Characterization of glutaraldehyde-immobilized chymotrypsin and an in-situ immobilized enzyme reactor using capillary electrophoresis-based peptide mapping

La digestion enzymatique des protéines est une méthode de base pour les études protéomiques ainsi que pour le séquençage en mode « bottom-up ». Les enzymes sont ajoutées soit en solution (phase homogène), soit directement sur le gel polyacrylamide selon la méthode déjà utilisée pour l’isolation de la protéine. Les enzymes protéolytiques immobilisées, c’est-à-dire insolubles, offrent plusieurs avantages tels que la réutilisation de l’enzyme, un rapport élevé d’enzyme-sur-substrat, et une intégration facile avec les systèmes fluidiques. Dans cette étude, la chymotrypsine (CT) a été immobilisée par réticulation avec le glutaraldehyde (GA), ce qui crée des particules insolubles. L’efficacité d’immobilisation, déterminée par spectrophotométrie d’absorbance, était de 96% de la masse totale de la CT ajouté. Plusieurs différentes conditions d’immobilisation (i.e., réticulation) tels que la composition/pH du tampon et la masse de CT durant la réticulation ainsi que les différentes conditions d’entreposage tels que la température, durée et humidité pour les particules GA-CT ont été évaluées par comparaison des cartes peptidiques en électrophorèse capillaire (CE) des protéines standards digérées par les particules. Les particules de GA-CT ont été utilisés pour digérer la BSA comme exemple d’une protéine repliée large qui requit une dénaturation préalable à la digestion, et pour digérer la caséine marquée avec de l’isothiocyanate de fluorescéine (FITC) comme exemple d’un substrat dérivé afin de vérifier l’activité enzymatique du GA-CT dans la présence des groupements fluorescents liés au substrat. La cartographie peptidique des digestions par les particules GA-CT a été réalisée par CE avec la détection par absorbance ultraviolet (UV) ou fluorescence induite par laser. La caséine-FITC a été, en effet, digérée par GA-CT au même degré que par la CT libre (i.e., soluble). Un microréacteur enzymatique (IMER) a été fabriqué par immobilisation de la CT dans un capillaire de silice fondu du diamètre interne de 250 µm prétraité avec du 3-aminopropyltriéthoxysilane afin de fonctionnaliser la paroi interne avec les groupements amines. Le GA a été réagit avec les groupements amine puis la CT a été immobilisée par réticulation avec le GA. Les IMERs à base de GA-CT étaient préparé à l’aide d’un système CE automatisé puis utilisé pour digérer la BSA, la myoglobine, un peptide ayant 9 résidus et un dipeptide comme exemples des substrats ayant taille large, moyenne et petite, respectivement. La comparaison des cartes peptidiques des digestats obtenues par CE-UV ou CE-spectrométrie de masse nous permettent d’étudier les conditions d’immobilisation en fonction de la composition et le pH du tampon et le temps de réaction de la réticulation. Une étude par microscopie de fluorescence, un outil utilisé pour examiner l’étendue et les endroits d’immobilisation GA-CT dans l’IMER, ont montré que l’immobilisation a eu lieu majoritairement sur la paroi et que la réticulation ne s’est étendue pas si loin au centre du capillaire qu’anticipée.

On The Center Sets and Center Numbers of Some Graph Classes

For a set S of vertices and the vertex v in a connected graph G, max x2S d(x, v) is called the S-eccentricity of v in G. The set of vertices with minimum S-eccentricity is called the S-center of G. Any set A of vertices of G such that A is an S-center for some set S of vertices of G is called a center set. We identify the center sets of certain classes of graphs namely, Block graphs, Km,n, Kn −e, wheel graphs, odd cycles and symmetric even graphs and enumerate them for many of these graph classes. We also introduce the concept of center number which is defined as the number of distinct center sets of a graph and determine the center number of some graph classes

Studies on scattering and thermodynamics of black holes in f(R) theory and Einstein's theory

