Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair

The extracellular matrix (ECM) is a key regulator of tissue morphogenesis and repair. However, its composition and architecture are not well characterized. Here, we monitor remodeling of the extracellular niche in tissue repair in the bleomycin-induced lung injury mouse model. Mass spectrometry quantified 8,366 proteins from total tissue and bronchoalveolar lavage fluid (BALF) over the course of 8 weeks, surveying tissue composition from the onset of inflammation and fibrosis to its full recovery. Combined analysis ofproteome, secretome, and transcriptome highlighted post-transcriptional events during tissue fibrogenesis and defined the composition of airway epithelial lining fluid. To comprehensively characterize the ECM, we developed a quantitative detergent solubility profiling (QDSP) method, which identified Emilin-2 and collagen-XXVIII as novel constituents of the provisional repair matrix. QDSP revealed which secreted proteins interact with the ECM, and showed drastically altered association of morphogens to the insoluble matrix upon injury. Thus, our proteomic systems biology study assigns proteins to tissue compartments and uncovers their dynamic regulation upon lung injury and repair, potentially contributing to the development of anti-fibrotic strategies

Bi-allelic variants in RNF170 are associated with hereditary spastic paraplegia.

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions

Symposium on the Scottish labour market

In the post-war period, up to the late 1960s, Britain enjoyed a modicum of unemployment and government policies which were geared to producing Full Employment were considered a success. It was simple - boost demand and more people would find work. But the mid 1970s the economic regency enjoyed by those advocating demand sided policies fell into disrepute as the OPEC nations raised prices dramatically and brought in a new era of both rising prices and unemployment. The laws of economics, which previously had viewed policy decisions as the choice between lower unemployment and higher inflation were now redundant. Both unemployment and inflation were moving in the same direction. The era of stagflation had begun

EAPP: Gatekeeper at the crossroad of apoptosis and p21-mediated cell-cycle arrest

We previously identified and characterized E2F-associated phospho-protein (EAPP), a nuclear phosphoprotein that interacts with the activating members of the E2F transcription factor family. EAPP levels are frequently elevated in transformed human cells. To examine the biological relevance of EAPP, we studied its properties in stressed and unstressed cells. Overexpression of EAPP in U2OS cells increased the fraction of G1 cells and lead to heightened resistance against DNA damage- or E2F1-induced apoptosis in a p21-dependent manner. EAPP itself becomes upregulated in confluent cells and after DNA damage and stimulates the expression of p21 independently of p53. It binds to the p21 promoter and seems to be required for the assembly of the transcription initiation complex. RNAi-mediated knockdown of EAPP expression brought about increased sensitivity towards DNA damage and resulted in apoptosis even in the absence of stress. Our results indicate that the level of EAPP is critical for cellular homeostasis. Too much of it results in G1 arrest and resistance to apoptosis, which, paradoxically, might favor cellular transformation. Too little EAPP seems to retard the expression not only of the p21 gene, but also of a number of other genes and ultimately results in apoptosis

Transcriptome and genome sequencing uncovers functional variation in humans

Summary Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome

Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo

The genetic architecture of type 2 diabetes

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of heritability. To test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole genome sequencing in 2,657 Europeans with and without diabetes, and exome sequencing in a total of 12,940 subjects from five ancestral groups. To increase statistical power, we expanded sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support a major role for lower-frequency variants in predisposition to type 2 diabetes

A Low-Frequency Inactivating Akt2 Variant Enriched in the Finnish Population is Associated With Fasting Insulin Levels and Type 2 Diabetes Risk

