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Live cell microscopy: From image to insight - raw data & analysis
<p>Accompanying raw and processed data as well as analysis scripts for the publication Biophysics Rev. 3, (2022); <a href="https://doi.org/10.1063/5.0082799">10.1063/5.0082799</a> "Live cell microscopy: From image to insight".</p>
Chloroplast redox state changes indicate cell-to-cell signalling during the hypersensitive response
<p>We performed detailed spatiotemporal analysis of chloroplast redox response to potato virus Y (PVY) infection in resistant <em>Ny</em><em>‐</em><em>1</em>-gene-bearing potato and its transgenic counterpart with impaired SA accumulation and compromised resistance. We found that the chloroplasts are highly oxidized in the cells adjacent to the cell death zone at different stages after virus inoculation in both genotypes. This hypothesis is further supported by highly induced formation of stroma filled tubules that extend from chloroplasts (stromules) in the cells adjacent to signalling cells. This dataset s a deposit of all the raw microscopy images of the study, plus the relevant metadata in ISA-tab compliant folder structure. </p> <p>After receiving reviews, we have made an additional experiment using a ROS inhibitor. The raw and processed data for this is in a separate file: _S_chlROS_inhibitor.zip</p> <p><br> <br> </p> <p><strong>Additional information about the microscopy images in this data deposit </strong></p> <ol> <li>Images of which name ends with <strong>ch00/ch01/ch02</strong> are maximum projections from Z-stacks for each ROI for each of three channels: chlorophyll fluorescence (ch00), GFP fluorescence after excitation with 405 nm laser line (ch01) and GFP fluorescence after excitation with 488 laser line (ch02), exported from Leica LAS X software.</li> <li>Only mesophyll cells are included in z-stack, except for the experiments with GFP-tagged PVY where additional z-stacks including both mesophyll and epidermal cells were produced ( »_2« added in the image name, see images with the comment »epidermal cells included in z-stack« in S_PhenodataRedox).</li> <li>Images of which name ends with <strong>ch00.tif</strong> and <strong>ch00.tif_ratio</strong> are analysed images, obtained using in-house Matlab script. These images were obtained by the analysis of ch00/ch01/ch02 images with the following steps: conversion to grayscale, filtering out pixels of low intensity, conversion to binary format using spatial adaptive thresholding, a round or erosion and dilation to remove single pixel noise around the chloroplasts, followed by size-based segmentation of individual chloroplasts. The ratios of fluorescence intensities 405/488 were then calculated for each pixel belonging to the chloroplast masks obtained in the previous step. Results were calculated per image (normalized to the fraction of pixels belonging to chloroplasts) and per individual chloroplast in each image.</li> <li>In the experiments with GFP-tagged PVY, due to high background signal in the cell death zone as a result of virus derived GFP fluorescence, signal in the 488 channel and 405/488 ratio (images named »scaled 488« and »405/488 in chloroplasts« in ch00.tif_ratio) in the cell death zone are not accurate.</li> <li>Figures of which name ends with <strong>ch00.tif_spatial_ROI1/ROI2</strong> show the 405/488 ratios in ROI1 or ROI2, determined for each pixel inside chloroplast masks for each Bin. See Image analysis in Methods for details.</li> <li>Experiment names and image names correspond to the names in S_PhenodataRedox. Details regarding experimental set-up and transgenic lines used are specified in S_PhenodataRedox.</li> </ol> <p>The Zenodo_reanalysis.zip file contains processed microscopic images in a new red/blue colormap. </p>
Extended Data Fig. 7 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 7 | Analysis of the inter-subunitshydrogen bondsat the interfaceof RBDandhACE2. Frequencyofformationofhydrogenbondsat theinterface of RBDandhACE2 intheknob (A), base (B), andtipregions (C). Theanalysisisperformedfor 9 different MDsimulations:3 replicatesof the SARS-CoV-2 (shadesof green),BANAL-236 (shadesof red), and BANAL-52/103 (shadesofblue) RBD–hACE2 complexes.
