Bundesministerium für Forschung, Technologie und Raumfahrt

Abstract Somatic mutations in cancer genomes are caused by multiple mutational processes, each of which generates a characteristic mutational signature 1 . Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium 2 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we characterized mutational signatures using 84,729,690 somatic mutations from 4,645 whole-genome and 19,184 exome sequences that encompass most types of cancer. We identified 49 single-base-substitution, 11 doublet-base-substitution, 4 clustered-base-substitution and 17 small insertion-and-deletion signatures. The substantial size of our dataset, compared with previous analyses 3–15 , enabled the discovery of new signatures, the separation of overlapping signatures and the decomposition of signatures into components that may represent associated—but distinct—DNA damage, repair and/or replication mechanisms. By estimating the contribution of each signature to the mutational catalogues of individual cancer genomes, we revealed associations of signatures to exogenous or endogenous exposures, as well as to defective DNA-maintenance processes. However, many signatures are of unknown cause. This analysis provides a systematic perspective on the repertoire of mutational processes that contribute to the development of human cancer.

Abstract Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale 1–3 . Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter 4 ; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation 5,6 ; analyses timings and patterns of tumour evolution 7 ; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity 8,9 ; and evaluates a range of more-specialized features of cancer genomes 8,10–18 .

Abstract Cancer develops through a process of somatic evolution 1,2 . Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes 3 . Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) 4 , we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.

Abstract A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes 1–7 . Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types 8 . Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions—as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2–7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and—in liver cancer—frequently activate the telomerase gene TERT . A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.

Abstract The discovery of drivers of cancer has traditionally focused on protein-coding genes 1–4 . Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium 5 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers 6,7 , raise doubts about others and identify novel candidates, including point mutations in the 5′ region of TP53 , in the 3′ untranslated regions of NFKBIZ and TOB1 , focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.

To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a "stopping solution." Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added [3H]adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract Transcript alterations often result from somatic changes in cancer genomes 1 . Various forms of RNA alterations have been described in cancer, including overexpression 2 , altered splicing 3 and gene fusions 4 ; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) 5 . Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis , of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed ‘bridged’ fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.

RelE of Escherichia coli is a global inhibitor of translation that is activated by nutritional stress. Activation of RelE depends on Lon-mediated degradation of RelB, the antagonist that neutralizes RelE. In vitro, RelE cleaves synthetic mRNAs positioned at the ribosomal A-site. We show here that in vivo overexpression of RelE confers cleavage of mRNA and tmRNA in their coding regions. RelE-mediated cleavage depended on translation of the RNAs and occurred at both sense and stop codons. RelE cleavage of mRNA and tmRNA was also induced by amino acid starvation. An ssrA deletion strain was hypersensitive to RelE, whereas overproduction of tmRNA counteracted RelE toxicity. After neutralization of RelE by RelB, rapid recovery of translation required tmRNA, indicating that tmRNA alleviated RelE toxicity by rescuing ribosomes stalled on damaged mRNAs. RelE proteins from Gram-positive Bacteria and Archaea cleaved tmRNA with a pattern similar to that of E. coli RelE, suggesting that the function and target of RelE may be conserved across the prokaryotic domains.

