PCA2GO: a new multivariate statistics based method to identify highly expressed GO-Terms

<p>Abstract</p> <p>Background</p> <p>Several tools have been developed to explore and search Gene Ontology (GO) databases allowing efficient GO enrichment analysis and GO tree visualization. Nevertheless, identification of highly specific GO-terms in complex data sets is relatively complicated and the display of GO term assignments and GO enrichment analysis by simple tables or pie charts is not optimal. Valuable information such as the hierarchical position of a single GO term within the GO tree (topological ordering), or enrichment within a complex set of biological experiments is not displayed. Pie charts based on GO tree levels are, themselves, one-dimensional graphs, which cannot properly or efficiently represent the hierarchical specificity for the biological system being studied.</p> <p>Results</p> <p>Here we present a new method, which we name PCA2GO, capable of GO analysis using complex multidimensional experimental settings. We employed principal component analysis (PCA) and developed a new score, which takes into account the relative frequency of certain GO terms and their specificity (hierarchical position) within the GO graph. We evaluated the correlation between our representation score <it>R </it>and a standard measure of enrichment, namely <it>p</it>-values to convey the versatility of our approach to other methods and point out differences between our method and commonly used enrichment analyses. Although <it>p </it>values and the <it>R </it>score formally measure different quantities they should be correlated, because relative frequencies of GO terms occurrences within a dataset are an indirect measure of protein numbers related to this term. Therefore they are also related to enrichment. We showed that our score enables us to identify more specific GO-terms i.e. those positioned further down the GO-graph than other common tools used for this purpose. PCA2GO allows visualization and detection of multidimensional dependencies both within the acyclic graph (GO tree) and the experimental settings. Our method is intended for the analysis of several experimental sets, not for one set, like standard enrichment tools. To demonstrate the usefulness of our approach we performed a PCA2GO analysis of a fractionated cardiomyocyte protein dataset, which was identified by enhanced liquid chromatography-mass spectrometry (GeLC-MS). The analysis enabled us to detect distinct groups of proteins, which accurately reflect properties of biochemical cell fractions.</p> <p>Conclusions</p> <p>We conclude that PCA2GO is an alternative efficient GO analysis tool with unique features for detection and visualization of multidimensional dependencies within the dataset under study. PCA2GO reveals strongly correlated GO terms within the experimental setting (in this case different fractions) by PCA group formation and improves detection of more specific GO terms within experiment dependent GO term groups than standard <it>p </it>value calculations.</p

Generating glycan variants for biological activity testing by means of parallel experimental design and multivariate analysis

For more than 20 years, the industry has mainly invested in productivity enhancements. Recently, the focus of cell-culture process development began to shift. The modulation of quality attributes of recombinant therapeutic protein has gained substantial interest as demonstrated by the plethora of recent publications describing the effect of cell culture media on post-translational modifications of recombinant proteins1. Focusing on glycosylation, our team has developed a toolbox of media design beyond the commonly known media components and a rational high-throughput experimental design method. We identified and tested a large variety of novel cell culture compatible chemical components in industrial relevant Chinese hamster ovary cell lines (CHO) expressing recombinant antibodies and antibody fusion molecules. The compounds were evaluated in five different parallel 96-DWP fed-batch experiments, considering their mode of biological action. Viable cell density, viability and product titer were monitored and purified supernatants underwent N-glycan analysis by 2AB-UPLC and site-specific glycan-peptide analysis. Multivariate analysis identified the best performing glycosylation modulators, which were confirmed in spin tubes. Intracellular nucleotide and nucleotide sugar levels were analyzed by capillary electrophoresis, the gene expression by next-generation sequencing technologies, and the impact of the generated glycan variants on the biological activity was assessed. Non-targeted metabolite profiling was carried out to build a multivariate model linking metabolites with the glycan fingerprint. The screening experiments in 96-DWP produced a large glycosylation distribution diversity2,3. Subsequent D-optimal quadratic design in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the glycosylation specifications was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications than the best 25% in 96-deepwell plates. Further enhancement enabled us to generate extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. The glycan variants induced significant responses in the respective in vitro biological activity assays. Moreover, metabolites correlating with time-dependent glycan profiling data were pinpointed and the glycan distribution of an external data set predicted. Our data highlight the great potential of cell culture medium optimization to modulate product quality and show the feasibility of the generation of a wide range of glycan variants suitable for biological activity testing. [1] Brühlmann D, Jordan M, Hemberger J, Sauer M, Stettler M and Broly H, Tailoring recombinant protein quality by rational media design, Biotechnology Progress 2015, 31:615–629. [2] Brühlmann D, Muhr A, Parker R, Vuillemin T, Bucsella B, Torre S, La Neve F, Lembo A, Haas T, Sauer M, Souquet J, Broly H, Hemberger J, Jordan M, Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation, Journal of Biotechnology 2017, 252:32-42. [3] Brühlmann D, Sokolov M, Butté A, Sauer M, Hemberger J, Souquet J, Broly H, Jordan M, Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve Biosimilar product quality, Biotechnology and Bioengineering 2017, 114(7):1363-1631

