Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Simple rules for passive diffusion through the nuclear pore complex.

Passive macromolecular diffusion through nuclear pore complexes (NPCs) is thought to decrease dramatically beyond a 30-60-kD size threshold. Using thousands of independent time-resolved fluorescence microscopy measurements in vivo, we show that the NPC lacks such a firm size threshold; instead, it forms a soft barrier to passive diffusion that intensifies gradually with increasing molecular mass in both the wild-type and mutant strains with various subsets of phenylalanine-glycine (FG) domains and different levels of baseline passive permeability. Brownian dynamics simulations replicate these findings and indicate that the soft barrier results from the highly dynamic FG repeat domains and the diffusing macromolecules mutually constraining and competing for available volume in the interior of the NPC, setting up entropic repulsion forces. We found that FG domains with exceptionally high net charge and low hydropathy near the cytoplasmic end of the central channel contribute more strongly to obstruction of passive diffusion than to facilitated transport, revealing a compartmentalized functional arrangement within the NPC

Mucosal Immunization with Surface-Displayed Severe Acute Respiratory Syndrome Coronavirus Spike Protein on Lactobacillus casei Induces Neutralizing Antibodies in Mice

Induction of mucosal immunity may be important for preventing SARS-CoV infections. For safe and effective delivery of viral antigens to the mucosal immune system, we have developed a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (PgsA) of Bacillus subtilis as an anchoring matrix. Recombinant fusion proteins comprised of PgsA and the Spike (S) protein segments SA (residues 2 to 114) and SB (residues 264 to 596) were stably expressed in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses, immunofluorescence microscopy, and flow cytometry. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides. More importantly, these antibodies exhibited potent neutralizing activities against severe acute respiratory syndrome (SARS) pseudoviruses. Orally immunized mice mounted a greater neutralizing-antibody response than those immunized intranasally. Three new neutralizing epitopes were identified on the S protein using a peptide neutralization interference assay (residues 291 to 308, 520 to 529, and 564 to 581). These results indicate that mucosal immunization with recombinant L. casei expressing SARS-associated coronavirus S protein on its surface provides an effective means for eliciting protective immune response against the virus

Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex*

Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (∼ 600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope

Molecular Architecture and Function of the SEA Complex, a Modulator of the TORC1 Pathway*

The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex comprises two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway

Echinochrome A Protects Mitochondrial Function in Cardiomyocytes against Cardiotoxic Drugs

Echinochrome A (Ech A) is a naphthoquinoid pigment from sea urchins that possesses antioxidant, antimicrobial, anti-inflammatory and chelating abilities. Although Ech A is the active substance in the ophthalmic and cardiac drug Histochrome®, its underlying cardioprotective mechanisms are not well understood. In this study, we investigated the protective role of Ech A against toxic agents that induce death of rat cardiac myoblast H9c2 cells and isolated rat cardiomyocytes. We found that the cardiotoxic agents tert-Butyl hydroperoxide (tBHP, organic reactive oxygen species (ROS) inducer), sodium nitroprusside (SNP; anti-hypertension drug), and doxorubicin (anti-cancer drug) caused mitochondrial dysfunction such as increased ROS level and decreased mitochondrial membrane potential. Co-treatment with Ech A, however, prevented this decrease in membrane potential and increase in ROS level. Co-treatment of Ech A also reduced the effects of these cardiotoxic agents on mitochondrial oxidative phosphorylation and adenosine triphosphate level. These findings indicate the therapeutic potential of Ech A for reducing cardiotoxic agent-induced damage

Varieties of State Aid and Technological Development : Government Support to the Pulp and Paper Industry, the 1970s to the 1990s

Countries promote the development of pulp and paper industry through industrial, technology and innovation policy measures. Direct interventions and regional and environmental policies, together with more general governmental measures on trade negotiations, taxation, labour policies, and infrastructure development (e.g. roads, energy) have also had an impact on shaping the geographical location of and investments in the pulp and paper industry. This chapter presents an historical overview of government support on pulp and paper industry in the Organisation for Economic Co-operation and Development (OECD) countries from roughly the 1970s to the 1990s. As the earlier literature suggests, in countries where this industry was a dominant line of business, attention was paid to create a favourable regulatory environment, as the companies had bargaining power in influencing governmental policies. The Nordic countries (Finland, Sweden and Norway) are the primary examples of this kind of pattern. On the contrary, in countries in which the pulp and paper industry did not play a significant role, state aid played a less important role. State aid also influenced on the technologicalPeer reviewe