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5′-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3′-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (α) and 40-kDa (β) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA. Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5′-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3′-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (α) and 40-kDa (β) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA. INTRODUCTIONHuman acid ceramidase ((AC) 1The abbreviations used are: ACacid ceramidaseFDFarber diseasebpbase pair(s)ACO(s)synthetic oligonucleotide(s)PAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reaction. N-acylsphingosine amidohydrolase, EC 3.5.1.23) catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (1Gatt S. J. Biol. Chem. 1963; 238: 3131-3133Abstract Full Text PDF PubMed Google Scholar). An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease (FD), also called Farber lipogranulomatosis (2Moser H.W. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 7th Ed. McGraw-Hill Inc., New York1995: 2589-2599Google Scholar). Patients with FD accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insufficiency, and a shortened life-span. The clinical diagnosis of FD is usually confirmed by biochemical methods, including the determination of lysosomal ceramide accumulation and/or the deficiency of AC activity. To date, seven FD subtypes have been described with varying degrees of clinical involvement; notably, a direct correlation between the amount of ceramide accumulation and the clinical severity of FD patients has recently been demonstrated (3Levade T. Moser H.W. Fensom A.H. Harzer K. Moser A.B. Salvayre R. J. Neurol. Sci. 1995; 134: 108-114Abstract Full Text PDF PubMed Scopus (47) Google Scholar).In addition to its central role in disease pathogenesis, sphingolipid biosynthesis, and membrane formation, ceramide is an important cell-signaling molecule involved in a variety of diverse processes such as neurite growth, monocyte differentiation, and Fas (Apo1/CD95)-induced apoptosis (for reviews, see Refs. 4Hannun Y.A. Obeid L.M. Trends Biochem. Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (570) Google Scholar and 5Kan C.C. Kolesnick R. Trends Genet. 1993; 5: 99-101Google Scholar). Moreover, sphingosine (the catabolic product of ceramide degradation) has been shown to inhibit protein kinase C activity and can exert a variety of effects on cell growth and differentiation (6Hannun Y.A. Loomis C.R. Merill Jr., A.H. Bell R.M. J. Biol Chem. 1986; 261: 12604-12609Abstract Full Text PDF PubMed Google Scholar, 7Wilson E. Olcott M.C. Merill Jr., A.H. Lambeth J.D. J. Biol. Chem. 1986; 261: 12616-12623Abstract Full Text PDF PubMed Google Scholar, 8Merill Jr., A.H. Sereni A.M. Stevens V.L. Hannun Y.A. Bell R.M. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 12610-12615Abstract Full Text PDF PubMed Google Scholar). Since ceramide degradation is the only catabolic source of intracellular sphingosine (9Rother J. van Echten G. Schwarzmann G. Sandhoff K. Biochem. Biophys. Res. Commun. 1992; 189: 14-20Crossref PubMed Scopus (130) Google Scholar, 10Wang E. Norred W.P. Bacon C.W. Riley R.T. Merill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar), AC activity may be the rate-limiting step in determining the intracellular levels of this compound.About 2 years ago, AC was purified to apparent homogeneity from human urine (11Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The final preparation contained a single polypeptide of ~50 kDa that could be resolved into two subunits (designated α (13 kDa) and β (40 kDa)) after treatment with sulfhydryl-reducing reagents. Treatment of the purified enzyme with N-glycanase F reduced the molecular mass of the β subunit to ~29 kDa, whereas the molecular mass of the α subunit remained unchanged. Polyclonal antibodies were raised against the purified urinary enzyme, and the biosynthesis of AC was studied in human skin fibroblasts. Metabolic labeling and immunoprecipitation analyses indicated that both subunits arose from a single precursor of ~55 kDa by proteolytic processing.This manuscript reports the isolation and characterization of a full-length cDNA encoding human AC and the identification of the first molecular lesion causing FD. The availability of this cDNA should facilitate further molecular genetic analysis of FD patients and stimulate additional studies defining the role of AC in ceramide metabolism, cell differentiation, and mammalian development.DISCUSSIONCeramide plays a critical role in normal cell function and disease pathogenesis. Thus, understanding the metabolism of this lipid is of critical importance to both basic biologists and physicians alike. Toward this end, we report the isolation and expression of a full-length cDNA encoding AC, the enzyme responsible for the hydrolysis of ceramide into free fatty acid and sphingosine. Authenticity of this cDNA was based on colinearity with 117 amino acid residues derived from purified AC, as well as transient expression studies in COS-1 cells. Moreover, a homoallelic point mutation was identified within the corresponding AC cDNA and genomic sequences from a patient with FD, which is the lysosomal storage disorder due to the deficiency of AC enzymatic activity.Initially, a putative full-length AC cDNA was obtained from a human skin fibroblast library. However, several features of this cDNA indicated that it may have been artifactual, including the facts that the first in-frame ATG was not followed by a signal peptide coding region, that there were no cysteine residues encoded within the 5′ region, which presumably contained the α-subunit sequences (11Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and that the molecular mass of the putative peptide encoded by this region did not agree with the known molecular mass (~13 kDa) of the authentic α-subunit. Analysis of a different AC cDNA isolated from a human pituitary library revealed that the 5′ end was different from that encoded by the fibroblast sequence; this cDNA had an extended 5′ open reading frame including three cysteine residues, an authentic signal coding region, and a 17-bp 5′-untranslated sequence. Except for these 95 nucleotides, the fibroblast and pituitary sequences were identical.Based on these findings, a hybrid AC cDNA was constructed that contained the 5′ pituitary sequence and the remaining fibroblast sequence. This full-length cDNA consisted of a 17-bp 5′-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3′-untranslated sequence containing three polyadenylation sites, and a 17-bp poly(A) tail (Fig. 1). The region from nucleotide 427 (i.e. the N-terminal codon of the β-subunit) to nucleotide 1185 encoded a peptide with a calculated mass of 29 kDa that contained six potential N-glycosylation sites and 5 