Objective: The objective of this study is to study the transcriptome of zebrafish embryos subjected to simulated microgravity and explore affected biological pathways, especially DNA damage response (DDR). The research question is whether simulated microgravity can have an impact on the basic biology of cell division, DNA repair, inflammation, and other vital cellular mechanisms. To validate that such experiments can yield relevant insights into human health and microgravity, we will correlate the found effects of simulated microgravity on zebrafish embryos with the reported effects of spaceflight on astronauts. Methods: 12 wild-type zebrafish embryos of both sexes, and of 3 to 12 months of age were microinjected with 2 nL (1 μg/μL) poly I:C or mock PBS buffer (0.5% phenol red, 240 mM KCl, and 40 mM HEPES at pH 7.4) using a microinjector followed by subjecting them immediately to the simulated microgravity conditions generated by RCCS or the normal gravity conditions in a cell culture dish. RNA-SEQ was performed on the samples according to the standard protocol. Raw gene counts data were obtained from the public domain (NASA Gene Labs) and subjected to further downstream analyses. Differential gene expression was performed using DESeq2. The results were annotated using pathway enrichment analysis (GSEA) on the KEGG pathway database and compared with the result of the NASA twin study. Result: Similar to previously published analysis, we found that a significant number of genes were differentially expressed under simulated microgravity conditions. We identified a total of 7542 genes out of 16532 when comparing expression between the groups: simulated microgravity and normal gravity (padj. value <0.05, log2 fold change in between -2 and 2). Out of these genes, 4504 were found to be up-regulated while 3038 were down-regulated compared to controls. Pathway enrichment analysis revealed that simulated microgravity has an effect on vital basic biological processes like DNA repair, peptide transport, and metabolism. Various other well-known signalling pathways like Notch signalling, wnt signalling, and p53 signalling were also significantly altered. These pathways are known to play an important role in DDR. To explore if the same pathways were also altered in humans, we explored the NASA twin study data and found that DDR was also significantly affected in the astronaut but due to ionizing radiation. Upon further investigation, we found that 62 genes belonging to the DDR pathway were mutually differentially expressed in Scott Kelly and the zebrafish embryos. However, there were 29 significantly differentially expressed genes belonging to the DDR pathway in zebrafish embryos that were not found to be differentially expressed in Scott Kelly. Out of these 29 genes, 14 were specific to zebrafish. Upon further investigation, we found that the DDR pathway is affected differently in simulated microgravity as compared to ionizing radiation. Conclusion: Simulated microgravity alters numerous biological pathways in zebrafish embryos, including DDR. But the nature of it is different from that of real spaceflight induced DDR. These observations should be investigated further to actually understand the nature of DNA damage response during spaceflights.
Experimental and Numerical Study of Capillary Driven Flow in Vane-type Tank under Normal Gravity and Microgravity Conditions