Extended Data Fig. 3 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 3 | Nucleotideandamino-acidalignmentsof thefurin cleavagesiteregion. Completenucleotideandamino-acidspikesequencesof representativebat SARS-CoV-2-likecoronavirusesweredownloadedfrom GenBankand GISAIDandaligned with MAFFT (G-INS-I parameter) (A & C). Alignmentsweremanuallyeditedas proposedby Zhou2 andLytras47 with CLC Main Workbench (Qiagen) (B & D). Alignmentsof thefurincleavageregionare presentedatthenucleotide (A & B) andtheamino-acid (C & D) level, respectively.
Extended Data Fig. 1 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 1 | Spike identity matricesatthe genus level of representativesarbecoviruses. Amino-acid (lower) andnucleotide (upper) identitymatricesof Laotianandrepresentativehuman,bat, andpangolin sarbecoviruses.Spike N-terminal (NTD), Receptor-binding (RBD) and S2 nucleotideandamino-acidsequenceswerealigned with MAFFT, andidentity matriceswereconstructedusing CLCMain Workbench 21.0.4 (Qiagen). Matriceswerecoloredaccordingtotheidentityscale,from 25% (red) to 100% (green) of nucleotideoramino-acididentity.
Extended Data Fig. 2 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 2 | Alignmentofthespike RBDdomain. Protein alignmentof the Receptor Binding Domain (RBD) of Laotianandrepresentative human, batandpangolinsarbecoviruses.Sequenceswerealignedwith MAFFT in G-iNS-Imode. Residuesinteractingwithhuman ACE2 receptorare highlighted ingrey.Thedomainusedforinteractionsmodeling,basedonthe X-raystructure 6M0J (residues T333 to G526), ishighlightedbyablackline.
Fig. 2 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Fig. 2 | Recombinationevents in the evolutionaryhistoryof sarbecoviruses. Representationof the 15 recombinant fragmentsof relevant Sarbecovirus genomescomparedtothe SARS-CoV-2 humanprototypestrain (NC_045512). Thecoordinatesof thebreakpointsrefertothenucleotide positioninthealignment.Wherepossible,theclosestviralsequenceis indicatedforeachfragment.Inothercases,MULTindicatesagroupof multiple sequences.Theasteriskmarksunresolvedfragment phylogeny (fragment 13, from positions 27,344 to 27,800 inthealignment).Sequencesarecolouredas in Fig. 1. Thecomplete phylogeneticanalysesarepresentedin Supplementary Fig.2.
Fig. 1 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Fig. 1 | Genomicdescriptionofbat-bornesarbecovirusesidentifiedin Laos. a, Mapof samplingsites.All BANALisolateswerecollectedfromthesamesite (site 1). Themapwasdownloadedfrom DIVA-GIS (https://www.diva-gis.org/ gdata). b, Phylogeneticanalysisof theproteinsequenceof the RBDof Laotian andrepresentativehuman,bat,andpangolinsarbecoviruses.Sequenceswere alignedwith MAFFT (ref.49) inautomode, andmaximum-likelihood phylogeneticreconstructionwasperformedwith PhyMLimplemented throughthe NGPhylogenyportal50 withthe LG + Gsubstitutionmodel.Branch supportwasevaluatedwiththeaBayes parameter.Batspeciesarespecifiedin thename of thesequences.Sequencesarecolouredaccordingto Fig. 1c. c, Similarityplotanalysisof Laotianandrepresentativebatandpangolin sarbecovirusesbasedonthefull-lengthgenomesequenceof the SARS-CoV-2 humanprototypestrain (NC_045512, Wuhan-Hu-1) usedasareference. Theanalysiswasperformedwiththe SimPlotprogramversion 3.5.1 (ref.51) with the Kimuratwo-parametermodel, awindowsizeof 1,000 basepairs,astepsize of 100 basepairs,a transition/transversionrate (T/t) of 2.0, anda Gap/Strip parameter: on51. nsp,non-structural protein;RdRP, RNA-dependent RNA polymerase.d, Heatmapof identitiesattheproteinlevelof representative human,batandpangolinsarbecovirusescomparedtohuman SARS-CoV-2 lineage B (NC_045512). Spikeproteinhasbeendividedintofunctionaldomains, andthesequencesareorderedaccordingtopercentageof identityof the RBD domain.Theasteriskmarkstheabsenceof afunctional ORF10 in Thaibat RacCS203 (accessionnumber MW251308). Theheatmapwascreatedusingthe gplotspackagein R (version 3.6.3). AA, aminoacid.
