominated 0°), which was wider in cells exposed to bidirectional compared to unidirectional flow . The directional persistence, which is a ratio of the Euclidean distance: accumulated contour distance travelled and therefore a measure of the uniformity of migration direction, was also significantly reduced in cells exposed to bidirectional compared to unidirectional flow . In addition to the loss of directionality in HUVEC under bidirectional flow, the average velocity (0.78 ± 0.10 µm/min) was also significantly reduced compared to cells under unidirectional flow . These data indicate that the direction and velocity of HUVEC migration were impeded downstream from the ridge. Thus we propose that EC in the upstream region move into free space effectively because their migration is promoted by unidirectional flow, whereas EC in the downstream region become ‘trapped’ because bidirectional flow at that site exerts opposing directional cues.These features were assessed by single cell tracking which revealed unified forward migration in the flow direction in cells exposed to unidirectional flow  or unidirectional flow (13 dyn/cm2) as a control. It was observed that HUVEC exposed to bidirectional flow exhibited delayed migration into cell-free space , a greater deviation in migration from the flow axis  and reduced velocity  compared to cells exposed to unidirectional flow. Treatment with pharmacological ROCK inhibitors (Y27632 or fasudil) promoted migration along the flow axis , and significantly enhanced directional persistence  in cells exposed to bidirectional flow whereas there was no effect on migration velocity . Similar observations were made using HCAEC which exhibited a greater deviation in migration from the flow axis, reduced directional persistence and reduced velocity compared to cells exposed to unidirectional flow . Inhibition of ROCK enhanced migration along the flow axis and increased directional persistence without altering migration velocity in HCAEC exposed to bidirectional flow . Similar effects were also generated by siRNA-mediated silencing of ROCK1/2  which promoted HUVEC migration along the flow axis  and enhanced directional persistence  in cells exposed to bidirectional flow without altering average velocity . Thus inhibition of ROCK promoted EC migration into cell-free space under bidirectional flow.We hypothesized that interventions that uncouple EC from flow-mediated directional cues may allow EC to migrate from regions of bidirectional flow into cell free space. This was tested using inhibitors of ROCK, a molecule that controls EC polarization in response to flow.Figure , compare top and bottom panels) and were capable of crossing the second ridge under ROCK inhibition . Similarly, single cell tracking analysis demonstrated that ROCK inhibition enhanced migration along the flow axis , enhanced directional persistence  and modestly heightened average migration velocity  in EC exposed to bidirectional flow downstream from ridges. We conclude that ROCK inhibitors enhance endothelialization of ridged surfaces exposed to flow by promoting EC migration through regions of bidirectional flow into cell-free space.The effects of ROCK inhibition were also studied using EC exposed to flow on ridged slides. EC treated with Y27632 migrated further downstream from the ridge compared to controls . Notably, pharmacological inhibition of ROCK reduced the levels of phosphorylated MLC and cofilin in cells exposed to bidirectional flow , thus simultaneously reducing MLC activity while enhancing cofilin activity. These changes led us to hypothesize that ROCK inhibition may alter migratory polarity which is intimately linked to actin dynamics. This was tested by immunofluorescent staining to determine the relative positions of the nucleus, MTOC, and the actin cytoskeleton. In polarized cells, the MTOC is positioned behind the nucleus along the axis of elongation as described.Figure ). Thus we conclude that ROCK inhibition promotes EC polarity under bidirectional flow associated with altered actin dynamics.The underlying molecular mechanism was studied by assessing the effects of shear stress and ROCK inhibition on the phosphorylation of MLC and cofilin which are regulators of actin dynamics and cell polarity.Figure S4ASupplementary material online, ) and vWF  levels in HCAEC exposed to bidirectional flow were not significantly altered by application of the ROCK inhibitor Y27632. Thus we conclude that ROCK inhibitors promote EC migration under disturbed flow without altering TFPI or vWF expression.We also examined whether ROCK inhibitors could influence the expression of TFPI and vWF since these regulators of haemostasis are known to be expressed in sheared endothelium.in vivo situation, we determined the ability of EC to migrate over Coroflex Blue stents (65 µm strut diameter) that were deployed in PDMS tubing and subsequently exposed to flow. This 3D flow system is advantageous because it allows assessment of EC responses to complex stent geometries including flow dividers and acutely angled struts, the effects of stent materials on endothelial physiology can be monitored, and PDMS tubing is compliant thus providing a better approximation of the vascular mechanical environment compared to an inflexible 2D surface.The ridged parallel-plate system that we developed is a highly reductionist model of EC responses to stent struts. To more closely mimic the Figure S5ASupplementary material online, ). Similar to the 2D parallel-plate system, WSS values are elevated (>40 dyn/cm2) on the ridges of the struts with low values just downstream and upstream of the strut . Bidirectional recirculation zones are restricted to strut orientations perpendicular to the flow direction and lead to complex streamlines lines . For example, the flow path labelled ‘a’ in Figure S5BSupplementary material online,  is associated with an average WSS value of 15 dyn/cm2 against the tubing walls. However, WSS is reduced to 0 dyn/cm2 just upstream of a ridge, followed by a sharp peak in WSS along the vertical plane peaking at > 50dyn/cm2 at the interface between the vertical and horizontal planes . WSS remains elevated along the ridge top, then decreases sharply along the downstream vertical plane. This pattern in WSS is repeated for each strut orientation.The influence of the flow was determined by CFD simulations using a 3D geometry generated via a PDMS cast followed by microCT imaging  and downstream from both the first and second struts at 42 h . We conclude that inhibition of ROCK enhanced endothelialization of stents exposed to flow under in vitro experimental conditions.We hypothesized that inhibition of ROCK will increase endothelialization of stented PDMS tubes. Thus Coroflex Blue stents were deployed in fibronectin-coated PDMS tubing under sterile conditions. HUVEC were then seeded immediately upstream from the first stent strut prior to the application of flow and subsequent assessment of EC coverage by brightfield microscopy. The study revealed that the application of a ROCK inhibitor (Y27632) significantly enhanced the number of cells that accumulated downstream from the first strut at 24 h . The influence of balloon angioplasty on endothelial coverage was assessed by en face staining using anti-PECAM-1 antibodies followed by confocal microscopy. It demonstrated that inflation of a balloon to approximately 1.2 times the artery diameter created vascular injury with loss of endothelial coverage and exposure of underlying smooth muscle cells, whereas intact endothelial monolayers were present in uninjured arteries . Thus angioplasty and stent placement in the left porcine carotid artery represent a useful model to study the processes that govern EC denudation and repair.Next we investigated whether inhibition of ROCK can promote re-endothelialization of stented arteries Figure ). Time-average calculations revealed a maximum velocity occurred at the centre of the lumen (0.28 m/s) with decreasing magnitude approaching the lumen surfaces. Mapping the WSS showed unidirectional highest values located upstream and downstream from the stent due to the smaller cross-sectional area at these sites . In the stented segment, WSS was approximately 15 dyne/cm2 at the inter-strut regions and the highest values (>80 dyne/cm2) were at the surface of the struts. Of particular note, sites downstream from the struts were associated with low WSS and recirculating flow  and streamlines (lower panel) at downstream and inter-strut sites). Our observation that stent placement in carotid arteries generated recirculating flow led us to hypothesize that inhibition of ROCK may promote endothelialization in vivo. To investigate this, we used fasudil to inhibit ROCK in vivo as it is tolerated well in pigs as well as humans.En face staining using anti-PECAM-1 antibodies demonstrated that the percentage of EC coverage was 58.8 ± 11.0% in the saline-treated control group at 3 days post-stent implantation, whilst intravenous delivery of fasudil resulted in significantly increased endothelialization of the stent . We conclude that inhibition of ROCK using fasudil significantly enhanced re-endothelialization of stented arteries in vivo.The influence of stent deployment on WSS was determined by CFD using a geometry generated by microCT imaging of a PDMS cast . However, EC positioned downstream from the ridge in a region exposed to flow recirculation migrated with non-uniform direction and did not move effectively into free space. The observation compliments data generated using a step-flow chamber, where EC migration distance was decreased within the flow recirculation zone.Figure 8). The 2D in vitro model contained ridges that were 100 μm high and thus approximated the dimensions of some stent struts used clinically. With the advancement in stent fabrication, stent strut size has reduced within the last decade and recent designs are approximately 60 μm in diameter.,in vitro model where stents were placed in fibronectin-coated tubing prior to seeding with EC and exposure to flow. This 3D system was used to validate observations made using the 2D parallel-plate chamber under with real stent geometries and materials under well-controlled experimental conditions.Here we used in vitro systems that we have developed represent useful platforms to test EC behaviour in response to different stent geometries, materials, or pharmacological agents. Here we used them to identify a novel pharmacological intervention, i.e. inhibition of ROCK, which promoted EC migration at sites of bidirectional flow by enhancing front-rear polarization. The ROCK family consists of two isoforms, ROCK1 and ROCK2, that act downstream from the small GTPase Ras homolog gene family member A (RhoA) to regulate cell polarity and migration by altering actin cytoskeletal organisation and dynamics.,,The 2D and 3D Figure 8). At a molecular level, the mechanism was shown to involve changes in migratory polarity and altered activity of MLC and cofilin. It was observed that EC exposed to bidirectional flow displayed reduced polarity associated with increased MLC activity compared to cells under unidirectional flow. Although MLC is required for the formation of a single protrusion in migrating cells, high MLC phosphorylation leads to the induction of multiple protrusions and stable adhesion thereby reducing polarized migration.,Live cell imaging studies revealed that ROCK inhibitors suppressed EC mechanotaxis by uncoupling the direction of migration from mechanical cues, whilst retaining kenotaxis which describes the capacity of cells to move into free space . Thus we propose that partial inhibition of ROCK enhances migratory polarity of EC under disturbed flow by altering the activity of MLC and cofilin, whilst retaining sufficient actinomyosin activity for the generation of traction forces.MLC phosphorylation is positively regulated by MLC kinase as well as ROCK.et al.While we have focused on shear forces, a recent study by Canver in vivo, endothelial injury was performed via balloon angioplasty prior to stent deployment. We chose to study the carotid artery because it has a relatively straight anatomy with minimal movement throughout a cardiac cycle, thus allowing accurate positioning of balloon injury and subsequent stent deployment via angiography. It was observed using this model that systemic administration of the ROCK inhibitor fasudil significantly enhanced re-endothelialization of stented carotid arteries. This observation compliments a previous study demonstrating that systemic administration of fasudil reduced neointimal formation in stented porcine coronary arteries by promoting vascular smooth muscle cell apoptosis and reducing inflammation.,,,In order to test the effect of ROCK inhibition on vascular repair in a stented artery Supplementary material is available at Cardiovascular Research online.