cysteine residues. The upstream region encoded a putative α-subunit with a predicted molecular mass of 13 kDa containing three cysteine residues but no N-glycosylation sites.Previous studies using purified AC have shown that the human enzyme was a heterodimeric protein consisting of an unglycosylated α-subunit (~13 kDa) and an N-glycosylated β-subunit (~40 kDa) (10Wang E. Norred W.P. Bacon C.W. Riley R.T. Merill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar). Complete deglycosylation resulted in the reduction of the molecular mass of the β-subunit by about 11 kDa, which corresponds to three or four N-linked oligosaccharide side chains. Biosynthetic studies carried out in cultured skin fibroblasts indicated that both subunits arose from a single glycosylated precursor protein of about 55 kDa by proteolytic processing (11Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar).After transfection of the full-length AC cDNA into COS-1 cells, metabolic labeling and immunoprecipitation studies using monospecific anti-AC antibodies raised against the purified enzyme revealed a protein pattern identical to that of human fibroblasts. Denaturing SDS gel electrophoresis showed that in the transfected COS cells, a 55-kDa precursor protein was processed into the 40-kDa β- and 13-kDa α-subunits. Endoglycosidase F treatment resulted in a reduced molecular mass for the precursor and the β-subunit (from 55 to 40 kDa and from 40 to 29 kDa, respectively), whereas the size of the α-subunit remained unchanged. Furthermore, COS-1 cells transfected with the full-length AC cDNA had up to 10-fold higher activities over the endogenous level of non-transfected and mock-transfected cells. The maximum of AC activity peaked between pH 4 and pH 5 and was not detectable at neutral or alkaline pH values (data not shown). These studies provided additional proof that the protein encoded by the full-length cDNA was AC.Further evidence was provided by the analysis of the AC sequence obtained from a patient with FD. Three nucleotide changes (M72V, I92V, and T222K) were identified in this patient as compared to the wild type sequence. Analysis of 14 unrelated normal individuals demonstrated that M72V and I92V were normal polymorphisms, whereas T222K was only found in the FD patient. Furthermore, analysis of the consanguineous parents revealed that both were heterozygous for the T222K mutation.This manuscript reports the first isolation of a full-length cDNA encoding AC and the first identification of a mutation causing FD. The availability of this cDNA allows us to generate the respective animal model and should help us to define a possible role of AC in signal transduction, as well as the identification of various molecular lesions underlying the different clinical subtypes of FD. INTRODUCTIONHuman acid ceramidase ((AC) 1The abbreviations used are: ACacid ceramidaseFDFarber diseasebpbase pair(s)ACO(s)synthetic oligonucleotide(s)PAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reaction. N-acylsphingosine amidohydrolase, EC 3.5.1.23) catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (1Gatt S. J. Biol. Chem. 1963; 238: 3131-3133Abstract Full Text PDF PubMed Google Scholar). An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease (FD), also called Farber lipogranulomatosis (2Moser H.W. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 7th Ed. McGraw-Hill Inc., New York1995: 2589-2599Google Scholar). Patients with FD accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insufficiency, and a shortened life-span. The clinical diagnosis of FD is usually confirmed by biochemical methods, including the determination of lysosomal ceramide accumulation and/or the deficiency of AC activity. To date, seven FD subtypes have been described with varying degrees of clinical involvement; notably, a direct correlation between the amount of ceramide accumulation and the clinical severity of FD patients has recently been demonstrated (3Levade T. Moser H.W. Fensom A.H. Harzer K. Moser A.B. Salvayre R. J. Neurol. Sci. 1995; 134: 108-114Abstract Full Text PDF PubMed Scopus (47) Google Scholar).In addition to its central role in disease pathogenesis, sphingolipid biosynthesis, and membrane formation, ceramide is an important cell-signaling molecule involved in a variety of diverse processes such as neurite growth, monocyte differentiation, and Fas (Apo1/CD95)-induced apoptosis (for reviews, see Refs. 4Hannun Y.A. Obeid L.M. Trends Biochem. Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (570) Google Scholar and 5Kan C.C. Kolesnick R. Trends Genet. 1993; 5: 99-101Google Scholar). Moreover, sphingosine (the catabolic product of ceramide degradation) has been shown to inhibit protein kinase C activity and can exert a variety of effects on cell growth and differentiation (6Hannun Y.A. Loomis C.R. Merill Jr., A.H. Bell R.M. J. Biol Chem. 1986; 261: 12604-12609Abstract Full Text PDF PubMed Google Scholar, 7Wilson E. Olcott M.C. Merill Jr., A.H. Lambeth J.D. J. Biol. Chem. 1986; 261: 12616-12623Abstract Full Text PDF PubMed Google Scholar, 8Merill Jr., A.H. Sereni A.M. Stevens V.L. Hannun Y.A. Bell R.M. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 12610-12615Abstract Full Text PDF PubMed Google Scholar). Since ceramide degradation is the only catabolic source of intracellular sphingosine (9Rother J. van Echten G. Schwarzmann G. Sandhoff K. Biochem. Biophys. Res. Commun. 1992; 189: 14-20Crossref PubMed Scopus (130) Google Scholar, 10Wang E. Norred W.P. Bacon C.W. Riley R.T. Merill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar), AC activity may be the rate-limiting step in determining the intracellular levels of this compound.About 2 years ago, AC was purified to apparent homogeneity from human urine (11Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The final preparation contained a single polypeptide of ~50 kDa that could be resolved into two subunits (designated α (13 kDa) and β (40 kDa)) after treatment with sulfhydryl-reducing reagents. Treatment of the purified enzyme with N-glycanase F reduced the molecular mass of the β subunit to ~29 kDa, whereas the molecular mass of the α subunit remained unchanged. Polyclonal antibodies were raised against the purified urinary enzyme, and the biosynthesis of AC was studied in human skin fibroblasts. Metabolic labeling and immunoprecipitation analyses indicated that both subunits arose from a single precursor of ~55 kDa by proteolytic processing.This manuscript reports the isolation and characterization of a full-length cDNA encoding human AC and the identification of the first molecular lesion causing FD. The availability of this cDNA should facilitate further molecular genetic analysis of FD patients and stimulate additional studies defining the role of AC in ceramide metabolism, cell differentiation, and mammalian development.

