Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations.

Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.This work was funded by BBSRC grant RG66581.This is the final version of the article. It was first available from Society for General Microbiology via http://dx.doi.org/10.1099/mic.0.00018

Studying minijets via the pTp_T dependence of two-particle correlation in azimuthal angle ϕ\phi

Following my previous proposal that two-particle correlation functions can be used to resolve the minijet contribution to particle production in minimum biased events of high energy hadronic interactions, I study the $p_T$ and energy dependence of the correlation. Using HIJING Monte Carlo model, it is found that the correlation $c(\phi_1,\phi_2)$ in azimuthal angle $\phi$ between two particles with $p_T>p_T^{cut}$ resembles much like two back-to-back jets as $p_T^{cut}$ increases at high colliding energies due to minijet production. It is shown that $c(0,0)-c(0,\pi)$, which is related to the relative fraction of particles from minijets, increases with energy. The background of the correlation for fixed $p_T^{cut}$ also grows with energy due to the increase of multiple minijet production. Application of this analysis to the study of jet quenching in ultrarelativistic heavy ion collisions is also discussed.Comment: 11 pages Latex text and 8 ps figures, LBL-3349

Space-time Structure of Initial Parton Production in Ultrarelativistic Heavy Ion Collisions

The space and time evolution of initial parton production in ultrarelativistic heavy ion collisions is investigated within the framework of perturbative QCD which includes both initial and final state radiations. Uncertainty principle is used to relate the life time of a radiating parton to its virtuality and momentum. The interaction time of each hard or semihard parton scattering is also taken into account. For central $Au+Au$ collisions at $\sqrt{s}=200$ GeV, most of the partons are found to be produced within 0.5 fm/c after the total overlap of the two colliding nuclei. The local momentum distribution is approximately isotropical at that time. The implication on how to treat correctly the the secondary scattering in an ultimate parton cascading model is also discussed.Comment: 19 pages in REVTEX with 12 figures in separate uuencoded postscript files, LBL-3415

Second-site mutation causing motility defects

Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.This work was funded by BBSRC grant RG66581

Multiple Interactions and the Structure of Beam Remnants

Recent experimental data have established some of the basic features of multiple interactions in hadron-hadron collisions. The emphasis is therefore now shifting, to one of exploring more detailed aspects. Starting from a brief review of the current situation, a next-generation model is developed, wherein a detailed account is given of correlated flavour, colour, longitudinal and transverse momentum distributions, encompassing both the partons initiating perturbative interactions and the partons left in the beam remnants. Some of the main features are illustrated for the Tevatron and the LHC.Comment: 69pp, 33 figure

Endothelial apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity

We recently cloned the full-length cDNA of a tumour-associated protein. The recombinant protein expressed in bacteria and referred to as angiocidin has potent antitumour activity in vivo and in vitro. Angiocidin inhibits tumour growth and angiogenesis by inducing apoptosis in endothelial cells. Based on the sequence similarity of angiocidin to S5a, one of the major polyubiquitin recognition proteins in eukaryotic cells, we postulated that the antiendothelial activity of angiocidin could be due in part to its polyubiquitin binding activity. In support of this hypothesis, we show that angiocidin binds polyubiquitin in vivo with high affinity and colocalises with ubiquitinated proteins on the surface of endothelial cells. Binding is blocked with an antiubiquitin antibody. Angiocidin treatment of endothelial cells transfected with a proteasome fluorescent reporter protein showed a dose-dependent inhibition of proteasome activity and accumulation of polyubiquitinated proteins. Full-length angiocidin bound polyubiquitin while three angiocidin recombinant proteins whose putative polyubiquitin binding sites were mutated either failed to bind polyubiquitin or had significantly diminished binding activity. The in vitro apoptotic activity of these mutants correlated with their polyubiquitin binding activity. These data strongly argue that the apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity

O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

BACKGROUND:Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium. METHODOLOGY/PRINCIPAL FINDINGS:Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2) or normoxic (21% O2) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3(rd) passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability. CONCLUSION/SIGNIFICANCE:We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells

Microvascular engineering in perfusion culture: immunohistochemistry and CLSM findings

BACKGROUND: One of the most challenging problems in tissue engineering is the establishment of vascular supply. A possible approach might be the engineering of microvasculature in vitro and the supply by engineered feeder vessels. METHODS: An in vitro model for a small-diameter vessel was developed and made from adipose tissue stromal cells and human umbilical vein endothelial cells in a tube-like gelatine scaffold. The number of "branches" emerging from the central lumen and the morphology of the central lumen of the vessel equivalent were assessed after 16 days of either pulsatile perfusion culture or culture in rotating containers by evaluation of immunohistochemically stained sections (n = 6 pairs of cultures). Intramural capillary network formation was demonstrated in five experiments with confocal laser scanning microscopy. RESULTS: Perfused specimens showed a round or oval lumen lined by a single layer of endothelial cells, whereas following rotation culture the lumen tended to collapse. Confocal laser scanning microscopy showed more extended network formation in perfused specimens as compared to specimens after rotation culture. Partially highly interconected capillary-like networks were imaged which showed orientation around the central lumen. Perfused specimens exhibited significantly more branches emerging from the central lumen. There were, however, hardly any capillary branches crossing the whole vessel wall. CONCLUSION: Pulsatile perfusion supports the development of vascular networks with physiological appearance. Advances in reactor development, acquisition of functional data and imaging procedures are however necessary in order to attain the ultimate goal of a fully functional engineered supplying vessel

Identification of guinea pig remains in the Pucará de Tilcara (Jujuy, Argentina): Evidence in favour of the presence of the Andean breed in the Quebrada de Humahuaca

In this article, we identified rodent remains found in the Pucará de Tilcara, an archaeological site from the Argentine Northwest that was occupied by humans from 1,100 ad until the Spanish conquest. The zooarchaeological analyses were carried out using anatomical descriptions and geometric morphometric analyses of the dorsal and ventral views of mandibular remains. The results and the archaeological context discussed showed that all the rodent remains could correspond to the Andean breed of domestic guinea pigs. The combination of the methods used here gave us a strong support to the taxonomical assignment. The presence of domestic guinea pigs in archaeological sites of the northwestern Argentina was never proposed. This approach allowed us to increase knowledge about the distribution of caviines in the region, and their relationship to anthropic processes.Fil: Lopez Geronazzo, Lautaro Nahuel. Universidad Nacional de Jujuy. Instituto de Ecorregiones Andinas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Ecorregiones Andinas; ArgentinaFil: Otero, Clarisa. Universidad Nacional de Jujuy. Instituto de Ecorregiones Andinas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Ecorregiones Andinas; Argentina. Universidad de Buenos Aires. Facultad de Filosofía y Letras; ArgentinaFil: Alvarez, Alicia. Universidad Nacional de Jujuy. Instituto de Ecorregiones Andinas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Ecorregiones Andinas; Argentina. Universidad Nacional de Jujuy. Instituto de Geología Minera; ArgentinaFil: Ercoli, Marcos Darío. Universidad Nacional de Jujuy. Instituto de Ecorregiones Andinas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Ecorregiones Andinas; Argentina. Universidad Nacional de Jujuy. Instituto de Geología Minera; ArgentinaFil: Cortés Delgado, Natalia. University of Illinois; Estados Unido