Quality interoperability within digital libraries: the DL.org perspective

Quality is the most dynamic aspect of DLs, and becomes even more complex with respect to interoperability. This paper formalizes the research motivations and hypotheses on quality interoperability conducted by the Quality Working Group within the EU-funded project DL.org (<a href="http://www.dlorg.eu">http://www.dlorg.eu/</a>). After providing a multi-level interoperability framework – adopted by DL.org - the authors illustrate key-research points and approaches on the way to the interoperability of DLs quality, grounding them in the DELOS Reference Model. By applying the DELOS Reference Model Quality Concept Map to their interoperability motivating scenario, the authors subsequently present the two main research outcomes of their investigation - the Quality Core Model and the Quality Interoperability Survey

IKZF1 Deletions with COBL Breakpoints Are Not Driven by RAG-Mediated Recombination Events in Acute Lymphoblastic Leukemia

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination

An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogenous malignancy with poor prognosis in relapsed adult patients. The genetic basis for relapse in aneuploid subtypes such as near haploid (NH) and high hyperdiploid (HeH) BCP-ALL is only poorly understood. Pathogenic genetic alterations remain to be identified. To this end, we investigated the dynamics of genetic alterations in a matched initial diagnosis-relapse (ID-REL) BCP-ALL cohort. Here, we firstly report the identification of the novel genetic alteration CYB5Aalt, an alternative transcript of CYB5A, in two independent cohorts. Methods: We identified CYB5alt in the RNAseq-analysis of a matched ID-REL BCP-ALL cohort with 50 patients and quantified its expression in various molecular BCP-ALL subtypes. Findings were validated in an independent cohort of 140 first diagnosis samples from adult BCP-ALL patients. Derived from patient material, the alternative open reading frame of CYB5Aalt was cloned (pCYB5Aalt) and pCYB5Aalt or the empty vector were stably overexpressed in NALM-6 cells. RNA sequencing was performed of pCYB5Aalt clones and empty vector controls followed by differential expression analysis, gene set enrichment analysis and complementing cell death and viability assays to determine functional implications of CYB5Aalt. Results: RNAseq data analysis revealed non-canonical exon usage of CYB5Aalt starting from a previously undescribed transcription start site. CYB5Aalt expression was increased in relapsed BCP-ALL and its occurrence was specific towards the shared gene expression cluster of NH and HeH BCP-ALL in independent cohorts. Overexpression of pCYB5Aalt in NALM-6 cells induced a distinct transcriptional program compared to empty vector controls with downregulation of pathways related to reported functions of CYB5A wildtype. Interestingly, CYB5A wildtype expression was decreased in CYB5Aalt samples in silico and in vitro. Additionally, pCYB5Aalt NALM-6 elicited a more resistant drug response. Conclusions: Across all age groups, CYB5Aalt was the most frequent secondary genetic event in relapsed NH and HeH BCP-ALL. In addition to its high subgroup specificity, CYB5Aalt is a novel candidate to be potentially implicated in therapy resistance in NH and HeH BCP-ALL. This is underlined by overexpressing CYB5Aalt providing first evidence for a functional role in BCL2-mediated apoptosis

PVT properties of an alternative biofuel: dimethyl ether

Dimethyl ether is an important chemical material and it has many engineering applications. It is a clean and economical alternative fuel and an ozone-friendly refrigerant. In this work, its PVT properties have been object of study. In particular, the experimental work was performed both in the two-phase region and in the superheated vapor region phase by means of the isochoric method. The isochoric measurements were carried out at temperatures from 219 K to 363 K and at pressures from 22 kPa up to 1,740 kPa. A total of 159 points, both in the two phase (71 points) and in the superheated vapor region (88 points) were obtained. The present experimental PVT data contribute to the deeper knowledge of the behaviour of the fluid both in the superheated vapour and in the saturation pressure region and to the development of a new equation of state

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Einfluss verschiedener Koeffektoren auf die differentielle Kohlenstoff Katabolitenregulation durch CcpA

