Research Containing: Genetics

A mutational analysis of Caenorhabditis elegans in space

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.

Spaceflight engages heat shock protein and other molecular chaperone genes in tissue culture cells of Arabidopsis thaliana

by cfynanon 9 June 2015in Biology & Biotechnology No comment

PREMISE OF THE STUDY: Gravity has been a major force throughout the evolution of terrestrial organisms, and plants have developed exquisitely sensitive, regulated tropisms and growth patterns that are based on the gravity vector. The nullified gravity during spaceflight allows direct assessment of gravity roles. The microgravity environments provided by the Space Shuttle and International Space Station have made it possible to seek novel insights into gravity perception at the organismal, tissue, and cellular levels. Cell cultures of Arabidopsis thaliana perceive and respond to spaceflight, even though they lack the specialized cell structures normally associated with gravity perception in intact plants; in particular, genes for a specific subset of heat shock proteins (HSPs) and factors (HSFs) are induced. Here we ask if similar changes in HSP gene expression occur during nonspaceflight changes in gravity stimulation. METHODS: Quantitative RT-qPCR was used to evaluate mRNA levels for Hsp17.6A and Hsp101 in cell cultures exposed to four conditions: spaceflight (mission STS-131), hypergravity (centrifugation at 3 g or 16 g), sustained two-dimensional clinorotation, and transient milligravity achieved on parabolic flights. KEY RESULTS: We showed that HSP genes were induced in cells only in response to sustained clinorotation. Transient microgravity intervals in parabolic flight and various hypergravity conditions failed to induce HSP genes. CONCLUSIONS: We conclude that nondifferentiated cells do indeed sense their gravity environment and HSP genes are induced only in response to prolonged microgravity or simulated microgravity conditions. We hypothesize that HSP induction upon microgravity indicates a role for HSP-related proteins in maintaining cytoskeletal architecture and cell shape signaling.

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp DNA Mini Kit, Reischl et al.'s method and FTA Elute. FTA Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions.

Toll mediated infection response is altered by gravity and spaceflight in Drosophila

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Space travel presents unlimited opportunities for exploration and discovery, but requires better understanding of the biological consequences of long-term exposure to spaceflight. Immune function in particular is relevant for space travel. Human immune responses are weakened in space, with increased vulnerability to opportunistic infections and immune-related conditions. In addition, microorganisms can become more virulent in space, causing further challenges to health. To understand these issues better and to contribute to design of effective countermeasures, we used the Drosophila model of innate immunity to study immune responses in both hypergravity and spaceflight. Focusing on infections mediated through the conserved Toll and Imd signaling pathways, we found that hypergravity improves resistance to Toll-mediated fungal infections except in a known gravitaxis mutant of the yuri gagarin gene. These results led to the first spaceflight project on Drosophila immunity, in which flies that developed to adulthood in microgravity were assessed for immune responses by transcription profiling on return to Earth. Spaceflight alone altered transcription, producing activation of the heat shock stress system. Space flies subsequently infected by fungus failed to activate the Toll pathway. In contrast, bacterial infection produced normal activation of the Imd pathway. We speculate on possible linkage between functional Toll signaling and the heat shock chaperone system. Our major findings are that hypergravity and spaceflight have opposing effects, and that spaceflight produces stress-related transcriptional responses and results in a specific inability to mount a Toll-mediated infection response.

Latent and lytic Epstein-Barr virus gene expression in the peripheral blood of astronauts

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Epstein-Barr virus (EBV) latent and replicative gene transcription was analyzed in peripheral blood B-lymphocytes from astronauts who flew on short-duration ( approximately 11 days) Shuttle missions and long-duration ( approximately 180 days) International Space Station (ISS) missions. Latent, immediate-early, and early gene replicative viral transcripts were detected in samples from six astronauts who flew on short-duration Shuttle missions, whereas viral gene transcription was mostly absent in samples from 24 healthy donors. Samples from six astronauts who flew on long-duration ISS missions were characterized by expanded expression of latent, immediate-early, and early gene transcripts and new onset expression of late replicative transcription upon return to Earth. These data indicate that EBV-infected cells are no longer expressing the restricted set of viral genes that characterize latency but are expressing latent and lytic gene transcripts. These data also suggest the possibility of EBV-related complications in future long-duration missions, in particular interplanetary travel.