To identify novel coding association signals and facilitate characterization of mechanisms influencing glycemic traits and type 2 diabetes risk, we analyzed 109,215 variants derived from exome array genotyping together with an additional 390,225 variants from exome sequence in up to 39,339 normoglycemic individuals from five ancestry groups. We identified a novel association between the coding variant (p.Pro50Thr) in AKT2 and fasting insulin, a gene in which rare fully penetrant mutations are causal for monogenic glycemic disorders. The low-frequency allele is associated with a 12% increase in fasting plasma insulin (FI) levels. This variant is present at 1.1% frequency in Finns but virtually absent in individuals from other ancestries. Carriers of the FI-increasing allele had increased 2-hour insulin values, decreased insulin sensitivity, and increased risk of type 2 diabetes (odds ratio=1.05). In cellular studies, the AKT2-Thr50 protein exhibited a partial loss of function. We extend the allelic spectrum for coding variants in AKT2 associated with disorders of glucose homeostasis and demonstrate bidirectional effects of variants within the pleckstrin homology domain of AKT2.Academy of Finland (129293, 128315, 129330, 131593, 139635, 139635, 121584, 126925, 124282, 129378, 258753); Action on Hearing Loss (G51); Ahokas Foundation; American Diabetes Association (#7-12-MN-02); Atlantic Canada Opportunities Agency; Augustinus foundation; Becket foundation; Benzon Foundation; Biomedical Research Council; British Heart Foundation (SP/04/002); Canada Foundation for Innovation; Commission of the European Communities, Directorate C-Public Health (2004310); Copenhagen County; Danish Centre for Evaluation and Health Technology Assessment; Danish Council for Independent Research; Danish Heart Foundation (07-10-R61-A1754-B838-22392F); Danish Medical Research Council; Danish Pharmaceutical Association; Emil Aaltonen Foundation; European Research Council Advanced Research Grant; European Union FP7 (EpiMigrant, 279143; FP7/2007-2013; 259749); Finland's Slottery Machine Association; Finnish Cultural Foundation; Finnish Diabetes Research Foundation; Finnish Foundation for Cardiovascular Research; Finnish Foundation of Cardiovascular Research; Finnish Medical Society; Finnish National Public Health Institute; Finska Läkaresällskapet; Folkhälsan Research Foundation; Foundation for Life and Health in Finland; German Center for Diabetes Research (DZD) ; German Federal Ministry of Education and Research; Health Care Centers in Vasa, Närpes and Korsholm; Health Insurance Foundation (2012B233) ; Helsinki University Central Hospital Research Foundation; Hospital districts of Pirkanmaa, Southern Ostrobothnia, North Ostrobothnia, Central Finland, and Northern Savo; Ib Henriksen foundation; Juho Vainio Foundation; Korea Centers for Disease Control and Prevention (4845–301); Korea National Institute of Health (2012-N73002-00); Li Ka Shing Foundation; Liv och Hälsa; Lundbeck Foundation; Marie-Curie Fellowship (PIEF-GA-2012-329156); Medical Research Council (G0601261, G0900747-91070, G0601966, G0700931); Ministry of Education in Finland; Ministry of Social Affairs and Health in Finland; MRC-PHE Centre for Environment and Health;Municipal Heath Care Center and Hospital in Jakobstad; Närpes Health Care Foundation; National Institute for Health Research (RP-PG-0407-10371); National Institutes of Health (U01 DK085526, U01 DK085501, U01 DK085524, U01 DK085545, U01 DK085584, U01 DK088389, RC2-DK088389, DK085545, DK098032, HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN, R01MH107666 and K12CA139160268201300050C, U01 DK062370, R01 DK066358, U01DK085501, R01HL102830, R01DK073541, PO1AG027734, R01AG046949, 1R01AG042188, P30AG038072, R01 MH101820, R01MH090937, P30DK020595, R01 DK078616, NIDDK K24 DK080140, 1RC2DK088389, T32GM007753); National Medical Research Council; National Research Foundation of Korea (NRF-2012R1A2A1A03006155); Nordic Center of Excellence in Disease Genetics; Novo Nordisk; Ollqvist Foundation; OrionFarmos Research Foundation; Paavo Nurmi Foundation; Perklén Foundation; Samfundet Folkhälsan; Signe and Ane Gyllenberg Foundation; Sigrid Juselius Foundation; Social Insurance Institution of Finland; South East Norway Health Authority (2011060); Swedish Cultural Foundation in Finland; Swedish Heart-Lung Foundation; Swedish Research Council; Swedish Research Council (Linné and Strategic Research Grant); The American Federation for Aging Research; The Einstein Glenn Center; The European Commission (HEALTH-F4-2007-201413); The Finnish Diabetes Association; The Folkhälsan Research Foundation; The Påhlssons Foundation; The provinces of Newfoundland and Labrador, Nova Scotia, and New Brunswick; The Sigrid Juselius Foundation; The Skåne Regional Health Authority; The Swedish Heart-Lung Foundation; Timber Merchant Vilhelm Bang’s Foundation; Turku University Foundation; Uppsala University; Wellcome Trust (064890, 083948, 085475, 086596, 090367, 090532, 092447, 095101/Z/10/Z, 200837/Z/16/Z, 095552, 098017, 098381, 098051, 084723, 072960/2/ 03/2, 086113/Z/08/Z, WT098017, WT064890, WT090532, WT098017, 098051, WT086596/Z/08/A and 086596/Z/08/Z). Detailed acknowledgment of funding sources is provided in the Additional Acknowledgements section of the Supplementary Materials