Catabolite control protein A (CcpA) represents a global transcriptional regulator in gram positive bacteria with low GC-content. In response to the available nutrient supply CcpA governs the expression of about 300 genes predominantly involved in carbon catabolism. Besides the binding of low-molecular-weight effectors, the interaction with the homologous phosphoproteins HPr-Ser46-P and Crh-Ser46-P is crucial for CcpA function. Associated allosteric relocations in the protein trigger binding of CcpA to so called catabolite response elements (cre) that finally result in differential gene regulation. The elucidation of the involved signal transduction pathways is part of this thesis. The E. coli/B. subtilis shuttle vector cloned in this study allows the generation of large ccpA mutant pools in E. coli that is crucial for random mutagenesis. Thus a novel screening system was established which is directly targeted towards the identification of differentially regulating ccpA mutants. This system relies on a B. subtilis strain carrying two independent reporter gene fusions whose expression is unequivocally detectable on appropriate selection plates. The screening system proved to be functional, but it was not yet applied in this study due to the low transformation efficiency of the B. subtilis ccpA deletion strain. HPr residue R17 bridges the N-terminal subdomains of CcpA by exerting Coulomb interactions with CcpA residues D85, D70` and D100`. The proper adjustment of CcpA subdomains is a prerequisite for the adoption of the DNA binding competent conformation. Accordingly, the CcpA triple mutant D70R-D85R-D100R fails to regulate the promoters of xynP, gnt, ackA and alsS. The respective single and double mutants, however, provoke differential regulation, indicating that this signal transduction pathway contributes differently to CCR of these promoters in Bacillus subtilis. HPrH15 and CcpAD297 were identified as determinants for specific stimulation of CcpA binding to HPr-Ser46-P by the glycolytic intermediates fructose 1,6-bisphosphate (FBP) and glucose 6-phosphate (Glc-6-P). Mutation of these residues to glutamine or alanine, respectively, does not affect protein-protein interaction or complex formation with xylAcre in SPR interaction analyses. The affinity, however, is no longer increased by FBP und Glc-6-P. The introduction of histidine at residue 15 in Crh renders the protein sensible for FBP or Glc-6-P stimulation. Thus position 15 in HPr and Crh discriminates for both phosphotransfer in the PTS and the stimulatory effect of glycolytic metabolites. HPr residues D11 and T20, which are also not conserved between HPr and Crh, are not involved in this mechanism. A contribution of glycolytic intermediates on CCR in vivo is not detectable under the conditions tested here. CcpA mutant D297A regulates the promoter of xynP, gnt, ackA and alsS similar to the wild type, the introduction of a negative charge in CcpAD297, however, provokes differential regulation. Bioinformatic investigations in the group of Prof. Sticht revealed an interaction of HPr-Ser46-P with the CcpA homolog transcription regulator RbsR. This interaction was confirmed experimentally. In gel mobility shift assays, HPr-Ser46-P mediated specific binding of RbsR to its operator sequence. The latter overlaps with a cre sequence for CcpA binding suggesting a competition of CcpA and RbsR for the coeffector HPr-Ser46-P and the mutual DNA binding site. However, the importance of this interaction in vivo remains unsettled. Though CcpA mediated CCR of the ribose transport operon was verified, RbsR seems not to act as repressor of the operon. Notwithstanding, the severe growth defect of a ptsH1/Δcrh double mutant in the presence of ribose compared to a ccpA deletion mutant implies that the phosphoproteins are involved in CcpA independent regulation of ribose transport and metabolism.Als globaler Transkriptionsregulator steuert das Katabolit-Kontroll Protein A (CcpA) in Gram-positiven Bakterien mit niedrigem GC-Gehalt die Expression von ca. 300 Genen, die vorwiegend in den Kohlenstoff Katabolismus involviert sind. Für die Funktion von CcpA ist neben der Bindung von niedermolekularen Molekülen die Interaktion mit den homologen Phosphoproteinen HPr-Ser46-P und Crh-Ser46-P essentiell. Damit verbundene allosterische Veränderungen im Protein erlauben die Bindung an so genannte catabolite response elements (cre), die CcpA letztlich zu differentieller Genregulation befähigt. Die Untersuchung der beteiligten Signaltransduktionswege ist Gegenstand dieser Arbeit. Der in dieser Arbeit klonierte E. coli/B. subtilis „Shuttle“-Vektor ermöglicht erstmals die Generierung großer ccpA Mutantenpools in E. coli, die für eine ungerichtete Mutagenese notwendig sind. Daher wurde ein neues Screeningsystem etabliert, mit dem direkt nach differentiell regulierenden ccpA Mutanten gesucht werden kann. Es beruht auf einem B. subtilis Stamm, der zwei voneinander unabhängige Reportergen-Fusionen trägt, deren Expression auf entsprechenden Selektionsplatten eindeutig erkennbar ist. Das Screeningsystem erwies sich als funktional, wurde jedoch aufgrund der schlechten Transformierbarkeit einer ccpA Deletionsmutante im Rahmen dieser Arbeit noch nicht angewandt. R17 in HPr vermittelt über Coulomb Wechselwirkungen mit den CcpA Resten D85, D70`und D100` die Verbrückung der N-terminalen Untereinheiten im CcpA Dimer. Die richtige Ausrichtung der CcpA Untereinheiten ist Voraussetzung für das Einnehmen der DNA bindenden Konformation. Demzufolge ist die CcpA Dreifachmutante D70R-D85R-D100R nicht mehr in der Lage, die Promotoren von xynP, gnt, ackA und alsS zu regulieren. Die jeweiligen Einzel- und Doppelmutanten bewirken hingegen differentielle Regulation - ein Hinweis darauf, dass dieser Signaltransduktionsweg abhängig vom untersuchten Promoter unterschiedlich zur Kohlenstoff-Katabolitenregulation (KKR) in Bacillus subtilis beiträgt. In SPR-Interaktionsanalysen wurden die Aminosäurereste HPrH15 und CcpAD297 als Determinaten für die spezifische Stimulation der Bindung von CcpA an HPr-Ser46-P durch die glykolytischen Intermediate Fruktose-1,6-bisphosphat (FBP) und Glukose-6-Phosphat (Glc-6-P) identifiziert. Die Mutation dieser Reste zu Glutamin bzw. Alanin hat weder Auswirkungen auf die Protein-Protein Interaktion noch auf die Komplexbildung mit xylAcre, die Affinität wird jedoch nicht mehr durch FBP und Glc-6-P verstärkt. Das Einführen von Histidin an Position 15 in Crh vermittelt Sensitivität gegenüber FBP und Glc-6-P. Neben dem Phosphotransfer im PTS diskriminiert Position 15 in HPr und Crh demnach auch für den stimulatorischen Effekt glykolytischer Metabolite. Die Aminosäurereste D11 und T20 in HPr, die im Crh ebenfalls nicht konserviert vorliegen, sind an diesem Mechanismus nicht beteiligt. Unter den gewählten Bedingungen ist ein Beitrag glykolytischer Intermediate auf die KKR in vivo nicht feststellbar. Die CcpA Mutante D297A reguliert die Promotoren xynP, gnt, ackA und alsS ähnlich dem Wildtyp, während die Einführung einer negativen Ladung in CcpAD297R zu differentieller Regulation führt. Die von der Arbeitsgruppe Sticht vorhergesagte Interaktion zwischen RbsR, einem zu CcpA homologen Transkriptionsregulator, und HPr-Ser46-P wurde in vitro bestätigt. In Gelmobilitätsassays vermittelte HPr-Ser46-P spezifisch die Bindung von RbsR an seine Operatorsequenz. Diese überlappt mit einer cre Sequenz, an die CcpA bindet, was einen Kompetitionsmechanismus zwischen CcpA und RbsR nahe legt. Die Bedeutung der RbsR–HPr-Ser46-P Interaktion in vivo ist dagegen noch unklar. Während die CcpA vermittelte KKR des rbs operons eindeutig nachgewiesen wurde, scheint RbsR wider Erwarten nicht als Repressor des Operons zu fungieren. Der deutliche Wuchsdefekt einer ptsH1/Δcrh Doppelmutante in Anwesenheit von Ribose gegenüber einer ccpA Deletionsmutante impliziert jedoch eine Beteiligung der Phosphoproteine an einer von CcpA unabhängigen Regulation des Ribose Transports und –Metabolismus

