Four Fallacies and an Oversight: Searching for Martian Life.

While it is anticipated that future human missions to Mars will increase the amount of biological and organic contamination that might be distributed on that planet, robotic missions continue to grow in capability and complexity, requiring precautions to be taken now to protect Mars, and particularly areas of Mars that might be Special Regions. Such precautionary cleanliness requirements for spacecraft have evolved over the course of the space age, as we have learned more about planetary environments, and are the subject of regular deliberations and decisions sponsored by the Committee on Space Research (COSPAR). COSPAR's planetary protection policy is maintained as an international consensus standard for spacecraft cleanliness that is recognized by the United Nations Committee on the Peaceful Uses of Outer Space. In response to the paper presented in this issue by Fairén et al. (2017), we examine both their concept of evidence for possible life on Mars and their logic in recommending that spacecraft cleanliness requirements be relaxed to access Special Regions "before it is too late." We find that there are shortcomings in their plans to look for evidence of life on Mars, that they do not support their contention that appropriate levels of spacecraft cleanliness are unaffordable, that there are major risks in assuming martian life could be identified by nucleic acid sequence comparison (especially if those sequences are obtained from a Special Region contaminated with Earth life), and that the authors do not justify their contention that exploration with dirty robots, now, is preferable to the possibility that later contamination will be spread by human exploration. We also note that the potential effects of contaminating resources and environments essential to future human occupants of Mars are both significant and not addressed by Fairén et al. (2017). Key Words: Mars-Special Region-Mission-Life detection-Planetary protection. Astrobiology 17, 971-974.

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

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.

Comparative analysis of Drosophila melanogaster and Caenorhabditis elegans gene expression experiments in the European Soyuz flights to the International Space Station.

The European Soyuz missions have been one of the main routes for conducting scientific experiments onboard the International Space Station, which is currently in the construction phase. A relatively large number of life and physical sciences experiments as well as technology demonstrations have been carried out during these missions. Included among these experiments are the Gene experiment during the Spanish "Cervantes" Soyuz mission and the ICE-1st experiment during the Dutch "Delta" mission. In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight. These experiments indicated relatively large scale changes in gene expression levels in response to spaceflight for two popular model systems, Drosophila melanogaster (Gene) and Caenorabditis elegans (ICE-1st). Here we report a comparative analysis of results from these two experiments. Finding orthologous genes between the fruit fly and the nematode was far from straightforward, reducing the number of genes that we could compare to roughly 20% of the full comparative genome. Within this sub-set of the data (2286 genes), only six genes were found to display identical changes between species (decreased) while 1809 genes displayed no change in either species. Future experiments using ground simulation techniques will allow producing a better, more comprehensive picture of the putative set of genes affected in multicellular organisms by changes in gravity and getting a deeper understanding of how animals respond and adapt to spaceflight.

Leiomodin and tropomodulin in smooth muscle.

Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

Leiomodins: larger members of the tropomodulin (Tmod) gene family.

The 64-kDa autoantigen D1 or 1D, first identified as a potential autoantigen in Graves' disease, is similar to the tropomodulin (Tmod) family of actin filament pointed end-capping proteins. A novel gene with significant similarity to the 64-kDa human autoantigen D1 has been cloned from both humans and mice, and the genomic sequences of both genes have been identified. These genes form a subfamily closely related to the Tmods and are here named the Leiomodins (Lmods). Both Lmod genes display a conserved intron-exon structure, as do three Tmod genes, but the intron-exon structure of the Lmods and the Tmods is divergent. mRNA expression analysis indicates that the gene formerly known as the 64-kDa autoantigen D1 is most highly expressed in a variety of human tissues that contain smooth muscle, earning it the name smooth muscle Leiomodin (SM-Lmod; HGMW-approved symbol LMOD1). Transcripts encoding the novel Lmod gene are present exclusively in fetal and adult heart and adult skeletal muscle, and it is here named cardiac Leiomodin (C-Lmod; HGMW-approved symbol LMOD2). Human C-Lmod is located near the hypertrophic cardiomyopathy locus CMH6 on human chromosome 7q3, potentially implicating it in this disease. Our data demonstrate that the Lmods are evolutionarily related and display tissue-specific patterns of expression distinct from, but overlapping with, the expression of Tmod isoforms.

Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.

The BMP-like signaling mediated by the ligands Dpp and Gbb is required to reinforce the development of most veins in the Drosophila wing. However, the formation of the cross veins is especially sensitive to reductions in BMP-like signaling. We show here that the formation of the definitive cross veins occurs after the initial specification of the longitudinal veins in a process that requires localized BMP-like activity. Since Dpp and Gbb levels are not detectably higher in the early phases of cross vein development, other factors apparently account for this localized activity. Our evidence suggests that the product of the crossveinless 2 gene is a novel member of the BMP-like signaling pathway required to potentiate Gbb of Dpp signaling in the cross veins. crossveinless 2 is expressed at higher levels in the developing cross veins and is necessary for local BMP-like activity. The Crossveinless 2 protein contains a putative signal or transmembrane sequence, and a partial Von Willebrand Factor D domain similar to those known to regulate the formation of intramolecular and intermolecular bonds. It also contains five cysteine-rich domains, similar to the cysteine-rich domains found in Chordin, Short Gastrulation and Procollagen that are known to bind BMP-like ligands. These features strongly suggest that Crossveinless 2 acts extracelluarly or in the secretory pathway to directly potentiate Dpp or Gbb signaling.

Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers.

PURPOSE: To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS: Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS: Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS: The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle.

Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo.

Cryostat tissue printing: an improved method for histochemical and immunocytochemical localization in soft tissues.

A modification of a previously described tissue print technique has been developed in which soft tissues are frozen and sectioned in a cryostat prior to direct collection on nitrocellulose or nylon membranes. The inexpensive embedding technique described here allows accurate orientation of specimens prior to mounting, and mounted material may be stored easily after initial sectioning for future reexamination. Standard hand tissue prints of soft specimens exhibit tissue distortion and uneven delivery of material to the membrane, which limits resolution and makes interpretation difficult. Cryostat sections retain tissue fragments in their original arrangement relative to each other during printing and deliver a consistent and quantitative amount of material from all parts of the specimen. The cryostat tissue print technique is applied here to immature floral buds, demonstrating the tissue-level histochemical localization of beta-glucuronidase in transgenic plants and immunolocalization of a novel protein present only in mutant plants. This modified technique is applicable for examining both plant and animal tissues.

How do alterations in plant mitochondrial genomes disrupt pollen development?

Cytoplasmic male sterility arises when mitochondrial activities are disrupted that are essential for pollen development. Rearrangements in the mitochondrial genome that create novel open reading frames are strongly correlated with CMS phenotypes in a number of systems. The morphological aberrations which indicate CMS-associated degeneration are frequently restricted to the male sporogenous tissue and a limited number of vegetative tissues. In several cases, this tissue specificity may result from interactions between the mitochondrial genome and nuclear genes that regulate mitochondrial gene expression. A molecular mechanism by which CMS might be caused has not been conclusively demonstrated for any system. Several hypotheses for general mechanisms by which mitochondrial dysfunction might disrupt pollen development are discussed, based on similarities between the novel CMS-associated genes from a number of systems.

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco.

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.

Tissue-Specific Protein Expression in Plant Mitochondria.

Although the physiological role of plant mitochondria is thought to vary in different tissues at progressive stages of development, there has been little documentation that the complement of mitochondrial proteins is altered in different plant organs. Because the phenomenon of cytoplasmic male sterility suggests an unusual function for mitochondria in floral buds, we examined the tissue-specific expression of mitochondrial proteins in petunia buds at several stages of development, using both fertile and cytoplasmic male sterile plants. On tissue prints of cryostat-sectioned buds, antibodies recognizing subunit A of the mitochondrial ATPase (ATPA) localized very differently from antibodies recognizing subunit II of the cytochrome oxidase (COXII), which indicated that mitochondria in the same tissue could differentially express mitochondrially encoded proteins. The petunia cytoplasmic male sterility-associated fused (pcf) gene encodes a protein that colocalized with ATPA and the nuclear-encoded mitochondrial alternative oxidase (AOA) in sporogenous tissues, where little COXII protein was found. These overlapping and differential localization patterns may provide clues to the molecular mechanism of cytoplasmic male sterility.