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71 results for “simulated microgravity”
Data from: Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight
<p>3-dimensional super-resolution microscopy volumes of human PBMCs recorded on a Zeiss LSM980 Airyscan2 laser scanning confocal microscope.</p> <p>Sample preparation and image capture:</p> <p>Live PBMCs were stained with 60 nM MitoTracker Red-CMX-Ros (ThermoFisher, Waltham, MA) either in 6-well plates or in the microgravity chambers for the last 2 hr of the microgravity simulation. At the end of the microgravity simulation cells were immediately fixed by 1:1 mixing the cell suspensions with 2× concentrated fixative (10% Sucrose (w/v) 120 mM KCl, 1% (w/v) glutaraldehyde, 8% (w/v) PFA pH 7.4) and incubated for 15 minutes at room temperature followed by 15 minutes on ice. Fixed cells were washed and stored in PBS until further staining for up to a week at 4 °C. 1 million fixed cells were resuspended in 1 mL of permeabilization solution (0.1% TritonX-100 in PBS) for 5 minutes. After twice washing in PBS, pellets were resuspended in 0.5 mL 1% BSA PBS containing Phalloidin-iFluor-488 (cat# ab176753, Abcam plc., Cambridge, UK) at the manufacturer’s recommended dilution, and were incubated for 90 minutes with gentle agitation. After washing in PBS, cells were stained with Hoechst 33342 (1 µg/mL in PBS) for 10 minutes. The fixed-stained cells were immobilized at 3 × 10<sup>5</sup> cells per well density in glass-bottom 96-well microplates (Greiner Bio-One, Monroe, NC), which were pre-coated with polyethyleneimine (1:15,000 (w/v)) for 16 hours in a 37 °C incubator, and washed twice with PBS. Microplates with the cell suspensions were centrifuged in a swing plate rotor centrifuge (Eppendorf 5810 R) at 400 × <em>g</em> and for 10 min and then fixed on the surface by adding an equal volume of 8% (w/v) PFA for 5 min. Finally, the fixative was replaced with 100 µL of antifade reagent (Vector Prolong Gold (ThermoFisher)). Samples were imaged immediately after this procedure on a Zeiss LSM980 Airyscan2 laser scanning confocal microscope (Carl Zeiss Microscopy, White Plains, NY). Single PBMCs were manually selected for recording based on low-resolution preview scans showing only nuclei. All singlet cells were selected in a small neighborhood to avoid biases. In each microscopy session, 24-40 cells were selected for recording in one well for each condition. This was performed in an interleaved manner, capturing 6-8 cells at a time, and then moving to the next well and then repeating this multiple times using the Experiment Designer module for automation. Super-resolution volumes of (358 × 358 × 70 pixels, 0.035 × 0.035 × 0.13 µm/voxel resolution) were recorded in the above-determined positions using Definite Focus autofocusing. A Plan-Apochromat 63 × 1.40 Oil lens, Airyscan2 SR (super-resolution) mode with optimal sampling and frame switching between 3 fluorescence channels to minimize spectral cross-bleed were used. MitoTracker Red, iFluor488, and Hoechs33342 were excited with 561, 488, and 405 nm solid-state lasers, respectively, using the optimal emission filter for each channel. 3D Airyscan2 processing was performed with standard filtering settings.</p> <p>File naming:</p> <p>Four zip files were deposited named as <Donor#id>.zip, where id goes from 1 to 4.</p> <p>Each zip file contains the following Zeiss Microscopy format image files: <Condition>_<Donor#id>_<Stain#batch>_<Cell>.czi</p> <p><Condition>:</p> <ul> <li>1G – Control culturing in 6-well plates for 25h</li> <li>uG – simulated microgravity culturing for 25h in NASA Rotating Wall Vessels</li> <li>1G+TLR – as above, with TLR 7/8 agonist (1 μM R848)</li> <li>uG+TLR– as above, with TLR 7/8 agonist (1 μM R848)</li> <li>1G+CyD– as above, with cytochalasin D</li> <li>uG+CyD– as above, with cytochalasin D</li> <li>1G+Q – as above, with quercetin 50µM</li> <li>uG+Q – as above, with quercetin 50µM</li> </ul> <p><Donor#id>: 1-4 indicates biological replicates</p> <p><Stain#batch>: 1-2 indicates experimental replicates of phalloidin staining and imaging session</p> <p><Cell>: arbitrary number to distinguish images within the same condition/donor/stain set.</p> <p>See image analysis pipelines used with these data at: https://github.com/gerencserlab/Superresolution-actin-and-mitochondria-analysis</p>
Female Pelvic Blood Flow Under Simulated Microgravity
ClinicalTrials.gov study NCT06633692. IPD Sharing: Not stated. Countries: 1. Publications: 6.
Time-course adaptive changes in hippocampal transcriptome and synaptic function induced by simulated microgravity associated with cognition
<p>Introduction: The investigation of cognitive function in microgravity, both short-term and long-term, remains largely descriptive. And the underlying mechanisms of the changes over time remain unclear. Methods: Behavioral tests, electrophysiological recording, and RNA sequencing were used to observe differences in behavior, synaptic plasticity, and gene expression. Results: Initially, we measured the performance of spatial cognition exposed to long-term simulated microgravity (SM). Both working memory and advanced cognitive abilities were enhanced. Somewhat surprisingly, the synaptic plasticity of the hippocampal CA3-CA1 synapse was impaired. To gain insight into the mechanism of changing regularity over time, transcriptome sequencing in the hippocampus was performed. The analysis identified 20 differentially expressed genes (DEGs) in the hippocampus after short-term modeling, 19 of which were up-regulated. Gene ontology (GO) analysis showed that these up-regulated genes were mainly enriched in synaptic-related processes, such as <em>Stxbp5l</em> and <em>Epha6</em>. This might be related to the enhancement of working memory performance under short-term SM exposure. Under exposure to long-term SM, 7 DEGs were identified in the hippocampus, all of which were up-regulated and related to oxidative stress and metabolism, such as <em>Depp1</em> and <em>Lrg1</em>. Compensatory effects occurred with increased modeling time. Discussion: To sum up, our current research indicates that the cognitive function under SM exposure is consistently maintained or potentially even being enhanced over both short and long durations. The underlying mechanisms are intricate and potentially linked to the differential expression of hippocampal-associated genes and alterations in synaptic function, with these effects being time-dependent. The present study will lay the experimental and theoretical foundation of the multi-level mechanism of cognitive function under space flight.</p>
['Comparative proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells']
['Microgravity is one of the most important features in spaceflight. Previous evidence has shown that significant changes to the musculoskeletal and immune systems occurred under microgravity. The present study was undertaken to explore the change in protein abundance in human colon colorectal cells that were incubated for 48 or 72 h either in normal conditions and µG simulated conditions. The comparative proteomic method based on the 18O labeling technique was applied to investigate the up-regulated proteins and down-regulated proteins in SH-SY5Y under simulated microgravity.']
Transcription profiling of mouse osteoblasts under static vs simulated microgravity
The total RNA was extracted from 2T3 pre-osteoblast cells exposed to static or simulated microgravity (Rotating Wall Vessel) conditions. The RNA was then sent to Affymetrix microarray core facility at Baylor College of Medicine (Houston TX) for microarray analysis.
The influence of simulated microgravity on the proteome of Daphnia magna
Background/Objectives: The waterflea Daphnia is an interesting candidate for bioregenerative life support systems (BLSS). These animals are particularly promising because of their central role in the limnic food web and its mode of reproduction. However the response of Daphnia to altered gravity conditions has to be investigated especially on the molecular level to evaluate the suitability of Daphnia for BLSS in space. Methods: In this study we applied a proteomic approach to identify key proteins and pathways involved in the response of Daphnia to simulated microgravity generated by a 2D-clinostat. We analysed 5 biological replicates using 2D-DIGE proteomic analysis. Results: We identified 109 protein spots differing in intensity (p < 0.05). Substantial fractions of these proteins are involved in actin microfilament organisation indicating the disruption of cytoskeletal structures during clinorotation. Furthermore proteins involved in protein folding were identified suggesting altered gravity induced breakdown of protein structures in general. In addition simulated microgravity increased the abundance of energy metabolism related proteins indicating an enhanced energy demand of Daphnia. Conclusion: The affected biological processes were also described in other studies using different organisms and systems either aiming to simulate microgravity conditions or providing real microgravity conditions. Moreover most of the Daphnia protein sequences are well conserved throughout taxa indicating that the response to altered gravity conditions in Daphnia follows a general concept.
Zebrafish larvae submitted to simulated microgravity on a clinostat (CLINO) at 5dpf for one day
The general objective of the study was to determine modulation of gene expression by environmental factors specifically simulation of microgravity with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a clinostat (CLINO) for 24 hours which was shown to simulate microgravity by the specific rotational movements it generates. We show that CLINO exposure causes a clear decrease of bone formation as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red by morphometric analysis of the resulting images. Thus a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.
Global gene expression profiles of cardiac progenitors differentiated from human pluripotent stem cells in 3D culture under simulated microgravity
Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. Methods: RNA-seq libraries were prepared using the Illumina TruSeq RNA kit and the TrueSeq method was employed for mRNA enrichment. The libraries were quantified and samples were multiplexed in each lane of the flowcell. Cluster generation was performed and then sequenced on the Illumina HiSeq1000 system. Reads were mapped on the Human Genome Reference and normalized expression table was generated. Results: Among differentially expressed genes 53 of them were up-regulated and 75 were down-regulated. Conclusions: Data demonstrate increased expression of genes associated with growth development and pro-survival in cardiac progenitors cultured under simulated microgravity compared with those cultured under standard gravity. RNA-sequencing analysis was performed to compare global gene expression profiles of cells at differentiation day 8 under simulated microgravity vs. standard gravity.
Effect of simulated microgravity on E. coli K12 MG1655 growth and gene expression
This study demonstrates simulated microgravity effects on E. coli K 12 MG1655 when grown on LB medium supplemented with glycerol. The results imply that E. coli readily reprograms itself to combat the multiple stresses imposed due to microgravity. Under these conditions it survives by upregulating oxidative stress protecting genes and simultaneously down regulating the membrane transporters and synthases to maintain cell homeostasis. In this study a clinostat that mimics microgravity conditions was used to investigate the effects of microgravity on E. coli grown in LB medium supplemented with glycerol to monitor the effects on growth and global gene expression using Affymetrix DNA microarrays.
A study of gene expression influenced by simulated microgravity in human lymphoblastoid cells
Here in this study we systematically examined the patterns of DNA methylation and hydroxy-methylation with its functional implications in gene regulation for the cultured TK6 lymphoblastoid cells upon exposure to micro-gravity conditions. The results reported here indicate that simulated microgravity alters methylation patterns in a limited way and subsequently the expression of genes involved in stress response like ATF3 FBXO17 MAP3K13 and VCL in TK6 cells. Overall design: Examination of RNA-seq with 2 replicates each for 1 cell type
Transcription profiling of Drosophila after exposure to microgravity in the International Space Station and in a microgravity simulator
Larvae-Pupae transition flies (Drosophila) were recovered and transport for 3 days at 12-14C to arrest development until the launch site then exposed to RT (18-20C) for some hours including the launch and trip to the International Space Station then pupae were exposed to microgravity in the ISS for 4 days and a half at 22C. Finally pupae were fixed on acetone and frozen until recovery on Earth. Four groups of samples: 1 ISS (+ground control) as described 2 RPM (microgravity simulator on Earth) as described 3 RPM without constrains (No MAMBA container and only 5 days exposure without cold transport) and 4 centrifuge 10g without constrains control.
Streptococcus mutans differential gene expression in response to simulated microgravity
Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight host factors that could contribute to oral diseases such as caries and periodontitis. However the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study High Aspect Ratio Vessel (HARV) S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles H2O2 resistance and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated >= 2-fold and 94 genes down-regulated >= 2-fold during simulated microgravity HARV growth. These included a number of genes located on extrachromosomal elements as well as genes involved in carbohydrate metabolism translation and stress responses. Collectively these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions. Overall design: Differential gene expression was compared between RNA from S. mutans grown in normal gravity HARVs (n=3 independent cultures) and RNA from S. mutans grown in simulated microgravity HARVs (n=3 independent cultures)
['RNA sequencing of zebrafish embryos under simulated microgravity conditions']
['To reveal the potential mechanisms involved in the dysfunction of antiviral immune responses under simulated microgravity conditions, we investigated the transcriptional changes related to the status of innate immune responses by RNA-seq with poly I:C or mock PBS treatment under Normal gravity or simulated microgravity conditions. Our results indicate that the retinoic acid inducible gene (RIG)-I-like receptor (RLR) and Toll-like receptor (TLR) signal pathways, which are both involved in the type-I interferon induction, are significantly inhibited by simulated microgravity effects.']
A study of alterations DNA epigenetic modifiincations (5mC and 5hmC) influenced by simulated microgravity in human lymphoblastoid cells
Here in this study we systematically examined the patterns of DNA methylation and hydroxy-methylation with its functional implications in gene regulation for the cultured TK6 lymphoblastoid cells upon exposure to micro-gravity conditions. The results reported here indicate that simulated microgravity alters methylation patterns in a limited way and subsequently the expression of genes involved in stress response like ATF3 FBXO17 MAP3K13 and VCL in TK6 cells. Overall design: Examination of 2 different DNA modifications with 2 replicates each for 1 cell type.
Global gene expression profiles of cardiac progenitors differentiated from human pluripotent stem cells in 3D culture under simulated microgravity
GEO Series GSE84582. Homo sapiens. 6 samples. Type: Expression profiling by high throughput sequencing.
SmallRNA analysis of simulated microgravity sensitized endothelial cells.
GEO Series GSE113800. Homo sapiens. 2 samples. Type: Non-coding RNA profiling by high throughput sequencing.
Single Cell Analysis Identifies Conserved Features of Immune Dysfunction in Simulated Microgravity and Spaceflight [RNA-Seq]
GEO Series GSE218935. Homo sapiens. 12 samples. Type: Expression profiling by high throughput sequencing.
Simulated Microgravity Triggers a Membrane Adaptation to Stress in E. coli REL606
GEO Series GSE284563. Escherichia coli. 64 samples. Type: Expression profiling by high throughput sequencing.
Streptococcus mutans differential gene expression in response to simulated microgravity
GEO Series GSE90166. Streptococcus mutans. 6 samples. Type: Expression profiling by high throughput sequencing.
Spaceflight and simulated microgravity conditions increase virulence of Serratia marcescens in the Drosophila melanogaster infection model
GEO Series GSE138116. Drosophila melanogaster. 12 samples. Type: Expression profiling by high throughput sequencing.
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