Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells.
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ABSTRACT:
SUBMITTER: Avilov S
PROVIDER: S-EPMC4792379 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveTo compare components of the embryo grading system with time for blastocyst formation (i.e., day of embryo transfer) for predicting live-birth rate in frozen embryo transfer cycles.DesignRetrospective cohort study.SettingUniversity-affiliated fertility clinic.PatientsFrom January 2015 to October 2018, 870 frozen embryos transferred in a total of 509 women and 728 cycles at our institution.InterventionsNone.Main outcome measuresProbability of live birth per cycle.ResultsIn unadjusted analysis of embryo grading components, both inner cell mass (ICM) and trophectoderm grades demonstrated a correlation with live-birth rates. However, this effect was lost in the ICM subgroup analysis by day of embryo transfer and preserved only in declining trophectoderm grades of day-6 transfers. In the adjusted analysis for prediction of live birth, only day of transfer was statistically significant. When assessing the composite score by Society for Assisted Reproductive Technology (SART) embryo grading, good embryos that blastulated on day 6 were statistically significantly less likely than day-5 embryos to result in live birth (risk ratio 0.70; 95% confidence interval, 0.58-0.85). Finally, in a predictive model adjusted for all individual components of embryo grade, the day of blastulation was the only statistically significant contributor.ConclusionsTime to blastulation is superior to other individual components of embryonic grading for prediction of live birth.
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Project description:It is estimated that two million new dental implants are inserted worldwide each year. Innovative implant materials are developed in order to minimize the risk of peri-implant inflammations. The broad range of material testing is conducted using standard 2D, terminal, and invasive methods. The methods that have been applied are not sufficient to monitor the whole implant surface and temporal progress. Therefore, we built a 3D peri-implant model using a cylindrical implant colonized by human gingival fibroblasts. In order to monitor the cell response over time, a non-toxic LIVE/DEAD staining was established and applied to the new 3D model. Our LIVE/DEAD staining method in combination with the time resolved 3D visualization using Scanning Laser Optical Tomography (SLOT), allowed us to monitor the cell death path along the implant in the 3D peri-implant model. The differentiation of living and dead gingival fibroblasts in response to toxicity was effectively supported by the LIVE/DEAD staining. Furthermore, it was possible to visualize the whole cell-colonized implant in 3D and up to 63 hours. This new methodology offers the opportunity to record the long-term cell response on external stress factors, along the dental implant and thus to evaluate the performance of novel materials/surfaces.
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Project description:We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.
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Project description:Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibroblasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered.
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2019-09-23 | GSE115908 | GEO
Project description:Background: The leishmaniases are neglected diseases that affect some of the most vulnerable populations in the tropical and sub-tropical world. The parasites are transmitted by sand flies and novel strategies to control this neglected vector-borne disease are needed. Blocking transmission by targeting the parasite inside the phlebotomine vector offers potential in this regard. Some experimental approaches can be best performed by longitudinal study of parasites within flies, for which non-destructive methods to identify infected flies and to follow parasite population changes are required. Methods: Lutzomyia longipalpis were reared under standard insectary conditions at the Wellcome Centre for Molecular Parasitology. Flies were artificially infected with L. tarentolae expressing green fluorescent protein (GFP. Parasite counts were carried out 5 days post-infection and the percentage of infected flies and survival of infected females was established up to days 5 post-infection. Whole living females were visualised using an epifluorescence inverted microscope to detect the presence parasites inferred by a localised green fluorescent region in the upper thorax. Confirmation of infection was performed by localised-fluorescence of dissected flies and estimates of the parasite population. Results : Leishmania tarentolae was successfully transfected and expressed GFP in vitro. L. tarentolae-GFP Infected flies showed similar parasite populations when compared to non-transfected parasites ( L. tarentolae-WT). Survival of non-infected females was higher than L. tarentolae-infected groups, (Log-rank (Mantel-Cox) test, p<0.05). L. tarentolae-GFP infected females displayed an intense localised fluorescence in the thorax while other specimens from the same infected group did not. Localised fluorescent flies were dissected and showed higher parasite populations compared to those that did not demonstrate high concentrations in this region (t-test, p<0.005). Conclusion : These results demonstrate the feasibility of establishing a safe non-human infectious fluorescent Leishmania-sand fly infection model by allowing non-destructive imaging to signal the establishment of Leishmania infections in living sand flies.
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