MAR elements and transposons for improved transgene integration and expression.

Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

Identification of novel elements of the Drosophila blisterome sheds light on potential pathological mechanisms of several human diseases.

Main developmental programs are highly conserved among species of the animal kingdom. Improper execution of these programs often leads to progression of various diseases and disorders. Here we focused on Drosophila wing tissue morphogenesis, a fairly complex developmental program, one of the steps of which - apposition of the dorsal and ventral wing sheets during metamorphosis - is mediated by integrins. Disruption of this apposition leads to wing blistering which serves as an easily screenable phenotype for components regulating this process. By means of RNAi-silencing technique and the blister phenotype as readout, we identify numerous novel proteins potentially involved in wing sheet adhesion. Remarkably, our results reveal not only participants of the integrin-mediated machinery, but also components of other cellular processes, e.g. cell cycle, RNA splicing, and vesicular trafficking. With the use of bioinformatics tools, these data are assembled into a large blisterome network. Analysis of human orthologues of the Drosophila blisterome components shows that many disease-related genes may contribute to cell adhesion implementation, providing hints on possible mechanisms of these human pathologies.

Elements of olfactory reception in adult Drosophila melanogaster.

The olfactory system of Drosophila has become an attractive and simple model to investigate olfaction because it follows the same organizational principles of vertebrates, and the results can be directly applied to other insects with economic and sanitary relevance. Here, we review the structural elements of the Drosophila olfactory reception organs at the level of the cells and molecules involved. This article is intended to reflect the structural basis underlying the functional variability of the detection of an olfactory universe composed of thousands of odors. At the genetic level, we further detail the genes and transcription factors (TF) that determine the structural variability. The fly's olfactory receptor organs are the third antennal segments and the maxillary palps, which are covered with sensory hairs called sensilla. These sensilla house the odorant receptor neurons (ORNs) that express one or few odorant receptors in a stereotyped pattern regulated by combinations of TF. Also, perireceptor events, such as odor molecules transport to their receptors, are carried out by odorant binding proteins. In addition, the rapid odorant inactivation to preclude saturation of the system occurs by biotransformation and detoxification enzymes. These additional events take place in the lymph that surrounds the ORNs. We include some data on ionotropic and metabotropic olfactory transduction, although this issue is still under debate in Drosophila.

Geochemistry and stable isotope composition of fresh alkaline porphyry copper tailings: Implications on sources and mobility of elements during transport and early stages of deposition

