Debris/ice/TPS assessment and integrated photographic analysis for Shuttle Mission STS-54

A Debris/Ice/TPS assessment and integrated photographic analysis was conducted for Shuttle Mission STS-54. Debris inspections of the flight elements and launch pad were performed before and after launch. Ice/frost conditions on the External Tank were assessed by the use of computer programs, nomographs, and infrared scanner data during cryogenic loading of the vehicle followed by on-pad visual inspection. High speed photography was analyzed after launch to identify ice/debris sources and evaluate potential vehicle damage and/or in-flight anomalies. This report documents the debris/ice/TPS conditions and integrated photographic analysis of Shuttle Mission STS-54, and the resulting effect on the Space Shuttle Program

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples

La spectrométrie de masse : un couteau suisse pour disséquer la structure et la fonction du spermatoprotéasome

Le protéasome est le complexe enzymatique protéolytique principal de la cellule. Son cœur catalytique (20S) est formé de quatre anneaux heptamériques. Son activité et sa spécificité de substrat peuvent être régulées par les complexes 19S, PA28alphaß, PA28gamma et PA200 ainsi que par des sous-unités 20S alternatives. La spermatogenèse est un processus de différenciation des cellules germinales mâles: les spermatogonies (SPG) se transforment en spermatocytes (SPC), en spermatides (SPT) puis en spermatozoïdes. Ce processus requière une protéolyse intense. Le spermatoprotéasome (spt20S) est spécifique des gamètes en développement et essentiel à la spermatogenèse. Il diffère du protéasome standard (std20S) par la sous-unité alpha4s qui remplace la sous-unité constitutive alpha4. Le spt20S joue un rôle important avec PA200 dans la progression de la méiose, mais les mécanismes qui le rendent différent du std20S restent inconnus. Nous avons établi des stratégies protéomiques complémentaires pour caractériser les complexes du protéasome immunopurifiés à partir de testicules. L'analyse Top-Down de protéasome purifié, nous a permis de montrer pour la première fois qu'alpha4s et alpha4 portent les mêmes MPTs. La protéomique Bottom-Up, nous a permis de comparer les immunopurifications (IPs) de protéasomes totaux avec celles obtenues avec un anticorps spécifique du stp20S que nous avons développé. Nous avons établi qu'alpha4 et alpha4s ne coexistent pas dans le même 20S, bien qu'ils soient presque également abondants dans les testicules. Nous avons également trouvé plus de 19S et de PA200 liés au spt20S qu'au std20S. Les autres protéines préférentiellement associées au spt20S incluent PI31 et Fbxo7 qui sont cruciales pour la fertilité et d'importants médiateurs du transport du protéasome et de l'ancrage des E3 ligases - deux processus qui semblent cruciaux pour la fonction du spt20S pendant la spermatogenèse. Nous avons ensuite obtenu des cellules germinales à différents stades de la différenciation et l'analyse protéomique des lysats ainsi que des IPs, nous a permis d'établir un interactome dynamique du protéasome tout au long de la spermatogenèse. Nous avons observé un changement total du std20S au spt20S entre les SPG pré-méiotiques et les SPC/SPT méiotiques et post-méiotiques. Un changement d'expression semble responsable, plutôt qu'une incorporation préférentielle d'alpha4s. En entrant dans la méiose, l'association de PA200 avec le protéasome a augmenté 7 fois, confirmant son importance dans le développement des gamètes. Bien que PA200 soit d'après la littérature le principal activateur du spt20S, nous montrons que le 19S est est en réalité majoritaire, lié à 60% des 20S dans les SPC - une activation du protéasome sans précédent. De nombreux partenaires du spt20S sont identifiés à la fois dans les cellules germinales et dans les testicules entiers, montrant la robustesse de nos méthodes. Ceux-ci incluent des protéines synaptonémales, de nombreuses protéines impliquées dans l'ubiquitylation, le cycle cellulaire et la progression méiotique ainsi que le transport cellulaire, en accord avec les fonctions du spt20S proposées dans la littérature. Le passage d'alpha4 à alpha4s semble crucial pour la méiose, mais quelles sont les bases moléculaires de cette transition ? L'échange hydrogène-deutérium nous a permis de montrer pour la première fois que les deux protéasomes présentent des interfaces d'interaction différentes : alpha4s contient des régions plus flexibles qu'alpha4. Cette découverte est confirmée par des pull-down in vitro montrant que le 19S se lie plus fortement au spt20S qu'au std20S, expliquant la hausse d'activité protéolytique pendant la méiose. L'activité trypsique du spt20S est plus élevée que celle du std20S in vitro, ce qui pourrait refléter la nécessité de dégradation des histones. Globalement, nos données révèlent un processus de