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.

Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift a large-scale analysis of mRNA expression levels was performed in the brains of 2 weeks control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extent all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes while the most down-regulated genes were those of the JAE class (Cell junction Adhesion Extracellular Matrix). TIC genes comprised 16% of those whose expression was altered including sodium channel nonvoltage-gated 1 beta (Scnn1b) glutamate receptor (Grin1) voltage-dependent anion channel 1 (Vdac1) calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by GeneMAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response learning and memory ion channels and cell junction. In particular data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore HU treatment seems to impact on key steps of synaptic plasticity and learning processes. We used brains of four control (C1 C2 C3 C4) and four tail-suspended hindlimb-unloaded (E1 E2 E3 E4) mice to ensure statistical relevance of the study. 60 ug of total RNA extracted in TRIzol from each brain was reversed transcribed into labeled cDNAs using fluorescent Cy5 or Cy3-dUTP (Amersham Biosciences NJ). Differently labeled cDNAs obtained from a pair of biological replicas were co-hybridized overnight at 50C with a microscope slide spotted with ~ 27,000 mouse cDNA sequences produced by the Microarray Core Facility of the Albert Einstein College of Medicine in the xef xbf xbdmultiple yellow xef xbf xbd design (i.e.: C1C2 C3C4 E1E2 E3E4) described in Iacobas DA Fan C Iacobas S et al. Transcriptomic changes in developing kidney exposed to chronic hypoxia. Biochem Biophys Res Commun. 2006 349(1):329-38).

Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift a large-scale analysis of mRNA expression levels was performed in the brains of 2 weeks control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extent all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes while the most down-regulated genes were those of the JAE class (Cell junction Adhesion Extracellular Matrix). TIC genes comprised 16% of those whose expression was altered including sodium channel nonvoltage-gated 1 beta (Scnn1b) glutamate receptor (Grin1) voltage-dependent anion channel 1 (Vdac1) calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by GeneMAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response learning and memory ion channels and cell junction. In particular data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore HU treatment seems to impact on key steps of synaptic plasticity and learning processes. We used brains of four control (C1 C2 C3 C4) and four tail-suspended hindlimb-unloaded (E1 E2 E3 E4) mice to ensure statistical relevance of the study. 60 ug of total RNA extracted in TRIzol from each brain was reversed transcribed into labeled cDNAs using fluorescent Cy5 or Cy3-dUTP (Amersham Biosciences NJ). Differently labeled cDNAs obtained from a pair of biological replicas were co-hybridized overnight at 50C with a microscope slide spotted with ~ 27,000 mouse cDNA sequences produced by the Microarray Core Facility of the Albert Einstein College of Medicine in the xef xbf xbdmultiple yellow xef xbf xbd design (i.e.: C1C2 C3C4 E1E2 E3E4) described in Iacobas DA Fan C Iacobas S et al. Transcriptomic changes in developing kidney exposed to chronic hypoxia. Biochem Biophys Res Commun. 2006 349(1):329-38).

Changes in the physical environment modulate cell responses and may lead to the impairment or even failure of tissue function as a result of mechanotransduction processes. It has been suggested that this situation occurs in some age-related diseases and some pathological conditions observed in space such as cardiovascular deconditioning bone loss muscle atrophy and impaired immune responses. All of these are associated with endothelial dysfunction but the precise mechanism is still unclear. We used the microarray approach to obtain insights into the mechanism responsible for endothelial dysfunction by taking advantage of the challenging environment of gravitational unloading onboard the International Space Station. The effects of gravitational unloading on HUVEC gene expression were investigated by means of cDNA microarray analyses of six randomly chosen samples (three for each of the two conditions of spaceflight and 1g) using Affymetrix Gene Human 1.0 ST Arrays

A combination therapy of electromagnetic fields (EMF) and simulated microgravity (SMG) has not been examined in regenerative medicine of cartilage. In the present study a bioreactor system using extremely low-frequency EMF and SMG was applied during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). It was hypothesized that a beneficial effect of EMF regarding chondrogenesis (COL2A) could be combined with an avoiding effect of SMG regarding hypertrophy (COLXA1) of cartilage. Pellet cultures of hMSCs formed cartilaginous tissue under the addition of growth factors (FGF; TGF-beta3). Pure SMG reduced COLXA1 expression but also COL2A expression of hMSCs. Pure EMF showed no gene expression changes of hMSCs during chondrogenic differentiation. Combining EMF/SMG resulted in a re-increase of COL2A but did not reach control levels. The COL2A to COLXA1 ratio of combined EMF/SMG was not significantly different from control levels. The combination therapy of EMF/SMG did not significantly improve the chondrogenic potential of hMSCs. chondrogenic differentiation electromagnetic stimulation-control 1 timepoint with/without stimulation.