Prevention of acid mine drainage (AMD) in sulfide-containing tailings requires the identification of the geochemical processes and element pathways in the early stages of tailing deposition. However, analyses of recently deposited tailings in active tailings impoundments are scarce because mineralogical changes occur near the detection limits of many assays. This study shows that a detailed geochemical study which includes stable isotopes of water (delta H-2, delta O-18), dissolved sulfates (delta S-34, delta O-18) and hydrochernical parameter (pH, Eh, DOC, major and trace elements) from tailings samples taken at different depths in rainy and dry seasons allows the understanding of weathering (oxidation, dissolution, sorption, and desorption), water and element pathways, and mixing processes in active tailings impoundments. Fresh alkaline tailings (pH 9.2-10.2) from the Cu-Mo porphyry deposit in El Teniente, Chile had low carbonate (0.8-1.1 Wt-% CaCO3 equivalent) and sulfide concentrations (0.8-1.3 wt.%, mainly as pyrite). In the alkaline tailings water, Mo and Cu (up to 3.9 mg/L Mo and 0.016 mg/L Cu) were mobile as MoO42- and Cu (OH)(2)(0). During the flotation, tailings water reached equilibrium with gypsum (up to 738 mg/L Ca and 1765 mg/ L SO4). The delta S-34 VS. delta O-18 covariations of dissolved sulfate (2.3 to 4.5% delta S-34 and 4.1 to 6.0 % delta O-18) revealed the sulfate sources: the dissolution of primary sulfates (12.0 to 13.2%. delta S-34, 7.4 to 10.9%.delta O-18) and oxidation of primary sulfides (-6.7 to 1.7%. delta S-34). Sedimented tailings in the tailings impoundment can be divided into three layers with different water sources, element pathways, and geochemical processes. The deeper sediments (> 1 m depth) were infiltrated by catchment water, which partly replaced the original tailings water, especially during the winter season. This may have resulted in the change from alkaline to near-neutral pH and towards lower concentrations of most dissolved elements. The neutral pH and high DOC (up to 99.4 mg/L C) of the catchment water mobilized Cu (up to 0.25 mg/L) due to formation of organic Cu complexes; and Zn (up to 130 mg/L) due to dissolution of Zn oxides and desorption). At I m depth, tailings pore water obtained during the winter season was chemically and isotopically similar to fresh tailings water (pH 9.8-10.6, 26.7-35.5 mg/L Cl, 2.3-6.0 mg/L Mo). During the summer, a vadose zone evolved locally and temporarily up to 1.2 m depth. resulting in a higher concentration of dissolved solids in the pore water due to evaporation. During periodical new deposition of fresh tailings, the geochemistry of the surface layer was geochemically similar to fresh tailings. In periods without deposition, sulfide oxidation was suggested by decreasing pH (7.7-9.5), enrichment of MoO42- and SO42-, and changes in the isotopic composition of dissolved sulfates. Further enrichment for Na, K, Cl, SO4, Mg, Cu, and Mo (up to 23.8 mg/L Mo) resulted from capillary transport towards the surface followed by evaporation and the precipitation of highly soluble efflorescent salts (e.g., mirabilite, syngenite) at the tailing surface during summer. (C) 2008 Elsevier B.V. All rights reserved.

A user's guide to the encyclopedia of DNA elements (ENCODE).

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.

A new chance-constrained maximum capture location problem

The paper presents a new model based on the basic Maximum Capture model,MAXCAP. The New Chance Constrained Maximum Capture modelintroduces astochastic threshold constraint, which recognises the fact that a facilitycan be open only if a minimum level of demand is captured. A metaheuristicbased on MAX MIN ANT system and TABU search procedure is presented tosolve the model. This is the first time that the MAX MIN ANT system isadapted to solve a location problem. Computational experience and anapplication to 55 node network are also presented.

Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements.

Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are nuclear hormone receptors that are activated by fatty acids and 9-cis-retinoic acid, respectively. PPARs and RXRs form heterodimers that activate transcription by binding to PPAR response elements (PPREs) in the promoter of target genes. The PPREs described thus far consist of a direct tandem repeat of the AGGTCA core element with one intervening nucleotide. We show here that the vitellogenin A2 estrogen response element (ERE) can also function as a PPRE and is bound by a PPAR/RXR heterodimer. Although this heterodimer can bind to several other ERE-related palindromic response elements containing AGGTCA half-sites, only the ERE is able to confer transactivation of test reporter plasmids, when the ERE is placed either close to or at a distance from the transcription initiation site. Examination of natural ERE-containing promoters, including the pS2, very-low-density apolipoprotein II and vitellogenin A2 genes, revealed considerable differences in the binding of PPAR/RXR heterodimers to these EREs. In their natural promoter context, these EREs did not allow transcriptional activation by PPARs/RXRs. Analysis of this lack of stimulation of the vitellogenin A2 promoter demonstrated that PPARs/RXRs bind to the ERE but cannot transactivate due to a nonpermissive promoter structure. As a consequence, PPARs/RXRs inhibit transactivation by the estrogen receptor through competition for ERE binding. This is the first example of signaling cross-talk between PPAR/RXR and estrogen receptor.

French version validation of the psychotic symptom rating scales (PSYRATS) for outpatients with persistent psychotic symptoms.

