The ability to clear bacterial infections is maintained in adult flies after spaceflight.

<p>Adults were infected with <i>E. coli</i> HB101 immediately after return from spaceflight. The overall capacity to clear the bacterial infection was measured by quantifying the live bacteria (CFU) remaining in the animal up to 3 days following infection. Flight adults cleared bacteria as effectively as controls immediately after flight, and more efficiently at days 2 and 3 post infection (<i>p-</i>value <0.05).</p

Spaceflight alters phagocytosis.

<p>A. The number of actively phagocytosing cells is reduced by spaceflight. At 15, 25, and 35 minutes, a significantly lower number of cells engulfed <i>E. coli</i> in hemocyte cultures from spaceflight larvae. The calculated <i>p</i>-values were 0.32, 2.16E-07, 3.38E-05, 3.86E-08, and 5.8E-04 for the 5, 15, 25, 35 and 45-minute time points respectively. The data points are tight and give calculated standard error values in the 10⁻² range, not visible on the graph. B. The phagocytic capacity of larval plasmatocytes is stunted. The number of Alexa594-labeled <i>E. coli</i> engulfed by larval plasmatocytes in culture was compared in samples from flight and ground up to 45 minutes after infection. Cells from both space- and ground-reared larvae had similar levels of activity up to 25 minutes post-infection, while after 35 minutes space larvae plasmatocytes had significantly reduced phagocytic capacity.</p

The constitutive expression of humoral immunity genes is altered in spaceflight larvae.

<p>Specific classes of genes involved in the humoral immune response that were altered by spaceflight included: the AMP response pathway, the stress factors turandot A and C, the lysozyme genes and the melanization gene black cells. Although some of the genes upstream of AMPs were upregulated, the target AMP effectors were all downregulated. The statistical significance of gene changes is shown by the adjusted <i>p</i>-value at an FDR threshold of less than 0.05.</p

Humoral responses are downregulated in spaceflight larvae.

<p>Gene Ontology (GO) categories of defense response show altered gene expression in flight larvae compared to ground-reared larvae: 30 out of the 37 classified as defense response are downregulated in flight. These include the majority of response to bacteria and in particular humoral response genes. Six experimental repeats of each of the samples (flight and ground) were used to extract RNA for microarray analysis and are represented in individual columns in the figure. Rows represent levels of individual gene expression in each of the experimental repeats. Data is shown following Z-score transformation. Red colors indicate Z-scores >0 (above mean), blue colors indicate Z-scores <0 (below mean). The gene names can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015361#pone-0015361-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015361#pone-0015361-t002" target="_blank">2</a>.</p

The expression of genes involved in hemocyte maturation and pathogen recognition and binding is downregulated in spaceflight larvae.

<p>Among the major categories of genes that were affected in larvae that developed in microgravity were the genes that mark the differentiation and maturation of plasmatocytes (l(3)mbn, Pxn, Pvf2,) and crystal cells (lz); genes involved in <i>E. coli</i> binding and phagocytosis; and the pattern recognition receptors GNBP3 and PGRPs which activate the humoral pathways. Gene expression level in spaceflight samples compared to ground controls is expressed as fold change. The statistical significance of the gene changes is shown by the adjusted <i>p</i>-value. The false discovery rate (FDR) cut-off was less than 0.05 for all genes.</p

The humoral response to <i>E. coli</i> infection in adults after spaceflight.

<p>The time course of gene expression after bacterial infection was compared between flight and ground-reared adult females using qPCR. Data was averaged from two experimental samples, with the exception of relish. Mock- infected flight RNA levels were normalized against ground control to obtain the pre-infection level, and all time course points for ground and flight were normalized against their respective pre-infection level.</p

Space-reared larvae have fewer plasmatocytes and are smaller.

<p>A. The average number of plasmatocytes per larva was significantly reduced in flight vs ground (<i>p</i>-value <0.008, n = 20). B. Larvae reared in space were significantly smaller than ground-reared larvae (<i>p</i>-value <0.0085, n = 10).</p

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

<div><p>The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen <i>Escherichia coli</i> and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.</p></div

Operations for the WetLab-2 system.

<p>Isolation and purification of RNA from biological tissues on–orbit starts with introduction of cells or tissues for lysis and homogenization into the Sample Preparation Module (SPM), followed by RNA binding to an affinity column, washing, and elution from the module. A Pipette Loader (PL) tool is provided for bubble free fluid transfer to a repeater pipette. This is then used to dispense accurate volumes of purified RNA into a centrifuge rotor/rack of lyophilized reagent tubes with enzymes and regents for reverse transcription and Taqman RT-qPCR. Data is available on-orbit within 3h of initiating the experiment, and transmitted by ISS to NASA Marshall Space Flight Center for emailing to investigators within 2 h of clean-up. A more detailed description of the process can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183480#pone.0183480.s001" target="_blank">S1 Text</a>.</p

<i>E</i>. <i>coli</i> and mouse liver assay validation using 100 ng control RNA.

<p><i>E</i>. <i>coli</i> and mouse liver assay validation using 100 ng control RNA.</p