Deposition within the vicinity of the Mid-Eifelian High: detailed sedimentological study and magnetic susceptibility of a mixed ramp-related system from the Eifelian Lauch and Nohn formations (Devonian; Ohlesberg,Eifel, Germany)

This study focuses on the base of the Eifelian stage and on the abandoned Ohlesberg quarry. The exposed section (92 m thick) is related to the Lauch and Nohn formations. Petrographic study leads to the definition of 11 microfacies which are integrated in a palaeogeographical model. It corresponds to a complex ramp setting where carbonate, mixed and siliciclastic deposits coexist. The microfacies evolution is interpreted in terms of bathymetric and lateral variations, showing a general shallowing-upward trend and transitions between carbonate-dominated and siliciclastic-dominated sedimentary domains. This interpretation is supported by trends in magnetic susceptibility data. Even if the proximity to emerged areas appears to be the major influence on magnetic susceptibility values, the influence of carbonate productivity and wave agitation is also noted. The Ohlesberg section clearly points to the local and regional complex facies architecture, and advocates to variegated depositional environments along the Mid-Eifelian High.     

Molecular scanner experiment with human plasma: improving protein identification by using intensity distributions of matching peptide masses

The development of high throughput utilities to identify proteins is a major challenge in present research in the field of proteomics. One such utility, the molecular scanner, uses proteins separated by two-dimensional polyacrylamide gel electrophoresis that are digested in the gel and during transfer onto a collecting membrane. After adding a matrix, the membrane is inserted into a matrix-assisted laser desorption/ionization-time of flight mass spectrometer and a peptide mass fingerprint (PMF) is measured for every scanned site. Since the spacing between scanned sites is much smaller than the size of the most abundant protein spots, there is a certain redundancy in the data that was used in an earlier experiment with Escherichia coli [1] to improve mass calibration and PMF identification results. It was observed that the signal intensity of a peptide mass as a function of the position on the membrane showed similar patterns if peptides stemmed from the same protein. Taking account of these similarities a clustering algorithm was used to find lists of experimental masses with similar intensity distributions, which provided clearer identification of the corresponding proteins. Here, these methods are applied to a human plasma scan, where proteins were highly modified and less separated. The presence of very abundant proteins like albumin and immunoglobulins added another difficulty. The calibration of the initial PMFs was not satisfactory and masses had to be recalibrated. After discarding chemical noise, the membrane was partitioned into regions and for each region protein identification was carried out separately. A new scoring method was used, where the PMF score was multiplied by a factor that measures the similarity of matching peptides. This method proved to be more robust than the method developed in [1] if the region where a protein was found had an extended, nonspherical shape and strong overlap with regions of other proteins. Many proteins annotated on the SWISS-2D PAGE human plasma master gel could be clearly identified and many interesting properties were observed.     

Uncovering Wolbachia Diversity upon Artificial Host Transfer

The common endosymbiotic Wolbachia bacteria influence arthropod hosts in multiple ways. They are mostly recognized for their manipulations of host reproduction, yet, more recent studies demonstrate that Wolbachia also impact host behavior, metabolic pathways and immunity. Besides their biological and evolutionary roles, Wolbachia are new potential biological control agents for pest and vector management. Importantly, Wolbachia-based control strategies require controlled symbiont transfer between host species and predictable outcomes of novel Wolbachia-host associations. Theoretically, this artificial horizontal transfer could inflict genetic changes within transferred Wolbachia populations. This could be facilitated through de novo mutations in the novel recipient host or changes of haplotype frequencies of polymorphic Wolbachia populations when transferred from donor to recipient hosts. Here we show that Wolbachia resident in the European cherry fruit fly, Rhagoletis cerasi, exhibit ancestral and cryptic sequence polymorphism in three symbiont genes, which are exposed upon microinjection into the new hosts Drosophila simulans and Ceratitis capitata. Our analyses of Wolbachia in microinjected D. simulans over 150 generations after microinjection uncovered infections with multiple Wolbachia strains in trans-infected lines that had previously been typed as single infections. This confirms the persistence of low-titer Wolbachia strains in microinjection experiments that had previously escaped standard detection techniques. Our study demonstrates that infections by multiple Wolbachia strains can shift in prevalence after artificial host transfer driven by either stochastic or selective processes. Trans-infection of Wolbachia can claim fitness costs in new hosts and we speculate that these costs may have driven the shifts of Wolbachia strains that we saw in our model system.     

Here Today,Gone Tomorrow – Adaptation to Change in Memory-Guided Visual Search

Visual search for a target object can be facilitated by the repeated presentation of an invariant configuration of nontargets (‘contextual cueing’). Here, we tested adaptation of learned contextual associations after a sudden, but permanent, relocation of the target. After an initial learning phase targets were relocated within their invariant contexts and repeatedly presented at new locations, before they returned to the initial locations. Contextual cueing for relocated targets was neither observed after numerous presentations nor after insertion of an overnight break. Further experiments investigated whether learning of additional, previously unseen context-target configurations is comparable to adaptation of existing contextual associations to change. In contrast to the lack of adaptation to changed target locations, contextual cueing developed for additional invariant configurations under identical training conditions. Moreover, across all experiments, presenting relocated targets or additional contexts did not interfere with contextual cueing of initially learned invariant configurations. Overall, the adaptation of contextual memory to changed target locations was severely constrained and unsuccessful in comparison to learning of an additional set of contexts, which suggests that contextual cueing facilitates search for only one repeated target location.     

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures.The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure.     

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.     

Extending the scope of the controlled logical clock

Event traces are helpful in understanding the performance behavior of parallel applications since they allow the in-depth analysis of communication and synchronization patterns. However, the absence of synchronized clocks on most cluster systems may render the analysis ineffective because inaccurate relative event timings may misrepresent the logical event order and lead to errors when quantifying the impact of certain behaviors or confuse the users of time-line visualization tools by showing messages flowing backward in time. In our earlier work, we have developed a scalable algorithm called the controlled logical clock that eliminates inconsistent inter-process timings postmortem in traces of pure MPI applications, potentially running on large processor configurations. In this paper, we first demonstrate that our algorithm also proves beneficial in computational grids, where a single application is executed using the combined computational power of several geographically dispersed clusters. Second, we present an extended version of the algorithm that—in addition to message-passing event semantics—also preserves and restores shared-memory event semantics, enabling the correction of traces from hybrid applications.     

Design,construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Designed ankyrin repeat proteins (DARPins) are well‐established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin‐target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase‐7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.     

N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.