Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

AbstractThe structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrPSc. These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex–Hexose, C6H12O6 carbohydrates, including mannnose and galactose; HexNAc—N-acetylhexosamine, C8H15NO6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc—N-acetylglucosamine; GalNAc—N-acetylgalactosamine; Fuc–Fucose; NeuAC—N-acetylneuraminic acid or sialic acid; TSE—Transmissible Spongiform Encephalopathy.

Wrapping Transition and Wrapping-Mediated Interactions for Discrete Binding along an Elastic Filament: An Exact Solution

The wrapping equilibria of one and two adsorbing cylinders are studied along a semi-flexible filament (polymer) due to the interplay between elastic rigidity and short-range adhesive energy between the cylinder and the filament. We show that statistical mechanics of the system can be solved exactly using a path integral formalism which gives access to the full effect of thermal fluctuations, going thus beyond the usual Gaussian approximations which take into account only the contributions from the minimal energy configuration and small fluctuations about this minimal energy solution. We obtain the free energy of the wrapping-unwrapping transition of the filament around the cylinders as well as the effective interaction between two wrapped cylinders due to thermal fluctuations of the elastic filament. A change of entropy due to wrapping of the filament around the adsorbing cylinders as they move closer together is identified as an additional source of interactions between them. Such entropic wrapping effects should be distinguished from the usual entropic configuration effects in semi-flexible polymers. Our results may be applicable to the problem of adsorption of proteins as well as synthetic nano-particles on semi-flexible polymers such as DNA.Comment: 24 pages, 12 figure

Progressive metabolic impairment underlies the novel nematicidal action of fluensulfone on the potato cyst nematode Globodera pallida

Background: Fluensulfone is a new nematicide with an excellent profile of selective toxicity against plant parasitic nematodes. Here, its effects on the physiology and biochemistry of the potato cyst nematode Globodera pallida have been investigated and comparisons made with its effect on the life-span of the free-living nematode Caenorhabditis elegans to provide insight into its mode of action and its selective toxicity. Results: Fluensulfone exerts acute effects (≤ 1 h; ≥ 100 μM) on stylet thrusting and motility of hatched second stage G. pallida juveniles (J2s). Chronic exposure to lower concentrations of fluensulfone (≥ 3 days; ≤ 30 μM), reveals a slowly developing metabolic insult in which G. pallida J2s sequentially exhibit a reduction in motility, loss of a metabolic marker for cell viability, high lipid content and tissue degeneration prior to death. These effects are absent in adults and dauers of the model genetic nematode Caenorhabditis elegans. Conclusion: The nematicidal action of fluensulfone follows a time-course which progresses from an early impact on motility through to an accumulating metabolic impairment, an inability to access lipid stores and death

On the nonlinear stability of viscous modes within the Rayleigh problem on an infinite flat plate

The stability has been investigated of the unsteady flow past an infinite flat plate when it is moved impulsively from rest, in its own plane. For small times the instantaneous stability of the flow depends on the linearized equations of motion which reduce in this problem to the Orr-Sommerfeld equation. It is known that the flow for certain values of Reynolds number, frequency and wave number is unstable to Tollmien-Schlichting waves, as in the case of the Blasius boundary layer flow past a flat plate. With increase in time, the unstable waves only undergo growth for a finite time interval, and this growth rate is itself a function of time. The influence of finite amplitude effects is studied by solving the full Navier-Stokes equations. It is found that the stability characteristics are markedly changed both by the consideration of the time evolution of the flow, and by the introduction of finite amplitude effects

DNA cruciform arms nucleate through a correlated but non-synchronous cooperative mechanism

Inverted repeat (IR) sequences in DNA can form non-canonical cruciform structures to relieve torsional stress. We use Monte Carlo simulations of a recently developed coarse-grained model of DNA to demonstrate that the nucleation of a cruciform can proceed through a cooperative mechanism. Firstly, a twist-induced denaturation bubble must diffuse so that its midpoint is near the centre of symmetry of the IR sequence. Secondly, bubble fluctuations must be large enough to allow one of the arms to form a small number of hairpin bonds. Once the first arm is partially formed, the second arm can rapidly grow to a similar size. Because bubbles can twist back on themselves, they need considerably fewer bases to resolve torsional stress than the final cruciform state does. The initially stabilised cruciform therefore continues to grow, which typically proceeds synchronously, reminiscent of the S-type mechanism of cruciform formation. By using umbrella sampling techniques we calculate, for different temperatures and superhelical densities, the free energy as a function of the number of bonds in each cruciform along the correlated but non-synchronous nucleation pathways we observed in direct simulations.Comment: 12 pages main paper + 11 pages supplementary dat

Larson's third law and the universality of molecular cloud structure

Larson (1981) first noted a scaling relation between masses and sizes in molecular clouds that implies that these objects have approximately constant column densities. This original claim, based upon millimeter observations of carbon monoxide lines, has been challenged by many theorists, arguing that the apparent constant column density observed is merely the result of the limited dynamic range of observations, and that in reality clouds have column density variations over two orders of magnitudes. In this letter we investigate a set of nearby molecular clouds with near-infrared excess methods, which guarantee very large dynamic ranges and robust column density measurements, to test the validity of Larson's third law. We verify that different clouds have almost identical average column densities above a given extinction threshold; this holds regardless of the extinction threshold, but the actual average surface mass density is a function of the specific threshold used. We show that a second version of Larson's third law, involving the mass-radius relation for single clouds and cores, does not hold in our sample, indicating that individual clouds are not objects that can be described by constant column density. Our results instead indicate that molecular clouds are characterized by a universal structure. Finally we point out that this universal structure can be linked to the log-normal nature of cloud column density distributions.Comment: 5 pages, 4 figures, A&A in press (letter

