Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications

<p>Abstract</p> <p>Background</p> <p>Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous <it>Phialophora verrucosa </it>were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of <it>P. verrucosa </it>and seven strains of <it>Phialophora americana </it>as the allied species.</p> <p>Results</p> <p>Intron-F's (inserted at L798 position) were found in 88% of <it>P. verrucosa </it>strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE.</p> <p>Conclusion</p> <p>The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of <it>P. verrucosa </it>and <it>P. americana </it>into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.</p

Comparative Analysis of Muscle Atrophy During Spaceflight, Nutritional Deficiency and Disuse in the Nematode Caenorhabditis elegans

While spaceflight is becoming more common than before, the hazards spaceflight and space microgravity pose to the human body remain relatively unexplored. Astronauts experience muscle atrophy after spaceflight, but the exact reasons for this and solutions are unknown. Here, we take advantage of the nematode C. elegans to understand the effects of space microgravity on worm body wall muscle. We found that space microgravity induces muscle atrophy in C. elegans from two independent spaceflight missions. As a comparison to spaceflight-induced muscle atrophy, we assessed the effects of acute nutritional deprivation and muscle disuse on C. elegans muscle cells. We found that these two factors also induce muscle atrophy in the nematode. Finally, we identified clp-4, which encodes a calpain protease that promotes muscle atrophy. Mutants of clp-4 suppress starvation-induced muscle atrophy. Such comparative analyses of different factors causing muscle atrophy in C. elegans could provide a way to identify novel genetic factors regulating space microgravity-induced muscle atrophy

The Effectiveness of RNAi in Caenorhabditis elegans Is Maintained during Spaceflight

PublishedJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Public Library of Science via the DOI in this record.BACKGROUND: Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. METHODS: Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at -80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. RESULTS: After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. CONCLUSIONS: Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Japan Society for the Promotion of Science, and “Ground-Based Research Announcement for Space Utilization” promoted by the Japan Space Forum. TE was supported by the Medical Research Council UK (G0801271). NJS was supported by the National Institutes of Health (NIH NIAMS ARO54342). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Loss of physical contact in space alters the dopamine system in <i>C.</i> <i>elegans</i>

Progressive neuromuscular decline in microgravity is a prominent health concern preventing interplanetary human habitation. We establish functional dopamine-mediated impairments as a consistent feature across multiple spaceflight exposures and during simulated microgravity in C. elegans. Animals grown continuously in these conditions display reduced movement and body length. Loss of mechanical contact stimuli in microgravity elicits decreased endogenous dopamine and comt-4 (catechol-O-methyl transferase) expression levels. The application of exogenous dopamine reverses the movement and body length defects caused by simulated microgravity. In addition, increased physical contact made comt-4 and dopamine levels rise. It also increased muscular cytoplasmic Ca2+ firing. In dop-3 (D2-like receptor) mutants, neither decrease in movement nor in body length were observed during simulated microgravity growth. These results strongly suggest that targeting the dopamine system through manipulation of the external environment (contact stimuli) prevents muscular changes and is a realistic and viable treatment strategy to promote safe human deep-space travel

Gene expression of the RNAi apparatus is unaltered by spaceflight.

<p>Adult hermaphrodites (2^nd generation) collected at 8 d during spaceflight showed no change in gene expression for components of the RNAi machinery, which were not different ground controls (<i>P</i>>0.05). mRNA expression values are the average of 18 separate probes over six microarrays, and are relative to an internal control (1G controls).</p

Degradation of α-actin is prevented by <i>asp-4</i> and <i>asp-6</i> RNAi in spaceflight and ground control (GC).

<p>Dauer animals treated for 4 d with RNAi vector control (VC) developed to adulthood. In both GC and spaceflight conditions animals displayed major loss of muscle specific α-actin following lysis in the absence of lysosomal protease inhibitors. Treatment with <i>asp-4</i> and <i>asp-6</i> RNAi for 4 d in GC and spaceflight resulted in a preservation of α-actin levels. A, representative immunoblot; B, average non-normalised quantification of three Western blots against α-actin. ** denotes significant difference from both GC and spaceflight VC conditions (<i>P</i><0.01).</p

RNAi against <i>gfp</i> reduces chromosomal GFP expression in spaceflight and ground control (GC).

<p>Animals fed RNAi vector control for 4 d from L1 larvae developed into normal adults in GC and spaceflight conditions. These animals also displayed GFP expression in oocytes and embryos in GC and spaceflight. Animals fed <i>gfp</i> RNAi for 4 d also developed normally to adulthood in GC and spaceflight, and demonstrated a loss of GFP expression in both GC and spaceflight. Scale bars represent 50 µm.</p