An automated algorithm for sequence confirmation of chemically modified oligonucleotides by tandem mass spectrometry

We have developed a tandem mass spectrometry (MS/MS) data analysis program for confirmation of sequence of chemically modified oligonucleotides. The method is based on the analysis of deconvoluted MS/MS data for fragment ions from three charge states and comparison of these data against a set of computer-generated masses from expected fragmentation patterns. The algorithm compares the experimental masses not only against the fragment set predicted for the expected sequence but also against a wider test set covering all next-neighbor position switches of the original sequence and all pairwise swaps of nucleosides, which in synthesis would result in molecules with masses within a preset mass tolerance. The algorithm is capable of identifying incorrect sequences that would not be distinguished by identity testing with electrospray ionization mass spectrometry. The method has been tested with permutations of the two 21-mer single strands of a chemically modified short interfering RNA containing 2′-O-methyl and phosphorothioate linkages. For both strands, challenge sequences were synthesized and tested with the premise that they were the original sequences. The algorithm correctly reported the locations of next-neighbor position switches and nucleoside swaps. The results confirm the approach as useful for MS/MS-based identity test methods for synthetic oligonucleotides.     

Enterococcus faecium stimulates human neutrophils via the formyl-peptide receptor 2

The human formyl-peptide receptor 2 (FPR2/ALX) senses phenol-soluble modulin (PSM) peptide toxins produced by pathogenic staphylococcal species and plays a crucial role in directing neutrophil influx during staphylococcal infection. However, it has remained unclear if FPR2 responds also to molecules from other bacterial pathogens. Here we analyzed a variety of gram-positive and gram-negative pathogens and found that apart from staphylococci only certain enterococcal strains have the capacity to stimulate FPR2/ALX. Most of the analyzed Enterococcus faecium but only sporadic Enterococcus faecalis strains released FPR2/ALX-stimulating molecules leading to neutrophil calcium ion fluxes, chemotaxis, and complement receptor upregulation. Among ten test strains vancomycin-resistant E. faecium had a significantly higher capacity to stimulate FPR2/ALX than vancomycin-susceptible strains, suggesting an association of strong FPR2/ALX activation with health-care associated strains. The enterococcal FPR2/ALX agonists were found to be peptides or proteins, which appear, however, to be unrelated to staphylococcal PSMs in sequence and physicochemical properties. Enterococci are among the most frequent invasive bacterial pathogens but the basis of enterococcal virulence and immune activation has remained incompletely understood. Our study indicates that previously unrecognized proteinaceous agonists contribute to Enterococcus-host interaction and underscores the importance of FPR2/ALX in host defense against major endogenous bacterial pathogens.     

Sesquiterpene Lactones Downregulate G2/M Cell Cycle Regulator Proteins and Affect the Invasive Potential of Human Soft Tissue Sarcoma Cells

Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27^Kip1. In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expression levels of matrix metalloproteinases (MMPs), which play a crucial role in extracellular matrix degradation and metastasis, were investigated by Luminex® technology and real-time RT-PCR. In the presence of costunolide, MMP-2 and -9 levels were significantly increased in SW-982 and TE-671 cells. Dehydrocostus lactone treatment significantly reduced MMP-2 and -9 expression in TE-671 cells, but increased MMP-9 level in SW-982 cells. In addition, the invasion potential was significantly reduced after treatment with both sesquiterpene lactones as investigated by the HTS FluoroBlock™ insert system.     

