Integrated Genomics Identifies Five Medulloblastoma Subtypes with Distinct Genetic Profiles, Pathway Signatures and Clinicopathological Features

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements in cure rates, prediction of disease outcome remains a major challenge and survivors suffer from serious therapy-related side-effects. Recent data showed that patients with WNT-activated tumors have a favorable prognosis, suggesting that these patients could be treated less intensively, thereby reducing the side-effects. This illustrates the potential benefits of a robust classification of medulloblastoma patients and a detailed knowledge of associated biological mechanisms. METHODS AND FINDINGS: To get a better insight into the molecular biology of medulloblastoma we established mRNA expression profiles of 62 medulloblastomas and analyzed 52 of them also by comparative genomic hybridization (CGH) arrays. Five molecular subtypes were identified, characterized by WNT signaling (A; 9 cases), SHH signaling (B; 15 cases), expression of neuronal differentiation genes (C and D; 16 and 11 cases, respectively) or photoreceptor genes (D and E; both 11 cases). Mutations in beta-catenin were identified in all 9 type A tumors, but not in any other tumor. PTCH1 mutations were exclusively identified in type B tumors. CGH analysis identified several fully or partly subtype-specific chromosomal aberrations. Monosomy of chromosome 6 occurred only in type A tumors, loss of 9q mostly occurred in type B tumors, whereas chromosome 17 aberrations, most common in medulloblastoma, were strongly associated with type C or D tumors. Loss of the inactivated X-chromosome was highly specific for female cases of type C, D and E tumors. Gene expression levels faithfully reflected the chromosomal copy number changes. Clinicopathological features significantly different between the 5 subtypes included metastatic disease and age at diagnosis and histology. Metastatic disease at diagnosis was significantly associated with subtypes C and D and most strongly with subtype E. Patients below 3 yrs of age had type B, D, or E tumors. Type B included most desmoplastic cases. We validated and confirmed the molecular subtypes and their associated clinicopathological features with expression data from a second independent series of 46 medulloblastomas. CONCLUSIONS: The new medulloblastoma classification presented in this study will greatly enhance the understanding of this heterogeneous disease. It will enable a better selection and evaluation of patients in clinical trials, and it will support the development of new molecular targeted therapies. Ultimately, our results may lead to more individualized therapies with improved cure rates and a better quality of life

Topside Ionosphere Sounding From the CHAMP, GRACE, and GRACE-FO Missions

Satellites in Low Earth Orbit (LEO) are essential for sounding the topside ionosphere. In this work, we present and validate a data set of Total Electron Content (TEC) and in situ electron density observations from the Gravity Recovery And Climate Experiment (GRACE) and GRACE-Follow-On missions as well as a TEC data set from the CHAllenging Minisatellite Payload mission. Concerning TEC, special emphasis is put to ensure optimal consistency to the already existing Swarm and Gravity field and steady-state ocean circulation explorer (GOCE) TEC data sets. The newly processed satellite missions allow covering two full solar cycles with LEO slant TEC. Furthermore, the twin satellite missions GRACE and GRACE-FO equipped with inter-satellite K-band ranging allows to derive the horizontal TEC and, due to the small inter-satellite distance of the satellite pairs, an approximation for local electron density. However, the derived value of electron density is relative and requires calibration using external information. In this work, the calibration is performed using the IRI-2016 model. Radar observations, as well as in situ electron density observations available from Swarm B Langmuir probes, are used for validation. Conjunctions between satellites are used to validate the TEC time series. The newly derived data set is shown to be highly consistent with the already existing data sets with standard deviations below 3 TECU for TEC (even 1 TECU was reached for low solar flux) and an offset below 7 × 1010 m−3 with a standard deviation near 1 × 1011 m−3 for the electron density.</p

High dissolved oxygen tension triggers outer membrane vesicle formation by Neisseria meningitidis