Non-Equilibrium Reaction Rates in the Macroscopic Chemistry Method for DSMC Calculations

The Direct Simulation Monte Carlo (DSMC) method is used to simulate the flow of rarefied gases. In the Macroscopic Chemistry Method (MCM) for DSMC, chemical reaction rates calculated from local macroscopic flow properties are enforced in each cell. Unlike the standard total collision energy (TCE) chemistry model for DSMC, the new method is not restricted to an Arrhenius form of the reaction rate coefficient, nor is it restricted to a collision cross-section which yields a simple power-law viscosity. For reaction rates of interest in aerospace applications, chemically reacting collisions are generally infrequent events and, as such, local equilibrium conditions are established before a significant number of chemical reactions occur. Hence, the reaction rates which have been used in MCM have been calculated from the reaction rate data which are expected to be correct only for conditions of thermal equilibrium. Here we consider artificially high reaction rates so that the fraction of reacting collisions is not small and propose a simple method of estimating the rates of chemical reactions which can be used in the Macroscopic Chemistry Method in both equilibrium and non-equilibrium conditions. Two tests are presented: (1) The dissociation rates under conditions of thermal non-equilibrium are determined from a zero-dimensional Monte-Carlo sampling procedure which simulates ‘intra-modal’ non-equilibrium; that is, equilibrium distributions in each of the translational, rotational and vibrational modes but with different temperatures for each mode; (2) The 2-D hypersonic flow of molecular oxygen over a vertical plate at Mach 30 is calculated. In both cases the new method produces results in close agreement with those given by the standard TCE model in the same highly nonequilibrium conditions. We conclude that the general method of estimating the non-equilibrium reaction rate is a simple means by which information contained within non-equilibrium distribution functions predicted by the DSMC method can be included in the Macroscopic Chemistry Method

Observational signatures of a non-singular bouncing cosmology

We study a cosmological scenario in which inflation is preceded by a bounce. In this scenario, the primordial singularity, one of the major shortcomings of inflation, is replaced by a non-singular bounce, prior to which the universe undergoes a phase of contraction. Our starting point is the bouncing cosmology investigated in Falciano et al. (2008), which we complete by a detailed study of the transfer of cosmological perturbations through the bounce and a discussion of possible observational effects of bouncing cosmologies. We focus on a symmetric bounce and compute the evolution of cosmological perturbations during the contracting, bouncing and inflationary phases. We derive an expression for the Mukhanov-Sasaki perturbation variable at the onset of the inflationary phase that follows the bounce. Rather than being in the Bunch-Davies vacuum, it is found to be in an excited state that depends on the time scale of the bounce. We then show that this induces oscillations superimposed on the nearly scale-invariant primordial spectra for scalar and tensor perturbations. We discuss the effects of these oscillations in the cosmic microwave background and in the matter power spectrum. We propose a new way to indirectly measure the spatial curvature energy density parameter in the context of this model.Comment: 40 pages, 5 figures, typos corrected and reference adde

Assessing risks and mitigating impacts of harmful algal blooms on mariculture and marine fisheries

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this nrecordAquaculture is the fastest growing food sector globally and protein provisioning from aquaculture now exceeds that from wild capture fisheries. There is clear potential for the further expansion of marine aquaculture (mariculture), but there are associated risks. Some naturally occurring algae can proliferate under certain environmental conditions, causing deoxygenation of seawater, or releasing toxic compounds (phycotoxins), which can harm wild and cultured finfish and shellfish, and also human consumers. The impacts of these so-called ‘harmful algal blooms’ (HABs) amount to approximately 8 $billion/yr globally, due to mass mortalities in finfish, harvesting bans preventing the sale of shellfish that have accumulated unsafe levels of HAB phycotoxins, and unavoided human health costs. Here we provide a critical review and analysis of HAB impacts on mariculture (and wild capture fisheries) and recommend research to identify ways to minimise their impacts to the industry. We examine causal factors for HAB development in inshore versus offshore locations and consider how mariculture itself, in its various forms, may exacerbate or mitigate HAB risk. From a management perspective, there is considerable scope for strategic siting of offshore mariculture and holistic Environmental Approaches for Aquaculture, such as offsetting nutrient outputs from finfish farming, via the co-location of extractive shellfish and macroalgae. Such pre-emptive, ecosystem-based approaches are preferable to reactive physical, chemical or microbiological control measures aiming to remove or neutralise HABs and their phycotxins. To facilitate mariculture expansion and long-term sustainability, it is also essential to evaluate HAB risk in conjunction with climate change.European Martime and Fisheries Fund (EMFF

Making the user more efficient: Design for sustainable behaviour

User behaviour is a significant determinant of a product’s environmental impact; while engineering advances permit increased efficiency of product operation, the user’s decisions and habits ultimately have a major effect on the energy or other resources used by the product. There is thus a need to change users’ behaviour. A range of design techniques developed in diverse contexts suggest opportunities for engineers, designers and other stakeholders working in the field of sustainable innovation to affect users’ behaviour at the point of interaction with the product or system, in effect ‘making the user more efficient’. Approaches to changing users’ behaviour from a number of fields are reviewed and discussed, including: strategic design of affordances and behaviour-shaping constraints to control or affect energyor other resource-using interactions; the use of different kinds of feedback and persuasive technology techniques to encourage or guide users to reduce their environmental impact; and context-based systems which use feedback to adjust their behaviour to run at optimum efficiency and reduce the opportunity for user-affected inefficiency. Example implementations in the sustainable engineering and ecodesign field are suggested and discussed