Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic Nanoparticles and Cells

Spatial modulation microscopy is a technique originally developed for quantitative spectroscopy of individual nano-objects. Here, a parallel implementation of the spatial modulation microscopy technique is demonstrated based on a line detector capable of demodulation at kHz frequencies. The capabilities of the imaging system are shown using an array of plasmonic nanoantennas and dendritic cells incubated with gold nanoparticles.Comment: 3 pages, 4 figure

Gold nanoparticles delivery in mammalian live cells: a critical review

Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, ‘nanoparticle and cell’ hit). Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable). To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping

PEG Branched Polymer for Functionalization of Nanomaterials with Ultralong Blood Circulation

Nanomaterials have been actively pursued for biological and medical applications in recent years. Here, we report the synthesis of several new poly(ethylene glycol) grafted branched-polymers for functionalization of various nanomaterials including carbon nanotubes, gold nanoparticles (NP) and gold nanorods (NRs), affording high aqueous solubility and stability for these materials. We synthesize different surfactant polymers based upon poly-(g-glutamic acid) (gPGA) and poly(maleic anhydride-alt-1-octadecene) (PMHC18). We use the abundant free carboxylic acid groups of gPGA for attaching lipophilic species such as pyrene or phospholipid, which bind to nanomaterials via robust physisorption. Additionally, the remaining carboxylic acids on gPGA or the amine-reactive anhydrides of PMHC18 are then PEGylated, providing extended hydrophilic groups, affording polymeric amphiphiles. We show that single-walled carbon nanotubes (SWNTs), Au NPs and NRs functionalized by the polymers exhibit high stability in aqueous solutions at different pHs, at elevated temperatures and in serum. Morever, the polymer-coated SWNTs exhibit remarkably long blood circulation (t1/2 22.1 h) upon intravenous injection into mice, far exceeding the previous record of 5.4 h. The ultra-long blood circulation time suggests greatly delayed clearance of nanomaterials by the reticuloendothelial system (RES) of mice, a highly desired property for in vivo applications of nanomaterials, including imaging and drug delivery

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 Commission’s 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

The Ternary Rab27a–Myrip–Myosin VIIa Complex Regulates Melanosome Motility in the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a–Myrip–MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery

Systematic Analysis of Pleiotropy in C. elegans Early Embryogenesis

Pleiotropy refers to the phenomenon in which a single gene controls several distinct, and seemingly unrelated, phenotypic effects. We use C. elegans early embryogenesis as a model to conduct systematic studies of pleiotropy. We analyze high-throughput RNA interference (RNAi) data from C. elegans and identify “phenotypic signatures”, which are sets of cellular defects indicative of certain biological functions. By matching phenotypic profiles to our identified signatures, we assign genes with complex phenotypic profiles to multiple functional classes. Overall, we observe that pleiotropy occurs extensively among genes involved in early embryogenesis, and a small proportion of these genes are highly pleiotropic. We hypothesize that genes involved in early embryogenesis are organized into partially overlapping functional modules, and that pleiotropic genes represent “connectors” between these modules. In support of this hypothesis, we find that highly pleiotropic genes tend to reside in central positions in protein-protein interaction networks, suggesting that pleiotropic genes act as connecting points between different protein complexes or pathways

Systematic Analysis of Pleiotropy in C. elegans Early Embryogenesis

Pleiotropy refers to the phenomenon in which a single gene controls several distinct, and seemingly unrelated, phenotypic effects. We use C. elegans early embryogenesis as a model to conduct systematic studies of pleiotropy. We analyze high-throughput RNA interference (RNAi) data from C. elegans and identify “phenotypic signatures”, which are sets of cellular defects indicative of certain biological functions. By matching phenotypic profiles to our identified signatures, we assign genes with complex phenotypic profiles to multiple functional classes. Overall, we observe that pleiotropy occurs extensively among genes involved in early embryogenesis, and a small proportion of these genes are highly pleiotropic. We hypothesize that genes involved in early embryogenesis are organized into partially overlapping functional modules, and that pleiotropic genes represent “connectors” between these modules. In support of this hypothesis, we find that highly pleiotropic genes tend to reside in central positions in protein-protein interaction networks, suggesting that pleiotropic genes act as connecting points between different protein complexes or pathways

