A flow cytometry based oligotrophic pollutant exposure test to detect bacterial growth inhibition and cell injury.

Toxicity of chemical pollutants in aquatic environments is often addressed by assays that inquire reproductive inhibition of test microorganisms, such as algae or bacteria. Those tests, however, assess growth of populations as a whole via macroscopic methods such as culture turbidity or colony-forming units. Here we use flow cytometry to interrogate the fate of individual cells in low-density populations of the bacterium Pseudomonas fluorescens SV3 exposed or not under oligotrophic conditions to a number of common pollutants, some of which derive from oil contamination. Cells were stained at regular time intervals during the exposure assay with fluorescent dyes that detect membrane injury (i.e., live-dead assay). Reduction of population growth rates was observed upon toxicant insult and depended on the type of toxicant. Modeling and cell staining indicate that population growth rate decrease is a combined effect of an increased number of injured cells that may or may not multiply, and live cells dividing at normal growth rates. The oligotrophic assay concept presented here could be a useful complement for existing biomarker assays in compliance with new regulations on chemical effect studies or, more specifically, for judging recovery after exposure to fluctuating toxicant conditions.

Struggles for meaning and struggles for control: the diffusion of bandwagon technology in two institutional environments

In this thesis, I examine the diffusion process for a complex medical technology, the PET scanner, in two different health care systems, one of which is more market-oriented (Switzerland) and the other more centrally managed by a public agency (Quebec). The research draws on institutional and socio-political theories of the diffusion of innovations to examine how institutional contexts affect processes of diffusion. I find that diffusion proceeds more rapidly in Switzerland than in Quebec, but that processes in both jurisdictions are characterized by intense struggles among providers and between providers and public agencies. I show that the institutional environment influences these processes by determining the patterns of material resources and authority available to actors in their struggles to strategically control the technology, and by constituting the discursive resources or institutional logics on which actors may legitimately draw in their struggles to give meaning to the technology in line with their interests and values. This thesis illustrates how institutional structures and meanings manifest themselves in the context of specific decisions within an organizational field, and reveals the ways in which governance structures may be contested and realigned when they conflict with interests that are legitimized by dominant institutional logics. It is argued that this form of contestation and readjustment at the margins constitutes one mechanism by which institutional frameworks are tested, stretched and reproduced or redefined.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

Free Technology Academy : a Joint Venture of Free Software and OER

The decision to publish educational materials openly and under free licenses brings up the challenge of doing it in a sustainable way. Some lessons can be learned from the business models for production, maintenance and distribution of Free and Open Source Software. The Free Technology Academy (FTA) has taken on these challenges and has implemented some of these models. We briefly review the FTA educational programme, methodologies and organisation, and see to which extent these models are proving successful in the case of the FTA.

The relationships between technology and open education in the development of a resilient higher education

The place of technology in the development of coherent educational responses to environmental and socio-economic disruption is here placed under scrutiny. One emerging area of interest is the role of technology in addressing more complex learning futures, and more especially in facilitating individual and social resilience, or the ability to manage and overcome disruption. However, the extent to which higher education practitioners can utilise technology to this end is framed by their approaches to the curriculum, and the socio-cultural practices within which they are located. This paper discusses how open education might enable learners to engage with uncertainty through social action within a form of higher education that is more resilient to economic, environmental and energy-related disruption. It asks whether open higher education can be (re)claimed by users and communities within specific contexts and curricula, in order to engage with an uncertain world.

The future of television : advertising, technology and the pursuit of audiences

La televisió està en crisi. Les noves tecnologies i els dispositius han fragmentat les audiències de televisió en segments més petits. En aquest informe, Marissa Gluck i Meritxell Roca Sales examinen l'explosió dels mitjans de comunicació que han impulsat aquesta fragmentació.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

CD209 in inflammatory bowel disease: a case-control study in the Spanish population

BACKGROUND The etiology of Ulcerative Colitis (UC) and Crohn's Disease (CD), considered together as Inflammatory Bowel Diseases (IBD), involves environmental and genetic factors. Although some genes are already known, the genetics underlying these diseases is complex and new candidates are continuously emerging. The CD209 gene is located in a region linked previously to IBD and a CD209 functional polymorphism (rs4804803) has been associated to other inflammatory conditions. Our aim was to study the potential involvement of this CD209 variant in IBD susceptibility. METHODS We performed a case-control study with 515 CD patients, 497 UC patients and 731 healthy controls, all of them white Spaniards. Samples were typed for the CD209 single nucleotide polymorphism (SNP) rs4804803 by TaqMan technology. Frequency comparisons were performed using chi2 tests. RESULTS No association between CD209 and UC or CD was observed initially. However, stratification of UC patients by HLA-DR3 status, a strong protective allele, showed that carriage of the CD209_G allele could increase susceptibility in the subgroup of HLA-DR3-positive individuals (p = 0.03 OR = 1.77 95% CI 1.04-3.02, vs. controls). CONCLUSION A functional variant in the CD209 gene, rs4804803, does not seem to be influencing Crohn's disease susceptibility. However, it could be involved in the etiology or pathology of Ulcerative Colitis in HLA-DR3-positive individuals but further studies are necessary.

Innovation strategies in the presence of technology markets: evidence from spanish innovative firms

The development of markets for technology has eased the acquisition of technology and reshaped the innovation strategies of firms that we classify as producers of innovations or as imitators. Innovative activities of firms include research, acquisition of technology and downstream activities. Within an industry, firms producing innovations tend to conduct more research and downstream activities than those imitating innovations. Acquisition of technology is equally important for both. To implement innovation strategies, firms producing innovations require both the capability to scan the external environment for technology and the capability to integrate new technology. Firms producing innovations require both, while firms imitating innovations require scan capabilities only

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

Passive lipoidal diffusion and carrier-mediated cell uptake are both important mechanisms of membrane permeation in drug disposition.

Recently, it has been proposed that drug permeation is essentially carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes the prevailing hypothesis of drug permeation through biological membranes, which integrates the contribution of multiple permeation mechanisms, including both carrier-mediated and passive lipoidal diffusion, depending on the compound's properties, membrane properties, and solution properties. The prevailing hypothesis of drug permeation continues to be successful for application and prediction in drug development. Proponents of the carrier-mediated only concept argue against passive lipoidal diffusion. However, the arguments are not supported by broad pharmaceutics literature. The carrier-mediated only concept lacks substantial supporting evidence and successful applications in drug development.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

Characterization of Adult Stem/Progenitor Cell Populations From Bone Marrow in a Three-Dimensional Collagen Gel Culture System.

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells which are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and non-adherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4 and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent, hematopoietic, mesenchymal and endothelial specific markers in vitro and can undergo osteogenic differentiation in vivo.