One of the interesting consequences of Einstein's General Theory of Relativity is the black hole solutions. Until the observation made by Hawking in 1970s, it was believed that black holes are perfectly black. The General Theory of Relativity says that black holes are objects which absorb both matter and radiation crossing the event horizon. The event horizon is a surface through which even light is not able to escape. It acts as a one sided membrane that allows the passage of particles only in one direction i.e. towards the center of black holes. All the particles that are absorbed by black hole increases the mass of the black hole and thus the size of event horizon also increases. Hawking showed in 1970s that when applying quantum mechanical laws to black holes they are not perfectly black but they can emit radiation. Thus the black hole can have temperature known as Hawking temperature. In the thesis we have studied some aspects of black holes in f(R) theory of gravity and Einstein's General Theory of Relativity. The scattering of scalar field in this background space time studied in the first chapter shows that the extended black hole will scatter scalar waves and have a scattering cross section and applying tunneling mechanism we have obtained the Hawking temperature of this black hole. In the following chapter we have investigated the quasinormal properties of the extended black hole. We have studied the electromagnetic and scalar perturbations in this space-time and find that the black hole frequencies are complex and show exponential damping indicating the black hole is stable against the perturbations. In the present study we show that not only the black holes exist in modified gravities but also they have similar properties of black hole space times in General Theory of Relativity. 2 + 1 black holes or three dimensional black holes are simplified examples of more complicated four dimensional black holes. Thus these models of black holes are known as toy models of black holes in four dimensional black holes in General theory of Relativity. We have studied some properties of these types of black holes in Einstein model (General Theory of Relativity). A three dimensional black hole known as MSW is taken for our study. The thermodynamics and spectroscopy of MSW black hole are studied and obtained the area spectrum which is equispaced and different thermo dynamical properties are studied. The Dirac perturbation of this three dimensional black hole is studied and the resulting quasinormal spectrum of this three dimensional black hole is obtained. The different quasinormal frequencies are tabulated in tables and these values show an exponential damping of oscillations indicating the black hole is stable against the mass less Dirac perturbation. In General Theory of Relativity almost all solutions contain singularities. The cosmological solution and different black hole solutions of Einstein's field equation contain singularities. The regular black hole solutions are those which are solutions of Einstein's equation and have no singularity at the origin. These solutions possess event horizon but have no central singularity. Such a solution was first put forward by Bardeen. Hayward proposed a similar regular black hole solution. We have studied the thermodynamics and spectroscopy of Hay-ward regular black holes. We have also obtained the different thermodynamic properties and the area spectrum. The area spectrum is a function of the horizon radius. The entropy-heat capacity curve has a discontinuity at some value of entropy showing a phase transition.

Impact of sward type, cutting date and conditioning temperature on mass and energy flows in the integrated generation of solid fuel and biogas from semi-natural grassland

