Life history analysis of integrative and conjugative element activation in growing microcolonies of Pseudomonas.

Integrative and conjugative elements (ICE) are in some ways parasitic mobile DNA that propagate vertically through replication with the bacterial host chromosome but at low frequencies can excise and invade new recipient cells through conjugation and reintegration (horizontal propagation). The factors that contribute to successful horizontal propagation are not very well understood. Here, we study the influence of host cell life history on the initiation of transfer of a model ICE named ICEclc in bacteria of the genus Pseudomonas. We use time-lapse microscopy of growing and stationary-phase microcolonies of ICEclc bearing cells in combination with physiological staining and gene reporter analysis in stationary-phase suspended cells. We provide evidence that cell age and cell lineage are unlikely to play a role in the decision to initiate the ICEclc transfer program. In contrast, cells activating ICEclc show more often increased levels of reactive oxygen species and membrane damage than nonactivating cells, suggesting that some form of biochemical damage may make cells more prone to ICEclc induction. Finally, we find that ICEclc active cells appear spatially at random in a microcolony, which may have been a selective advantage for maximizing ICEclc horizontal transmission to new recipient species.

Compositional data analysis in archaeometry

We shall call an n × p data matrix fully-compositional if the rows sum to a constant, and sub-compositional if the variables are a subset of a fully-compositional data set1. Such data occur widely in archaeometry, where it is common to determine the chemical composition of ceramic, glass, metal or other artefacts using techniques such as neutron activation analysis (NAA), inductively coupled plasma spectroscopy (ICPS), X-ray fluorescence analysis (XRF) etc. Interest often centres on whether there are distinct chemical groups within the data and whether, for example, these can be associated with different origins or manufacturing technologies

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Neutron diffraction analysis on FINEMET alloys

Neutron diffraction has been used to study in situ the nanocrystallization process of Fe73.5Cu1Nb3Si22.5-xBx (x = 5, 9, and 12) amorphous alloys. Nanocrystallization results in a decrease of both the silicon content and the grain size of the Fe(Si) phase with increasing value of x. By comparing the radial distribution function peak areas with those predicted for ideal bcc and DO3 structure, it can be concluded that the ordering in DO3 Fe(Si) crystals increases with the silicon content.

Assessment of dissolution techniques for the analysis of ceramic samples by plasma spectrometry

In this study, 13 ceramic samples were subjected to dissolution using three different procedures: (a) acid attack in open PTFE vessels with a mixture of HF-HClO4, (b) fusion of the sample with lithium metaborate and (c) microwave digestion in PTFE bombs. The samples used in the study had been previously analyzed by neutron activation analysis (NAA), X-ray fluorescence (XRF) and X-ray diffraction (XRD) and they cover a wide range of ceramics fired in different atmospheres and temperatures as well as different mineralogical and chemical compositions. The effectiveness of each procedure is evaluated in terms of its ability to dissolve the various mineralogical phases of the samples, of the number of elements that can be determined and of the time needed for the whole scheme of analysis to be completed.

Analysis of bulk and surface contributions in the neutron skin of nuclei

The neutron skin thickness of nuclei is a sensitive probe of the nuclear symmetry energy and has multiple implications for nuclear and astrophysical studies. However, precision measurements of this observable are difficult to obtain. The analysis of the experimental data may imply some assumptions about the bulk or surface nature of the formation of the neutron skin. Here we study the bulk or surface character of neutron skins of nuclei following from calculations with Gogny, Skyrme, and covariant nuclear mean-field interactions. These interactions are successful in describing nuclear charge radii and binding energies but predict different values for neutron skins. We perform the study by fitting two-parameter Fermi distributions to the calculated self-consistent neutron and proton densities. We note that the equivalent sharp radius is a more suitable reference quantity than the half-density radius parameter of the Fermi distributions to discern between the bulk and surface contributions in neutron skins. We present calculations for nuclei in the stability valley and for the isotopic chains of Sn and Pb.

Analysis of SKI-1/S1P prodomain provides insight on maturation and activation of SKI-1/S1P zymogen