Abstract Background Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria and can be used as vaccines. Often, detergents are used to promote release of OMVs and to remove the toxic lipopolysaccharides. Lipopolysaccharides can be detoxified by genetic modification such that vesicles spontaneously produced by bacteria can be directly used as vaccines. The use of spontaneous OMVs has the advantage that no separate extraction step is required in the purification process. However, the productivity of spontaneous OMVs by bacteria at optimal growth conditions is low. One of many methods for increasing OMV formation is to reduce the linkage of the outer membrane to the peptidoglycan layer by knocking out the rmpM gene. A previous study showed that for Neisseria meningitidis this resulted in release of more OMVs. Furthermore, cysteine depletion was found to trigger OMV release and at the same time cause reduced growth and oxidative stress responses. Here we study the effect of growth rate and oxidative stress on OMV release. Results First, we identified using chemostat and accelerostat cultures of N. meningitidis that increasing the growth rate from 0.03 to 0.18 h−1 has a limited effect on OMV productivity. Thus, we hypothesized that oxidative stress is the trigger for OMV release and that oxidative stress can be introduced directly by increasing the dissolved oxygen tension of bacterial cultures. Slowly increasing oxygen concentrations in a N. meningitidis changestat showed that an increase from 30 to 150% air saturation improved OMV productivity four-fold. Batch cultures controlled at 100% air saturation increased OMV productivity three-fold over batch cultures controlled at 30% air saturation. Conclusion Increased dissolved oxygen tension induces the release of outer membrane vesicles in N. meningitidis cultures. Since oxygen concentration is a well-controlled process parameter of bacterial cultures, this trigger can be applied as a convenient process parameter to induce OMV release in bacterial cultures. Improved productivity of OMVs not only improves the production costs of OMVs as vaccines, it also facilitates the use of OMVs as adjuvants, enzyme carriers, or cell-specific drug delivery vehicles

High dissolved oxygen tension triggers outer membrane vesicle formation by Neisseria meningitidis

Background: Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria and can be used as vaccines. Often, detergents are used to promote release of OMVs and to remove the toxic lipopolysaccharides. Lipopolysaccharides can be detoxified by genetic modification such that vesicles spontaneously produced by bacteria can be directly used as vaccines. The use of spontaneous OMVs has the advantage that no separate extraction step is required in the purification process. However, the productivity of spontaneous OMVs by bacteria at optimal growth conditions is low. One of many methods for increasing OMV formation is to reduce the linkage of the outer membrane to the peptidoglycan layer by knocking out the rmpM gene. A previous study showed that for Neisseria meningitidis this resulted in release of more OMVs. Furthermore, cysteine depletion was found to trigger OMV release and at the same time cause reduced growth and oxidative stress responses. Here we study the effect of growth rate and oxidative stress on OMV release. Results: First, we identified using chemostat and accelerostat cultures of N. meningitidis that increasing the growth rate from 0.03 to 0.18 h-1 has a limited effect on OMV productivity. Thus, we hypothesized that oxidative stress is the trigger for OMV release and that oxidative stress can be introduced directly by increasing the dissolved oxygen tension of bacterial cultures. Slowly increasing oxygen concentrations in a N. meningitidis changestat showed that an increase from 30 to 150% air saturation improved OMV productivity four-fold. Batch cultures controlled at 100% air saturation increased OMV productivity three-fold over batch cultures controlled at 30% air saturation. Conclusion: Increased dissolved oxygen tension induces the release of outer membrane vesicles in N. meningitidis cultures. Since oxygen concentration is a well-controlled process parameter of bacterial cultures, this trigger can be applied as a convenient process parameter to induce OMV release in bacterial cultures. Improved productivity of OMVs not only improves the production costs of OMVs as vaccines, it also facilitates the use of OMVs as adjuvants, enzyme carriers, or cell-specific drug delivery vehicles.</p

Quality of sOMV and eOMV vaccines.