In cancer, the primary tumour's organ of origin and histopathology are the strongest determinants of its clinical behaviour, but in 3% of cases a patient presents with a metastatic tumour and no obvious primary. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we train a deep learning classifier to predict cancer type based on patterns of somatic passenger mutations detected in whole genome sequencing (WGS) of 2606 tumours representing 24 common cancer types produced by the PCAWG Consortium. Our classifier achieves an accuracy of 91% on held-out tumor samples and 88% and 83% respectively on independent primary and metastatic samples, roughly double the accuracy of trained pathologists when presented with a metastatic tumour without knowledge of the primary. Surprisingly, adding information on driver mutations reduced accuracy. Our results have clinical applicability, underscore how patterns of somatic passenger mutations encode the state of the cell of origin, and can inform future strategies to detect the source of circulating tumour DNA.

Multi-omics datasets represent distinct aspects of the central dogma of molecular biology. Such high-dimensional molecular profiles pose challenges to data interpretation and hypothesis generation. ActivePathways is an integrative method that discovers significantly enriched pathways across multiple datasets using statistical data fusion, rationalizes contributing evidence and highlights associated genes. As part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumor types, we integrated genes with coding and non-coding mutations and revealed frequently mutated pathways and additional cancer genes with infrequent mutations. We also analyzed prognostic molecular pathways by integrating genomic and transcriptomic features of 1780 breast cancers and highlighted associations with immune response and anti-apoptotic signaling. Integration of ChIP-seq and RNA-seq data for master regulators of the Hippo pathway across normal human tissues identified processes of tissue regeneration and stem cell regulation. ActivePathways is a versatile method that improves systems-level understanding of cellular organization in health and disease through integration of multiple molecular datasets and pathway annotations.