SKI-l/SlP protease is a member of the proprotein convertase family, with several functions in cellular metabolism and homeostasis. It is responsible for the processing of several cellular substrates, including ATF6, SREBPs, and GlcNAc-1- phosphotranspherase. Furthermore, SKI-1/SlP is also responsible for maturation of arenavirus surface glycoprotein into GP1 and GP2 subunits. This processing is a strict requirement in order to achieve fully mature and fusion-competent virions. Furthermore, SKI-1/SlP itself is synthesized as an inactive zymogen, requiring sequential autocatalytic processing at several sites (B'/B and C) in its prodomain in order to mature and become fully active. Our project focused on the analysis of SKI- 1/S1P prodomain in the biogenesis of the active enzyme. In this context we have additionally developed and characterized a novel cell-based sensor for assessment of cellular activity of the enzyme, with a potential application in screening for novel SKI- 1/S1P inhibitors. In a first aim we have analysed the relevance of cleavage motifs found in the enzyme prodomain. Using molecular and biochemistry tools we have identified and characterized a novel C' maturation site. Furthermore, we found that SKI-1/SlP autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. Contrasting with other proprotein convertases, incompletely matured intermediates of SKI-1/SlP exhibit full catalytic activity toward selected substrates. In a second aim, we turned our attention to the structural basis of SKI-1/SlP N- terminus assisted folding. Studying the folding and activity of prodomain-truncated forms of the enzyme we found that a minimal folding unit is contained in the AB region. Deletion of the BC sequence affected auto-maturation but not folding, and partial activity was retained. However, the BC region seemed required for complete and full activity. Phylogenetic analyses showed that the AB sequence is highly conserved, while the BC fragment is variable in sequence and length. Specifically, replacement of the human prodomain with that of Drosophila, resulted in a fully mature and active chimeric enzyme, suggesting an evolution process of SKI-1/SlP prodomain towards a more complex arrangement and steps of activation. Overall, the additional data we have produced might provide fundamental knowledge crucial for the development of novel SKI-1/SlP inhibitors while also providing new SKI- 1/S1P variants with potential use in crystallization purpose. -- SKI-l/SlP est une protéase membre de la famille des proprotéines convertases (PCs), avec plusieurs fonctions dans le métabolisme cellulaire et de l'homéostasie. Il est responsable pour la maturation de plusieurs substrats cellulaires, y compris ATF6, SREBPs et GlcNAc-1-phosphotranspherase. SKI-l/SlP est également responsable pour la maturation de la glycoprotéine des arénavirus, une exigence stricte pour atteindre des virions infectieuse. Synthétisé comme un zymogène inactif, SKI-l/SlP nécessite d'un traitement autocatalytique séquentiel sur plusieurs sites (B'/B et C) de son prodomaine afin de devenir pleinement active. Notre projet était axé sur l'analyse de SKI-l/SlP prodomaine dans la biogenèse de l'enzyme. Dans ce contexte, nous avons développé un nouveau senseur-cellulaire pour l'évaluation de l'activité de l'enzyme. Ce dernier pourrait avoir une potentielle application dans l'identification de nouveaux inhibiteurs de SKI-l/SlP. Premièrement, nous avons analysé la pertinence des motifs de clivage trouvés dans le prodomaine de l'enzyme. En utilisant des outils moléculaires et biochimiques, nous avons identifié et caractérisé un nouveau site de maturation (C'). Aussi, nous avons constaté que la maturation de SKI-l/SlP a des intermédiaires dont le domaine catalytique reste associé à des fragments du prodomaine de différentes longueurs. Contrastant avec d'autres PCs, les intermédiaires partiellement matures de SKI-1 / SIP présentent une activité catalytique complète envers des substrats spécifiques. Dans un deuxième but nous avons tourné notre attention sur la base structurelle du pliage de SKI-l/SlP assisté par son N-terminus: En étudiant l'activité et pliage des formes tronquées dans le prodomaine de l'enzyme, nous avons constaté qu'une unité de pliage minimale est contenue dans la région de l'AB. La suppression de la séquence d'auto-BC affecte la maturation mais pas le pliage, et l'activité partielle est maintenue. Cependant, la région BC semble nécessaire pour une activité complète. Les analyses phylogénétiques ont montré que la séquence AB est fortement conservée, tandis que le fragment de BC est variable en longueur et en séquence. En particulier, le remplacement du prodomaine humain avec celui de la drosophile, a donné lieu à une enzyme chimérique complètement mature et active. Suggérant un processus d'évolution du prodomaine vers un arrangement et des mesures d'activation plus complexe. Globalement, ces donnees supplémentaires augment les connaissances fondamentales cruciales pour le développement de nouveaux inhibiteurs de SKI-1/ SIP, tout en offrant de nouvelles variantes SKI-1 / SIP dans le but d'obtenir la structure cristallographique de l'enzyme.

Dual activation of pathways regulated by steroid receptors and peptide growth factors in primary prostate cancer revealed by Factor Analysis of microarray data

Background: We use an approach based on Factor Analysis to analyze datasets generated for transcriptional profiling. The method groups samples into biologically relevant categories, and enables the identification of genes and pathways most significantly associated to each phenotypic group, while allowing for the participation of a given gene in more than one cluster. Genes assigned to each cluster are used for the detection of pathways predominantly activated in that cluster by finding statistically significant associated GO terms. We tested the approach with a published dataset of microarray experiments in yeast. Upon validation with the yeast dataset, we applied the technique to a prostate cancer dataset. Results: Two major pathways are shown to be activated in organ-confined, non-metastatic prostate cancer: those regulated by the androgen receptor and by receptor tyrosine kinases. A number of gene markers (HER3, IQGAP2 and POR1) highlighted by the software and related to the later pathway have been validated experimentally a posteriori on independent samples. Conclusion: Using a new microarray analysis tool followed by a posteriori experimental validation of the results, we have confirmed several putative markers of malignancy associated with peptide growth factor signalling in prostate cancer and revealed others, most notably ERRB3 (HER3). Our study suggest that, in primary prostate cancer, HER3, together or not with HER4, rather than in receptor complexes involving HER2, could play an important role in the biology of these tumors. These results provide new evidence for the role of receptor tyrosine kinases in the establishment and progression of prostate cancer.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes.

Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).

Compositional data analysis in archaeometry

We shall call an n × p data matrix fully-compositional if the rows sum to a constant, and sub-compositional if the variables are a subset of a fully-compositional data set1. Such data occur widely in archaeometry, where it is common to determine the chemical composition of ceramic, glass, metal or other artefacts using techniques such as neutron activation analysis (NAA), inductively coupled plasma spectroscopy (ICPS), X-ray fluorescence analysis (XRF) etc. Interest often centres on whether there are distinct chemical groups within the data and whether, for example, these can be associated with different origins or manufacturing technologies

Measurement of thermal neutron cross section and resonance integral for the (165)Ho(n, gamma)(166g)Ho reaction

The ground state thermal neutron cross section and the resonance integral for the (165)Ho(n, gamma)(166)Ho reaction in thermal and 1/E regions, respectively, of a thermal reactor neutron spectrum have been measured experimentally by activation technique. The reaction product, (166)Ho in the ground state, is gaining considerable importance as a therapeutic radionuclide and precisely measured data of the reaction are of significance from the fundamental point of view as well as for application. In this work, the spectrographically pure holmium oxide (Ho(2)O(3)) powder samples were irradiated with and without cadmium covers at the IEA-RI reactor (IPEN, Sao Paulo), Brazil. The deviation of the neutron spectrum shape from 1/E law was measured by co-irradiating Co, Zn, Zr and Au activation detectors with thermal and epithermal neutrons followed by regression and iterative procedures. The magnitudes of the discrepancies that can occur in measurements made with the ideal 1/E law considerations in the epithermal range were studied. The measured thermal neutron cross section at the Maxwellian averaged thermal energy of 0.0253 eV is 59.0 +/- 2.1 b and for the resonance integral 657 +/- 36b. The results are measured with good precision and indicated a consistency trend to resolve the discrepant status of the literature data. The results are compared with the values in main libraries such as ENDF/B-VII, JEF-2.2 and JENDL-3.2, and with other measurements in the literature.

Numerical analysis of an incremented statistical sampling procedure in MCNP

MCNP has stood so far as one of the main Monte Carlo radiation transport codes. Its use, as any other Monte Carlo based code, has increased as computers perform calculations faster and become more affordable along time. However, the use of Monte Carlo method to tally events in volumes which represent a small fraction of the whole system may turn to be unfeasible, if a straight analogue transport procedure (no use of variance reduction techniques) is employed and precise results are demanded. Calculations of reaction rates in activation foils placed in critical systems turn to be one of the mentioned cases. The present work takes advantage of the fixed source representation from MCNP to perform the above mentioned task in a more effective sampling way (characterizing neutron population in the vicinity of the tallying region and using it in a geometric reduced coupled simulation). An extended analysis of source dependent parameters is studied in order to understand their influence on simulation performance and on validity of results. Although discrepant results have been observed for small enveloping regions, the procedure presents itself as very efficient, giving adequate and precise results in shorter times than the standard analogue procedure. (C) 2007 Elsevier Ltd. All rights reserved.

A comparison between neutron-fluence measurements using metal-activation monitors and standard glasses calibrated via thin uranium-fission monitors and via ηq method

The two fundamental approaches to fission-track dating involve either an explicit determination of the thermal neutron fluence (φ-method) or a calibration against age standards (ζ-method). The neutron fluence measurements are carried out with metal-activation monitors or with uranium-fission monitors, co-irradiated with the samples. Uranium-fission monitors consist of either a thin mono-atomic) film, or a thick fission source (standard uranium glass) irradiated against a muscovite external track detector. In this work, different techniques for performing neutron-fluence measurements were compared: based on thin-film calibration, based on thick-source calibration, and based on gamma spectrometry of co-irradiated metal monitors (Au, Co). The results suggest that more experiments are needed to make all calibrations consistent, including new measurements of the length of etched induced tracks in mica. Also the standard glass calibration carried out with thin films should be confirmed with a greater number of calibrating irradiations. © 2013 Elsevier Ltd. All rights reserved.

Multivariate curve resolution analysis applied to time-resolved synchrotron X-ray Absorption Spectroscopy monitoring of the activation of copper alumina catalyst

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