régulation du spt20S qui est plus complexe que ce qui avait été suggéré précédemment et jettent les bases des différences structurales et fonctionnelles entre le spt20S et le std20S.The proteasome is the main enzymatic complex for targeted proteolysis in the cell. Its core complex (20S) consists of four stacked heptameric rings and requires activator complexes: 19S, PA28alphaß, PA28gamma and PA200, which regulate 20S activity and substrate specificity. Alternative 20S subunits exist to further modulate the proteasome activity. Spermatogenesis is a process of male germ cell differentiation, where spermatogonia (SPG) transform through spermatocyte (SPC), then spermatid (SPT) stages, to become spermatozoa. This process requires intense proteolysis. The spermatoproteasome (spt20S) is specific to the developing gametes and essential for spermatogenesis. It differs from the standard proteasome (std20S) by only one subunit - alpha4s, which replaces the constitutive alpha4 subunit. Together with PA200, the spt20S plays an important role in meiosis progression, however, the mechanisms that make it different compared to std20S remain unknown. We established complementary proteomic pipelines for characterisation of proteasome complexes in the testes, combining immunopurification (IP) and mass spectrometry (MS) analysis. Our Top-Down analysis of purified proteasome, showed for the first time that both alpha4 and alpha4s carry the same PTMs. Using Bottom-Up proteomics we compared the interactome of total proteasomes with that of the spt20S only, obtained using a specific antibody we developed for this purpose. We established that alpha4 and alpha4s do not co-exist in the same 20S, although they are almost equally abundant in the testes.  We also measured that 19S and PA200 regulators were bound in higher ratios to the spt20S compared to std20S. Among other preferentially-associated proteins of spt20S were PI31 and Fbxo7, both shown to be crucial for fertility. They mediate proteasome transport and docking of E3 ligases - both processes that could be crucial for spt20S function. We then obtained germ cells at different differentiation stages and performed a proteomics analysis of both lysates and IP-ed proteasome complexes, to establish a dynamic image of the proteasome throughout spermatogenesis. We observed a complete shift from std20S to spt20S between pre-meiotic SPG and meiotic and post-meiotic SPC and SPT cells. We explained this by a shift in expression, rather than preferential incorporation of alpha4s. Upon entering meiosis, the PA200 association with core proteasome increased 7-fold, marking its importance in gamete development. Although PA200 was represented in literature as the main spt20S interactor, we show that 19S was undoubtedly stoichiometrically dominant, occupying 60% of the existing 20S in SPCs - an unprecedented proteasome activation. Identified spt20S-interacting proteins largely correlated with previous interactome analysis on the whole testes, showing robustness of our methods. We identified synaptonemal proteins bound exclusively to spt20S and numerous proteins involved in ubiquitylation, cell cycle and meiotic progression as well as cellular transport, which fits the current model of spt20S role, proposed by earlier work. The shift from alpha4 to alpha4s in meiosis was shown to be crucial, but what is the molecular basis for this transition? The hydrogen-deuterium exchange experiment coupled to MS helped us to show for the first time that the two proteasomes exhibit different binding interfaces: alpha4s contains regions that are more flexible compared to alpha4. We further supported this finding with pull-down assays, which showed that 19S binds more strongly to the spt20S than to std20S, which would explain the increase in proteolytic activity required during meiosis. The spt20S showed a higher tryptic activity compared to the std20S in vitro, which might reflect a particular need for histone degradation. Altogether, our data reveal a more complex process of spt20S regulation than previously suggested and set the basis for structural and functional differences between the spt20S and std20S

Monoclonal Antibodies

Monoclonal antibodies are established in clinical practice for the treatment of cancer, and autoimmune and infectious diseases. The first generation of antibodies has been dominated by classical IgG antibodies, however, in the last decade, the field has advanced, and, nowadays, a large proportion of antibodies in development have been engineered. This Special Issue on "Monoclonal Antibodies" includes original manuscripts and reviews covering various aspects related to the discovery, analytical characterization, manufacturing and development of therapeutic and engineered antibodies

Wide-Field InfrarRed Survey Telescope-Astrophysics Focused Telescope Assets WFIRST-AFTA 2015 Report