Energy production from biomass and the conservation of ecologically valuable grassland habitats are two important issues of agriculture today. The combination of a bioenergy production, which minimises environmental impacts and competition with food production for land with a conversion of semi-natural grasslands through new utilization alternatives for the biomass, led to the development of the IFBB process. Its basic principle is the separation of biomass into a liquid fraction (press fluid, PF) for the production of electric and thermal energy after anaerobic digestion to biogas and a solid fraction (press cake, PC) for the production of thermal energy through combustion. This study was undertaken to explore mass and energy flows as well as quality aspects of energy carriers within the IFBB process and determine their dependency on biomass-related and technical parameters. Two experiments were conducted, in which biomass from semi-natural grassland was conserved as silage and subjected to a hydrothermal conditioning and a subsequent mechanical dehydration with a screw press. Methane yield of the PF and the untreated silage was determined in anaerobic digestion experiments in batch fermenters at 37°C with a fermentation time of 13-15 and 27-35 days for the PF and the silage, respectively. Concentrations of dry matter (DM), ash, crude protein (CP), crude fibre (CF), ether extract (EE), neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent ligning (ADL) and elements (K, Mg, Ca, Cl, N, S, P, C, H, N) were determined in the untreated biomass and the PC. Higher heating value (HHV) and ash softening temperature (AST) were calculated based on elemental concentration. Chemical composition of the PF and mass flows of all plant compounds into the PF were calculated. In the first experiment, biomass from five different semi-natural grassland swards (Arrhenaterion I and II, Caricion fuscae, Filipendulion ulmariae, Polygono-Trisetion) was harvested at one late sampling (19 July or 31 August) and ensiled. Each silage was subjected to three different temperature treatments (5°C, 60°C, 80°C) during hydrothermal conditioning. Based on observed methane yields and HHV as energy output parameters as well as literature-based and observed energy input parameters, energy and green house gas (GHG) balances were calculated for IFBB and two reference conversion processes, whole-crop digestion of untreated silage (WCD) and combustion of hay (CH). In the second experiment, biomass from one single semi-natural grassland sward (Arrhenaterion) was harvested at eight consecutive dates (27/04, 02/05, 09/05, 16/05, 24/05, 31/05, 11/06, 21/06) and ensiled. Each silage was subjected to six different treatments (no hydrothermal conditioning and hydrothermal conditioning at 10°C, 30°C, 50°C, 70°C, 90°C). Energy balance was calculated for IFBB and WCD. Multiple regression models were developed to predict mass flows, concentrations of elements in the PC, concentration of organic compounds in the PF and energy conversion efficiency of the IFBB process from temperature of hydrothermal conditioning as well as NDF and DM concentration in the silage. Results showed a relative reduction of ash and all elements detrimental for combustion in the PC compared to the untreated biomass of 20-90%. Reduction was highest for K and Cl and lowest for N. HHV of PC and untreated biomass were in a comparable range (17.8-19.5 MJ kg-1 DM), but AST of PC was higher (1156-1254°C). Methane yields of PF were higher compared to those of WCD when the biomass was harvested late (end of May and later) and in a comparable range when the biomass was harvested early and ranged from 332 to 458 LN kg-1 VS. Regarding energy and GHG balances, IFBB, with a net energy yield of 11.9-14.1 MWh ha-1, a conversion efficiency of 0.43-0.51, and GHG mitigation of 3.6-4.4 t CO2eq ha-1, performed better than WCD, but worse than CH. WCD produces thermal and electric energy with low efficiency, CH produces only thermal energy with a low quality solid fuel with high efficiency, IFBB produces thermal and electric energy with a solid fuel of high quality with medium efficiency. Regression models were able to predict target parameters with high accuracy (R2=0.70-0.99). The influence of increasing temperature of hydrothermal conditioning was an increase of mass flows, a decrease of element concentrations in the PC and a differing effect on energy conversion efficiency. The influence of increasing NDF concentration of the silage was a differing effect on mass flows, a decrease of element concentrations in the PC and an increase of energy conversion efficiency. The influence of increasing DM concentration of the silage was a decrease of mass flows, an increase of element concentrations in the PC and an increase of energy conversion efficiency. Based on the models an optimised IFBB process would be obtained with a medium temperature of hydrothermal conditioning (50°C), high NDF concentrations in the silage and medium DM concentrations of the silage.

Transport impact of in urban planning: the case of Transmilenio in Bogota

The growth and expansion of cities during the last century can’t be seen without taking into account the important role they have assumed in the transport infrastructure. In Bogota have passed trough streetcar, buses and automóviles was deciding for the city, because all this periods marked a drastic change in growth morphology. This article studies the planning of Mass Transit System –MTS– Transmilenio and its interaction with the urban structure, in light of the current city planning framework. It also specifies the behavior of land use in the construction of the system first phase. One of the most important findings of this study is that there is no articulation between land and transport use system, despite the decisions taken in the ordering model. Similarly, local and intermediate planning exhibits Peak articulation.The existing infrastructure of the MTS reiterates the tendency to concentrate the accessibility in one place: the center has the largest accessibility whereas periphery fails to overcome its limitations of accessibility. While the city continued with this trend the existence of a coordinated planning system for Bogota and the ability to meet expectations of planning model is questionable, however it is something that depends not only on MTS.

