**3. Discussion**

The Space Shuttle and the International Space Station (ISS) are engineering miracles [63]. However, the biological importance of the ISS remains mired in controversy over academic and commercial priorities and funding. Do microgravity models provide specialized biological conditions that can be exploited for translational application in the commercial healthcare sector? This is a matter of critical importance to national funding priorities, international competitiveness of the United States, and the health status of Americans and our allies [64]. Not humble or simple questions.

To quote Gene Kranz, the Johnson Space Center Flight Director best known for his leadership during the Apollo13 crisis: "Let's look at this from the point of view of status"[65]. To plan strategically, we need to understand the data we have, the timing of samples, the models studied, and the specifics of the analysis.

This article aims to summarize the status of genome-wide microarray studies in models of microgravity and the true microgravity of space: the data available, the timing of samples, the models studied, and the specifics of the analysis. Specifically, identifying the genes and pathways that may be of central importance in microgravity, may direct areas of commercial and health translation.

With genome-wide array technologies, it becomes possible to study microgravity effects on living organisms at the cellular and molecular levels and in much greater detail, which is key to elucidation of the molecular mechanisms of this environmental factor. Since 2011, there has been both an increase in the number of relevant publications and an increase in the quality of retrievable data. This has allowed us to expand our list of putative "major space genes" from the initial number of eight (from our initial attempt toward the identification of the major space genes using literature data [4]) to the present number of 129 genes, at the comparable confi‐ dence level. Because of the increase in the number of relevant studies, we were able to go beyond the level of four or more studies in the selection of putative major space genes which was the highest level possible in our previous report [4]. We proceeded to go two levels higher to identify genes found differentially regulated in five or more and six or more studies to be 35 genes and 13 genes, respectively (Table 2). These two additional levels of higher stringency for the selection of putative major space genes enable a significantly higher level of confidence. Our further bioinformatics analysis on the differentially regulated genes showed interesting connections among many of the putative major space genes and several key pathways.

**Species**

106 Biotechnology

Mouse

Mouse

**Cell**

calvarial osteoblasts

Dermal Fibroblast

Rat gastrocnemius muscles

**3. Discussion**

Not humble or simple questions.

and health translation.

studied, and the specifics of the analysis.

**µg**

**Duration**

**CTGF**

**CYR61**

**EGR1**

**FN1**

Mouse hepatocytes RWV 24h + [7]

Human Muscle BR 60d + + + [25] Mouse Soleus Muscle HLS 7d - [56]

BR= bed rest HLS = Hind limb suspension ISS = International Space Station ML = Magneto Levitation PF = Parabolic flight RPM = Random Positioning Machine RWV = Rotating Wall Vessel STS = Space Shuttle. "+" Indicates up-regula‐ tion; "-" Indicates down-regulation.

**Table 7.** The top 13 putative space genes organized according to types of microgravity. The columns under the gene

The Space Shuttle and the International Space Station (ISS) are engineering miracles [63]. However, the biological importance of the ISS remains mired in controversy over academic and commercial priorities and funding. Do microgravity models provide specialized biological conditions that can be exploited for translational application in the commercial healthcare sector? This is a matter of critical importance to national funding priorities, international competitiveness of the United States, and the health status of Americans and our allies [64].

To quote Gene Kranz, the Johnson Space Center Flight Director best known for his leadership during the Apollo13 crisis: "Let's look at this from the point of view of status"[65]. To plan strategically, we need to understand the data we have, the timing of samples, the models

This article aims to summarize the status of genome-wide microarray studies in models of microgravity and the true microgravity of space: the data available, the timing of samples, the models studied, and the specifics of the analysis. Specifically, identifying the genes and pathways that may be of central importance in microgravity, may direct areas of commercial

With genome-wide array technologies, it becomes possible to study microgravity effects on living organisms at the cellular and molecular levels and in much greater detail, which is key

symbols show the direction of differential regulation in each cell type and microgravity condition.

**FOS**

**HSPA1A**

RWV 5d - - - - [44]

RWV 2d/7d - [50]

HLS 16d + [55]

**ITGB4**

**KPNA2**

**MT1**

**MT2**

**MYC**

**TUBA4A**

**TXNIP**

**Citation**

Perhaps the most important insights gleaned from this analysis are the limitations of the current data, which in turn suggests vectors for future analysis [64]. The animal and cellular ground control models are diverse, and incompletely characterized for advantages and limitations and the durations of exposure to true microgravity or simulations, are broadly spread. The analysis platforms vary, but are similar in scope and sensitivity. Despite these limitations, some themes emerge. The effects of radiation are apparent, as are changes in redox potential in the response to microgravity. These are both pathways relevant to tumors.