Laser-In-Situ Monitoring of Combustion Processes

Several examples of laser in situ monitoring of combustion processes are presented. Using a frequency modulated 13CO2 waveguide laser, in situ concentrations of NH3 down to 1 ppm were measured at temperatures up to 600°C in waste incinerators and power or chemical plants. Following ignition of CH3OH-O2 mixtures by a TEA CO2 laser, gas temperature profiles were measured using rapid scanning tunable diode laser spectroscopy of CO molecules. In laminar CH4-air counterflow diffusion flames at atmospheric pressure absolute concentrations, temperatures, and collisional lifetimes of OH radicals were determined by 2-D and picosecond LIF and absorption spectroscopy. Two-dimensional LIF and Mie scattering were used to observe fuel injection and combustion in a diesel engine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86767/1/Sick52.pd

A doublet of 3" cylindrical silicon drift detectors in the CERES/NA45 experiment

We report on the performance of a doublet of 3" cylindrical silicon drift detectors installed as an upgrade of the CERES/NA45 electron pair spectrometer for the Pb-beam at the CERN SPS. The silicon detectors provide external particle tracking and background rejection of conversions and close Dalitz pairs. Results on vertex reconstruction and rejection from Pb test-run in 1994 are presented

Intercomparison of thermal diffusivity measurements on CuCrZr and PMMA

The results of an inter laboratory comparison of thermal diffusivity measurements on two different materials, namely a copper alloy (CuCrZr) and a polymer (PMMA), are presented here. Both materials were selected with respect to their different thermal conductivity, since the copper alloy belongs to the family of good metallic conductors whereas the polymer is characterized by a low thermal conductivity. The measurements of the thermal diffusivity have been performed within a temperature range from RT to 500°C for the copper alloy and from RT to 100°C for the PMMA, respectively

Anti-Listeria Activities of Galleria mellonella Hemolymph Proteins ▿ †

We report the use of antimicrobial hemolymph proteins from the model host Galleria mellonella as an inhibitor for various Listeria strains, providing a novel source for antilisterial therapeutics. We also have shown that specific virulence-associated genes known to mediate antimicrobial resistance of Listeria in mammalian models indicated a similar function in Galleria

Comparative genome-wide analysis of small RNAs of major Gram-positive pathogens

In the recent years, the number of drug- and multi-drug-resistant microbial strains has increased rapidly. Therefore, the need to identify innovative approaches for development of novel anti-infectives and new therapeutic targets is of high priority in global health care. The detection of small RNAs (sRNAs) in bacteria has attracted considerable attention as an emerging class of new gene expression regulators. Several experimental technologies to predict sRNA have been established for the Gram-negative model organism Escherichia coli. In many respects, sRNA screens in this model system have set a blueprint for the global and functional identification of sRNAs for Gram-positive microbes, but the functional role of sRNAs in colonization and pathogenicity for Listeria monocytogenes, Staphylococcus aureus, Streptococcuspyogenes, Enterococcus faecalis and Clostridium difficile is almost completely unknown. Here, we report the current knowledge about the sRNAs of these socioeconomically relevant Gram-positive pathogens, overview the state-of-the-art high-throughput sRNA screening methods and summarize bioinformatics approaches for genome-wide sRNA identification and target prediction. Finally, we discuss the use of modified peptide nucleic acids (PNAs) as a novel tool to inactivate potential sRNA and their applications in rapid and specific detection of pathogenic bacteria

QDPR homologues in Danio rerio regulate melanin synthesis, early gliogenesis, and glutamine homeostasis.

Dihydropteridine reductase (QDPR) catalyzes the recycling of tetrahydrobiopterin (BH4), a cofactor in dopamine, serotonin, and phenylalanine metabolism. QDPR-deficient patients develop neurological symptoms including hypokinesia, truncal hypotonia, intellectual disability and seizures. The underlying pathomechanisms are poorly understood. We established a zebrafish model for QDPR deficiency and analyzed the expression as well as function of all zebrafish QDPR homologues during embryonic development. The homologues qdpra is essential for pigmentation and phenylalanine metabolism. Qdprb1 is expressed in the proliferative zones of the optic tectum and eye. Knockdown of qdprb1 leads to up-regulation of pro-proliferative genes and increased number of phospho-histone3 positive mitotic cells. Expression of neuronal and astroglial marker genes is concomitantly decreased. Qdprb1 hypomorphic embryos develop microcephaly and reduced eye size indicating a role for qdprb1 in the transition from cell proliferation to differentiation. Glutamine accumulation biochemically accompanies the developmental changes. Our findings provide novel insights into the neuropathogenesis of QDPR deficiency