Description of International Caenorhabditis elegans Experiment first flight (ICE-FIRST)

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Traveling, living and working in space is now a reality. The number of people and length of time in space is increasing. With new horizons for exploration it becomes more important to fully understand and provide countermeasures to the effects of the space environment on the human body. In addition, space provides a unique laboratory to study how life and physiologic functions adapt from the cellular level to that of the entire organism. Caenorhabditis elegans is a genetic model organism used to study physiology on Earth. Here we provide a description of the rationale, design, methods, and space culture validation of the ICE-FIRST payload, which engaged C. elegans researchers from four nations. Here we also show C. elegans growth and development proceeds essentially normally in a chemically defined liquid medium on board the International Space Station (10.9 day round trip). By setting flight constraints first and bringing together established C. elegans researchers second, we were able to use minimal stowage space to successfully return a total of 53 independent samples, each containing more than a hundred individual animals, to investigators within one year of experiment concept. We believe that in the future, bringing together individuals with knowledge of flight experiment operations, flight hardware, space biology, and genetic model organisms should yield similarly successful payloads.

Oxidative stress and antioxidant capacity in barley grown under space environment

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The gene expression and enzyme activity of superoxide dismutase, catalase, and ascorbate peroxidase in the space-grown barley were not significantly different from those of the ground-grown barley. Cu2+ reducing and radical scavenging activities in an extract of the space-grown barley were lower than those of the ground-grown barley by 0.7 fold, suggesting that the space environment does not induce oxidative stress, and reduces antioxidant capacity in plants.

A possible involvement of autophagy in amyloplast degradation in columella cells during hydrotropic response of Arabidopsis roots

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Seedling roots display not only gravitropism but also hydrotropism, and the two tropisms interfere with one another. In Arabidopsis (Arabidopsis thaliana) roots, amyloplasts in columella cells are rapidly degraded during the hydrotropic response. Degradation of amyloplasts involved in gravisensing enhances the hydrotropic response by reducing the gravitropic response. However, the mechanism by which amyloplasts are degraded in hydrotropically responding roots remains unknown. In this study, the mechanistic aspects of the degradation of amyloplasts in columella cells during hydrotropic response were investigated by analyzing organellar morphology, cell polarity and changes in gene expression. The results showed that hydrotropic stimulation or systemic water stress caused dramatic changes in organellar form and positioning in columella cells. Specifically, the columella cells of hydrotropically responding or water-stressed roots lost polarity in the distribution of the endoplasmic reticulum (ER), and showed accelerated vacuolization and nuclear movement. Analysis of ER-localized GFP showed that ER redistributed around the developed vacuoles. Cells often showed decomposing amyloplasts in autophagosome-like structures. Both hydrotropic stimulation and water stress upregulated the expression of AtATG18a, which is required for autophagosome formation. Furthermore, analysis with GFP-AtATG8a revealed that both hydrotropic stimulation and water stress induced the formation of autophagosomes in the columella cells. In addition, expression of plastid marker, pt-GFP, in the columella cells dramatically decreased in response to both hydrotropic stimulation and water stress, but its decrease was much less in the autophagy mutant atg5. These results suggest that hydrotropic stimulation confers water stress in the roots, which triggers an autophagic response responsible for the degradation of amyloplasts in columella cells of Arabidopsis roots.

Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 muM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 muM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.

Antibiotic efficacy and microbial virulence during space flight

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Human space flight is a complex undertaking that entails numerous technological and biomedical challenges. Engineers and scientists endeavor, to the extent possible, to identify and mitigate the ensuing risks. The potential for an outbreak of an infectious disease in a spacecraft presents one such concern, which is compounded by several components unique to an extraterrestrial environment. Various factors associated with the space flight environment have been shown to potentially compromise the immune system of astronauts, increase microbial proliferation and microflora exchange, alter virulence and decrease antibiotic effectiveness. An acceptable resolution of the above concerns must be achieved to ensure safe and efficient space habitation. To help bring this about, scientists are employing advances in biotechnology to better characterize the relevant variables and establish appropriate solutions. Because many of these clinical concerns are also relevant in terrestrial society, this research will have reciprocal benefits back on Earth.

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Research Containing: Genetics

A mutational analysis of Caenorhabditis elegans in space

Spaceflight engages heat shock protein and other molecular chaperone genes in tissue culture cells of Arabidopsis thaliana

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

Toll mediated infection response is altered by gravity and spaceflight in Drosophila

Latent and lytic Epstein-Barr virus gene expression in the peripheral blood of astronauts

Description of International Caenorhabditis elegans Experiment first flight (ICE-FIRST)

Oxidative stress and antioxidant capacity in barley grown under space environment

A possible involvement of autophagy in amyloplast degradation in columella cells during hydrotropic response of Arabidopsis roots

Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments

Antibiotic efficacy and microbial virulence during space flight