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Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars. Abstract. Bibliografija: str. 1348-1350.

Networks of transcriptional and post-transcriptional regulators are critical for bacterial survival and adaptation to environmental stressors. While transcriptional regulators provide rapid activation and/or repression of a wide-network of genes, post-transcriptional regulators, such as small RNAs (sRNAs), are also important to fine-tune gene expression. However, the mechanisms of sRNAs remain poorly understood, especially in less-studied bacteria. Deinococcus radiodurans is a gram-positive bacterium resistant to extreme levels of ionizing radiation (IR). Although multiple unique regulatory systems (e.g., the Radiation and Desiccation Response (RDR)) have been identified in this organism, the role of post-transcriptional regulators has not been characterized within the IR response. In this study, we have characterized an sRNA, PprS (formerly Dsr2), as a post-transcriptional coordinator of IR recovery in D. radiodurans. PprS showed differential expression specifically under IR and knockdown of PprS resulted in reduced survival and growth under IR, suggesting its importance in regulating post-radiation recovery. We determined a number of potential RNA targets involved in several pathways including translation and DNA repair. Specifically, we confirmed that PprS binds within the coding region to stabilize the pprM (DR_0907) transcript, a RDR modulator. Overall, these results are the first to present an additional layer of sRNA-based control in DNA repair pathways associated with bacterial radioresistance.

Ionizing radiation-induced tissue damage recruits monocytes into the exposed area where they are differentiated to macrophages. These implement phagocytic removal of dying cells and elicit an acute inflammatory response, but can also facilitate tumorigenesis due to production of anti-inflammatory cytokines. Using primary human monocyte-derived macrophages (MDMs) and the THP1 monocytic cell line, we demonstrate that gamma radiation triggers monocyte differentiation toward the macrophage phenotype with increased expression of type I interferons (IFN-I) and both pro- and anti-inflammatory macrophage activation markers. We found that these changes correlate with significantly upregulated expression of 622 retroelements from various groups, particularly of several clades of human endogenous retroviruses (HERVs). Elevated transcription was detected in both sense and antisense directions in the HERV subgroups tested, including the most genetically homogeneous clade HML-2. The level of antisense transcription was three- to five-fold higher than of the sense strand levels. Using a proximity ligation assay and immunoprecipitation followed by RNA quantification, we identified an increased amount of the dsRNA receptors MDA-5 and TLR3 bound to an equivalent number of copies of sense and antisense chains of HERVK HML-2 RNA. This binding triggered MAVS-associated signaling pathways resulting in increased expression of IFN-I and inflammation related genes that enhanced the cumulative inflammatory effect of radiation-induced senescence. HML-2 knockdown was accompanied with reduced expression and secretion of IFNα, pro-inflammatory (IL-1β, IL-6, CCL2, CCL3, CCL8, and CCL20) and anti-inflammatory (IL10) modulators in irradiated monocytes and MDMs. Taken together, our data indicate that radiation stress-induced HERV expression enhances the IFN-I and cytokine response and results in increased levels of pro-inflammatory modulators along with expression of anti-inflammatory factors associated with the macrophage tumorigenic phenotype., Author summary Ionizing radiation is a powerful stressogenic factor that induces massive cell damage. The signals released from radiation-damaged tissues recruit the monocytes, which are differentiated into macrophages that remove dying cells via phagocytosis and facilitate inflammation but can also contribute to tumorigenesis through anti-inflammatory and regenerative activities. The mechanism of this dual response of macrophages to irradiation is not fully understood. Using primary human macrophages and a monocytic cell line, we demonstrated that gamma radiation doses activate expression of various human endogenous retroviruses (HERVs). At the molecular level, we have shown that increased numbers of sense and antisense transcripts of tested HERV subgroups bind to double-stranded RNA receptors inducing the expression of type I interferons, multiple pro-inflammatory and some anti-inflammatory factors. At the phenotypic level, polarized macrophages exhibit a potent inflammatory response along with potentially tumorigenic characteristics. Our data suggest that endogenous retroviruses represent an important contributor of the macrophage-mediated inflammation in response to radiation-induced stress but may also indirectly influence tumorigenesis via biased macrophage polarization.

