Evidence for two interaction regions for phosphatidylinositol(4,5)-bisphosphate on mammalian profilin I

AbstractThe binding of phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2) to profilin at a region distinct from the actin interaction surface is demonstrated by experiments with covalently cross-linked profilin:β-actin. The result is in agreement with observations made with several mutant profilins and provides strong evidence for two regions on mammalian profilin mediating electrostatic interaction with phosphatidylinositol lipids; one close to the binding site for poly(L-proline), and one partially overlapping with the actin-binding surface. Congruent with this, two plant profilins, which have a reduced number of positive amino acids in one of these regions, displayed a dramatically lower binding to PI(4,5)P2 compared to human profilin I

Analysis of intact ladderane phospholipids, originating from viable anammox bacteria, using RP-LC-ESI-MS

Since the discovery of the anaerobic ammonium oxidizing (anammox) bacteria, many attempts have been made in order to identify these environmentally important bacteria in natural environments. Anammox bacteria contain a unique class of lipids, called ladderane lipids and here we present a novel method to detect viable anammox bacteria in sediments and waste water treatment plants based on the use of a ladderane lipid biomarker. Intact ladderane phosphatidylcholine (PC) lipids are analyzed using reversed-phase liquid chromatography–electrospray ionization–mass spectrometry. Following extraction from the complex sediment matrix, reversed-phase LC is used to separate ladderane PC lipids based on their tail group hydrophobicity as well as their ether or ester link to the glycerol backbone in the sn-2 position. We investigate the presence of intact ladderane lipids in natural sediments displaying anammox activity and illustrate the use of a specific intact membrane forming PC lipid as a biomarker for viable anammox bacterial cells. The presented method can be used to elucidate the whereabouts of viable anammox bacteria, subsequently enabling an estimation of anammox activity. This will greatly increase the knowledge of anammox bacteria and their importance in the global nitrogen cycle

Light-Weight Techniques for Improving the Controllability and Efficiency of ISA-Level Fault Injection Tools

ISA-level fault injection, i.e. the injection of bit-flip faults in Instruction Set Architecture (ISA) registers and main memory words, is widely used for studying the impact of transient and intermittent hardware faults. ISA-level fault injection tools can be characterized by different properties such as repeatability, observability, reachability, intrusiveness, efficiency and controllability. This paper presents two pre-injection analysis techniques that improve controllability and efficiency using object code analysis. To improve controllability, we propose a technique for identifying the type of data that is stored in a potential target location. This allows the user to selectively direct fault injections to addresses, data and/or control information. Experimental results show that the data type of 84-100% of the targets locations in 8 programs were successfully identified by this technique. The second technique improves efficiency by fault pruning, i.e., by avoiding injection of faults that is known a priori to be detected by the tested system. This technique leverage the fact that faults in certain bits in the program counter and the stack pointer are always detected by machine exceptions. We show that exclusion of these bits from the fault space could significantly prune the fault space and reduce the time it takes to conduct a fault injection campaign

The contribution of insect prey to the total nitrogen content of sundews (Drosera spp.) determined in situ by stable isotope analysis

The contribution of insect prey to total N in the carnivorous plants, Drosera rotundifolia and D. intermedia, was quantified in situ and without any experimental manipulation using natural abundance stable isotope analysis. Samples of D. rotundifolia and D. intermedia, insects and noncarnivorous reference plants were collected from three contrasting locations across Britain. The proportion of Drosera nitrogen obtained from insect prey was calculated by a mixing model using δ<sup>15</sup>N values from the different plant groups. The mean proportion of Drosera N derived from prey was 50%. There were significant differences in this proportion between sites, and significant differences within sites. There were significant differences between plant tissues and a significant negative relationship between the proportion of N derived from prey and the C : N ratio of Drosera tissues. There was little evidence of differences in prey capture/utilisation in response to N availability, possibly due to a limited range in available N between the sites. However, evidence of a positive benefit of prey capture was apparent through the decrease in C : N ratio with increasing prey N concentrations in the plants

Cavitation dynamics and underwater radiated noise signature of a ship with a cavitating propeller

The paper presents SSPA’s work in the EU project AQUO to predict underwater radiated noise (URN) generated by a coastal tanker with a cavitating propeller. A CFD method, consisting of a multi-phase Delayed Detached Eddy Simulation (DDES) and a Ffowcs Williams-Hawkings (FWH) acoustic analogy, is applied to predict the cavitation, pressure pulses and radiated noise for the ship at model and full scale. In comparison with the data obtained from the model test and full scale measurement, it is found that the predicted sheet cavity correlates quite well with the observed ones in the experiment and sea trial. Some success is made in predicting the collapse and rebound of tip vortex cavitation (TVC) at model scale, yet the extension of TVC is under-predicted.The predicted pressure pulses agree reasonably well with the measured ones at the first three harmonics, deviation becomes larger at higher harmonics.The tonal noise has fairly good agreement with the measured signal at both scales up to 5th harmonics. The simulation however under-predicts part of broadband noise that is caused by the TVC, mainly due to an under-resolution of the flow in the tip region and the propeller wake. The agreement with the data for the model scale case is slightly better than that for the full scale case

Learning field Archaeology - Student Integrating Methods in Tertiary Education

The aim of this paper is to present a pedagogical development project which focuses upon student integrating methods for learning archaeology in tertiary education. The project was initiated by the teachers of the department because of the gap experienced between academic reflexivity and archaeological practice during our field courses. The students seemed to suffer from cognitive overload when they suddenly found themselves in the field so in order to bridge the gap we wanted to develop methods which would facilitate the students verbalisation of their field experience and observations, and which would improve communicating about it during field work. The results are that students become more active when the opportunity to develop a voice starts early in their education and they become more visible as actors in their education. A general improvement in the learning environment is noticed, too. We position this project, its results and potentials in a progressively emergent international discourse on pedagogy in tertiary education in archaeology

Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by <it>S</it>. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of <it>S</it>. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches.</p> <p>Results</p> <p>In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B₁₂, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX.</p> <p>Conclusion</p> <p>Using a multi-step digest approach the LPI™ technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI™ technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for <it>Salmonella </it>Typhimurium.</p

Minimizing tillage modifies fungal denitrifier communities, increases denitrification rates and enhances the genetic potential for fungal, relative to bacterial, denitrification

Nitrous oxide (N2O) emissions from arable soils are predominantly caused by denitrifying microbes, of which fungal denitrifiers are of particular interest, as fungi, in contrast to bacteria, terminate denitrification with N2O. Reduced tillage has been shown to increase gaseous nitrogen losses from soil, but knowledge of how varying tillage regimes and associated soil physical and chemical alterations affect fungal denitrifiers is limited. Based on results from a long-term (>40 years) tillage experiment, we show that non-inversion tillage resulted in increased potential denitrification activity in the upper soil layers, compared to annual or occasional (every 4-5 years) conventional inversion tillage. Using sequence-corrected abundance of the fungal nirK gene, we further identified an increased genetic potential for fungal denitrification, compared to that caused by bacteria, with decreasing tillage intensity. Differences in the composition and diversity of the fungal nirK community imply that different tillage regimes select for distinct fungal denitrifiers with differing functional capabilities and lifestyles, predominantly by altering carbon and nitrogen related niches. Our findings suggest that the creation of organic hotspots through stratification by non-inversion tillage increases the diversity and abundance of fungal denitrifier communities and modifies their composition, and thus their overall relevance for N2O production by denitrification, in arable soils

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or ‘classical’ carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits ‘non-classical’ carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.publishedVersio