247 research outputs found
Membrane identity and GTPase cascades regulated by toggle and cut-out switches
Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5-to-Rab7 conversion is rather based on a newly characterized âcut-out switch' analogous to an electrical safety-breaker. Both designs require cooperativity of auto-activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut-out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut-out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes
Membrane identity and GTPase cascades regulated by toggle and cut-out switches
Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5-to-Rab7 conversion is rather based on a newly characterized âcut-out switch' analogous to an electrical safety-breaker. Both designs require cooperativity of auto-activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut-out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut-out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes
A Magnetic Bead-Integrated Chip for the Large Scale Manufacture of Normalized esiRNAs
The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications
Pyruvate Dehydrogenase Kinase 4: Regulation by Thiazolidinediones and Implication in Glyceroneogenesis in Adipose Tissue
OBJECTIVEâPyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled by PDK
False negative rates in Drosophila cell-based RNAi screens: a case study
<p>Abstract</p> <p>Background</p> <p>High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.</p> <p>Results</p> <p>We performed a meta-analysis of several genome-wide, cell-based <it>Drosophila </it>RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.</p> <p>Conclusions</p> <p>RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.</p
CellCognition : time-resolved phenotype annotation in high-throughput live cell imaging
Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Methods 7 (2010): 747-754, doi:10.1038/nmeth.1486.Fluorescence time-lapse imaging has become a powerful tool to investigate complex
dynamic processes such as cell division or intracellular trafficking. Automated
microscopes generate time-resolved imaging data at high throughput, yet tools for
quantification of large-scale movie data are largely missing. Here, we present
CellCognition, a computational framework to annotate complex cellular dynamics.
We developed a machine learning method that combines state-of-the-art classification
with hidden Markov modeling for annotation of the progression through
morphologically distinct biological states. The incorporation of time information into
the annotation scheme was essential to suppress classification noise at state
transitions, and confusion between different functional states with similar
morphology. We demonstrate generic applicability in a set of different assays and
perturbation conditions, including a candidate-based RNAi screen for mitotic exit
regulators in human cells. CellCognition is published as open source software,
enabling live imaging-based screening with assays that directly score cellular
dynamics.Work in the Gerlich
laboratory is supported by Swiss National Science Foundation (SNF) research grant
3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young
Investigator (EURYI) award of the European Science Foundation, an EMBO YIP
fellowship, and a MBL Summer Research Fellowship to D.W.G., an ETH TH grant, a
grant by the UBS foundation, a Roche Ph.D. fellowship to M.H.A.S, and a Mueller
fellowship of the Molecular Life Sciences Ph.D. program Zurich to M.H. M.H. and
M.H.A.S are fellows of the Zurich Ph.D. Program in Molecular Life Sciences. B.F.
was supported by European Commissionâs seventh framework program project
Cancer Pathways. Work in the Ellenberg laboratory is supported by a European
Commission grant within the Mitocheck consortium (LSHG-CT-2004-503464). Work
in the Peter laboratory is supported by the ETHZ, Oncosuisse, SystemsX.ch (LiverX)
and the SNF
Perrault Aux Prises Avec la Fontaine: Imitation, Compétition et Correction Dans Les Fables de Faërne (1699)
Known especially for his fairy tales, Charles Perrault is also the author of the Fables de FaĂ«rne (1699). In this French translation of the Neo-Latin volume Fabulae Centum (1564), written by the Italian humanist Gabriel Faerno, Perrault had to position himself against his renowned predecessor Jean de La Fontaine, who had been dominating fable literature for decades. Perrault could either imitate his famous example, or evade it, due to anxiety of influence. To illustrate this inner struggle, we systematically compare both authorsâ fables, concentrating our analysis on versification (metre and rhyme), vocabulary and apostrophe. In our comparison, we constantly verify whether any of the resemblances could be attributable to other French, versified fable books read by both Perrault and La Fontaine. Occasionally, this seems to be the case for the anonymous collection LâEsbatement moral des animaux (1578).Vakpublicati
Vortex nozzle interaction in solid rocket motors: A scaling law for upstream acoustic response
In solid rocket motors, vortex nozzle interactions can be a source of large-amplitude pressure pulsations. Using a two-dimensional frictionless flow model, a scaling law is deduced, which describes the magnitude of a pressure pulsation as being proportional to the product of the dynamic pressure of the upstream main flow and of vortex circu- lation. The scaling law was found to be valid for both an integrated noz- zle with surrounding cavity and a nozzle geometry without surrounding cavity that forms a right angle with the combustion chamber side wall. Deviations from the scaling law only occur when unrealistically strong circulations are considered
Trocar-guided total tension-free vaginal mesh repair of post-hysterectomy vaginal vault prolapse
Contains fulltext :
81076.pdf (publisher's version ) (Closed access)INTRODUCTION AND HYPOTHESIS: The objective of this study was to report 1 year anatomical and functional outcomes of trocar-guided total tension-free vaginal mesh (Prolift) repair for post-hysterectomy vaginal vault prolapse with one continuous piece of polypropylene mesh. METHODS: We conducted a prospective observational cohort study of 46 patients. A minimum sample size of 35 patients was needed to detect a recurrence rate of less than 20% at 12 months. Instruments of measurement used were pelvic organ prolapse quantification and validated questionnaires. RESULTS: Overall anatomical success was 91% (95% confidence interval 83-99), with significant improvement in experienced bother and quality of life. Mesh exposure occurred in seven patients (15%). No adverse effects on sexual function could be detected. CONCLUSIONS: Trocar-guided total tension-free vaginal mesh (Prolift) repair with one continuous piece of mesh for post-hysterectomy vaginal vault prolapse is well tolerated and anatomically and functionally highly effective. Results of controlled trials will determine its position in the operative armamentarium
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