Recently, a class of endogenous species of RNA called circular RNA (circRNA) has been shown to regulate gene expression in mammals and their role in cellular function is just beginning to be understood. To investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of primary sites, peritoneal and lymph node metastases from three patients with stage IIIC ovarian cancer. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-Seq libraries. This pipeline revealed thousands of circular isoforms in Epithelial Ovarian Carcinoma (EOC). These circRNAs are enriched for potentially effective miRNA seed matches. A significantly larger number of circRNAs are differentially expressed between tumor sites than mRNAs. Circular and linear expression exhibits an inverse trend for many cancer related pathways and signaling pathways like NFkB, PI3k/AKT and TGF-β typically activated for mRNA in metastases are inhibited for circRNA expression. Further, circRNAs show a more robust expression pattern across patients than mRNA forms indicating their suitability as biomarkers in highly heterogeneous cancer transcriptomes. The consistency of circular RNA expression may offer new candidates for cancer treatment and prognosis. Epithelial ovarian carcinoma (EOC) is the fifth most common cancer among women and one of the most lethal gynecological tumors, being responsible for more deaths than any other cancer of the female reproductive system . Most paCurrent tumor models fail to reflect their outcomes when applied to clinical samples, as the intra-tumor heterogeneity of multiple genotypes and phenotypes is a major obstacle to robust, cross-patient analysis. The tumors represent a complex ecosystem composed of cells with distinct phenotypes, genotypes and epigenetic states which co-exist and evolve simultaneously . ConventWe have previously described copy number variation (CNV) and gene expression analysis of matched ovarian primary tumors and peritoneal metastases, and reported targeting of several specific pathways that play a role in ovarian cancer metastasis both at genomic and transcriptomic levels , 8. ManyHomo sapiens and Mus musculus [CDR1 and Sry genes have been shown to bind miR-7 and miR-138, respectively, at multiple binding sites without getting degraded, making them excellent candidates for competing endogenous RNA activity [Recently, a novel class of RNA termed circular RNAs (circRNA) is increasingly being recognized as an abundant class of regulatory transcripts primarily derived from protein coding exons and widely expressed across eukaryotic organisms including musculus –17. Thesmusculus  in a promusculus , 19, 20.musculus . Furtheractivity , 21, 22.activity , 24 or pTo investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of nine ovarian cancer samples from three patients at primary ovarian tumor and its matched peritoneum and lymph node metastatic stages. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-seq data. Our results show that circRNAs are widely expressed in epithelial ovarian cancer with thousands of circular isoforms present in primary ovarian tumor and its peritoneum and lymph node metastases. These candidate circRNAs are enriched for miRNA seed matches and potentially capable for competing endogenous RNA activity. Moreover, as against a highly heterogeneous linear transcriptome in ovarian cancer, we report a robust expression pattern of circRNAs across patients and tumor stages.Total RNA was collected from the biopsies of the primary tumor, as well as peritoneum and lymph node metastases of three ovarian cancer patients presenting with stage IIIc papillary serous adenocarcinomas . Tumor sWe assessed the extent of alignment artifacts or DNA sequence redundancies towards the backsplice junction identification process and therefore any inadvertent discovery of circRNA candidates. To this end, we first randomized the DNA sequence of each of the exons for all exon pairs used in our circRNA detection pipeline, and aligned the randomized exome to the paired end RNA-seq data to look for any backsplice junctions, and as expected no junction was found. To make the DNA sequence less random, in a second step only the downstream exon (Exon2) was flipped without randomizing the DNA sequence and again aligned to the paired end RNA-seq data to look for any head-to-head spliced (Exon1-flipExon2) junctions, we found only 33 such junctional events having zero overlap with our circRNA candidates. This convincingly indicates that mis-alignments and DNA sequence redundancies have been genuinely ruled out and do not contribute towards candidate circRNA identification process. To estimate the sensitivity and precision of our circRNA detection pipeline, we applied it to a simulated dataset of 50bp paired-end reads for ~200 randomly selected “backsplices” mixed with a background of their linear canonical transcripts generated using mason software . Figure In order to validate that observed circRNA candidates are not the misidentified linear products of trans-splicing events , we desiCanonical alternative splicing of primary RNA transcripts is an important biological process that allows a gene to have multiple RNA and protein isoforms with related or distinct functions. With more than 90% of the human genes undergoing alternative splicing, and a large fraction producing appreciable levels of two or more distinct populations of mRNA isoforms , we sougMicroRNAs (miRNAs) are ~22nt long post-transcriptional regulators of gene expression that guide an effector silencing complex containing the Argonaut protein to the 3′ untranslated regions (3′ UTR) of target mRNAs resulting in their destabilization and translational repression –44. Therhttp://www.bioconductor.org/). For estimating significant differences, low expression loci were filtered out and only loci that were expressed at a minimum of three read counts in atleast three of the samples were taken for differential expression analysis. This reduced the number of pre-filtered genes and circRNAs for differential expression analysis to 2166;2683 for ovary vs peritoneum and 2803;2613 for ovary vs lymph node respectively. Table Gene expression products in ovarian cancer could represent candidate biomarkers and potential oncogenic factors involved in tumorogenesis and disease progression. We analyzed gene expression for both mRNAs and circRNAs and evaluated their abundances in an integrated manner to uncover crosstalk between networks of circRNAs, miRNAs, and mRNAs. Gene expression was estimated after aligning the RNA-seq data to reference genome (GRCh38) in a paired end aware manner using tophat spliced read mapper  and only®, QIAGEN Redwood City, www.qiagen.com/ingenuity) revealed an overwhelming enrichment of differentially expressed circRNA genes for cancer related diseases  of differentially expressed circRNAs for each of the potential target genes (see Methods). These potential gene targets were obtained through microRNA target filter module of QIAGEN's Ingenuity® Pathway Analysis  application as either experimentally observed or high confidence predicted targets of miRNAs and are highly enriched for cancer related pathways. This resulted in identifying 233 and 246 differentially expressed circRNAs containing binding sites for 1016 and 1070 miRNAs, and associated with 1140 and 1572 downstream gene targets in primary ovarian tumor versus peritoneal or lymph node metastases respectively  were cultured in DMEM  supplemented with 10% FBS (Sigma), 1% antibiotic - Antimitotic solution . RNA isolations were performed after culturing the cell lines for at least one month using these standard conditions.Total RNA was isolated using Qiagen RNAeasy mini kit according to manufacturer's protocols including oncolumn DNAse digestion. For RNase R treatment, 2 micrograms of total RNA was briefly heated to 70°C to denature, and then cooled to 40°C on a thermo cycler. 20units of RNAse R (Epicenter) and 1 unit/microliter murine Ribonuclease Inhibitor (New England Biolabs) were added to the denatured RNA samples and incubated at 40°C for 1 hr.100 ng of total RNA of SKOV3 and OVCAR3 cells treated with RNAse R (as described above) and untreated samples were used for library preparation. cDNA synthesis was done using Ovation RNA-Seq system v2 (NuGEN) with SPIA amplification. RNA-Seq libraries were prepared by using Truseq library preparation protocol (Illumina). Sequencing was performed on an illumina Hiseq instrument with 100bp paired end reads.StepOnePlus™ Real-Time PCR Systems (Life Technologies). Quantification of circRNA enrichment in comparison with linear housekeeping genes like Beta actin (ACTB) and GAPDH is also done. For confirmation of circular junctions a PCR reaction was performed using one step RT PCR kit (Qiagen) with convergent circ Primer pair was used for template amplification.Total RNA was isolated from SKOV3 and OVCAR3 cells. cDNA synthesis was done by using First Strand cDNA Synthesis Kit for RT-PCR (AMV) from ROCHE using either random hexamer or oligo dT as indicated. Primers used in qRT-PCR were designed as convergent primers to detect circular junctions, and to cross the backsplice junction . Real-tiFor each of the four circRNAs to be sequenced, primers were designed in regions about 50 bp to either side of the junction, with one primer pair on each of the exons in the noncanonical junction. Each primer pair was divergent, overlapping by only 6 bp, and a Tm of about 60°C. Reverse transcription was carried out on pooled OVCAR RNA using Thermoscript RT (Life Technologies) according to directions, with a pool of the four primers that would amplify template strand circRNAs. The resultant cDNA was subjected to PCR amplification with each primer pair. The bands were gel purified and submitted for Sanger sequencing on a Applied Biosystems 3130xl Genetic analyzer, using each of the four primers designed against that circle.A non-canonical mode of RNA splicing results in a scrambled junction, in which a downstream exon (Exon2) is “backspliced” in a head-to-tail fashion to an upstream exon (Exon1) into a circular RNA molecule placing Exon2 upstream of Exon1. We created a reference scrambled exome for all possible pairs of intragenic non-linear combinations of exons, as well as single exon “backsplices”, by either joining the 3′ and 5′ ends of a single exon or joining these ends from a downstream to an upstream exon. This represents the sample space for all possible exon-junction structures that can occur through circularization events. We aligned the RNA-seq data to this reference scrambled exome with Bowtie2 . In a suhttp://www.bioconductor.org). For estimating significant differences, only loci that were expressed at reasonable levels of a minimum of three read counts in atleast three of the samples were taken for differential expression analysis. Before estimating the differential expression, raw count data was normalized for variation in sequencing depths between the samples and adjusted for RNA composition effects using the default Trimmed Mean of M-values (TMM) method. A design matrix was then defined based on an additive linear model with patient as the blocking factor and estimates for trended and tagwise dispersion parameters were computed. Finally, a negative binomial generalized linear model was fitted to determine differential expression using the likelihood ratio test which computes p-value and the Benjamini-Hochberg adjusted p-values (FDR) for each tag and ranks them in order of evidence for differential expression.For each circRNA candidate, expression was summarized as the aggregate total number of paired-end fragments that mapped to a scrambled exon junction in an orientation supporting a head-to-tail “backsplice” instead of a “linear splice”. We included both reads spanning actual junctions  and divergent paired-ends that supported (“supported”) a circular structure. Finally considering samples from three patients as biological replicates statistical significance of differential expression was estimated at a positive predictive value of 98% (FDR < 0.02) with a minimum of two fold change in expression between primary ovarian tumor and metastatic lesions, using edgeR package  from BioGene expression was estimated after aligning the RNA-seq data to reference genome (GRCh38) in a paired end aware manner using tophat spliced read mapper  and onlyWeighted Expression of circRNAs targeting a given microRNA (miRNA-x) is calculated as,N is the number of circRNAs targeting miRNA-x, xin the number of seed matches on ith circRNA for miRNA-x and ix is the fold change expression of the ith circRNA. An Average Ecirc is then computed for each of the downstream target genes that shows either an experimentally observed interaction with miRNAs or contains highly significant predicted binding sites for miRNAs.Where circ = AverageAverage E (circE1 + circE2 + … + circEN)The data for cell-lines has been submitted to Sequence Read Archive (NCBI SRA) under accession numbers SRR1772957, SRR1772257, SRR1777309 and SRR1777310.


Runx genes encode transcription factors that play essential but distinct and lineage-specific roles in development. These sequence-specific DNA binding proteins share a common binding cofactor (CBFβ) that confers protein stability and high affinity for target DNA on its RUNX partners. An important link to cancer was first realised through identification of both RUNX1 and CBFB as frequent targets for chromosomal translocations in human leukaemia. Early studies suggested that RUNX1 is a tumour suppressor subject to dominant negative inhibition by its fusion oncoprotein derivatives and to loss-of-function mutations in AML  leukemias where TEL-RUNX1 compromises RUNX functions [Our recent study  sheds fuunctions .nullRunx1 progenitors, notwithstanding the observation that Runx1 can bind directly at ribosomal gene loci [At first sight our findings contrast with a recent report that Runx1 deficiency in normal haematopoietic progenitors leads to reduced cell size due to down-regulation of genes involved in ribosome biogenesis (Ribi). Moreover, Runx1 deficient progenitor cells displayed a stress-resistant, pro-survival phenotype that has been suggested as an explanation for susceptibility to transformation . In contene loci . Howeverene loci .in vivo. This is very encouraging with regard to the prospects for treating primary lymphomas, which are more likely to have intact p53. Whether loss of p53 is sufficient to explain the ability of Eμ-Myc cells to survive without Runx1 is as yet unclear. However, this finding highlights another relevant feature of the potent collaboration between Runx and Myc which appears to suppress p53 function in lymphoma cells in vivo, providing a paradigm for collaborating oncogenes that act synergistically by neutralising the cell's failsafe responses to oncogene over-activity [While established Eμ-Myc lymphoma cells that express Runx1 have a clear selective advantage over excised cells, they are much less Runx-dependent than primary lymphomas activity . One of


The influenza A virus genome consists of eight RNA segments. RNA structures within these segments and complementary (cRNA) and protein-coding mRNAs may play a role in virus replication. Here, conserved putative secondary structures that impose significant evolutionary constraints on the gene segment encoding the surface glycoprotein hemagglutinin (HA) were investigated using available sequence data on tens of thousands of virus strains. Structural constraints were identified by analysis of covariations of nucleotides suggested to be paired by structure prediction algorithms. The significance of covariations was estimated by mutual information calculations and tracing multiple covariation events during virus evolution. Covariation patterns demonstrated that structured domains in HA RNAs were mostly subtype-specific, whereas some structures were conserved in several subtypes. The influence of RNA folding on virus replication was studied by plaque assays of mutant viruses with disrupted structures. The results suggest that over the whole length of the HA segment there are local structured domains which contribute to the virus fitness but individually are not essential for the virus. Existence of subtype-specific structured regions in the segments of the influenza A virus genome is apparently an important factor in virus evolution and reassortment of its genes. Influenza A virus is an important pathogen, causing severe disease outbreaks in humans and domestic animals, in particular poultry and pigs1in vitro5678910111215Similar to other viral RNA genomes, the influenza A virus genome may encode RNA structures which play a role in virus replicationin vitroA genome-wide analysis of influenza virus RNA segments suggested that negative-sense vRNA is globally less structured than the positive-sense counterpart818198Multiple functional structural elements are expected in the influenza vRNA. The efficient packaging of all eight vRNP segments requires a network of interactions, involving both the main packaging signals at the segment ends and the central domains2223242526181922242529263031In this work, we used a comparative RNA structure analysis to explore possible subtype-specific RNA structures in segment 4 (HA) of the influenza A virus genome, which encodes the important viral surface glycoprotein. Distinct subtypes of the HA protein exist (H1–H16), and enormous numbers of HA sequences from different strains, available from public nucleotide databases, can help in the detection of conserved structural motifs. In this analysis, we identified RNA motifs that impose significant structural constraints on the evolution of the HA segments of different subtypes. Such constraints are reflected in RNA-specific patterns of sequence diversity with coordinated variations (covariations) of the nucleotides paired in the conserved structures.From previous work in the search for structured regions in the influenza virus RNA segments815181933Usage of different sequence datasets  gave the opportunity to generate alternative secondary structure models for each of the HA subtypes. The differences between alternative models for a given subtype were variable, depending on intra-subtype similarity levels. For instance, in three predictions of H1 HA structures only 31% of predicted base pairs were common (149 out of 481), and 31–53% of predicted base pairs were the same in pairwise comparisons of the models, while in the predictions for less diverse H15 HA segments more than 99% of predicted base pairs were present in all three models. The overlaps between alternative models were considered as putative conserved functional local structures. In order to distinguish functional structural RNA motifs that exhibited significant constraints on HA sequences from potential artifacts of folding predictions due to sequence similarity of protein coding regions, the analysis of covariations in conserved local structures was performed.1(xy) = M(xy)/H(x) and R2(xy) = M(xy)/H(y), which reflect correlations between substitutions of bases involved in putative base pairs, taking both mutual information M(xy) and variabilities (entropies) at two positions into account. Covariations with at least one correlation value R1(xy) or R2(xy) higher than 0.8 are listed in The predictions of putative subtype-specific secondary structures by the RNAalifold and RNAz algorithms in HA segments, followed by mutual information calculations as described in Materials and Methods, yielded a relatively small number of significant covariations that could be attributed to RNA structural constraints. The significances were evaluated by ratios RThree structures that contained more than one covariation characterized by a score of at least 0.8 were identified: in human H3N2 strains, in H7 viruses and in H15 viruses . The preFour covariations, each having a correlation value of at least 0.7, were identified in the stem-loop structure predicted in the region 794–839 of HA segments from human H3N2 strains , Fig. 1.35One of the covariations in the 794–839 domain (807–820) seems to be determined by non-canonical base pairing . In the 1(xy) = 0.56; R2(xy) = 0.48) than those in human strains (1(xy) = 0.42; R2(xy) = 0.44) between AU and GC, similar to human strains. Thus, in all H3 HA segments six independent covariation events are observed in this domain with its interior part apparently adopting alternative conformations  is 0.76, which is relatively high. Furthermore, it should be noted that the H7 and