Microgravity leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast is the multinucleated bone resorbing cell. In this study we used NASA developed ground based Rotary Wall Vessel Bioreactor (RWV) Rotary Cell Culture System (RCCS) to simulate microgravity (uXg) conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to ground based control (Xg) mouse bone marrow cultures. We further determined the gene expression profiling of RAW 264.7 osteoclast progenitor cells in microgravity by agilent microarray analysis. Gene expression pattern was functional group clustered by transcriptome analysis using gene ontology tree machine (GOTM) for cell proliferation/survival differentiation and function. We confirm the microgravity modulated gene expression critical for osteoclast differentiation by real-time RT-PCR and Western blot analysis in murine bone marrow cultures. We identify transcription factors such as c-Jun c-Fos PU-1 critical for osteoclast differentiation is up-regulated in microgravity conditions. In addition microgravity resulted in 2.3 and 2.0-fold increase in the level of cathepsin K and MMP-9 matrix metalloproteinase expression in preosteoclast cells involved in the bone resorption process respectively. We also demonstrate a significant increase in the expression levels of M-CSF receptor c-Fms and PLCy2 and S100A8 molecules that play an important role in Ca2+ signaling essential for osteoclast function. Further microgravity stimulated preosteoclast cells showed elevated cytosolic Ca2+ levels compared to ground based control cells. Thus microgravity regulated gene expression profiling in preosteoclast cells provide new insights in to molecular mechanisms and therapeutic targets of osteoclast differentiation/activation responsible for bone loss and fracture risk in astronauts during space flight mission. Microgravity associated with space flight is a challenge for normal bone homeostasis. Astronauts experience 10-15% bone loss during a space flight mission. We aimed to determine the effect of simulated microgravity on osteoclast preosteoclasts cells. RAW264.7 cells (1.5 x 106 /ml) were loaded in RCCS with DMEM containing 10% FBS for 24 h. The cells were stimulated with RANKL (80ng/ml) for 24 h to obtain preosteoclasts in parallel with ground based control cells. Total RNA was isolated using RNAzol reagent (Biotecx Labs Houston TX) from control (Xg) and microgravity (uXg) subjected cells and hybridized with Agilent whole mouse genome 4x44K array system. Slides were washed and scanned on an Agilent G2565 microarray scanner. Data obtained were analyzed with Agilent feature extraction and GeneSpring GX v7.3.1 software packages (Genus biosystem Inc. Northbrook IL USA).

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in microgravity (uG). Immunosuppression during spaceflight is a major barrier to safe long-term human space habitation and travel. The goals of these experiments were to prove that uG was the cause of impaired T cell activation during spaceflight as well as understand the mechanisms controlling early T cell activation. T cells from 4 human donors were stimulated with concanavalin A (ConA) and anti-CD28 onboard the International Space Station (ISS). An onboard centrifuge was used to generate a 1g simultaneous control to isolate the effects of uG from other variables of spaceflight. Microarray expression analysis after 1.5 hours of activation demonstrated that mg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly differentially down-regulated in uG. Importantly several key immediate early genes were inhibited in uG. T cells were isolated from human volunteers. T cells from each donor were kept separate and loaded into individual chambers in separate cassettes for the following treatments: uG non-activated uG activated and 1g activated. Therefore samples represent biological triplicates. Experimental units were launched into space and placed into the KUBIK facility onboard the International Space Station. The 1g units were placed in the central centrifuge positions and centrifuged with an applied 1g force. The uG units were place in the static positions for continued uG exposure. After 30 minutes of pre-incubation uG non-activated units were fixed by addition of RNALater (QIAGEN Valencia CA) removed from the incubator and stored in 4 xc2 xb0C. The uG and 1g activated units were injected with final concentration 10mg/ml Con A and 4mg/ml anti-CD28. These cassettes were replaced into KUBIK on either the centrifuge or static positions and activated for 1.5 hours. Activation was stopped with the addition of RNALater and the units were then moved to 4 xc2 xb0C storage. All units were returned to Earth for analysis.