Streptococcus mitis Strains Causing Severe Clinical Disease in Cancer Patients

The genetically diverse viridans group streptococci (VGS) are increasingly recognized as the cause of a variety of human diseases. We used a recently developed multilocus sequence analysis scheme to define the species of 118 unique VGS strains causing bacteremia in patients with cancer; Streptococcus mitis (68 patients) and S. oralis (22 patients) were the most frequently identified strains. Compared with patients infected with non–S. mitis strains, patients infected with S. mitis strains were more likely to have moderate or severe clinical disease (e.g., VGS shock syndrome). Combined with the sequence data, whole-genome analyses showed that S. mitis strains may more precisely be considered as >2 species. Furthermore, we found that multiple S. mitis strains induced disease in neutropenic mice in a dose-dependent fashion. Our data define the prominent clinical effect of the group of organisms currently classified as S. mitis and lay the groundwork for increased understanding of this understudied pathogen

Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis.

Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis

Mass spectra showing that CovR is phosphorylated on threonine 65.

<p>Recombinant CovR was incubated with Stk_KD and then subjected to chymotrypsin digestion. (A) Mass spectra of the MS/MS fragmentation of a 2⁺ ion of the CovR-derived peptide EVTphosRRLQTEKTTY. Fragment marked with blue arrow shows the peptide that has lost phosphoric acid, thus decreasing the apparent mass from 852.92 to 804.27. The peptide marked with the red star corresponds to the y₁₀²⁺ fragment (RRLQTEKTTY) and has not lost phosphoric acid. Thus, T3 must be the position of phosphorylation. (B) Mass spectra of the MS/MS/MS fragmentation of the y₁₀²⁺ ion confirmed that the y₁₀²⁺ fragment is indeed RRLQTEKTTY. Fragments of corresponding b or y ions are indicated.</p