H10 covariations are separate events with covariation R1(xy) scores of 0.78 and 0.74  = 0.59 at base pair 1036–1057 in H5 numbering  = 0.54), with an AU pair predominantly in Eurasia and GC in America  = 0.67) and exhibited even higher correlation values when computed for the whole cluster: R1 = 0.75 (38The folding of the domain bottom stem in the strains of the H5/H6/H2/H1 cluster  is suppo) = 0.75 . Compari= 0.75 38. In the = 0.75 38.Highly pathogenic avian influenza A (HPAI) viruses are characterized by multibasic cleavage motifs in the HA protein, which are created in H5 and H7 strains by inserting one or several codons coding for basic amino acids in the cleavage site region37 of −18.6 kcal/mol in the positive-sense RNA, still comparable to the values calculated for homologous structures in low pathogenic avian influenza (LPAI) H5 HA genes of the lineages closely related to the gs/GD/96-like segmentsThe stem-loop structure in the ancestor gs/GD/96 HA segment, with an insertion of 12 nucleotides , has a fAnalysis of HA RNA sequences of the strains belonging to multiple clades that evolved from the gs/GD/96 lineage during 1996–2014Similar to the insertions in the HA segments of HPAI H5 strains, the insertions in H7 HPAI segments seem to occur in the hairpin loops of LPAI H7 HA RNA structures. In the cleavage site region of H7 HA segments, different alternative structures were predicted . Close vA few structures with significant covariations were revealed in the minimal packaging signals of the HA vRNA in virions. In H1 HA segments, these signals include both vRNA termini, containing 3′ and 5′ untranslated regions (UTRs) and extending to the coding part by 45 and 80 nucleotides from the 3′- and 5′-ends, respectively2(xy) = 0.31) at another base pair  replaced the relatively rarely used (in human genes) GUA codon  by another rare one UUA (Leu), while silent substitution C1 replaced commonly used CUG Leu codon by less frequent CUU. This was also reflected in the estimate of heterologous gene expression in 293 T cells according to the Codon Adaptation Index (CAI), computed using the CAI calculator4748No obvious correlation was found between the observed behavior of mutants and the introduced changes in codon usage according to available data on codon frequencies in human or influenza virus genomesOther designed HA mutants in this and other HA domains exhibited no significant replication deficiency which could be attributed to RNA structure disruption . Even diThe nucleotide covariation analysis of conserved RNA structures in the HA segment of the influenza A virus genome, presented here, suggests a number of local structured domains exist, which are specific for certain HA subtypes only and are not conserved in all 16 known subtypes. In contrast to the NS, M and NP segments, where several structures conserved in all influenza A strains have been identified in the coding regions58A given covariation can serve as reliable evidence for a structural model only if it is likely that the observed bias at the putative base pair is indeed determined by RNA structure rather than by other evolutionary pressures or chance alone. Evaluation of covariation-based evidence for functional coevolution of two sites in a biopolymer is not straightforward5051An inadequacy of a covariation metric alone in the discrimination between different mechanisms of nucleotide coevolution can be compensated by taking the phylogenetic history of covariations into account50515253Indeed, several RNA structures supported by more than one covariation event were predicted in HA segments, pointing to important constraints determined by secondary structure. Interestingly, the most reliably supported structures were detected in the central regions of the HA segment, in particular, the region coding for the cleavage site motif in the HA protein. These structures turned out to be conserved in different HA subtypes, with independent covariations occurring at homologous positions.On the basis of covariation data it is impossible to conclude whether predicted local hairpins are functional in the positive-sense RNA (mRNA or cRNA) or negative-sense vRNA, and folding free energy differences cannot be used in support of a conclusion because of the influence of NP binding on RNA structures within RNPs1819252626303156The insertions coding for multibasic cleavage site motifs in the HA segments of highly pathogenic H5 and H7 viruses occur in the hairpin loops of stem-loop structures flanking the cleavage site region so that the folding topology remains the same . Further58The conserved H5 and H7 structures in the central HA domain , folded A small hairpin, predicted to fold in the region essential for vRNA packaging , exhibitin vivo while no effect in cell culture was seen562425A strong effect of the specific combination of mutations in the 794–839 domain of human H3N2 viruses on virus viability  seems to56Certain correlations between covariation events in the structures of HA segments from human H3N2 viruses  and 3 an3535Presumably, RNA secondary structures displaying nucleotide covariations only partially represent functional RNA folding in the influenza A virus HA segment. The covariation analysis reveals the importance of RNA folding in the HA segment evolution, but the most conserved RNA structures could be hidden from this consideration in genome regions that are less prone to mutations. Predictions of consensus RNA structures by the RNAalifold algorithm contained a number of potential hairpins with (nearly) invariant stems within a given HA subtype. Such hairpins could be formed by sequences containing clusters of conserved HA codons that are thought to determine the segment packaging signals263031Initial predictions of potential structured RNA domains in the influenza A virus HA segments were carried out using two algorithms: RNAalifold1 = M(xy)/H(x) and R2 = M(xy)/H(y) were calculated. In the estimates of covariations in RNA structures, these measures are less sensitive to dataset bias than M(xy) scores15Significance of covariations was first evaluated by mutual information calculations using all available sequences of the HA segment of a given subtype, taking positional entropies of sequence alignment into account, as described previously in the study of NP segment structuresMDCK and 293 T cells were cultured as described previouslyThe HA gene segment of A/Anhui/1/05 (H5N1) was cloned into the BsmBI sites of the previously described modified pHW2000 plasmid64Recombinant viruses were produced using the calcium phosphate method as described previouslyVirus replication of the mutant viruses was determined using a plaque assay. The plaque assay was performed as described previouslyHow to cite this article: Gultyaev, A. P. et al. Subtype-specific structural constraints in the evolution of influenza A virus hemagglutinin genes. Sci. Rep.6, 38892; doi: 10.1038/srep38892 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The objective was to examine whether: (1) statin use was associated with muscle related outcomes at age 60–64, (2) these associations were modified by 25-hydroxyvitamin D (25(OH)D) status and explained by inflammation, body-size or lifestyle in a British birth cohort. Markers of myalgia (intrusive body pain) and myopathy (self-reported and performance-based measures) were examined in 734 men and 822 women . Statin use was associated with intrusive body pain, difficulty climbing stairs and slower chair rise speed. Some associations were modified by 25(OH)D e.g. the association with intrusive body pain was evident in the insufficient (13–20 ng/l) and deficient (<13 ng/l) 25(OH)D status groups  but not in those with status >20 ng/l  (p = 0.003 for interaction). Associations were maintained in fully adjusted models of intrusive body pain and difficulty climbing stairs, but for chair rise speed they were fully accounted for by inflammation, body-size and lifestyle. In a nationally representative British population in early old age, statin use was associated with lower limb muscle-related outcomes, and some were only apparent in those with 25(OH)D status below 20 ng/l. Given 25(OH)D is modifiable in clinical practice, future studies should consider the links between 25(OH)D status and muscle related outcomes. This is reflected in recent guidelines from NICE3 that suggest that statins should be prescribed to anyone with a 10% ten-year risk of a cardiovascular event. As a result, approximately one-quarter of the population aged between 30 and 85 years old could be prescribed a statin. However, there remain serious concerns about the potential adverse effects of statins if prescribed to such a large population5.There is clear and compelling evidence that statins have a beneficial effect on cardiovascular risk factors6. Data from large randomised controlled trials (RCT) suggest that adverse effects are similar to placebo. In a review of the trial data, Finegold (2014) concluded that only a minority of symptoms were genuinely due to statin use7. Nevertheless, it should be noted that the trials do not typically monitor for more indolent muscle conditions. For instance, the influential CTT collaboration meta-analysis of trials explored a small set of adverse effects that only included acute muscle conditions 1. As chronic muscle conditions have not generally been recorded in RCTs, it cannot be assumed that an absence of evidence is evidence of absence.There is a discrepancy between the data from randomised controlled trials (RCT) and observational studies, therefore the incidence of adverse effects of statins continues to be debated8. Similarly, in a survey of over ten thousand statin users, a quarter of users reported muscle related side effects9. Cross sectional population data from the National Health and Nutrition Examination Survey (NHANES 1999–2002) suggest 22% of participants taking a statin reported musco-skeletal pain compared with 18% participants not taking a statin10. Other large observational studies, some with up to 9 years of follow up11, report a higher prevalence of muscle related events in statin users compared with non statin users12. Data from UK general practices are unclear; there are reports of an increased risk of moderate to severe myopathic