OMR-Arena: Automated Measurement and Stimulation System to Determine Mouse Visual Thresholds Based on Optomotor Responses

Measurement of the optomotor response is a common way to determine thresholds of the visual system in animals. Particularly in mice, it is frequently used to characterize the visual performance of different genetically modified strains or to test the effect of various drugs on visual performance. Several methods have been developed to facilitate the presentation of stimuli using computer screens or projectors. Common methods are either based on the measurement of eye movement during optokinetic reflex behavior or rely on the measurement of head and/or body-movements during optomotor responses. Eye-movements can easily and objectively be quantified, but their measurement requires invasive fixation of the animals. Head movements can be observed in freely moving animals, but until now depended on the judgment of a human observer who reported the counted tracking movements of the animal during an experiment. In this study we present a novel measurement and stimulation system based on open source building plans and software. This system presents appropriate 360 stimuli while simultaneously video-tracking the animal''s head-movements without fixation. The on-line determined head gaze is used to adjust the stimulus to the head position, as well as to automatically calculate visual acuity. Exemplary, we show that automatically measured visual response curves of mice match the results obtained by a human observer very well. The spatial acuity thresholds yielded by the automatic analysis are also consistent with the human observer approach and with published results. Hence, OMR-arena provides an affordable, convenient and objective way to measure mouse visual performance.     

The Role of Extrinsic Rewards and Cue-Intention Association in Prospective Memory in Young Children

The current study examined, for the first time, the effect of cue-intention association, as well as the effects of promised extrinsic rewards, on prospective memory in young children, aged 5-years-old (n = 39) and 7-years-old (n = 40). Children were asked to name pictures for a toy mole, whilst also having to remember to respond differently to certain target pictures (prospective memory task). The level to which the target picture was associated with the intention was manipulated across two conditions (low- or high-association) for all participants, whilst half of the participants were promised a reward for good prospective memory performance. Results showed a main effect of age, with the 7-year-olds outperforming the 5-year-olds. Furthermore, there was a main effect of reward, with those promised a reward performing better than those who were not. No effect was found for cue-association, with the participants of both age groups performing equally well in both association conditions. No significant interactions were found between any of the variables. The potentially important role of reward in young children’s everyday prospective memory tasks, and possible reasons for the lack of a reflexive-associative effect, are discussed.     

Influence of hyperbaric oxygen tension on growth parameters of Candida tropicalis and Rhodococcus erythropolis

Summary Oxygen-limited growth was avoided by means of oxygen-enriched aeration in aerobic fermentation processes. Studies were carried out with Candida tropicalis (Cast.) Berkhout and Rhodococcus erythropolis (DSM 43215). The effect of hyperbaric dissolved oxygen tension on growth parameters was examined by varying the dissolved oxygen concentration and the carbon source (glucose, ethanol, and n-alkanes). Up to an oxygen concentration of 40 mg/l in the culture suspension no impairment of the economic coefficients and no promotion of cell lysis was found. It was observed that raised oxygen concentrations in the aeration gas led to enhanced specific growth rates. At cell concentrations above 20 g/l dry weight an uncoupling of carbon source dissimilation and biomass production was observed even at non-limiting oxygen concentrations.     

Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

An understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogen Ustilago maydis. However, mfe2 mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in the had1 gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog, had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of an mfe2Δ mutant. We also show that short-chain fatty acids induce cell death in U. maydis and that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms by U. maydis that includes potential metabolic contributions to proliferation in planta and an effect on virulence-related morphogenesis.     

Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal

Annexin A1 is an intracellular calcium/phospholipid-binding protein that is involved in membrane organization and the regulation of the immune system. It has been attributed an anti-inflammatory role at various control levels, and recently we could show that annexin A1 externalization during secondary necrosis provides an important fail-safe mechanism counteracting inflammatory responses when the timely clearance of apoptotic cells has failed. As such, annexin A1 promotes the engulfment of dying cells and dampens the postphagocytic production of proinflammatory cytokines. In our current follow-up study, we report that exposure of annexin A1 during secondary necrosis coincided with proteolytic processing within its unique N-terminal domain by ADAM10. Most importantly, we demonstrate that the released peptide and culture supernatants of secondary necrotic, annexin A1-externalizing cells induced chemoattraction of monocytes, which was clearly reduced in annexin A1- or ADAM10-knockdown cells. Thus, altogether our findings indicate that annexin A1 externalization and its proteolytic processing into a chemotactic peptide represent final events during apoptosis, which after the transition to secondary necrosis contribute to the recruitment of monocytes and the prevention of inflammation.