Extended Data Fig. 5 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 5 | Additionalanalysisof the MDsimulationsof the BANAL-52/103 RBD–hACE2 complex. Comparisonof thetimeseriesof interface RMSDduringthecourseof two MDsimulationsof the BANAL-52/103 RBD–hACE2 complexwithshort (BANAL-52/103-CoV.1) andlong (BANAL- 52/103-CoV.1*) equilibrationphase.Thelargefluctuationsof theinterface RMSDareduetotheflexibilityof the RBDloopbetweenresidues S443 and Y449 (insets, inyellow).Whentheseresidueswerenotincludedinthe calculationof theinterface RMSD, thetimeseriesdisplayedamorestable behavior (BANAL-52/103-CoV.1-Land BANAL-52/103-CoV.1*-L).
Fig. 4 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Fig. 4 | BANAL-236 entryandpropagationinhumancells. a, Resultsof spike-pseudotyped BANAL-236 (squares) and SARS-CoV-2 (strain BetaCoV/ France/IDF0372/2020, GISAIDaccessionnumber EPI_ISL_406596,diamonds) pseudovirusentryassayin HEK-293T cellsexpressing (purple lines) ornot (grey lines) thehACE2 receptor,showninrelativeluminescenceunits (RLUs) producedbythefireflyluciferasepresentinthelentiviralbackbone andthe Bright-Gloluciferasesubstrate.Asingleexperimentperformedintriplicate representativeof twoexperiments isshown.Centrevaluesrepresentthe averageof thethreereplicatesanderrorbarsindicates.d. b, Resultsof spike-pseudotyped BANAL-236 (black) and SARS-CoV-2 (strain BetaCoV/ France/IDF0372/2020, grey) neutralizationassayexpressedas apercentageof neutralizationofluciferaseactivityintheabsenceof serum.Seraneutralizing SARS-CoV-2 werefrompatientswithconfirmedinfectionswhereas non-neutralizingserasampleswerecollectedbeforethespreadof SARS-CoV-2 Laos.Thedashedlinemarkstheneutralizationthreshold.Asingleexperiment representativeof threeindependentexperimentsisshown. c, Humancelllines expressingendogenous ACE2,Calu-3 (blue lines) and Caco-2 (green lines),were infectedatan MOIof 0.01 withthe BANAL-236 virus (squares) andthevirusfirst detectedin Wuhan (diamonds).VeroE6 cellswereinfectedatan MOI of 0.0001 with BANAL-236 virus (squares) and SARS-CoV-2 (strain BetaCoV/France/ IDF0372/2020, diamonds) pre-incubatedwith (grey lines) orwithout (pink lines) solublehACE2 (sACE2) at 25 Μgml−1 for 30 min. Genomecopynumberwas determinedbyquantitative RT-PCRinthesupernatantsrecovered 3 and 4 days post-infection. Asingleexperimentperformedintriplicateisshown.Centre valuesrepresenttheaverageof thethreereplicatesanderrorbarsindicates.d.
Fig. 3 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Fig. 3 | Dynamicsof thebindingof hACE2 to bat-sarbecovirus-borne RBDs andinsightintothestructureofthecomplex. a, Biolayerinterferometry (BLI) bindinganalysis of thehACE2 peptidasedomaintoimmobilized BANAL-52/103,BANAL-236 or SARS-CoV-2 RBDs.Black linescorrespondto aglobalfitof thedatausinga 1:1 bindingmodel.b, Frequencyofformationof saltbridgesclosetothe RBD–ACE2 interface (fromlefttoright:D30/K417, E35/ K493, D38/K493, K31/E35 and D38/K353) duringthecourseof the MD simulations.Theanalysiswasperformedforninedifferent MDsimulations (threereplicatesforeachcomplex) ofhACE2 incomplexwith RBDs from SARS-CoV-2 (SARS-CoV-2.1,SARS-CoV-2.2 and SARS-CoV-2.3, shadesof green), BANAL-236 (BANAL 236-CoV.1, BANAL 236-CoV.2 and BANAL 236-CoV.3,shadesof red) and BANAL-52/103 (BANAL 52/103-CoV.1, BANAL 52/103-CoV.2 and BANAL 52/103-CoV.3, shadesofblue). c, Ribbon representationsof thecrystalstructuresof thehACE2 peptidase domain (cyan) incomplexwith SARS-CoV-2 (PDB 6M0J) or BANAL-236 (this study,PDB 7PKI) RBDs (pink). Blackarrowsintheoverallstructuresindicatethestructural differencebetweenthetwocomplexesatthelevelofhelix H4. The magnificationsshowthemaininteractionsinthe ACE2–RBD interfaces. Residuesinthereceptor-bindingmotif alteredbetween SARS-CoV-2 and BANAL-236 arehighlighted withcolouredoutlines.Numbers 1, 2 and 3 indicate thethreemainclustersof interactionsbetweenhACE2 and RBDs.
Extended Data Fig. 4 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 4 | Analysisof thestability and conformational heterogeneityofRBD–hACE2 complexes. Timeseries (leftcolumn) and violinplots (rightcolumn) ofbackbone Root Mean Square Deviation (RMSD) fromtheinitial,energy-minimizedmodelcalculatedontheresiduesin RBD (A), hACE2 (B), attheinterface of RBDandhACE2 (C), andontheentirecomplex (D). Intheviolinplots,thewhitecirclecorrespondstothemedianvalue,the blackrectangleextendsfromthefirsttothethirdquantiles,andthethinblack linerepresentsthe 95% confidenceintervals.Populationof the 3 most significantclustersvisitedduringthecourseof the MDsimulations (E). Theanalysisisperformedfor 9 different MDsimulations:3 replicatesof the SARS-CoV-2 (shadesof green),BANAL-236 (shadesof red), and BANAL-52/103 (shadesofblue) RBD–hACE2 complexes.
LiBforSecUse Data Release - Impedance spectra of life cycle tests of commercial 18650 cells
<p>The EMPIR project LiBforSecUse aimed to develop empirical measurement models to estimate the residual capacity of second-use Li-ion battery cells with impedance-based measurement and evaluation methods. The models have been established based on a series of life cycle tests of commercial 18650 (graphite/NMC) cells including regular impedance spectroscopy and capacity measurements. The measured data are made publicly available here. They can be downloaded to verify the models established within the project and they may be used for further investigations. However, the user is asked to pay tribute to the project and the researchers providing the data by citing this data source. A pdf file is added to give more detailed information on the data.</p>
Extended Data Fig. 8 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 8 | Stick representation. Stick representation of segments D364-S375 of BANAL-236 (left panel) and SARS-CoV-2 (right panel) RBDs.A 2Fo-Fc composite omit map (contoured at 3σ) is shown for this region in BANAL-236 RBD.
Extended Data Fig. 6 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 6 | Estimationof RBD–hACE2 bindingenergy. Time series (leftcolumn) andviolinplots (rightcolumn) of the RBD–hACE2 binding energyestimatedusing ROSETTA (A) and FoldX (B). Intheviolinplots,the whitecirclecorresponds tothemedianvalue,theblackrectangleextendsfrom thefirsttothethirdquantiles,andthethinblacklinerepresentsthe 95% confidenceintervals.Theanalysisisperformedfor 9 different MDsimulations: 3 replicatesof the SARS-CoV-2 (shadesof green), BANAL-236 (shadesof red), and BANAL-52/103 (shadesofblue) RBD–hACE2 complexes.