We present details of the construction and characterization of the coaddition of the Sloan Digital Sky Survey (SDSS) Stripe 82 ugriz imaging data. This survey consists of 275 deg2 of repeated scanning by the SDSS camera over −50° ⩽ α ⩽ 60° and −125 ⩽ δ ⩽ +125 centered on the Celestial Equator. Each piece of sky has ∼20 runs contributing and thus reaches ∼2 mag fainter than the SDSS single pass data, i.e., to r ∼ 23.5 for galaxies. We discuss the image processing of the coaddition, the modeling of the point-spread function (PSF), the calibration, and the production of standard SDSS catalogs. The data have an r-band median seeing of 11 and are calibrated to ⩽1%. Star color–color, number counts, and PSF size versus modeled size plots show that the modeling of the PSF is good enough for precision five-band photometry. Structure in the PSF model versus magnitude plot indicates minor PSF modeling errors, leading to misclassification of stars as galaxies, as verified using VVDS spectroscopy. There are a variety of uses for this wide-angle deep imaging data, including galactic structure, photometric redshift computation, cluster finding and cross wavelength measurements, weak lensing cluster mass calibrations, and cosmic shear measurements.

Long non-coding RNAs (lncRNAs) are a growing focus of cancer genomics studies, creating the need for a resource of lncRNAs with validated cancer roles. Furthermore, it remains debated whether mutated lncRNAs can drive tumorigenesis, and whether such functions could be conserved during evolution. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we introduce the Cancer LncRNA Census (CLC), a compilation of 122 GENCODE lncRNAs with causal roles in cancer phenotypes. In contrast to existing databases, CLC requires strong functional or genetic evidence. CLC genes are enriched amongst driver genes predicted from somatic mutations, and display characteristic genomic features. Strikingly, CLC genes are enriched for driver mutations from unbiased, genome-wide transposon-mutagenesis screens in mice. We identified 10 tumour-causing mutations in orthologues of 8 lncRNAs, including LINC-PINT and NEAT1, but not MALAT1. Thus CLC represents a dataset of high-confidence cancer lncRNAs. Mutagenesis maps are a novel means for identifying deeply-conserved roles of lncRNAs in tumorigenesis.

Nucleotides, nucleosides and nucleobases belong to the non-protein-nitrogen (NPN) fraction of milk. The largest amounts of ribonucleosides and ribonucleotides--ribose forms only were considered in this review--were measured directly after parturition in bovine milk and other ruminants as well as in the milk of humans. Generally, concentrations of most of the nucleos(t)ides tend to decrease gradually with advancing lactation period or nursing time. The species-specific pattern of these minor constituents in milk from different mammals is a remarkable property and confirms, at least, the specific physiological impact of these minor compounds in early life. The physiological capacity of these compounds in milk is given by the total potentially available nucleosides. The main dietary sources of nucleos(t)ides are nucleoproteins and nucleic acids which are converted in the course of intestinal digestion into nucleosides and nucleobases the preferred forms for absorption in the intestine. Thus, nucleosides and nucleobases are suggested to be the acting components of dietary and/or supplemented nucleic acid-related compounds in the gut. They are used by the body as exogenous trophochemical sources and can be important for optimal metabolic functions. Up to 15 % of the total daily need for a breast-fed infant was calculated to come from this dietary source. Concerning their biological role they not only act as metabolites but are also involved as bioactive substances in the regulation of body functions. Dietary nucleotides affect immune modulation, e.g. they enhance antibody responses of infants as shown by a study with more than 300 full-term healthy infants. Dietary nucleos(t)ides are found to contribute to iron absorption in the gut and to influence desaturation and elongation rates in fatty acid synthesis, in particular long-chain polyunsaturated fatty acids in early stages of life. The in vitro modulation of cell proliferation and apoptosis has been described by ribonucleosides, in particular by modified components using human cell culture models. Due to the bio- and trophochemical properties of dietary nucleos(t)ides, the European Commission has allowed the use of supplementation with specific ribonucleotides in the manufacture of infant and follow-on formula. From the technochemical point of view, the ribonucleoside pattern is influenced by thermal treatment of milk. In addition ribonucleosides are useful indicators for quantifying adulterations of milk and milk products.