A Microcosm of the Biomedical Research Experience for Upper-level Undergraduates

The skill set required of biomedical researchers continues to grow and evolve as biology matures as a natural science. Science necessitates creative yet critical thinking, persuasive communication skills, purposeful use of time, and adeptness at the laboratory bench. Teaching these skills can be effectively accomplished in an inquiry-based, active-learning environment at a primarily undergraduate institution. Cell Biology Techniques, an upper-level cell biology laboratory course at St. John Fisher College, features two independent projects that take advantage of the biology of the nematode Caenorhabditis elegans, a premier yet simple model organism. First, students perform a miniature epigenetic screen for novel phenotypes using RNA interference. The results of this screen combined with literature research direct students toward a singe gene that they attempt to subclone in the second project. The biology of the chosen gene/protein also becomes an individualized focal point with respect to the content of the laboratory. Progress toward course goals is evaluated using written, oral, and group-produced assignments, including a concept map. Pre- and postassessment indicates a significant increase in the understanding of broad concepts in cell biological research

The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking

The effectiveness of the Austrian disease management programme for type 2 diabetes: a cluster-randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>Disease management programmes (DMPs) are costly and impose additional work load on general practitioners (GPs). Data on their effectiveness are inconclusive. We therefore conducted a cluster-randomised controlled trial to evaluate the effectiveness of the Austrian DMP for diabetes mellitus type 2 on HbA1c and quality of care for adult patients in primary care.</p> <p>Methods</p> <p>All GPs of Salzburg-province were invited to participate. After cluster-randomisation by district, all patients with diabetes type 2 were recruited consecutively from 7-11/2007. The DMP, consisting mainly of physician and patient education, standardised documentation and agreement on therapeutic goals, was implemented in the intervention group while the control group received usual care. We aimed to show superiority of the intervention regarding metabolic control and process quality. The primary outcome measure was a change in HbA1c after one year. Secondary outcomes were days in the hospital, blood pressure, lipids, body mass index (BMI), enrolment in patient education and regular guideline-adherent examination. Blinding was not possible.</p> <p>Results</p> <p>92 physicians recruited 1489 patients (649 intervention, 840 control). After 401 ± 47 days, 590 intervention-patients and 754 controls had complete data. In the intention to treat analysis (ITT) of all 1489 patients, HbA1c decreased 0.41% in the intervention group and 0.28% in controls. The difference of -0.13% (95% CI -0.24; -0.02) was significant at p = 0.026. Significance was lost in mixed models adjusted for baseline value and cluster-effects (adjusted mean difference -0.03 (95% CI -0.15; 0.09, p = 0.607). Of the secondary outcome measures, BMI and cholesterol were significantly reduced in the intervention group compared to controls in ITT after adjustments (-0.53 kg/m²; 95% CI -1.03;-0.02; p = 0.014 and -0.10 mmol/l; 95% CI -0.21; -0.003; p = 0.043). Additionally, more patients received patient education (49.5% vs. 20.1%, p < 0.0001), eye- (71.0% vs. 51.2%, p < 0.0001), foot examinations (73.8% vs. 45.1%, p < 0.0001), and regular HbA1c checks (44.1% vs. 36.0%, p < 0.01) in the intervention group.</p> <p>Conclusion</p> <p>The Austrian DMP implemented by statutory health insurance improves process quality and enhances weight reduction, but does not significantly improve metabolic control for patients with type 2 diabetes mellitus. Whether the small benefit seen in secondary outcome measures leads to better patient outcomes, remains unclear.</p> <p>Trial Registration</p> <p>Current Controlled trials Ltd., ISRCTN27414162.</p