This report describes the 2014 study by the Science Definition Team (SDT) of the Wide-Field Infrared Survey Telescope (WFIRST) mission. It is a space observatory that will address the most compelling scientific problems in dark energy, exoplanets and general astrophysics using a 2.4-m telescope with a wide-field infrared instrument and an optical coronagraph. The Astro2010 Decadal Survey recommended a Wide Field Infrared Survey Telescope as its top priority for a new large space mission. As conceived by the decadal survey, WFIRST would carry out a dark energy science program, a microlensing program to determine the demographics of exoplanets, and a general observing program utilizing its ultra wide field. In October 2012, NASA chartered a Science Definition Team (SDT) to produce, in collaboration with the WFIRST Study Office at GSFC and the Program Office at JPL, a Design Reference Mission (DRM) for an implementation of WFIRST using one of the 2.4-m, Hubble-quality telescope assemblies recently made available to NASA. This DRM builds on the work of the earlier WFIRST SDT, reported by Green et al. (2012) and the previous WFIRST-2.4 DRM, reported by Spergel et. (2013). The 2.4-m primary mirror enables a mission with greater sensitivity and higher angular resolution than the 1.3-m and 1.1-m designs considered previously, increasing both the science return of the primary surveys and the capabilities of WFIRST as a Guest Observer facility. The addition of an on-axis coronagraphic instrument to the baseline design enables imaging and spectroscopic studies of planets around nearby stars.Comment: This report describes the 2014 study by the Science Definition Team of the Wide-Field Infrared Survey Telescope mission. 319 pages; corrected a misspelled name in the authors list and a typo in the abstrac

MALDI-ToF mass spectrometry biomarker profiling via multivariate data analysis application in the biopharmaceutical bioprocessing industry

PhD ThesisMatrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) is a technique by which protein profiles can be rapidly produced from biological samples. Proteomic profiling and biomarker identification using MALDI-ToF MS have been utilised widely in microbiology for bacteria identification and in clinical proteomics for disease-related biomarker discovery. To date, the benefits of MALDI-ToF MS have not been realised in the area of mammalian cell culture during bioprocessing. This thesis explores the approach of ‘intact-cell’ MALDI-ToF MS (ICM-MS) combined with projection to latent structures – discriminant analysis (PLS-DA), to discriminate between mammalian cell lines during bioprocessing. Specifically, the industrial collaborator, Lonza Biologics is interested in adopting this approach to discriminate between IgG monoclonal antibody producing Chinese hamster ovaries (CHO) cell lines based on their productivities and identify protein biomarkers which are associated with the cell line productivities. After classifying cell lines into two categories (high/low producers; Hs/Ls), it is hypothesised that Hs and Ls CHO cells exhibit different metabolic profiles and hence differences in phenotypic expression patterns will be observed. The protein expression patterns correlate to the productivities of the cell lines, and introduce between-class variability. The chemometric method of PLS-DA can use this variability to classify the cell lines as Hs or Ls. A number of differentially expressed proteins were matched and identified as biomarkers after a SwissProt/TrEMBL protein database search. The identified proteins revealed that proteins involved in biological processes such as protein biosynthesis, protein folding, glycolysis and cytoskeleton architecture were upregulated in Hs. This study demonstrates that ICM-MS combined with PLS-DA and a protein database search can be a rapid and valuable tool for biomarker discovery in the bioprocessing industry. It may help in providing clues to potential cell genetic engineering targets as well as a tool in process development in the bioprocessing industry. With the completion of the sequencing of the CHO genome, this study provides a foundation for rapid biomarker profiling of CHO cell lines in culture during recombinant protein manufacturing.Lonza Biologics

Unmanned Aerial Vehicle (UAV)-Enabled Wireless Communications and Networking

The emerging massive density of human-held and machine-type nodes implies larger traffic deviatiolns in the future than we are facing today. In the future, the network will be characterized by a high degree of flexibility, allowing it to adapt smoothly, autonomously, and efficiently to the quickly changing traffic demands both in time and space. This flexibility cannot be achieved when the network’s infrastructure remains static. To this end, the topic of UAVs (unmanned aerial vehicles) have enabled wireless communications, and networking has received increased attention. As mentioned above, the network must serve a massive density of nodes that can be either human-held (user devices) or machine-type nodes (sensors). If we wish to properly serve these nodes and optimize their data, a proper wireless connection is fundamental. This can be achieved by using UAV-enabled communication and networks. This Special Issue addresses the many existing issues that still exist to allow UAV-enabled wireless communications and networking to be properly rolled out