ABSTRACT: BACKGROUND: Most scales that assess the presence and severity of psychotic symptoms often measure a broad range of experiences and behaviours, something that restricts the detailed measurement of specific symptoms such as delusions or hallucinations. The Psychotic Symptom Rating Scales (PSYRATS) is a clinical assessment tool that focuses on the detailed measurement of these core symptoms. The goal of this study was to examine the psychometric properties of the French version of the PSYRATS. METHODS: A sample of 103 outpatients suffering from schizophrenia or schizoaffective disorders and presenting persistent psychotic symptoms over the previous three months was assessed using the PSYRATS. Seventy-five sample participants were also assessed with the Positive And Negative Syndrome Scale (PANSS). RESULTS: ICCs were superior to .90 for all items of the PSYRATS. Factor analysis replicated the factorial structure of the original version of the delusions scale. Similar to previous replications, the factor structure of the hallucinations scale was partially replicated. Convergent validity indicated that some specific PSYRATS items do not correlate with the PANSS delusions or hallucinations. The distress items of the PSYRATS are negatively correlated with the grandiosity scale of the PANSS. CONCLUSIONS: The results of this study are limited by the relatively small sample size as well as the selection of participants with persistent symptoms. The French version of the PSYRATS partially replicates previously published results. Differences in factor structure of the hallucinations scale might be explained by greater variability of its elements. The future development of the scale should take into account the presence of grandiosity in order to better capture details of the psychotic experience.

Surviving in a competitive spatial market: The threshold capture model

Most facility location decision models ignore the fact that for a facility to survive it needs a minimum demand level to cover costs. In this paper we present a decision model for a firm thatwishes to enter a spatial market where there are several competitors already located. This market is such that for each outlet there is a demand threshold level that has to be achievedin order to survive. The firm wishes to know where to locate itsoutlets so as to maximize its market share taking into account the threshold level. It may happen that due to this new entrance, some competitors will not be able to meet the threshold and therefore will disappear. A formulation is presented together with a heuristic solution method and computational experience.

Intervenció educativa basada en la promoció dels elements facilitadors del desenvolupament positiu de menors subjectes de mesures judicials

Al llarg dels darrers mesos i amb la participació de bona part del col·lectiu d’educadors i tècnics de medi obert del Departament de Justícia de la Generalitat de Catalunya, s’ha fet un treball d’aprofundiment entorn del paper que tenen els elements facilitadors del desenvolupament positiu dels joves que es troben en mesures penals juvenils, com a veritables desencadenants de processos de millora i d’inclusió social. Hem tractat de fer una classificació i guia per identificar aquests elements i una metodologia concreta per determinar-ne la presència o absència. Finalment, hem intentat, comptant amb l’experiència del col·lectiu , elaborar una guia d’eines i estratègies per a la promoció d’aquests elements positius, que poden afavorir el desenvolupament dels menors i facilitar la intervenció del tècnic. Després d’intensos i interessants debats sorgits en les aportacions al Fòrum del portal, i també en les reunions del grup d’entusiastes, hem acabat per definir el concepte, el que entenem, allò a què ens referim quan parlem d’elements facilitadors. És així que,finalment, els definirem com: Tots els recursos personals, familiars, socials i institucionals que promouen el desenvolupament exitós de l’adolescent, o bé que fan disminuir el risc d’aparèixer un desenvolupament alterat. La pretensió del “producte” elaborat no és constituir-se en un document de caràcter teòric, més aviat hem volgut donar-hi un sentit pràctic que l’apropi més a una eina de treball que no pas a un marc teòric de caire general. És per això que, un cop delimitat el concepte i els objectius del producte, el treball ha consistit en la relació i classificació d’aquests elements facilitadors del desenvolupament positiu que trobem en els menors i joves amb qui treballem, i en l’observació de la seva presència, absència o necessitat.