events with statin use13 but other reports show no difference in myositis between statin users and non users14. The few studies to include measures of motor performance did not find an association between statin use and reduced physical performance18 or response to physical training19. In both the clinical trials and observational studies there is little consistency in what constitutes a statin related adverse effect which makes direct comparison challenging.Observational studies of patients on statins typically focus on chronic muscle conditions and report a much higher rate of adverse effects than the RCTs. In a retrospective study of patients in routine care in Boston (USA), myalgia (muscle pain) and myopathy (muscle weakness) were the most commonly documented reasons for statin discontinuation, affecting 27% of patients21 and is a cause of myopathy and myalgia. Previous studies report that low 25-hydroxyvitamin D status may exacerbate musculoskeletal symptoms experienced with statin use24. Indeed, in a small series of 128 patients with statin induced myalgia, 25-hydroxyvitamin D was significantly lower in statin users than non users and responded positively to replacement in the majority of cases25. Overall this highlights the potential interaction between low 25-hydroxyvitamin D status and statin use in relation to adverse effects.There are many additional factors that could contribute to an increased risk of muscle related conditions in statin users compared with non users in observational studies. For instance, low 25-hydroxyvitamin D is prevalent in the UK population27. Statin users tend to have higher levels of inflammatory markers such as Interleukin 6 (IL-6) and C-reactive protein (CRP) and elevated levels of these markers have also been associated with poorer motor performance28, strength29, BMI30 and lifestyle31.Higher body mass index, adverse lifestyle factors such as smoking, and socioeconomic conditions (SEC) are associated with muscle-related outcomes and have been shown to be associated with 25-hydroxyvitamin D levels24) and accounted for by inflammatory status, prior body mass index (BMI) and osteoarthritis (OA), smoking and SEC32. Testing these hypotheses in a nationally representative British cohort addresses a significant gap within the literature that directly informs the implementation of the NICE guidelines.We investigated whether statin use was associated with a range of muscle related outcomes in a large nationally representative British birth cohort of men and women followed up to early old age. We assessed intrusive body pain as a marker of myalgia. In addition, we used both self-reported and performance-based measures of limb function as markers of myopathy. We hypothesized that any association between statin use and these outcomes would be modified by current 25-hydroxyvitamin D  participants, of whom 2474 completed a postal questionnaire and 2229 had a clinical assessment (1690 at a CRF and 539 at home). No attempt was made to contact the remaining study members: 718 (13.4%) had already died, 594 (11.1%) had previously withdrawn from the study, 567 (10.6%) lived abroad and 320 (5.9%) had been untraceable for more than ten years. The study received ethical approval for the data collection at age 60–64 from the Central Manchester Local Research Ethics Committee and the Scotland A Research Ethics Committee and is compliant with the appropriate guidelines and regulations. Informed consent was provided by participants.The MRC National Survey of Health and Development (NSHD) has followed a nationally representative sample, originally of 5,362 individuals, born in mainland Britain in March 1946. At the 23rd follow up at 60–64 years, study members first received postal questionnaires to complete and were then invited to attend one of six clinical research facilities (CRFs) or to have a research nurse visit their home34. The data for responses were recoded from a scale of 0  to 4 (extremely) into a binary variable which indicated if intrusive body pain was absent (0) or present (1–4).The definition of myalgia is broad. We used a question from the SF-36  that asked about intrusive body pain 35.Muscle disorders predominantly affect the proximal muscle groups. We therefore used measures that represent proximal limb function and compared them with measures of distal limb function. Assessments of function were conducted during the clinical assessment at age 60–64 with trained nurses following standardised protocols for the performance-based tests36.As a subjective indicator of proximal muscle function participants were asked if they had difficulty going up and down a flight of 12 stairs (coded Yes vs No)Participants were asked if they had difficulty gripping heavy objects like a full kettle (coded Yes vs No). This was used as a subjective indicator of distal limb function.38, by taking the sex-specific mean of the bottom fifth.Chair rise speed was used as an objective indicator of proximal lower limb function. Chair rise speed per minute was calculated from the time taken to rise from a sitting to a standing position with straight back and legs, and then sit down again as fast as possible ten times (chair rise speed per minute = (10/chair rise time)*60). Where a test was not completed for reasons of poor health (n = 136), values were imputed as in previous analyses39. The maximum strength achieved during 6 attempts (three in each hand) was used. Where a test was not completed for reasons of poor health (n = 49), values were imputed as in previous analyses38, by taking the sex-specific mean of the bottom fifth.Grip strength was used as an objective indicator of distal limb function and was measured isometrically using an electronic handgrip dynamometerStudy members were asked to report all regularly prescribed medications on the postal questionnaire and by the nurse at their clinical assessment. These reports were coded according to the British National Formulary (BNF) formulary and a variable denoting statin use (yes vs no) was derived.40. Analyses of IL-6 and 25-hydroxyvitamin D were undertaken by the British Heart Foundation Research Centre in Glasgow (BHFRC) the latter using LC MS/MS (Liquid chromatography–mass spectrometry). Inflammatory markers were positively skewed and therefore transformed using the natural logarithm to achieve normality of their distributions40 and used as continuous variables.Overnight fasting blood samples were taken during the clinical assessment and initially processed at the CRF laboratories. Aliquots were frozen and stored prior to being transferred to the MRC Human Nutrition Research (HNR) laboratory in Cambridge where analyses of CRP was processed according to standardised protocolsa priori to adopt a categorical approach for the following reasons. First, supporting literature, particularly the guidelines for clinicians (for instance the US reference ranges41), typically consider 25-hydroxyvitamin D as categories. Second, treating 25-hydroxyvitamin D as a continuous variable assumes interactions are linear and ignores the cellular42, observational43 and clinical44 evidence that supports a threshold effect of 25-hydroxyvitamin D status.We carefully considered how to model 25-hydroxyvitamin D in these analyses and despite the acknowledged loss of statistical power we decided 24, we hypothesized that the effect of statin on the muscle outcomes would be exacerbated by low 25-hydroxyvitamin D.Therefore 25-hydroxyvitamin D levels were split into thirds; normal (>20 ng/l); insufficient (13–20 ng/l); and deficient (<13 ng/l). This is broadly similar to the US reference ranges (insufficient 25-hydroxyvitamin D is considered <12 ng/l). A priori, based upon published data45 or others47 and which were also hypothesized to be associated with statin use.We included those covariates that have been shown to be associated with the muscle outcome variables in this cohort48. We used the American College of Rheumatology criteria for the clinical diagnosis of idiopathic knee OA age 53, namely knee pain in either knee on most days for at least 1 month in the last year and at least two of the following: stiffness, crepitus, bony tenderness and bony enlargement. These items were assessed through a clinical examination conducted by a trained research nurse; further details have been published elsewhere49.We included knee OA given its importance to proximal lower limb performance and intrusive body pain and because it is also associated with statin use2). Own occupational class was chosen to represent adult SEC based on the study member’s occupation at age 53 . The Registrar General’s six-group social classification was collapsed into three groups: professional and intermediate (I and II); skilled non-manual (IIINM) or skilled manual (IIIM); and semi-skilled and unskilled manual (IV and V). The highest educational qualification obtained by age 26 was collapsed into three groups: degree level or advanced secondary qualifications ; ordinary secondary level ; no formal qualifications.Information on life time cigarette smoking up to age 60–64 was classified into three categories: never, former and current smoker. Height and weight were measured by the research nurse according to standard protocols at a home visit at age 53, and body mass index (BMI) was calculated (kg/mhttp://www.R-project.org/).All statistical analyses were conducted using R  as appropriate. The associations of statin use and 25-hydroxyvitamin D status with each muscle outcome were investigated in the combined sample of men and women as there was no evidence for interactions between sex and statin use with any of the outcomes. We used logistic regression for binary outcomes  and linear regression for continuous outcomes (chair rise speed and grip strength). In the first set of models, we examined the associations of statin use and 25-hydroxyvitamin D with each of the muscle outcomes separately, adjusted only for sex. In the second set of models, we tested for interactions between statin use and 25-hydroxyvitamin D status using likelihood ratio tests with 25-hydroxyvitamin D first modelled categorically and then rerun modelled continuously. This was to formally examine whether low 25-hydroxyvitamin D exacerbated the risk of muscle