<p>In addition to yield, quality of the sOMV and eOMV vaccines is compared. It was previously demonstrated that both vaccine types provide low toxicity and high functional immunogenicity in mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054314#pone.0054314-vandeWaterbeemd1" target="_blank">[11]</a>. (A) Protein composition of eOMV reference vaccines is comparable to sOMV vaccines after cysteine depletion (time points I, K, M). Each lane contains 4 µg total protein, except sOMV at time points D and F (low protein concentration due to a low yield; maximal sample volume is loaded). PorA antigen (∼41 kD) has a major contribution to total protein content (>60%) in all vaccines. (B) Dynamic light scattering analysis reveals that sOMV vaccines have a slightly broader size distribution and minor aggregation compared to the eOMV references, indicating that the purification procedure is not yet fully consistent. X-axis represents vesicle size distribution (nm).</p

Implications for vaccine development.

<p>A novel approach for the production of sOMV vaccine against N. meningitidis serogroup B is explored by utilizing the effect of cysteine depletion. (A) Biomass concentration (closed circles) is monitored in bioreactor cultivations (time points A to N). Time point G marks onset of stationary growth, caused by depletion of cysteine (open circles). (B) Yield of purified sOMV vaccine (black bars) is compared with eOMV reference vaccine, which uses detergent-free biomass extraction to improve yield (white bars). Several time points before (D, F) and after (I, K, M) cysteine depletion are included. After cysteine depletion, sOMV yield increases gradually to quantities that are comparable to the eOMV reference (no significant difference at time point M). Significant yield differences are indicated with asterikses (p<0.05). ‘NS’ indicates a non-significant difference.</p

Cysteine is the growth-limiting medium component.

<p>Previous work demonstrated that vesicle release by <i>N. meningitidis</i> increased during the stationary growth phase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054314#pone.0054314-vandeWaterbeemd2" target="_blank">[23]</a>. Nutrient analysis of the chemically defined medium indicated that only arginine and cysteine were depleted during early stationary growth. Therefore, concentrations of both components were systematically lowered to identify the growth-limiting nutrient. Black lines and black intersected lines represent growth curves on media with normal and low arginine concentration, respectively. Normal and low cysteine concentrations are marked with circles and triangles. Cultivation time t = 0 represents the expected onset of stationary growth on reference medium with normal amounts of cysteine and arginine. The results indicate that cysteine, not arginine is the growth-limiting component. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054314#pone.0054314.s002" target="_blank">Figure S2</a> for corresponding nutrient data.</p

Proteome Analysis Is a Valuable Tool to Monitor Antigen Expression during Upstream Processing of Whole-Cell Pertussis Vaccines

Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. <i>Bordetella pertussis</i> bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors. Magnesium sulfate was added during the steady state of the culture in order to diminish the expression of virulence proteins. Changes in gene expression and antigen composition were measured by microarrays, mass spectrometry and ELISA. Transcriptome and proteome data revealed high similarity between the biological triplicates demonstrating consistent cultivation of <i>B. pertussis</i>. The addition of magnesium sulfate resulted in an instant downregulation of the virulence genes in <i>B. pertussis</i>, but a gradual decrease of virulence proteins. The quantity of virulence proteins concurred highly with the potency of the corresponding whole-cell pertussis vaccines, which were determined by the Kendrick test. In conclusion, proteome analysis provided detailed information on the composition and proportion of virulence proteins present in the whole-cell preparations of <i>B. pertussis</i>. Moreover, proteome analysis is a valuable method to monitor the production process of whole-cell biomass and predict the product quality of whole-cell pertussis vaccines

The Swarm Satellite Constellation Application and Research Facility (SCARF) and Swarm data products

International audiencewith the Earth system, is expected to be launched in late 2013. The objective of the Swarm mission is to providethe best ever survey of the geomagnetic field and its temporal evolution, in order to gain new insights into theEarth system by improving our understanding of the Earth’s interior and environment. In order to derive advancedmodels of the geomagnetic field (and other higher-level data products) it is necessary to take explicit advantageof the constellation aspect of Swarm. The Swarm SCARF (Satellite Constellation Application and ResearchFacility) has been established with the goal of deriving Level-2 products by combination of data from the threesatellites, and of the various instruments. The present paper describes the Swarm input data products (Level-1band auxiliary data) used by SCARF, the various processing chains of SCARF, and the Level-2 output data productsdetermined by SCARF