Is the capture success of orchid bees (Hymenoptera, Apoidea) influenced by different baited trap designs? A case study from southern Brazil

Orchid bees are increasingly applied on Neotropical biomonitoring and bioindication studies due to the relative easiness of sampling and identification when compared to other bee groups. A considerable number of orchid bee community studies have been adopting baited traps as a sampling method, especially for replication purposes. However, the trap attributes are variable, and hitherto no evaluation of different designs was carried out. Here, five attributes of baited traps were tested: trap volume, number of entrance holes, presence of landing platform, kind of landing platform, and fixation content. We use Mann-Whitney tests to access differences in richness and abundance capture rates for each trap design. We found that volume, number of entrance holes, and fixation content do not influence orchid bees capture. However, the design without landing platforms had a significantly higher capture rate for richness when compared with sanded landing platforms. On the other hand, analyzing the kind of landing platform, we detected a significantly higher richness and abundance for the trap with landing platforms glued with sand. Despite the fact that the effects of different designs tested here were very punctual, we consider that results from samples taken with different baited trap designs are comparable. Some adjustments on trap design can be done according to the particularities of future studies.

DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region.

The three subtypes of the peroxisome proliferator-activated receptors (PPARalpha, beta/delta, and gamma) form heterodimers with the 9-cis-retinoic acid receptor (RXR) and bind to a common consensus response element, which consists of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1). As a first step toward understanding the molecular mechanisms determining PPAR subtype specificity, we evaluated by electrophoretic mobility shift assays the binding properties of the three PPAR subtypes, in association with either RXRalpha or RXRgamma, on 16 natural PPAR response elements (PPREs). The main results are as follows. (i) PPARgamma in combination with either RXRalpha or RXRgamma binds more strongly than PPARalpha or PPARbeta to all natural PPREs tested. (ii) The binding of PPAR to strong elements is reinforced if the heterodimerization partner is RXRgamma. In contrast, weak elements favor RXRalpha as heterodimerization partner. (iii) The ordering of the 16 natural PPREs from strong to weak elements does not depend on the core DR1 sequence, which has a relatively uniform degree of conservation, but correlates with the number of identities of the 5'-flanking nucleotides with respect to a consensus element. This 5'-flanking sequence is essential for PPARalpha binding and thus contributes to subtype specificity. As a demonstration of this, the PPARgamma-specific element ARE6 PPRE is able to bind PPARalpha only if its 5'-flanking region is exchanged with that of the more promiscuous HMG PPRE.

How many diagnosis fields are needed to capture safety events in administrative data? Findings and recommendations from the WHO ICD-11 Topic Advisory Group on Quality and Safety

OBJECTIVE: As part of the WHO ICD-11 development initiative, the Topic Advisory Group on Quality and Safety explores meta-features of morbidity data sets, such as the optimal number of secondary diagnosis fields. DESIGN: The Health Care Quality Indicators Project of the Organization for Economic Co-Operation and Development collected Patient Safety Indicator (PSI) information from administrative hospital data of 19-20 countries in 2009 and 2011. We investigated whether three countries that expanded their data systems to include more secondary diagnosis fields showed increased PSI rates compared with six countries that did not. Furthermore, administrative hospital data from six of these countries and two American states, California (2011) and Florida (2010), were analysed for distributions of coded patient safety events across diagnosis fields. RESULTS: Among the participating countries, increasing the number of diagnosis fields was not associated with any overall increase in PSI rates. However, high proportions of PSI-related diagnoses appeared beyond the sixth secondary diagnosis field. The distribution of three PSI-related ICD codes was similar in California and Florida: 89-90% of central venous catheter infections and 97-99% of retained foreign bodies and accidental punctures or lacerations were captured within 15 secondary diagnosis fields. CONCLUSIONS: Six to nine secondary diagnosis fields are inadequate for comparing complication rates using hospital administrative data; at least 15 (and perhaps more with ICD-11) are recommended to fully characterize clinical outcomes. Increasing the number of fields should improve the international and intra-national comparability of data for epidemiologic and health services research, utilization analyses and quality of care assessment.