related outcomes as hypothesized a priori. We then further adjusted the models in which 25-hydroxyvitamin D was modelled categorically for inflammatory factors, BMI and knee OA, and finally for smoking status and adult SEC.In sensitivity analyses we compared the estimates from the sex adjusted models in the sample with complete data and in the samples (maximum n = 2380) for whom statin use and at least one muscle related outcome was known.NS affirms that the manuscript is an honest, accurate, and transparent account of the study being reported and that no important aspects of the study have been omitted.Bona fide researchers can apply to access the NSHD data via a standard application procedure .A fifth of participants 19.9%) were taking statins . Statin users also had higher mean levels of IL-6, higher mean BMI and were more likely to have knee OA (women only).Statin use, but not 25-hydroxyvitamin D, was associated with greater odds of reporting intrusive body pain Table . The estStatin use and being in the deficient 25-hydroxyvitamin D group were both associated with greater odds of difficulty going up/down stairs Table  Model A.Neither statin use or 25-hydroxyvitamin D were associated with the odds of reporting difficulty gripping heavy objects in the sex-adjusted model, or in any subsequent regression model (Supplementary Table Statin use and being in the lower 25-hydroxyvitamin D groups were associated with slower chair rise speed (see Table There was no association between statin use and grip strength in the sex adjusted model (difference in mean grip strength (kg) = −0.5, 95% CI −1.8 to 0.8, p = 0.4), or in any subsequent regression models (Supplementary Table 2). There was no evidence of an interaction between statin use and 25-hydroxyvitamin D on grip strength p = 0.59).There was no difference in prevalence estimates of statin use when comparing the sub-sample with complete data (n = 1556) (19.9%) with the larger sample for whom statin use and at least one muscle related outcome was known (n = 2380) (20.7%). While there were some differences between the analytical sample and the sample of study members who were assessed at age 60–64 but did not have complete data and so were excluded from our analyses , the estimates from sex adjusted models rerun using the maximum available samples were similar to the estimates reported.In a nationally representative British population, statin use was associated in sex-adjusted models with increased odds of intrusive body pain and difficulty climbing stairs and with slower chair rise speed. The associations were strongest in those with insufficient or deficient 25-hydroxyvitamin D status. For chair rise speed, these associations were fully accounted for by inflammation, prior BMI, knee OA, smoking and SEC. For intrusive body pain and difficulties with stairs, there remained associations with statin use for those of insufficient and/or deficient 25-hydroxyvitamin D status in fully-adjusted models.18 have typically focused on one or another of the broad range of adverse side effects of statins. We instead examined a range of chronic muscle related outcomes including proximal and distal limb function (both self-reported and performance-based), and intrusive pain. Importantly, consistent with others18, we find no association between statin use and grip strength. Muscle related disorders tend to affect the proximal rather than distal muscles50 adding biological plausibility to our results. Our results support the view that the relationship between 25-hydroxyvitamin D status and health outcomes is non linear and is subject to a threshold effect43. This is evidenced by the weaker or absence of an interaction between 25-hydroxyvitamin D status and statin use when 25-hydroxyvitamin D is modeled continuously rather than categorically. This is consistent with clinical recommendations which typically use a threshold for treatment.Previous studies23. However in a more select group of statin users - those who wished to switch to another statin - there did not appear to be an association between 25-hydroxyvitamin D status and myalgia51. In a small series of 128 patients25 with statin induced myalgia, 25-hydroxyvitamin D was significantly lower in statin users than non users. Importantly, in this study, the statin induced myalgia responded to 25-hydroxyvitamin D replacement in 92% of the cases. It should be noted that low 25-hydroxyvitamin D status is itself a cause of myopathy and myalgia50.For intrusive body pain, the associations with statin use were only apparent in those with insufficient 25-hydroxyvitamin D status. These associations remained, albeit weakened, after adjustment for covariates. Previous cross sectional studies have suggested the involvement of 25-hydroxyvitamin D in statin related myalgia. Data from the NHANES, report greater myalgia in statin users in the low 25-hydroxyvitamin D groupset al. found no differences between statin users and non users in a battery of tests assessed repeatedly over six years including 6-min walk test performance, repeated chair stands and grip strength 52. Statin use did not substantially increase decline in gait speed in community-dwelling older adults . Some studies, such as the Three City Cohort , even find that statin users have better physical performance than non users54. Notably however, 25-hydroxyvitamin D (or inflammatory markers and previous knee OA and SEC) are not typically included in these studies. Small interventional studies suggest that 25-hydroxyvitamin D supplementation can ameliorate statin induced intolerance of myopathy 56.There was weak evidence in this study, that statin use was associated with difficulty going up and down stairs only for those with insufficient and/or deficient 25-hydroxyvitamin D status. Again these associations were only partially explained by the covariates. Statin use was associated with poorer objective motor performance (as indicated by slower chair rise speed) but the differences did not vary significantly across the 25-hydroxyvitamin D groups; and these associations were fully attenuated after adjustment for BMI, knee OA, smoking and SEC. There is conflicting evidence on whether statin use is associated with poor motor performance. Gray 57.Given our findings, both the inverse associations between 25-hydroxyvitamin D status and certain muscle-related outcomes, and evidence that 25-hydroxyvitamin D status may modify the associations of statin use on these outcomes needs to be further investigated. Ideally a trial is required to address whether treatment of a low 25-hydroxyvitamin D prior to starting a statin prevents myalgia and any adverse change in proximal motor performance. Such studies may be redundant if a broader strategy to treat low 25-hydroxyvitamin D status in the general population is adopted. Pragmatically some would suggest that a low 25-hydroxyvitamin D is sufficient grounds for replacement given the pleiotropic effects 25-hydroxyvitamin D may have on other outcomes including cognitive function, cardiac disease and bone43. While it is recognised that low 25-hydroxyvitamin D is a risk factor for statin related adverse side effects59, we are not aware of a consensus statement regarding best practice with regards to statin use. The first author’s personal practice (NS) is to check 25-hydroxyvitamin D when considering a statin – the timing of replacement and statin initiation depends on the clinical situation; and to check and replace 25-hydroxyvitamin D in patients who appear to be intolerant of statins or report chronic side effects. No doubt practice varies depending upon the setting (primary versus secondary care) and the patient population. Clinicians should be aware of the possible relationship between low 25-hydroxyvitamin D and adverse side effects of statin use, and investigate and treat based upon local practice and individual patient circumstances.What are the implications for clinical practice? As stated above, a trial may be required but may be redundant if clinicians feel there is sufficient grounds to treat low 25-hydroxyvitamin D due to the broad health benefits60. Trial data focus almost exclusively on the more acute side effects of statins : out of 42 trials, 16 do not even report the frequency of the more insidious chronic muscle problems. Only one trial asked about chronic muscle problems61. Trials may not be structured to identify these adverse effects. 25-hydroxyvitamin D status is not typically considered in trials but one would expect low 25-hydroxyvitamin D status to be balanced across treatment and placebo groups. Nevertheless, it may be prudent for future statin trials to assess 25-hydroxyvitamin D status and incorporate it into the randomization process.In RCTs, what constitutes a statin related adverse effect is poorly defined and varies between trials. In 26 trials, broadly defined ‘muscle problems’ occurred in 12.7% of statin users versus 12.4% in the placebo group64. A strength of NSHD is that the sample includes individuals with a range of health conditions and remains broadly representative of the population born in mainland Britain straight after the second world war65. However, a third of participants had one or more missing variables which may have affected its representativeness, although the proportion of statin users and the associations we observed between statin and non statin users were similar in the maximum and the complete data samples. Further, statin use and 25-hydroxyvitamin D status was comparable to other representative samples of those in early old age67. Nevertheless, our findings need to be replicated in older and younger cohorts to see whether they are generalizable to the whole adult population.Another relevant aspect of RCTs is that patients with severe hypertension, chronic obstructive pulmonary disease (COPD), renal disease and poorly controlled diabetes are typically excluded; and these patients may be more likely to have low 25-hydroxyvitamin D status11. Given low 25-hydroxyvitamin D status increases with age68 and is associated with telomere length69, further work is needed to explore whether this partly explains the age related association between statin use and muscle-related outcomes.Another strength of this study is that