The Agendas of The Stream

The new tech has introduced the traits of the hypermedia, interactivity and convergence in the journalism field. Moreover, it has involved the emergence of new media and changes in the informative contents. In 2006 Al Jazeera in English was created for covering the underreported regions, inasmuch as new media arise, new contents do so, thus, the research examines the agendas of The Stream, the TV show from Al Jazeera that relies heavily on social networks.

The gas-phase reactions Of SiCl4 and Si2Cl6 with CH3OH and C2H5OH: an investigation by mass spectrometry and matrix-isolation infrared spectroscopy

The gas phase reactions Of SiCl4 and Si2Cl6 With CH3OH and C2H5OH have been investigated using both mass spectrometry and matrix isolation techniques. SiCl4 reacts with both CH3OH and C2H5OH upon mixing of the vapours for times in excess of 3 h to generate the HCl-elimination products SiCl3OR (R = CH3 or C2H5). The identity of these products is confirmed by deuteration experiments and by ab initio calculations at the HF/6-31G(d) level. Further products are generated when the mixture is passed through a tube heated to 750degreesC. Si2Cl6 reacts with CH3OH and C2H5OH via a different mechanism in which the Si-Si bond is cleaved to yield SiCl3OR and HCl. Other products of the type SiCl4-n(OCH3)(n) are tentatively identified by a combination of mass spectrometric and matrix isolation measurements. These latter products indicate further replacement of Cl atoms by OR groups as a result of reaction of CH3OH or C2H5OH with the initial product.

Vibrio parahaemolyticus associated with mass mortality of postlarval abalone, Haliotis diversicolor supertexta (L.), in Sanya, China

Outbreaks of mass mortality in postlarval abalone, Haliotis diversicolor supertexta (L.), have swept across south China since 2002 and in turn have resulted in many abalone farms closing. Twenty-five representative bacterial isolates were isolated from a sample of five diseased postlarval abalone, taken 15 d postfertilization during an outbreak of postlarval disease in Sanya, Hainan Province, China in October 2004. A dominant isolate, referred to as Strain 6, was found to be highly virulent to postlarvae in an experimental challenge test, with a 50% lethal dose (LD50) value of 3.2 x 10(4) colony forming units (CFU)/mL, while six of the other isolates were weakly virulent with LD50 values ranging from 1 x 10(6) to 1 x 10(7) CFU/mL, and the remaining 18 isolates were classified as avirulent with LD50 values greater than 1 x 10(8) CFU/mL. Using both an API 20E kit and 16S recombinant DNA sequence analysis, Strain 6 was shown to be Vibrio parahaemolyticus. It was sensitive to 4 and intermediately sensitive to 5 of the 16 antibiotics used when screening the antibiotic sensitivities of the bacterium. Extracellular products (ECPs) prepared from the bacterium were lethal to postlarvae when used in a toxicity test at a concentration of 3.77 mg protein/mL, and complete liquefaction of postlarvae tissues occurred within 24 h postexposure. Results from this study implicate V. parahaemolyticus as the pathogen involved in the disease outbreaks in postlarval abalone in Sanya and show that the ECPs may be involved in the pathogenesis of the disease.

Liquid AP-UV-MALDI enables stable ion yields of multiply charged peptide and protein ions for sensitive analysis by mass spectrometry