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.

ABSTRACT Physical activity is not only beneficial to physical health but also to cognitive functions. In particular, executive functions that are closely related to learning achievement can be improved by acute and recurring physical activity. We examined the effects of a single 30‐min physical education program in contrast to a 5‐min movement break on working memory, cognitive flexibility, and inhibition of attention and behavioral tendencies of eighty‐one 13‐ to 14‐year‐old students in grade 7 in Germany. Results indicate that the maintenance of on‐task attention in the face of distraction was improved by an aerobic endurance exercise‐based physical education program but not by a short aerobic movement break. This suggests that the duration of a school sports program is decisive for improving students' executive attention.

Abstract Cancers require telomere maintenance mechanisms for unlimited replicative potential. They achieve this through TERT activation or alternative telomere lengthening associated with ATRX or DAXX loss. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium , we dissect whole-genome sequencing data of over 2500 matched tumor-control samples from 36 different tumor types aggregated within the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium to characterize the genomic footprints of these mechanisms. While the telomere content of tumors with ATRX or DAXX mutations (ATRX/DAXX trunc ) is increased, tumors with TERT modifications show a moderate decrease of telomere content. One quarter of all tumor samples contain somatic integrations of telomeric sequences into non-telomeric DNA. This fraction is increased to 80% prevalence in ATRX/DAXX trunc tumors, which carry an aberrant telomere variant repeat (TVR) distribution as another genomic marker. The latter feature includes enrichment or depletion of the previously undescribed singleton TVRs TTCGGG and TTTGGG, respectively. Our systematic analysis provides new insight into the recurrent genomic alterations associated with telomere maintenance mechanisms in cancer.

The coordination of activity across neocortical areas is essential for mammalian brain function. Understanding this process requires simultaneous functional measurements across the cortex. In order to dissociate direct cortico-cortical interactions from other sources of neuronal correlations, it is furthermore desirable to target cross-areal recordings to neuronal subpopulations that anatomically project between areas. Here, we combined anatomical tracers with a novel multi-area two-photon microscope to perform simultaneous calcium imaging across mouse primary (S1) and secondary (S2) somatosensory whisker cortex during texture discrimination behavior, specifically identifying feedforward and feedback neurons. We find that coordination of S1-S2 activity increases during motor behaviors such as goal-directed whisking and licking. This effect was not specific to identified feedforward and feedback neurons. However, these mutually projecting neurons especially participated in inter-areal coordination when motor behavior was paired with whisker-texture touches, suggesting that direct S1-S2 interactions are sensory-dependent. Our results demonstrate specific functional coordination of anatomically-identified projection neurons across sensory cortices.

Organic C, N, clay, and carbonate content are basic soil properties, which are commonly assessed by different analytical methods. This study aimed at utilizing temperature‐induced dynamics of weight losses to determine these parameters simultaneously without losing precision and accuracy. For this purpose, thermogravimetric profiles of 52 soil samples were recorded and compared with C, N, and textural data obtained from conventional analyses using dry combustion and pipette methods. The samples originated from different climatic regions, land‐use systems and parent materials. The results showed that thermal weight losses correlated closely ( P < 0.001) with the content of organic C ( R 2 = 0.98), total N ( R 2 = 0.94), clay ( R 2 = 0.87) and CO 3 –C ( R 2 = 0.99) for mineral soil samples, while no such relationships were found in organic soil horizons. For mineral soils, each soil property correlated with a specific interval of temperature‐induced weight losses. Application of thermogravimetry to independent additional sets of 250 samples confirmed a reliable estimation of organic C, total N, clay and CO 3 –C of mineral soils. It is concluded that thermogravimetry allows a simultaneous, rapid and reliable screening of these four properties in mineral soils.