We found thioredoxin-interacting protein (TXNIP) to be one of the putative space genes that was most frequently differentially expressed in microgravity (Table 2). TXNIP was upregulated by 10.5 fold in human umbilical vein endothelial cells (HUVECS) in the ISS, making it the most significantly altered gene expression in that study of true microgravity. In micro‐ gravity emulated by the random positioning machine (RPM), TXNIP in endothelial cells was down regulated by more than 4-fold after five days and slightly up-regulated after 7 days [35]. However, the same group found TXNIP to be up-regulated in thyroid cancer cells exposed to emulated microgravity [33]. TXNIP is a tumor suppressor in thyroid cells [66]. Up-regulation of this tumor suppressor gene may explain why Grosse et al found that thyroid cancer cells became less aggressive when grown in emulated microgravity [33]..

Metallotionin I and II (MT-I and –II) are also among the top 13 putative major space genes affected (Table 2). These isoforms function primarily in metal ion homeostasis, scavenging of ROS, redox status, immune defense responses, cell proliferation and cell death [67, 68].

Changes in redox-related genes were also identified using fitness profiling of yeast deletion series grown in spaceflight and ground [69] using next generation sequencing. Techniques such as next generation sequencing technology, offer the potential for far more nuanced and detailed analysis of the whole genome, and secondary pathway analysis of the sequence data generated [69]. The genome-wide sensitivity profiles obtained from spaceflight were queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. The effects of spaceflight have high concordance with the effects of changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

The redox state of tumor cells is frequently disrupted and this is difficult to reproduce in ground-based cultures [70-72]. Hence, redox-dependent drug metabolism in tumors may be uniquely modeled in microgravity. Many of the genes most commonly associated with microgravity-related changes have been identified to have roles in cell cycling, which is critical for both carcinogenesis and responses to radiation damage. EGR1 (Early growth response protein 1; also referred to as Zif268, zinc finger protein 225; and NGFIA, nerve growth factorinduced protein A) is a tumor suppressor transcription factor for differentiation and mitogen‐ esis. MYC encodes for a transcription factor with roles in cell cycle progression, apoptosis and cellular transformation [73]. Karyopherin alpha 2 (KPNA2) promotes tumorigenicity through up-regulation of c-MYC [74]. In endothelial cells grown in microgravity KPNA2 was shown to up-regulated in both adherent cells and multicellular conglomerates at 5 days in simulated microgravity, but down-regulated in multicellular conglomerates at 7 days [35]. HSP70 family members such as HSPA1A have been found to be critical to cellular homeostasis and cancer cell survival[75]. Integrin, Beta 4 (ITGB4) is the receptor for laminin and has been found to be up-regulated in thyroid cancer cells and MCTS grown on RPM for 24 hours [33].

Similar to the results of the current meta-analysis, Nislow et al. 2014 found spaceflight has subtle but significant effects on core cellular processes including growth control via RNA and ribosomal biogenesis, metabolism, modification and decay pathways. Furthermore, significant roles for DNA repair and replication, response to pH signaling, control of gene expression, and mitochondrial function were observed. The yeast chemogenetic analysis of spaceflight samples strongly implicates DNA and RNA damage as the major ground based analogs of spaceflight stress. Given the unique, and substantial radiation exposure in space, this is consistent with major radiation-mediated effects which may mimic cancer related effects.

Suppression of the immune system has been thought to be an important side-effect of micro‐ gravity exposure [1, 2, 31, 76]. Recently, a global gene expression analysis of human T cells after 1.5 h of stimulation by Con A and anti-CD28 in the LEUKIN spaceflight has identified immediate early genes whose transcription are inhibited in microgravity [31]. The transcrip‐ tion of immediate early genes is inhibited in T cells activated in microgravity, which may be involved in the molecular basis of spaceflight immunosuppression. NF-κB is known to regulate transcription in most mammalian cells and plays a key role in immune responses to antigens, cytokines, UV radiation, oxidized LDL, free radicals, etc. [77-81]. CREB, a cAMP-responsive transcription factor, regulates immune genes including IL-2, IL-6, IL-10, and TNF-α. CREB also promotes survival and proliferation to T-cells, monocytes, and macrophages [82]. EGR1 and MYC, which are among the 48 most significantly down-regulated by microgravity in the T cell activation study are identified as the putative major space genes in the current study (Table 2).

This analysis shows a commonality of gene changes and pathways between different micro‐ gravity models. As data is systematically accumulated, this type of analysis will allow even more meaningful analysis. A key question is whether the unique environment of the ISS induces biological changes of commercial translational value to enhance ground-based health care? In which areas does ISS provide a specific advantage over ground-based biological simulations to direct strategic planning of space based biological science? Within and between ground-based microgravity simulations, can we identify areas where specific techniques are best suited for health care applications? This approach will place space-based science at the center of academic medical center activity [83], and translate to commercial applications.