In biological mass spectrometry (MS), two ionization techniques are predominantly employed for the analysis of larger biomolecules, such as polypeptides. These are nano-electrospray ionization [1, 2] (nanoESI) and matrix-assisted laser desorption/ionization [3, 4] (MALDI). Both techniques are considered to be “soft”, allowing the desorption and ionization of intact molecular analyte species and thus their successful mass-spectrometric analysis. One of the main differences between these two ionization techniques lies in their ability to produce multiply charged ions. MALDI typically generates singly charged peptide ions whereas nanoESI easily provides multiply charged ions, even for peptides as low as 1000 Da in mass. The production of highly charged ions is desirable as this allows the use of mass analyzers, such as ion traps (including orbitraps) and hybrid quadrupole instruments, which typically offer only a limited m/z range (< 2000–4000). It also enables more informative fragmentation spectra using techniques such as collisioninduced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) in combination with tandem MS (MS/MS). [5, 6] Thus, there is a clear advantage of using ESI in research areas where peptide sequencing, or in general, the structural elucidation of biomolecules by MS/MS is required. Nonetheless, MALDI with its higher tolerance to contaminants and additives, ease-of-operation, potential for highspeed and automated sample preparation and analysis as well as its MS imaging capabilities makes it an ionization technique that can cover bioanalytical areas for which ESI is less suitable. [7, 8] If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and large-scale proteomic analyses based on MALDI MS(/MS) would become feasible.

Are superhumps good measures of the mass ratio for AM CVn systems?

We extend recent work that included the effect of pressure forces to derive the precession rate of eccentric accretion discs in cataclysmic variables to the case of double degenerate systems. We find that the logical scaling of the pressure force in such systems results in predictions of unrealistically high primary masses. Using the prototype AM CVn as a calibrator for the magnitude of the effect, we find that there is no scaling that applies consistently to all the systems in the class. We discuss the reasons for the lack of a superhump period to mass ratio relationship analogous to that known for SU UMa systems and suggest that this is because these secondaries do not have a single valued mass-radius relationship. We highlight the unreliability of mass-ratios derived by applying the SU UMa expression to the AM CVn binaries.

Tracking the momentum flux of a CME and quantifying its influence on geomagnetically induced currents at Earth

We investigate a coronal mass ejection (CME) propagating toward Earth on 29 March 2011. This event is specifically chosen for its predominately northward directed magnetic field, so that the influence from the momentum flux onto Earth can be isolated. We focus our study on understanding how a small Earth-directed segment propagates. Mass images are created from the white-light cameras onboard STEREO which are also converted into mass height-time maps (mass J-maps). The mass tracks on these J-maps correspond to the sheath region between the CME and its associated shock front as detected by in situ measurements at L1. A time series of mass measurements from the STEREO COR-2A instrument is made along the Earth propagation direction. Qualitatively, this mass time series shows a remarkable resemblance to the L1 in situ density series. The in situ measurements are used as inputs into a three-dimensional (3-D) magnetospheric space weather simulation from the Community Coordinated Modeling Center. These simulations display a sudden compression of the magnetosphere from the large momentum flux at the leading edge of the CME, and predictions are made for the time derivative of the magnetic field (dB/dt) on the ground. The predicted dB/dt values were then compared with the observations from specific equatorially located ground stations and showed notable similarity. This study of the momentum of a CME from the Sun down to its influence on magnetic ground stations on Earth is presented as a preliminary proof of concept, such that future attempts may try to use remote sensing to create density and velocity time series as inputs to magnetospheric simulations.

The geomagnetic mass spectrometer— mass and energy dispersions of ionospheric ion flows into the magnetosphere

NASA's Dynamics Explorer (DE) mission was designed to study the coupling between the Earth's magnetosphere, ionosphere and neutral thermosphere1. One area of major interest is the outflow of ionospheric plasma into the magnetosphere, the scale and significance of which is only now becoming apparent with the advent of mass-resolving, low-energy ion detectors. Here we compare observations of ion flows in the polar magnetosphere, made by the retarding ion mass spectrometer (RIMS)2 on DE1, with those made simultaneously in the topside ionosphere by the ion drift meter (IDM)3 on the lower-altitude DE2 spacecraft. The results show the dayside auroral ionosphere to be a significant and highly persistent source of plasma for the magnetosphere. The upwelling ionospheric ions are spatially dispersed, according to both their energy and mass, by the combined actions of the geomagnetic field and the dawn-to-dusk convection electric field, in an effect analogous to the operation of an ion mass spectrometer.