Background Amifostine is a potent scavenger of reactive oxygen species that is used for the salivary gland protection during therapy with radioactive iodine for thyroid cancer. There are no data on the potential effect of amifostine on thyroid cancer cells. Methods We investigated the effects of the active form of amifostine (WR-1065) on the response of thyroid cancer cells to treatment with DNA-damaging agents. WR-1065 was examined in human thyroid cancer cell lines (FTC133, TPC1, BCPAP and C643) and embryonic fibroblast cells NIH3T3. DNA damage was induced by exposure to H2O2 (0.1 mM), by treatment with the radiomimetic neocarzinostatin (NCS 250 ng/mL) and by γ-radiation (6 Gy). DNA damage, cell viability and apoptosis were examined. Results We demonstrated the selective action of WR-1065 (0.1 mM), which prevented oxidative stress–induced DNA damage in fibroblasts, but did not protect thyroid cancer cells from DNA damage and apoptosis documented by caspase-3 and PARP cleavage after exposure to H2O2, NCS and γ-radiation. Prolonged exposure to WR-1065 (0.1 mM for 24 h) was toxic for thyroid cancer cells; this treatment decreased the number of viable cells by 8% in C643 cells, 47% in TPC cells, 92% in BCPAP cells and 82% in FTC 133 cells. The cytotoxic effects of WR-1065 were not associated with induction of apoptosis. Conclusions Our data show that amifostine has no protective effect on thyroid cancer cells against DNA-damaging agents in vitro and suggest that amifostine will not attenuate the efficacy of radioiodine treatment in patients with thyroid cancer.

Exposure to chronic ionizing radiation (CIR) from nuclear power plant accidents, acts of terrorism, and space exploration poses serious threats to humans. Fungi are a group of highly radiation-resistant eukaryotes, and an understanding of fungal CIR resistance mechanisms holds the prospect of protecting humans. We compared the abilities of 95 wild-type yeast and dimorphic fungal isolates, representing diverse Ascomycota and Basidiomycota, to resist exposure to five environmentally-relevant stressors: CIR (long-duration growth under 36 Gy/h) and acute (10 kGy/h) ionizing radiation (IR), heavy metals (chromium, mercury), elevated temperature (up to 50 °C), and low pH (2.3). To quantify associations between resistances to CIR and these other stressors, we used correlation analysis, logistic regression with multi-model inference, and customized machine learning. The results suggest that resistance to acute IR in fungi is not strongly correlated with the ability of a given fungal isolate to grow under CIR. Instead, the strongest predictors of CIR resistance in fungi were resistance to chromium (III) and to elevated temperature. These results suggest fundamental differences between the mechanisms of resistance to chronic and acute radiation. Convergent evolution towards radioresistance among genetically distinct groups of organisms is considered here.

Despite concerted functional genomic efforts to understand the complex phenotype of ionizing radiation (IR) resistance, a genome sequence cannot predict whether a cell is IR-resistant or not. Instead, we report that absorption-display electron paramagnetic resonance (EPR) spectroscopy of nonirradiated cells is highly diagnostic of IR survival and repair efficiency of DNA double-strand breaks (DSBs) caused by exposure to gamma radiation across archaea, bacteria, and eukaryotes, including fungi and human cells. IR-resistant cells, which are efficient at DSB repair, contain a high cellular content of manganous ions (Mn2+) in high-symmetry (H) antioxidant complexes with small metabolites (e.g., orthophosphate, peptides), which exhibit narrow EPR signals (small zero-field splitting). In contrast, Mn2+ ions in IR-sensitive cells, which are inefficient at DSB repair, exist largely as low-symmetry (L) complexes with substantially broadened spectra seen with enzymes and strongly chelating ligands. The fraction of cellular Mn2+ present as H-complexes (H-Mn2+), as measured by EPR of live, nonirradiated Mn-replete cells, is now the strongest known gauge of biological IR resistance between and within organisms representing all three domains of life: Antioxidant H-Mn2+ complexes, not antioxidant enzymes (e.g., Mn superoxide dismutase), govern IR survival. As the pool of intracellular metabolites needed to form H-Mn2+ complexes depends on the nutritional status of the cell, we conclude that IR resistance is predominantly a metabolic phenomenon. In a cross-kingdom analysis, the vast differences in taxonomic classification, genome size, and radioresistance between cell types studied here support that IR resistance is not controlled by the repertoire of DNA repair and antioxidant enzymes.