Characterization of circular RNAs in human, mouse and rat hearts.

Deep sequencing techniques and advanced data analysis methods recently enabled the characterization of thousands of circular RNA isoforms (circRNAs) from a number of tissues and organisms. There is emerging evidence that some circRNAs may have important biological functions or serve as diagnostic biomarkers in disease conditions. In order to analyze circRNA expression in the heart and its changes in different conditions we performed RNA-Seq analysis of ribosome-depleted libraries from rats (neonatal and adult), mice (sham or after transverse aortic constriction, TAC) and humans (failing, non-failing). All samples were sequenced after a treatment with exonuclease RNase R or a mock treatment and >9000 candidate circRNAs were detected for each species. Additionally, we performed separate isolation of nuclear and cytoplasmic RNA and co-immunoprecipitated RNA interacting with endogenous argonaute 2 (Ago2) in primary cardiac myocytes. We found circRNAs to be significantly enriched in the cytoplasm compared to linear transcripts and to have a similar level of association with Ago2. Notably in all three species we observed dozens of circRNAs arising from the titin (Ttn) gene, which is known to undergo highly complex alternative splicing during heart maturation. Correspondingly we observed extensive differential regulation of Ttn circRNAs between neonatal and adult rat hearts, suggesting that circRNA formation could be involved in the regulation of titin splicing. We expect that our inventory of cardiac circRNAs, as well as the information on their conservation and differential expression will provide an important basis for further studies addressing their function and suitability as biomarkers

SP8 promotes an aggressive phenotype in hepatoblastoma via FGF8 activation,

Hepatoblastoma (HB) is the most common malignant liver tumor in childhood and it generally has a good prognosis. However, if associated with aggressive metastatic disease, outcome is still poor. The molecular mechanisms leading to metastatic spread in HB patients are still unknown. By combining RNA-sequencing and a genome-wide methylome analysis, we identified the transcription factor SP8 and the growth factor FGF8 among the most strongly upregulated genes in metastatic HB cases, with a concomitant robust demethylation of the respective promoter regions. Of note, high expression of both candidates was associated with the aggressive C2 subtype of the 16-gene signature and poor survival. Chromatin immunoprecipitation revealed a direct transcriptional regulation of FGF8 through binding of SP8 to theFGF8promoter. Gain- and loss-of-function experiments proved promoting effects of SP8 on motility, self-renewal, migration, and the invasive potential of HB cells. Moreover, stable overexpression of SP8 in Hep3B cells resulted in the acquisition of a mesenchymal phenotype and a strong upregulation of epithelial-mesenchymal transition-associated genes. Using KRAB-mediated CRISPR-dCas9 interference directed against FGF8, we could show that FGF8 is essential for the SP8-mediated aggressive tumor behavior. Treatment of HB cell lines with the pan SP family inhibitor mithramycin A resulted in a significant inhibition of their clonogenic growth. In summary, we identified SP8 and FGF8 as key players in aggressive traits of HB and propose SP8 inhibiting drugs as a new effective treatment strategy especially for metastatic tumors