DIFFUSION OF MAGNETIC FIELD AND REMOVAL OF MAGNETIC FLUX FROM CLOUDS VIA TURBULENT RECONNECTION

The diffusion of astrophysical magnetic fields in conducting fluids in the presence of turbulence depends on whether magnetic fields can change their topology via reconnection in highly conducting media. Recent progress in understanding fast magnetic reconnection in the presence of turbulence reassures that the magnetic field behavior in computer simulations and turbulent astrophysical environments is similar, as far as magnetic reconnection is concerned. This makes it meaningful to perform MHD simulations of turbulent flows in order to understand the diffusion of magnetic field in astrophysical environments. Our studies of magnetic field diffusion in turbulent medium reveal interesting new phenomena. First of all, our three-dimensional MHD simulations initiated with anti-correlating magnetic field and gaseous density exhibit at later times a de-correlation of the magnetic field and density, which corresponds well to the observations of the interstellar media. While earlier studies stressed the role of either ambipolar diffusion or time-dependent turbulent fluctuations for de-correlating magnetic field and density, we get the effect of permanent de-correlation with one fluid code, i.e., without invoking ambipolar diffusion. In addition, in the presence of gravity and turbulence, our three-dimensional simulations show the decrease of the magnetic flux-to-mass ratio as the gaseous density at the center of the gravitational potential increases. We observe this effect both in the situations when we start with equilibrium distributions of gas and magnetic field and when we follow the evolution of collapsing dynamically unstable configurations. Thus, the process of turbulent magnetic field removal should be applicable both to quasi-static subcritical molecular clouds and cores and violently collapsing supercritical entities. The increase of the gravitational potential as well as the magnetization of the gas increases the segregation of the mass and magnetic flux in the saturated final state of the simulations, supporting the notion that the reconnection-enabled diffusivity relaxes the magnetic field + gas system in the gravitational field to its minimal energy state. This effect is expected to play an important role in star formation, from its initial stages of concentrating interstellar gas to the final stages of the accretion to the forming protostar. In addition, we benchmark our codes by studying the heat transfer in magnetized compressible fluids and confirm the high rates of turbulent advection of heat obtained in an earlier study.

The effect of a single supernova explosion on the cuspy density profile of a small-mass dark matter halo

Some observations of galaxies, and in particular dwarf galaxies, indicate a presence of cored density profiles in apparent contradiction with cusp profiles predicted by dark matter N-body simulations. We constructed an analytical model, using particle distribution functions (DFs), to show how a supernova (SN) explosion can transform a cusp density profile in a small-mass dark matter halo into a cored one. Considering the fact that an SN efficiently removes matter from the centre of the first haloes, we study the effect of mass removal through an SN perturbation in the DFs. We find that the transformation from a cusp into a cored profile occurs even for changes as small as 0.5 per cent of the total energy of the halo, which can be produced by the expulsion of matter caused by a single SN explosion.

Characterisation of anthracyclines from a cosmomycin D-producing species of Streptomyces by collisionally-activated dissociation and ion mobility mass spectrometry

Cultures of cosmomycin D-producing Streptomyces olindensis ICB20 that were propagated for many generations underwent mutations that resulted in production of a range of related anthracyclines by the bacteria. The anthracyclines that retained the two trisaccharide chains of the parent compound were separated by HPLC. Exact mass determination of these compounds revealed that they differed from cosmomycin D (CosD) in that they contained one to three fewer oxygen atoms (loss of hydroxyl groups). Some of the anthracyclines that were separated by HPLC had the same mass. The location from which the hydroxyl groups had been lost relative to CosD (on the aglycone and/or on the sugar residues) was probed by collisionally-activated dissociation using an electrospray ionisation linear quadrupole ion trap mass spectrometer. The presence of anthracyclines with the same mass, but different structure, was confirmed using an electrospray ionisation travelling wave ion mobility mass spectrometer.