A rapid microwell fluorescence immunoassay for cellular protein detection

In this paper, we describe a simple, rapid, specific, sensitive, and reliable method, the FICP method (Fluorescence Immunoassay for Cellular Protein detection) which is readily applicable to the detection of proteins directly on cells cultured in 96-well plates. In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21CIP1/WAF1, in untreated and 2-cyclopenten-1-one treated breast cancer cells. When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics. By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis. Therefore, we believe this screening assay could be very useful for detecting poorly expressed proteins and for drug development

Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

Background: Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure. Methodology/Principal Findings: To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and-independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels. Conclusions: These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperatureindependen

Raman-modes of index-identified free-standing single-walled carbon nanotubes

Using electron diffraction on free-standing single-walled carbon nanotubes we have determined the structural indices (n,m) of tubes in the diameter range from 1.4 to 3nm. On the same free-standing tubes we have recorded Raman spectra of the tangential modes and the radial breathing mode. For the smaller diameters (1.4-1.7nm) these measurements confirm previously established radial breathing mode frequency versus diameter relations, and would be consistent with the theoretically predicted proportionality to the inverse diameter. However, for extending the relation to larger diameters, either a yet unexplained environmental constant has to be assumed, or the linear relation has to be abandoned.Comment: 4 pages, 4 figures, +additional materials (select PostScript to obtain it

Four quasars above redshift 6 discovered by the Canada-France High-z Quasar Survey

The Canada-France High-z Quasar Survey (CFHQS) is an optical survey designed to locate quasars during the epoch of reionization. In this paper we present the discovery of the first four CFHQS quasars at redshift greater than 6, including the most distant known quasar, CFHQS J2329-0301 at z=6.43. We describe the observational method used to identify the quasars and present optical, infrared, and millimeter photometry and optical and near-infrared spectroscopy. We investigate the dust properties of these quasars finding an unusual dust extinction curve for one quasar and a high far-infrared luminosity due to dust emission for another. The mean millimeter continuum flux for CFHQS quasars is substantially lower than that for SDSS quasars at the same redshift, likely due to a correlation with quasar UV luminosity. For two quasars with sufficiently high signal-to-noise optical spectra, we use the spectra to investigate the ionization state of hydrogen at z>5. For CFHQS J1509-1749 at z=6.12, we find significant evolution (beyond a simple extrapolation of lower redshift data) in the Gunn-Peterson optical depth at z>5.4. The line-of-sight to this quasar has one of the highest known optical depths at z~5.8. An analysis of the sizes of the highly-ionized near-zones in the spectra of two quasars at z=6.12 and z=6.43 suggest the IGM surrounding these quasars was substantially ionized before these quasars turned on. Together, these observations point towards an extended reionization process, but we caution that cosmic variance is still a major limitation in z>6 quasar observations.Comment: 15 pages, 9 figures, AJ, in press, minor changes to previous versio

High fragmentation characterizes tumour-derived circulating DNA.

BACKGROUND: Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments. METHODOLOGY: In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients. CONCLUSION/SIGNIFICANCE: Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60-100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16)

Blood contains circulating cell-free respiratory competent mitochondria.

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases

ZOOTAXA

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Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

La résonance lectorale

À quoi tient qu'une œuvre littéraire nous émeut, nous touche, nous donne à penser ? L’incursion dans l’imaginaire de l’autre, dans ses fantasmes, la reconnaissance des éléments de l’histoire (la grande et la petite) convoqués dans le roman, la pièce de théâtre ou le poème viennent élargir notre expérience. Toute création est sans doute dépassement d’un donné arrière-textuel par la confrontation avec la langue, ses contraintes et ses potentialités. Mais que se passe-t-il si l’on replace l’acte créatif dans la relation littéraire comme co-création ? C’est ce phénomène d’écho qu’explore, sous le nom de résonance, le présent volume, dixième de la collection Approches Interdisciplinaires de la Lecture, entre harmonie et dissonance, dans la confrontation des espaces socioculturels liés à la production du texte et à ses lectures successives, dans la recherche, par-delà les problèmes de longueur d’onde, d’un noyau de vérité à exhumer ou à faire advenir