Understanding chronic ionizing radiation (CIR) effects is of utmost importance to protecting human health and the environment. Diverse bacteria and fungi inhabiting extremely radioactive waste and disaster sites (e.g. Hanford, Chernobyl, Fukushima) represent new targets of CIR research. We show that many microorganisms can grow under intense gamma-CIR dose rates of 13-126 Gy/h, with fungi identified as a particularly CIR-resistant group of eukaryotes: among 145 phylogenetically diverse strains tested, 78 grew under 36 Gy/h. Importantly, we demonstrate that CIR resistance can depend on cell concentration and that certain resistant microbial cells protect their neighbors (not only conspecifics, but even radiosensitive species from a different phylum), from high-level CIR. We apply a mechanistically-motivated mathematical model of CIR effects, based on accumulation/removal kinetics of reactive oxygen species (ROS) and antioxidants, in bacteria (3 Escherichia coli strains and Deinococcus radiodurans) and in fungi (Candida parapsilosis, Kazachstania exigua, Pichia kudriavzevii, Rhodotorula lysinophila, Saccharomyces cerevisiae, and Trichosporon mucoides). We also show that correlations between responses to CIR and acute ionizing radiation (AIR) among studied microorganisms are weak. For example, in D. radiodurans, the best molecular correlate for CIR resistance is the antioxidant enzyme catalase, which is dispensable for AIR resistance; and numerous CIR-resistant fungi are not AIR-resistant. Our experimental findings and quantitative modeling thus demonstrate the importance of investigating CIR responses directly, rather than extrapolating from AIR. Protection of radiosensitive cell-types by radioresistant ones under high-level CIR is a potentially important new tool for bioremediation of radioactive sites and development of CIR-resistant microbiota as radioprotectors.

The genetic platforms of Deinococcus species remain the only systems in which massive ionizing radiation (IR)-induced genome damage can be investigated in vivo at exposures commensurate with cellular survival. We report the whole genome sequence of the extremely IR-resistant rod-shaped bacterium Deinococcus ficus KS 0460 and its phenotypic characterization. Deinococcus ficus KS 0460 has been studied since 1987, first under the name Deinobacter grandis, then Deinococcus grandis. The D. ficus KS 0460 genome consists of a 4.019 Mbp sequence (69.7% GC content and 3894 predicted genes) divided into six genome partitions, five of which are confirmed to be circular. Circularity was determined manually by mate pair linkage. Approximately 76% of the predicted proteins contained identifiable Pfam domains and 72% were assigned to COGs. Of all D. ficus KS 0460 proteins, 79% and 70% had homologues in Deinococcus radiodurans ATCC BAA-816 and Deinococcus geothermalis DSM 11300, respectively. The most striking differences between D. ficus KS 0460 and D. radiodurans BAA-816 identified by the comparison of the KEGG pathways were as follows: (i) D. ficus lacks nine enzymes of purine degradation present in D. radiodurans, and (ii) D. ficus contains eight enzymes involved in nitrogen metabolism, including nitrate and nitrite reductases, that D. radiodurans lacks. Moreover, genes previously considered to be important to IR resistance are missing in D. ficus KS 0460, namely, for the Mn-transporter nramp, and proteins DdrF, DdrJ and DdrK, all of which are also missing in Deinococcus deserti. Otherwise, D. ficus KS 0460 exemplifies the Deinococcus lineage. Electronic supplementary material The online version of this article (doi:10.1186/s40793-017-0258-y) contains supplementary material, which is available to authorized users.

Karstic caves often support white, yellow, grey or pink microbial colonies that are termed ‘cave silver’ by speleologists. Using various sample pre-treatments and culture media, a wide variety of bacteria associated with these colonies were recovered from a cave in Slovenia, Pajsarjeva jama. Decreasing the inoculum size resulted in significant increases in viable counts, while pre-treatments had the opposite effect with the exception of microwave irradiation. While all growth media yielded viable counts, the maximal counts were observed on a low-nutrient TWA medium. Based on the 16S rRNA gene sequence of OTU representatives, the majority of the 80 isolates examined belonged to Streptomyces (25%), Micrococcus (16%) and Rhodococcus (10%) Other abundant groups were Pseudomonas (9%), Agrobacterium (8%), Lysobacter (6%) and Paenibacillus (5%), while members of genera Microbacterium, