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Transcription profiling of rat to study the effect of hindlimb unloading on healing of medial collateral ligaments 3 weeks after injury

A recent physiological study established that hindlimb unloading of rats at 3 and 7 weeks inhibits healing of injured ligaments resulting in a badly aligned discontinuous collagen matrix. Using tissue from these rats we focused on the 3-week time point employing microarray analysis to identify what cellular processes or lack of processes could account for these observed deficiencies. We used the Affymetrix RG_U34A GeneChip and performed image analysis with Microarray Suite 5.0. For normalization we used the MAS global normalization protocol with a default target mean signal of 500. Gene expression in medial collateral ligament tissue under 4 different treatment conditions was measured: loaded control loaded wound healing unloaded control and unloaded wound healing. From our results it appears that unloaded tissue lags behind loaded tissue in its progression through the healing process and at 3 weeks is still engaged in the proliferative phase whereas loaded tissue is actively remodeling its collagen matrix.

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.

Dissecting transcriptional responses of nucleolin mutants to red light stimulation and darkness in ground reference conditions

Understanding plant adaptive responses to the space environment is a requisite for enabling space farming. Spaceflight produce deleterious effects on plant cells particularly affecting ribosome biogenesis a complex stress-sensitive process coordinated with cell division and differentiation known to be activated by red light. Here we have used mutants from the two nucleolin genes in Arabidopsis (NUC1 and NUC2) encoding the main regulator of the ribosome biogenesis in the nucleolus in order to better understand their role in adaptive response mechanisms to stress. Thus we show that nucleolin stress-related gene NUC2 can compensate the environmental stress provided by darkness in nuc1 plants while nuc2 plants are not able to provide a complete response to red light. These ground control findings as part of the ESA/NASA Seedling Growth spaceflight experiments will determine the basis for the identification of a genetic background enabling an adaptive advantage for plants in future space experiments.

Spatially resolved transcriptional analysis of hearts from mice flown on the RR-3 mission

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c 18-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight ground controls and basal groups had blood collected then were euthanized had one hind limb removed and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection. Spatially resolved transcriptional profiles were generated from hearts from three flight and three ground control animals as follows. Hearts were cryosectioned longitudinally onto an array of capture probes that bind RNA fixed stained and visualized. Heart sections were then permeabilized to release RNA onto the capture probes and cDNA synthesized on the chip so that its spatial arrangement is encoded within a set of molecular barcodes. cDNA was then released and sequenced. Four to five levels of each heart was analyzed in this manner to allow a 3D reconstruction of the transcriptome.

Transcription profiling of Drosophila exposed to a levitation magnet for different lengths of time

Drosophila samples were exposed to the levitation magnet inside a 25mm diameter tubes with 3 ml of yeast-based Drosophila food in the bottom and a chamber of only 5 mm of height over the food. This small region is needed in order to guarantee that all the flies were located in the effective g area so a maximum of 35 to 40 imagos or pupa can be exposed to each condition per experiment. All experiments were carried out with a parallel 1g external control in a temperature regulated incubator outside the magnet. Three experiments of different duration were performed inside the magnet system to analyze the effect of strong magnetic fields and magnetic levitation during different stages of the Drosophila development

['Relevance of Unfolded Protein Response to Spaceflight-Induced Transcriptional Reprogramming in Arabidopsis']

['Plants are primary producers of food and oxygen on Earth and will likewise be indispensable to the establishment of large-scale sustainable ecosystems and human survival in space. To contribute to the understanding of how plants respond to spaceflight stresses, we examined the relevance of the unfolded protein response (UPR), a conserved signaling cascade that responds to a number of unfavorable environmental stresses, in the model plant species Arabidopsis thaliana. To do so, we compared the transcriptional responses of wild type and UPR-defective seedlings to spaceflight during the SpaceX-CRS12 mission to the International Space Station. We established that orbital culture substantially altered the expression of hundreds of stress related genes compared to ground control conditions. Although many of these genes were differentially regulated in the UPR mutants in the ground control conditions compared to wild type, their expression was largely equalized in all genotypes by flight. Our results have yielded new information on how plants respond to growth in orbit and support the hypothesis that spaceflight induces the activation of signaling pathways that compensate for the loss of UPR regulators in the control of downstream transcriptional regulatory networks.']

Transcription profiling of activated human T cells induced by microgravity to identify apoptotic genes and other immune response genes

The purpose of this study was to search for microgravity-sensitive genes specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. Experiment Overall Design: Two-group design with paired samples i.e. one 1G and one MMG culture came from the same donor. Therefore 6 samples came from 3 different donors. Experiment Overall Design: Donor 1 :GSM96146,GSM96147 Experiment Overall Design: Donor 2: GSM96148 GSM96149 Experiment Overall Design: Donor 3: GSM96150,GSM96151 Experiment Overall Design: Total RNA was submitted to and then labeled hybridized and data generated by the Baylor College Medicine Microarray Core Facility (333E One Baylor Plaza Houston TX 77030).