our sample was homogeneous for age which enabled us to examine associations free from the confounding effect of age. In samples heterogeneous for age, it is consistently found to be one of the strongest predictors of statin related adverse effectsThis study also has additional limitations. First, statin use was self-reported and we do not have information on the type of statin, the dose or its duration. Second, we cannot directly assess pre and post statin function and cannot comment on acute adverse side effects such as myositis. Third, we do not have information on how many study members have tried and stopped statins in the past. Fourth, residual confounding may remain from unmeasured factors, especially as the covariates used did at least partly attenuate the associations observed between statin use and the muscle related outcomes.In conclusion, we find that statin use was associated with a range of non acute muscle related outcomes in men and women in early old age. These associations were confined or more apparent in those with low 25-hydroxyvitamin D status, and partly explained by other risk factors such as BMI and knee OA. Replication in older and younger populations is required given recent NICE guidelines that call for more widespread use of statins. Although a trial is required to determine if treatment of low 25-hydroxyvitamin D prevents statin related adverse effects, if there are clear clinical grounds for treating a low 25-hydroxyvitamin D then it may be reasonable to do so before starting statin treatment. The recommendations from the UK Government 25-hydroxyvitamin D group are awaited.Supplementary Information


Eukaryotic cells contain translation-associated mRNA surveillance pathways which prevent the production of potentially toxic proteins from aberrant mRNA translation events. We found that loss of mRNA surveillance pathways in mutants deficient in nonsense-mediated decay (NMD), no-go decay (NGD) and nonstop decay (NSD) results in increased protein aggregation. We have isolated and identified the proteins that aggregate and our bioinformatic analyses indicates that increased aggregation of aggregation-prone proteins is a general occurrence in mRNA surveillance mutants, rather than being attributable to specific pathways. The proteins that aggregate in mRNA surveillance mutants tend to be more highly expressed, more abundant and more stable proteins compared with the wider proteome. There is also a strong correlation with the proteins that aggregate in response to nascent protein misfolding and an enrichment for proteins that are substrates of ribosome-associated Hsp70 chaperones, consistent with susceptibility for aggregation primarily occurring during translation/folding. We also identified a significant overlap between the aggregated proteins in mRNA surveillance mutants and ageing yeast cells suggesting that translation-dependent protein aggregation may be a feature of the loss of proteostasis that occurs in aged cell populations. It is often classified into two general categories: amyloid and amorphous. The amyloid state is a highly structured, insoluble, fibrillar deposit, usually consisting of many repeats of the same protein2. It is central to the pathology of many neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s. Amorphous protein aggregation is the unordered aggregation of proteins, where proteins aggregate without forming a specific higher order structure. The aggregation of amyloid proteins has been more extensively studied since it is implicated in the pathogenesis of several neurological diseases, whereas, less is known regarding amorphous aggregation. It is not clearly established whether aggregation of amorphous proteins is mechanistically and functionally any different to amyloid proteins in terms of impact to the physiological state of the cell. Additionally, whether the proteins in disease settings are in fact amyloid or amorphous has not been well characterised. Proteins may also form amyloid progressively from initial disordered aggregates3.Protein aggregation is the abnormal association of misfolded proteins into aggregated, insoluble protein structures5. Such conditions disrupt the native folding of proteins, exposing hydrophobic regions leading to the formation of high molecular weight aggregates. Newly synthesized proteins are particularly vulnerable to misfolding and aggregation which is potentially toxic to cells8. A wide-range of proteins are prone to aggregation during normal unstressed conditions, with higher protein abundances and higher translation rates suggested as being the main determinants11. Aggregated proteins are also often substrates of ribosome-associated Hsp70 chaperones indicating that they are likely to be susceptible to aggregation during translation and folding9. Cellular stresses appear to reduce the general threshold for protein aggregation resulting in the aggregation of proteins which do not possess distinct kinds of physicochemical properties13. Proteins in stress-induced aggregates tend to have lower abundances, slower translation rates and increased sizes relative to unstressed protein aggregates. This suggests that protein aggregation is a normal physiological event, but conditions which act to perturb cellular homeostasis, can increase the burden of protein aggregation10. Several aggregation-prone yeast proteins have human homologues that are implicated in misfolding diseases, highlighting that similar mechanisms may apply in disease and non-disease settings. However, the relevance of widespread protein aggregation in disease is unknown.Protein aggregation occurs following protein misfolding, which arises as a consequence of translational errors during stress and ageing conditions, as well as a consequence of mutations or lack of oligomeric assembly partners16. There are three translation-associated mRNA surveillance pathways that target mRNAs for degradation to prevent the production of potentially toxic proteins. Nonsense-mediated decay (NMD) functions to detect premature stop codons and prevents the expression of truncated proteins. No-go decay (NGD) recognises transcripts on which ribosomes have stalled during translation17. Nonstop decay (NSD) is a quality control system for nonstop mRNAs which recognises stalled ribosome at the 3′ end of mRNAs and targets them for rapid degradation18. All of these specialised pathways use the same decay enzymes responsible for degrading normal transcripts; however, they differ in the mechanism by which they recognise and target defective mRNAs for degradation19.The process of mRNA decay is recognised as a major contributor to the regulation of gene expression, as well as maintaining regulatory responses crucial for cellular homeostasis. mRNA turnover plays critical roles in assessing the accuracy of mRNA biogenesis and in the degradation of aberrant transcripts. These aberrant transcripts are recognised and targeted for rapid degradation by mRNA surveillance mechanisms. These surveillance mechanisms block the expression of defective mRNAs, which can have deleterious consequences for the cellThe importance that mRNA surveillance pathways might have in regulating proteostasis is currently not well understood. The aim of this study was to determine whether loss of NSD, NMD or NGD, would have any effects on proteostasis by examining protein aggregation in strains defective in mRNA surveillance pathways. We show that defects in mRNA quality control systems result in the production of aberrant proteins that tend to misfold and form aggregates. The aggregated proteins share common biophysical properties with aggregation-prone proteins, suggesting that mRNA surveillance pathways normally function to suppress defective protein production arising from errors in translation.dom34, hbs1), NMD , NSD (ski7) and the Ski complex (ski8)20. We monitored sites of protein aggregation using a fluorescently-tagged Hsp104 chaperone. Hsp104-RFP is predominantly observed as diffuse cytoplasmic fluorescence in cells under normal growth conditions, whereas, it accumulates at the sites of protein aggregation following protein misfolding23. Diffuse cytoplasmic Hsp104-RFP fluorescence was detected in the majority of wild-type cells examined and only 3% of cells contained visible puncta marking sites of protein aggregation  mutants with approximately 16–18% of cells examined contained visible Hsp104 puncta  and NGD (hbs1) mutants along with ski7 and ski8 mutants. Our analysis identified 246 (wild-type), 428 (ski7), 399 (ski8), 429 (upf2) and 428 (hbs1) aggregated proteins. More aggregated proteins were identified in the mRNA surveillance mutants compared with the wild-type strain but there was generally a large overlap in the proteins that aggregated in both the wild-type and mutant strains  compared with the wild-type strain (wild-type = 3–5%). There was also a large overlap (198 proteins) in the proteins that aggregated in all five strains, indicating that most proteins aggregate because they are aggregation-prone rather than arising due to any mutant specific-effects  suggesting that a few proteins do aggregate in an mRNA surveillance pathway-specific manner.The proteins that aggregate following loss of mRNA surveillance pathways were identified using mass spectrometry. We focused on single NMD , ski7 (428), ski8 (399), upf2 (429) and hbs1 (349) mutant strains were compared. Our analysis revealed that the proteins within the aggregates isolated from all strains could generally be grouped into two major overarching categories: protein synthesis and proteins with binding function or cofactor requirement .We next assessed the physicochemical properties of the aggregated proteins within our datasets to determine whether they possess particular properties which make them aggregation-prone. For this analysis, we compared the Common-set consisting of 198 proteins which aggregate in all strains including the wild-type  compared with the MS-set , more highly expressed  and are translated at a higher rate  of the aggregated proteins were observed  to determine whether they possess any particular properties which might cause their aggregation to be mutant-specific. No significant enrichments for any functional categories or subcellular