No abstract

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of oligosaccharides and gangliosides normally causes loss of sialic acid, particularly when alpha-cyano-4-hydroxycinnamic acid is used as the matrix. In addition, the potential signal is split because both positive and, to a greater extent, negative ions are formed while signals are frequently complicated as the result of partial alkali-salt formation. In order to stabilize the sialic acid moieties under MALDI conditions and to divert all of the signal into the positive-ion mode, a method involving their conversion into methyl esters has been developed. The method is relatively rapid and produces strong positive-ion signals from N-linked oligosaccharides containing sialic acid and from gangliosides. The latter compounds are stable, even in the presence of alpha-cyano-4-hydroxycinnamic acid. They give abundant molecular (MNa+) ions, but with sufficient residual in-source fragmentation to allow the sequence of the sugar chain to be determined. The sialic acid residue is stable after methylation, irrespective of its linkage to the parent molecule.

SummaryBackgroundStaphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection.MethodsIn this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants.FindingsBetween Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18–45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference −1·4%, 95% CI −7·0 to 4·3; hazard ratio 0·96, 0·68–1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3–4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005).InterpretationAdjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia.FundingUK National Institute for Health Research Health Technology Assessment.

This review outlines the use of modern methods of mass spectrometry, mainly based on electrospray ionization and matrix-assisted lased desorption/ionization, for the identification of protein-bound carbohydrates. The techniques are briefly described together with methods for glycan release and purification prior to mass spectrometry. Fragmentation of glycans, produced under various conditions, is described in the context of obtaining structural information at the sensitivity required for proteomic work.

In a new, versatile approach to fun­ction­alizing recombinant spider silk, L‐azidohomoalanine is introduced residue‐specifically in the minispidroin protein 4RepCT through expression in an E. coli methionine auxotroph. Both fluorophores and the antibiotic levofloxacin are attached to this bio‐orthogonal amino acid using copper‐catalyzed click chemistry, either before or after the silk fibers are self‐assembled.

The SEC18 gene product is 48% identical to mammalian NSF (N-ethylmaleimide-sensitive fusion protein), and both proteins encode cytoplasmic ATPases which are essential for membrane traffic in yeast and mammalian cells, respectively. A wealth of biochemical analysis has led to the description of a model for the action of NSF; through its interaction with SNAPs (soluble NSF attachment proteins), NSF can associate with SNAP receptors (SNAREs) on intracellular membranes, forming 20S complexes. SNAPs then stimulate the intrinsic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is based almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membrane fusion is conserved in all eukaryotic cells. If so, the crucial biochemical properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here that Sec18p can specifically interact not only with Sec17p but also with its mammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-SNAP stimulation of NSF, although differences in temperature and N-ethylmaleimide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNAP to stimulate regulated exocytosis in mammalian cells. We conclude that the mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved from yeast to mammals.

Four chemical plume-tracking algorithms have been compared using a mobile robot. These algorithms are based upon hypotheses proposed to explain the plume-tracking behavior of flying insects. They all use information from a wind sensor and a single chemical sensor to determine how the agent should move to locate the source of the chemical plume. The performance of the robot using each of the algorithms was tested in a wind tunnel under a range of wind speeds (0.55, 0.95, and 1.4 m/s) using a model chemical (ionized air). The robot was capable of tracking the ion plume to its source effectively with each algorithm, having an overall success rate of over 85%. The simplest implemented algorithm, surge anemotaxis, was found to be the fastest. However, the shape of the tracking paths observed indicated that this simple algorithm may not explain the plume-tracking behavior of certain insects as well as the other algorithms tested. Further tests are required to see if the surge anemotaxis algorithm remains the most efficient under more realistic wind conditions.

Beyond the identification of proteins involved in a particular physiological situation, many aspects of proteomics require more detailed characterization of the proteins involved. Post-translational modifications (PTMs) of proteins are a common means to target proteins, regulate their activities and to mediate communication between proteins and cells. Owing to the much higher analytical complexity of glycan analysis compared to e.g. protein identification, PTM analysis in general and glycosylation analysis in particular is largely neglected in proteomics. In this review, the current technological status of global and site-specific glycosylation analysis of gel-separated proteins is described and the way in which the available technology can be employed in proteomics is critically discussed.