Transcription profiling of rat response to changes in developmental stage - 3 types of tissue 3 gravity conditions 2 developmental conditions

Transcriptional crosstalk between mammary gland liver and adipose tissue Experiment Overall Design: Pregnant and Lactating rats exposed to 3 gravity conditions

Transcription profiling of Arabidopsis seedings exposed to UV-B irradiation

Interaction of COP1 and UVR8 which regulate UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis thaliana.

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.

An environment with strong gravitational and magnetic field alterations synergizes to promote variations in Arabidopsis thaliana callus global transcriptional state

Using diamagnetic levitation we have exposed A. thaliana in vitro callus cultures to five environments with different levels of effective gravity (from levitation i.e. simulated mg* to 2g*) and magnetic fields (10.1 to 16.5 Tesla) and we have compared the results with those of similar experiments done in a Random Position Machine (simulated micro g) and a Large Diameter Centrifuge (2g) free of high magnetic fields. Microarray analysis indicates that there are changes in overall gene expression of the cultured cells exposed to these unusual environments but also that gravitational and magnetic field produce synergic variations in the steady state of the transcriptional profile of A. thaliana. Significant changes in the expression of structural abiotic stress and secondary metabolism genes were observed into the magnet field. These results confirm that the strong magnetic field both at micro g* or 2g* has a significant effect on the expression of these genes but subtle gravitational effects are still observable. These subtle responses to microgravity environments are opposite to the ones observed in a hypergravity one. seven-condition experiment MM2D Arabidopsis culture callus control vs. Treatment (altered gravity simulation GBF). Three GBF were used (LDC (2g) + control RPM (mg) + control and Magnet (mg* 0.1g* 1g* 1.9g* 2g*) + control). Biological replicates: 3 replicates in all conditions and controls except 1.9g* (2 replicates)

Salmonella Typhimurium transcription profiles in space flight

Salmonella transcription profiles were obtained from samples flown on space shuttle mission STS-115 and compared to profiles from Salmonella grown under identical conditions on the ground. Keywords: stress response transcriptional profile Triplicate experimental samples were hybridized to slides that contain three identical Salmonella ORF arrays. Each hybridization was performed with Cy3 labeled total RNA and Cy5 labeled gDNA as control.

['Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men']

['Physical inactivity is a risk factor for insulin resistance. We examined the effect of nine days of bed rest on basal and insulin stimulated expression of genes potentially involved in insulin action by applying hypothesis-generating microarray in parallel with candidate gene real-time PCR approaches in 20 healthy, young men. Furthermore, we investigated whether bed rest affected DNA methylation in the promoter region of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) gene. Subjects were re-examined after four weeks of retraining. Findings: Bed rest induced insulin resistance and altered the expression of more than 4,500 genes. These changes were only partly normalized after four weeks of retraining. Pathway analyses revealed significant down-regulation of 34 pathways, predominantly those of genes associated with mitochondrial function including PPARGC1A. Despite induction of insulin resistance, bed rest resulted in a paradoxically increased response to acute insulin in the general expression of genes, particularly those involved in inflammation and endoplasmatic reticulum (ER) stress. Furthermore, bed rest changed gene expressions of several insulin resistance and diabetes candidate genes. We also observed a trend toward increased PPARGC1A DNA methylation after bed rest. Conclusions: Impaired expression of PPARGC1A and other genes involved in mitochondrial function as well as a paradoxically increased response to insulin of genes involved in inflammation and ER stress may contribute to the development of insulin resistance induced by bed rest. Lack of complete normalization of changes after four weeks of exercise retraining underscores the importance of maintaining a minimum of daily physical activity.']

Transcriptional analysis of soleus from mice preserved with the Rapid Freeze hardware