localizations were identified in these datasets. It should be noted that this may not be surprising given the relatively small sizes of these datasets. However, unlike the aggregated protein datasets used above (proteins which aggregate in mRNA surveillance mutant strains but not in the wild-type strain using pair-wise comparisons) there was no enrichment for highly expressed , abundant (molecules per cell) or stable (T1/2) proteins compared with the MS-set  which is competitively incorporated into nascent proteins in place of proline. AZC alters the conformation of the polypeptide backbone resulting in decreased thermal stability and misfoldingnts Fig. . We addiome Fig. . In contnts Fig. . There wnts Fig. . Similarnts Fig. .upf2 mutant34. However, no correlation was found between the up-regulated mRNAs and the proteins which aggregate in mRNA surveillance mutants including the upf2 mutant  complex, which recognises stalled proteins and promotes their ubiquitination and degradation, similarly results in protein aggregation36.mRNA quality control mechanisms recognise and degrade aberrant mRNAs including mRNAs containing premature stop codons (PTCs), mRNAs lacking termination codons and mRNAs containing structures which promote pausing during translation elongationupf2 mutant. However, this may simply indicate that any mRNAs that accumulate, may do so at relatively low levels. Alternatively, an accumulation of stalled/aberrant polypeptides in mRNA surveillance mutants could sequester chaperones leading to the aggregation of other proteins. These proteins may aggregate themselves as well as act to nucleate the aggregation of other proteins.Our analysis was performed using cells grown under non-stress conditions indicating that mRNA surveillance pathways normally act to prevent aberrant mRNAs driving translation-dependent aggregation during normal growth and metabolism. There are a number of possible mechanisms by which loss of surveillance pathways might promote aggregation. For example, aberrant or truncated proteins may be produced by translation of mRNAs which would normally be degraded in surveillance mutants. We did not see any accumulation of mRNAs encoding aggregated proteins in a Our analysis identified a significant overlap in the proteins that aggregate in different mRNA surveillance pathway mutants. Not surprisingly therefore, the intracellular localisation of the proteins that aggregate in mRNA surveillance mutants is similar to the localisation of the proteins that aggregate in a wild-type strain. Similarly, the protein functional categories that might be affected as a result of protein aggregation titrating proteins away from their normal soluble forms is predominantly the same in wild-type and mRNA surveillance mutant strains. This is consistent with the idea that the increased protein aggregation detected in mRNA surveillance pathway mutants predominantly arises due to the increased aggregation of a core set of what are already aggregation-prone proteins, rather than aggregation occurring in a mutant-specific manner.39. In agreement with this idea, the aggregated proteins identified in mRNA surveillance mutants were commonly enriched for hydrophobicity. Aggregation-prone proteins also tend to be more abundant and highly expressed, compared with unaggregated proteins12. Similarly, the aggregated proteins identified in mRNA surveillance mutants were enriched for highly translated and abundant proteins. The threat of aggregation may be unavoidable in highly concentrated proteins as they pose a bigger challenge for the proteostasis network, particularly when the proteostasis machinery is compromised. Estimates have suggested that the aggregation tendency of globular proteins may be as high as 20% in diverse proteomes40. This may result from the crowded and complex environment that highly expressed nascent proteins encounter resulting in a greater chance of specific and non-specific molecular interactions, making aggregation more likely to occur41.Many studies have identified hydrophobicity as a driving force for protein aggregation. The burial of hydrophobic segments within protein structures prevents exposed hydrophobic regions from interacting with other exposed hydrophobic regions leading to aberrant protein-protein interactionsThere was a significant overlap in the proteins that aggregate in mRNA surveillance mutants compared with the proteins that aggregate in response to nascent protein misfolding, suggesting that protein aggregation in mRNA surveillance mutants may primarily occur during protein synthesis and folding. The commonly aggregated proteins were also enriched for co-translational Ssb2 substrates further emphasising that protein misfolding may occur co-translationally. Taken together, our data are consistent with the idea that mRNA surveillance pathways normally act to suppress the production of abnormal proteins resulting from aberrant translation events.42. Two types of mRNA transcripts may be targets for surveillance pathways: those where genetic features make them a constitutive surveillance target (e.g. because of long 3′ UTRs or PTCs) and those where random damage produces aberrant features that are recognised by surveillance pathways. The balance between these two types of target may shift during ageing where higher levels of random damage may be expected to influence the fidelity of translation43. There was a strong correlation with the proteins that aggregate in response to nascent protein misfolding, whereas, there was little correlation with the proteins that aggregate following a denaturing stress caused by elevated temperature, further emphasising that aberrant translational events most likely underlie the high levels of aggregation observed in mRNA surveillance mutants. An increased understanding of the roles of mRNA surveillance pathways in protecting against the formation of non-functional RNAs and the subsequent production of abnormal proteins will be important given the established links between the degradation of aberrant mRNAs and human diseases46.The fact that there was a significant overlap with the proteins that aggregate in ageing yeast cells suggests that translation-dependent aggregation may be a particular problem as cells ageMATa ade1–14 ura3–52 leu2–3,112 trp1–289 his3–200) including ski7::HIS3, ski8::HIS3, upf1::HIS3, upf2::HIS3, dom34::HIS3 and hbs1::HIS3. Strains were grown at 30 °C (180 rpm) in SCD medium .Yeast strains used were isogenic derivatives of the wild-type 74D-694  glycerol, 1 mM PMSF and 1 × complete mini protease cocktail (Roche). Cells were resuspended in 300 μl ALB and spheroplasts were generated following treatment with lyticase (1 mg/ml) for 30 min at 30 °C. Cell breakage was achieved by sonication  and samples were adjusted to equal protein concentrations before isolation of protein aggregates by centrifugation at 13000 rpm, 4 °C for 20 min. Insoluble fractions were resuspended in a buffer containing ALB buffer with 2% (v/v) Igepal CA-630 (Sigma) through sonication . Samples were centrifuged at 13000 rpm for 20 min at 4 °C and the detergent wash was repeated. Residual detergent was removed by two washes with ALB and the pellet was resuspended by sonication. The final insoluble fraction was resuspended in 80 μl ALB and 20 μl reducing 4 × protein loading buffer, separated by reducing SDS-PAGE (10% gels) and visualised by silver staining using the Bio-Rad silver stain plus kit.All strains were harvested at the same cell optical density  and Insoluble protein aggregates isolated exactly as previously describedwww.matrixscience.com) to search the NCBInr and Swissprot databases. These results were put into Scaffold software, (https://www.proteomesoftware.com/products/free-viewer/) that conducts a statistical validation of the data, at both the peptide and protein levels. We selected proteins that had at least 95% statistical confidence of being present in our samples. Final datasets for each condition were determined by selecting proteins that were identified in at least two of the three replicates.Aggregated proteins were identified by mass spectrometry  in triplicate for each condition. For protein identification, protein samples were run a short distance into SDS-PAGE gels and stained using Instant Blue protein stain (Expedeon). Total proteins were excised, trypsin digested, and identified using liquid chromatography-mass spectrometry (LC-MS). Proteins were identified using the Mascot mass fingerprinting programme (Protein extracts were electrophoresed under reducing conditions on SDS-PAGE minigels and electroblotted onto PVDF membrane (Amersham Pharmacia Biotech). Bound antibody was visualized using LI-COR Odyssey-FC. Primary antibodies used were Pgk1  and Hsp104 .21. Cells expressing Hsp104-RFP were washed and immobilised on 10% poly-L-lysine-coated slides. All images were acquired on a Delta Vision (Applied Precision) restoration microscope using a 100 × /NA 1.42 Plan Apo objective and TRITC filter  from the Sedat filter set (Chroma). Raw images were then deconvolved using the Softworx software and maximum intensity projections of these deconvolved images are shown in the results. At least 100 cells were visualized in triplicate experiments for each strain and the percentage of cells containing visible puncta scored.The plasmid containing fluorescently tagged Hsp104 has been described previouslyp-value > 0.01; Bonferroni corrected) of functional categories (MIPS database) using FunCat (available at http://www.helmholtz-muenchen.de/en/ibis). Protein abundance data was retrieved from the PaxDB integrated dataset (available at http://pax-db.org). The proteins that aggregate in aged-yeast, AZC, hydrogen peroxide and heat stressed yeast have been described previously33 and statistical significance of the overlap was evaluated with a hypergeometric test. Venn diagrams and visualization of the distribution of protein hits between conditions were made using Bio Venn (http://www.biovenn.nl). Physicochemical data, translation rates and chaperone interactions were evaluated with pair wise Mann-Whitney-Wilcoxon U-tests using GraphPad Prism.Datasets for each condition were assessed for functional enrichment (Supplementary Fig. 1 and Fig. 2Supplementary Table 1