The spaceflight experiment was carried out using male C57BL/10J mice (8 weeks old at launch). Wild type mice (n=3) were launched by Space Shuttle Discovery and housed on the International Space Station (ISS) for 91 days. They returned to the Earth by Space Shuttle Atlantis. But only one mouse returned to the Earth alive. Whole brain was sampled from the mouse killed by inhalation of carbon dioxide at the Life Sciences Support Facility of Kennedy Space Center within 3-4 hours after landing. After the spaceflight experiment the on-ground experiment was also carried out at the Advanced Biotechnology Center in Genova Italy. A mouse with the same species sex and age was housed in mice drawer system (MDS) which was utilized for the spaceflight (SF) mice for 3 months as the ground control (GC). Another mouse was housed in normal vivarium cage as the laboratory control (LC). Amount of food and water supplementation and environmental conditions were simulated as the flight group. After 3 months brain was sampled from one mouse in group GC and LC respectively. Comprehensive analyses of gene expression were performed in the right brain. Total of 4,000 genes were analyzed. The expression levels of 60 genes significantly changed in response to SF compared with LC and/or GC. The 15 and 16 genes were up- (> 2 folds) and down-regulated (< 0.5 folds) respectively following SF vs. GC. The levels of 58 genes were significantly altered by housing in MDS in space and/or on the ground. Forty seven and 11 genes were significantly up- and down-regulated vs. LC. Twenty seven out of these genes responded to caging in MDS both in space and on the ground. Further 31 genes were influenced by housing in MDS on the Earth. Responses of the characteristics of brain to long-term gravitational unloading were investigated in mice.

In March 2006 murine Bone Marrow Stromal Cells (BMSC) were flown in the Soyuz 12S to the International Space Station to investigate the effects of microgravity on their osteogenic potential in a three-dimensional environment. BMSC were grown in porous bioceramic Skelite disks (dia 9 mm x T 1.2 mm). The constructs were exposed to microgravity for ca. 8 days then fixed for RNA extraction. While the flight experiment was performed in fully automated hardware inside the KUBIK incubator one group of control samples were incubated inside manually operated hardwares (flight control) and the other control group was incubated under routine laboratory conditions (lab control). The altered gene expression profile was analyzed by Mouse Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. Each one of the 28853 genes is represented on the array by approximately 26 probes spread across the full length of the gene providing a more complete and more accurate picture of gene expression than the 3 based expression array design. A few days of microgravity were sufficient to determinate at least at the molecular level an effect in the BMSC; this response expressed a stress condition able to determinate consequences on several compartments and cellular functions. In particular it seems to promote a gene expression known to be associated with neurogenic activity (e.g. axon guidance) perhaps promoting the BMSC capability to be committed in that direction. The osteo-induction by dexamethasone-based medium due to the short duration of stimulation did not have the possibility to manifest itself at the phenotypic level but only partially at the molecular level.

In March 2006 murine Bone Marrow Stromal Cells (BMSC) were flown in the Soyuz 12S to the International Space Station to investigate the effects of microgravity on their osteogenic potential in a three-dimensional environment. BMSC were grown in porous bioceramic Skelite disks (? 9 mm x T 1.2 mm). The constructs were exposed to microgravity for ca. 8 days then fixed for RNA extraction. While the flight experiment was performed in fully automated hardware inside the KUBIK incubator one group of control samples were incubated inside manually operated hardwares (flight control) and the other control group was incubated under routine laboratory conditions (lab control). The altered gene expression profile was analyzed by Mouse Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. Each one of the 28853 genes is represented on the array by approximately 26 probes spread across the full length of the gene providing a more complete and more accurate picture of gene expression than the 3 xc3 x94 xc3 xb8 xce xa9 based expression array design. A few days of microgravity were sufficient to determinate at least at the molecular level an effect in the BMSC; this response expressed a stress condition able to determinate consequences on several compartments and cellular functions. In particular it seems to promote a gene expression known to be associated with neurogenic activity (e.g. axon guidance) perhaps promoting the BMSC capability to be committed in that direction. The osteo-induction by dexamethasone-based medium due to the short duration of stimulation did not have the possibility to manifest itself at the phenotypic level but only partially at the molecular level.