Data from the NASA Rodent Research-1 (RR-1) mission showed that gene-expression levels in mouse livers are different depending on what tissue preservation protocol is used and that slow freezing is not an effective method for preserving signals in gene-expression data. In response to these and other observations the Rapid Freeze hardware was built for use on the International Space Station. The Rapid Freeze hardware freezes mouse tissues (Glovebox freezer) and whole carcasses (Cryochiller) at rates closely mimicking those attained with immersion in liquid nitrogen. Because this hardware will be used extensively on future rodent research missions it is crucial to understand whether or not it preserves signals in gene expression data in order to maximize the value of these rare and expensive spaceflight experiments. Therefore this study was designed with three goals: 1) To evaluate the temperature profile of the Cryochiller and Glovebox freezer cartridges (Rapid Freeze hardware) over time during mock on-orbit procedures; 2) To determine the freezing profiles of tissues and carcasses using Rapid Freeze hardware at both optimal and sub-optimal temperatures (to mimic on-orbit operations) compared with those frozen in liquid nitrogen (the laboratory gold standard) or frozen at -80 C (the current standard method); 3) To identify gene expression changes in a) tissues that were frozen via the Glovebox freezer and b) tissues dissected from whole or partial carcasses that were frozen via the Cryochiller versus tissues that were frozen via control methods (liquid nitrogen or -80C slow freeze) to assess how the Rapid Freeze hardware compares with laboratory gold standard practices and our current standard methods.

The Heat-Inducible Transcription Factor HsfA2 Enhances Anoxia Tolerance in Arabidopsis

Anoxia induces several heat shock proteins and a heat pre-treatment can acclimatize Arabidopsis seedlings to a subsequent anoxic treatment. In this work we analyzed the response of Arabidopsis seedlings to anoxia heat and a combined heat+anoxia stress. A significant overlapping between the anoxic and heat shock responses has been observed by whole-genome microarray analysis. Overall design: We treated Arabidopsis seedling 4-days old dark germinated with: Control (23C dark liquid Murashige-Skoog medium containing 30mM sucrose). Heat-treated (38C for 90 minutes dark liquid Murashige-Skoog medium containing 30mM sucrose). Anoxia-treated (23C under anoxia for 6h dark liquid Murashige-Skoog medium containing 30mM sucrose). Combined heat+Anoxia-treatment (23C treated at 38C for 90 min and thereafter under anoxia for 6h dark liquid Murashige-Skoog medium containing 30mM sucrose). Two biological replicates for each condition.

Transcriptional analysis of livers from mice preserved with the Rapid Freeze hardware

Spatially resolved transcriptional analysis of brains from mice flown on the RR-3 mission

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c 18-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age and matching sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight ground controls and basal groups had blood collected then were euthanized had one hind limb removed and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection. Brain samples from three flight and three ground control animal groups were cut in half between hemispheres. One hemisphere of each brain was used for generating spatially resolved transcriptional profiling data. Hemispheres were cryosectioned so that 2 consecutive sections from the hippocampus of each brain was placed on Visium Gene Expression arrays. Samples were fixed stained with Hematoxylin and Eosin and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently following the 10XGenomics Visium Gene Expression protocol Spatial Transcriptomics RNA-seq libraries were prepared and sequenced.

Transcription profiling of human peripheral blood to development gene expression signatures for practical radiation biodosimetry

To further development of our gene expression approach to biodosimetry we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish radiation dose across an exposure range relevant for medical decision-making in a radiological emergency. Human peripheral blood from healthy donors was irradiated ex vivo and a 74-gene consensus signature was identified that distinguished between four radiation doses (0.5 2 5 and 8 Gy) and control samples. The same set of genes separated samples by exposure level at both six and 24 hours after treatment with overlap evident only at the highest two doses (5 and 8 Gy). Expression of five genes (CDKN1A FDXR SESN1 BBC3 and PHPT1) from this signature was quantified in the same RNA samples by real-time PCR confirming low variability between donors as well as the predicted radiation response pattern. Experiment Overall Design: Radiation induced gene expression in human blood was measured at 6 and 24 hours after exposure to doses of 0 0.5 2 5 and 8 Gy g-rays. Five independent experiments were performed at each time (6 or 24 hours) using different donors for each experiment

Transcription profiling of atm mutant adm mutant and wild type whole plants and roots of Arabidopsis after gamma ray irradiation in a time series

Whole seedlings of wild type (4d) and atm mutants (4d) have been analyzed after a gamma ray irradiation of 0.75h 1.5h 3h & 5h (time course). Roots of wt (4d) atm (3d) and atr (4d) mutants have been analyzed after a 1h irradiation. Ataxia Telangiectasia Mutated (ATM) encodes a large protein with a phosphatidylinositol 3-kinase (PI3K)-like domain at the C terminus (reviewed by Rotman and Shiloh 1998). PI3K-related proteins make up a large family of Ser-Thr protein kinases numerous members of which are involved in the regulation of cell cycle progression responses to DNA damage and the maintenance of genomic stability (Hoekstra 1997). AtATM plays an essential role in meiosis and in the somatic response to DNA damage in plants similar to the function of ATM in mammals and other eukaryotes. Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation transmitting DNA damage signals to downstream effectors of cell-cycle progression.