Slow evolution toward "Super-Aggregation" of the oligomers formed through the swapping of RNase A N-Termini: a wish for amyloidosis?

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 degrees C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits' N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not "amyloid-like" derivatives. This confirms that RNase A acts as an "auto-chaperone", although it displays many amyloid-prone short segments, including the 16-22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers' dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

BACKGROUND: Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. RESULTS: A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. CONCLUSIONS: Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed

List of Figures / List of Tables / Acknowledgement

In the context of Turkey’s accession to the EU, the issue of potential migration from Turkey and its impact upon European labor markets became one of the concerns of the EU, considering Turkey’s growing population and young labor force. In 2011, half a century after the bi-lateral agreement between Turkey and Germany on labor recruitment in 1961, migration plays a key role in relations of Turkey with the EU and will even increase its significance – not necessarily for the next fifty years but certainly for the next decade. This book touches upon various aspects of the ongoing debate about the effects of Turkey’s accession to the EU upon the migration flows and sheds light on various dimensions of current panorama, addresses policy implications as well as future challenges and opportunities.In the context of Turkey’s accession to the EU, the issue of potential migration from Turkey and its impact upon European labor markets became one of the concerns of the EU, considering Turkey’s growing population and young labor force. In 2011, half a century after the bi-lateral agreement between Turkey and Germany on labor recruitment in 1961, migration plays a key role in relations of Turkey with the EU and will even increase its significance – not necessarily for the next fifty years but certainly for the next decade. This book touches upon various aspects of the ongoing debate about the effects of Turkey’s accession to the EU upon the migration flows and sheds light on various dimensions of current panorama, addresses policy implications as well as future challenges and opportunities

Determination of Serotypes of Shiga Toxin-Producing Escherichia coli Isolates by Intact Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿

Shiga toxin-producing Escherichia coli (STEC) isolates representing the serotypes O165:H25, O26:H11/H32, and O156:H25 were analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectra of whole cells, a procedure also known as intact cell mass spectrometry (ICMS or IC-MALDI MS) or MALDI-typing. We demonstrate that within the given species the three serotypes can be well discriminated by ICMS. Conditions for the construction of serotype-specific prototypic mass spectra were systematically optimized by filtering out masses that do not contribute to the discrimination of the serotypes. Binary distances between prototypic spectra and sample spectra were used to determine serotypes of unknown samples. With parameters optimized, only 0.7% of the assignments were incorrect compared to 31% when distances were calculated from alignments of unfiltered mass spectra. Within the different serotypes, clusters of genetically related E. coli most probably originating from single clones could be distinguished by restriction fragment length polymorphism analysis. Since ICMS did not reproduce these clusters, we conclude that the power of ICMS is just sufficient to discriminate E. coli serotypes under certain conditions but fails for the differentiation of E. coli below this level

Integration of Chlorella protothecoides production in wastewater treatment plant: From lab measurements to process design

Abstract The exploitation of microalgae in a wastewater treatment process is currently an open issue, and its actual applicability is still under investigation. In this work the effects of temperature, day/night irradiation and bacterial competition were studied on growth and nutrient removal of Chlorella protothecoides cultivated in real and unsterilized primary urban wastewater. C. protothecoides showed a linear dependence of growth rate on temperature under continuous irradiation with a maximum at 30 \ufffdC. Continuous flow experiment under day\u2013night irradiation condition showed that a cyclic steady state was achieved, with significant differences in biomass concentration and nutrient removal after dark and light periods. The presence of a native microflora in wastewater did not affect the microalgal growth, both in batch and continuous flow experiments. N and P were efficiently removed in all conditions tested, while the \{COD\} was not consumed by microalgal biomass. Thus, in view of a large scale application, a two step depuration process would be required, where the photobioreactor will remove nutrients as N and P and an activated sludge reactor downstream will reduce the organic matter. The experimental data obtained were used to design a possible process of this type

A Conserved Insertion in Protein-primed DNA Polymerases is Involved in Primer Terminus Stabilisation

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or α-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of ø29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3′–5′ exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in ø29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.This investigation has been aided by research grant 5R01 GM27242-23 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigación Cientı́fica y Técnica, by grant ERBFMRX CT97 0125 from the European Union, and by an institutional grant from Fundación Ramón Areces to the Centro de Biologı́a Molecular “Severo Ochoa”. E.D. was a post-doctoral fellow of the European Union.Peer reviewe