Extended Data Fig. 9 in Bat coronaviruses related to SARS-CoV-2 and infectious for human cells
Extended Data Fig. 9 | Isolationof BANAL-236 on VeroE6 cells. (A) CPE observedon VeroE64 daysafterinoculationatan MOIof 10-4 fromthe C1. (B) Uninfected VeroE6 cellslayer.(C) Plaqueassayperformedfromthe C2 stock on VeroE6 cells.(D) Comparative CPEobservedon VeroE6 cellsinfectedwith SARS-CoV-2 ('Wuhan', toppanel) or BANAL-236 (bottom panel) inabsence (left) orinpresence (right) of soluble ACE2. Photosweretakenwitha EVOSXLCore microscopeatx10. Asingleexperimentperformedintriplicateisshownfor eachcellline.
Data generated by the model presented in the research article entitled "Simulation of mass and heat transfer in an evaporatively cooled PEM fuel cell"
<p>This repository provides all the data and scripts necessary to reproduce the line plots shown in the manuscript entitled "Simulation of mass and heat transfer in an evaporatively cooled PEM fuel cell".</p>
Lipogenesis mediated by OGR1 regulates metabolic adaptation to acid stress in cancer cells via autophagy
<p>Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype is a cellular adaption to extracellular acidity. LD marker PLIN2, is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing GPCR, OGR1 expressed highly in breast tumors. OGR1 depletion inhibited acid induced lipid accumulation while activation by synthetic agonist triggered LD formation. Inhibition of OGR1 downstream signaling abrogated the lipogenic phenotype which could be rescued with OGR1 ectopic expression. OGR1 depleted cells showed growth inhibition under acidic growth conditions in vitro and tumor formation in vivo. Isotope tracing showed that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1 depleted cells were defective in endoplasmic reticulum stress response and autophagy, hence failed to accumulate LDs affecting survival under acidic stress.</p>
Fig. 2 in Macrophages And Pigment Cells In The Liver Of Pelophylax Ridibundus (Anura)
Fig. 2. Mitosis in the precursor cell of the macrophage line (A) and macrophages of varying degrees of maturity (B, C, D, E, F) on the smears of the frog lake liver. Coloring according to Pappenheim, x900.
Fig. 1 in Histological, Topographical And Ultrastructural Organization Of Different Cells Lining The Olfactory Epithelium Of Red Piranha, Pygocentrus Nattereri (Characiformes, Serrasalmidae)
Fig. 1. Photomicrographs of the olfactory epithelium of Pygocentrus nattereri by scanning electron microscopy (SEM) and histological architecture stained with Delafield's Haematoxylin-Eosin (HE) and Mallory's triple (MT) stain. A — oval shaped olfactory rosette showing olfactory lamellae (OL) radiating from median raphe (R). Note tongue shaped structure (arrow heads) on the apical end of the lamellae (SEM) ×50. B — sensory olfactory epithelium (OEP) lined with receptor cells. Note the presence of blood vessels (BV) in the central core (CC) which is distinguished from OEP by basement membrane (BM). Arrow heads indicate basal cells above BM (MT) ×400. C — higher magnification of OEP showing a large number of primary receptor cells (RC) with conspicuous nuclei (N), secondary recptor cells (broken arrows), microvillous cells (MV) intermingled with supporting cells (SC). Note the presence of BV in CC and BC (arrow heads) near CC. Solid arrow indicates the axons of secondary RC communicate to CC (MT) ×1000. D — OEP exhibiting cylindrical RC with knob like vesicles (black arrow heads), ciliated supporting cells (solid arrows), non-ciliated supporting cells (white arrow heads) and BC above CC. Broken arrows mark the cilia of supporting cells on the epithelial surface (HE) ×400. E — tuft of receptor cells (RC) in between supporting cells (SC) (SEM) ×4000. F — dendrite patches of RC (broken arrows) and microvillous cells (solid arrows) in between stratified epithelial cells (SEC). Note the opening of mucous cells (arrow heads) in between SEC (SEM) ×2500.
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
ABODe is a University of Edinburgh DataShare dataset for behavior classification in group-housed mice using home-cage video, identities, bounding boxes, ground-plate positions, and annotator labels.
DANDI Archive for NWB datasets
DANDI is a BRAIN Initiative archive for publishing and sharing neurophysiology data, including electrophysiology, optophysiology, and behavioral data packaged as NWB and related standards.
International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.