Microarray analysis of whole genome expression of intracellular Mycobacterium tuberculosis

Analysis of the changing mRNA expression profile of Mycobacterium tuberculosis though the course of infection promises to advance our understanding of how mycobacteria are able to survive the host immune response. The difficulties of sample extraction from distinct mycobacterial populations, and of measuring mRNA expression profiles of multiple genes has limited the impact of gene expression studies on our interpretation of this dynamic infection process. The development of whole genome microarray technology together with advances in sample collection have allowed the expression pattern of the whole M. tuberculosis genome to be compared across a number of different in vitro conditions, murine and human tissue culture models and in vivo infection samples. This review attempts to produce a summative model of the M. tuberculosis response to infection derived from or reflected in these gene expression datasets. The mycobacterial response to the intracellular environment is characterised by the utilisation of lipids as a carbon source and the switch from aerobic/microaerophilic to anaerobic respiratory pathways. Other genes induced in the macrophage phagosome include those likely to be involved in the maintenance of the cell wall and genes related to DNA damage, heat shock, iron sequestration and nutrient limitation. The comparison of transcriptional data from in vitro models of infection with complex in vivo samples, together with the use of bacterial RNA amplification strategies to sample defined populations of bacilli, should allow us to make conclusions about M. tuberculosis physiology and host microenvironments during natural infection

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis

Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profilin

Distinct phosphorylation clusters determines the signalling outcome of the free fatty acid receptor FFA4/GPR120

It is established that long-chain free fatty acids including ω-3 fatty acids mediate an array of biological responses through members of the free fatty acid receptor family, which includes FFA4. However, the signalling mechanisms and modes of regulation of this receptor class remain unclear. Here we employ mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C terminal tail, designated cluster 1 (Thr347, Thr349 and Ser350) and cluster 2 (Ser357 and Ser361). Mutation of these phospho-acceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to ERK1/2 activation. Rather an inhibitor of Gq/11 proteins completely prevented receptor signalling to ERK1/2. In contrast, the recruitment of arrestin 3, receptor internalization and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signalling was extended further by selective mutations of the phospho-acceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phospho-acceptor sites within cluster 1 had no effect on receptor internalization and a less extensive effect on arrestin 3 recruitment, but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signalling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signalling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C-terminus of the receptor

Evidence for Recent Wet-Based Crater Glaciation in Tempe Terra, Mars.

[Introduction] Mars’ mid-latitudes host abundant putative debris-covered water-ice glaciers (viscous flow features; VFF). Eskers emerging from 110-150 Myr-old VFF in Phlegra Montes and Tempe Terra provide evidence for rare occurences of past, localized basal melting of their parent VFF, despite the cold climates of the late Amazonian (see this conf.). Eskers are sinuous ridges comprising glaciofluvial sediment deposited by meltwater flowing through tunnels within glacial ice. Here, we describe a population of sinuous ridges emerging from VFF in an unnamed ~45 km-diameter crater (38.47 N, 72.43 W) in Tempe Terra, ~600 km from the VFF-linked esker identified by Butcher et al. We consider two working hypotheses for the formation of the sinuous ridges; that they are either (1) eskers formed by melting of the glaciers from which they emerge, or (2) topographically inverted fluvial channels which formed prior to glaciation of the crater. We present observations from preliminary geomorphic mapping of the crater to start to test those hypotheses

Absolute calibration of the LOPES antenna system

Radio emission in extensive air showers arises from an interaction with the geomagnetic field and is subject of theoretical studies. This radio emission has advantages for the detection of high energy cosmic rays compared to secondary particle or fluorescence measurement methods. Radio antennas like the LOPES30 antenna system are suited to investigate this emission process by detecting the radio pulses. The characteristic observable parameters like electric field strength and pulse length require a calibration which was done with a reference radio source resulting in an amplification factor representing the system behavior in the environment of the KASCADE-Grande experiment. Knowing the amplification factor and the gain of the LOPES antennas LOPES30 is calibrated absolutely for systematic analyses of the radio emission.Comment: 5 pages, Proceedings of International Workshop on Acoustic and Radio EeV Neutrino detection Activities: ARENA, May 17-19, 2005, DESY Zeuthe

Multipole nonlinearity of metamaterials

We report on the linear and nonlinear optical response of metamaterials evoked by first and second order multipoles. The analytical ground on which our approach bases permits for new insights into the functionality of metamaterials. For the sake of clarity we focus here on a key geometry, namely the split-ring resonator, although the introduced formalism can be applied to arbitrary structures. We derive the equations that describe linear and nonlinear light propagation where special emphasis is put on second harmonic generation. This contribution basically aims at stretching versatile and existing concepts to describe light propagation in nonlinear media towards the realm of metamaterials.Comment: 7 pages, 3 figure

On the General Analytical Solution of the Kinematic Cosserat Equations

Based on a Lie symmetry analysis, we construct a closed form solution to the kinematic part of the (partial differential) Cosserat equations describing the mechanical behavior of elastic rods. The solution depends on two arbitrary analytical vector functions and is analytical everywhere except a certain domain of the independent variables in which one of the arbitrary vector functions satisfies a simple explicitly given algebraic relation. As our main theoretical result, in addition to the construction of the solution, we proof its generality. Based on this observation, a hybrid semi-analytical solver for highly viscous two-way coupled fluid-rod problems is developed which allows for the interactive high-fidelity simulations of flagellated microswimmers as a result of a substantial reduction of the numerical stiffness.Comment: 14 pages, 3 figure