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

A COMPARISON OF THREE RACKET SKILLS EXECUTED SY NOVICE AND EXPERIENCED PERFORMERS

INTRODUCTIONT he purpose of this investigation was to compare an adult novice (NOV) with an experienced (EXP) adult performing a badminton deep serve, a racquetball forehand, and a racquetball drive serve. Movements were videotaped by 4cameras at 120 Hz and the 3-D data were analyzed using the PEAK5 motion measurement system. Range of motion (ROM), sequence of motion, and temporal values were assessed on the following angular movements: absolute pelvic (P) and upper torso (UT) rotation, and relative humeral (H), elbow (E), and wist (W) rotation. RESULTS For each skill and performer, the segmental sequence and ROM are presented. This information is followed by the range of lag times (LT) between segments within the sequence and the total time (TT) (backswing to contact)over which the skill occurred. Badminton Serve EXP P(37.4"),UT(69.6"), H(76.3"), W(59.7"), E(16.0°).LT(.02-.I 8s). TT(.23s).NOV UT(30.4"), P(l 3.0°), H(67.0°),W(10.2"), E(34.2").LT(.OI -.14~)T. T(.2Os).Racquetball Forehand EXP H(68.8"),P(57.g0),U T(71. I") , W(ll .go), E(12.8").LT(.02-.1 0s). TT(. II S).NOV P(34.8"), UT(69.2"), W(59.4"),E(8.7"), H(10.7").LT(.03-. 1 3s). TT(.20s).Racquetball Serve U(P P(92.4"),UT(122.0°), H(154.0°), E(75.3"),W(41.8"). LT(.00-. 10s). TT(. 1 8s).NOV P(64.3"), UT(77.g0), W(81 .OO),H(87.0°), E (0.8").LT(.OI -. 14s). TT(. 194.CONCLUSIONS1) Results for the badminton serve indicated the EXP exhibited greater ROM than the NOV for all angles except E. The NOV constrained the W to possibly allow for the greater E movement.2) In the forehand and in the racquetball serve, the NOV constrained the elbow more than the EXP which was consistent with Southard's (1 987) results; however, the great W ROM was inconsistent and appeared to be a last resort to generatevelocity.3) The EXP was more compact performing the forehand than the racquetball serve. During the serve, the lack of initial ball velocity required greater ROM to achieve the goal.4) In all 3 tasks, the EXP displayed essentially the same sequence of propulsive segmental initiation (proximal to distal). The patterns exhibited by the NOV were more simultaneous and the sequences varied for each task. REFERENCES Bird, M., Hills, L., & Hudson, J. L.(1 991 ). Intersegmental coordination: an exploration of context. In C. Tant, P.Patterson, & S. York (Eds.),Biomechanics in Sworts IX: Proceedings of the 9th International Symposium on Biomechanics in Sports (pp. 233-237).Ames, IA: Iowa State University.Southard, D. & Higgins,T,. (1 987).Changing movement patterns: effects of demonstration and practice. Research Quarterlv for Exercise and Sport, 58(1),77-80

Linear response functions for a vibrational configuration interaction state

Linear response functions are implemented for a vibrational configuration interaction state allowing accurate analytical calculations of pure vibrational contributions to dynamical polarizabilities. Sample calculations are presented for the pure vibrational contributions to the polarizabilities of water and formaldehyde. We discuss the convergence of the results with respect to various details of the vibrational wave function description as well as the potential and property surfaces. We also analyze the frequency dependence of the linear response function and the effect of accounting phenomenologically for the finite lifetime of the excited vibrational states. Finally, we compare the analytical response approach to a sum-over-states approac

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

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

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

Methionine sulfoximine resistance in Mycobacterium tuberculosis is due to a single nucleotide deletion resulting in increased expression of the major glutamine synthetase, GlnA1

We investigated the effect of methionine sulfoximine (MetSox), a potent inhibitor of glutamine synthetase, on Mycobacterium tuberculosis. M. tuberculosis encodes four glutamine synthetases, of which MetSox targets the type I enzyme encoded by glnA1. Trancriptional profiling revealed that glutamate synthetase (gltB) and a type II glutamine synthetase (glnA3) were induced after exposure to MetSox. In addition, we observed a high rate (10−5) of spontaneous resistance to MetSox. All resistant strains had a single-nucleotide deletion in the 5’ region of glnA1, and Western analysis revealed that GlnA1 expression was increased in resistant as compared with sensitive strains. These data show that M. tuberculosis can respond to the effect of MetSox inhibition either by up-regulation of GlnA3 or by GlnA1. The high frequency of resistance suggests that MetSox and other compounds specifically targeting GlnA1 are not likely to become successful anti-mycobacterial agents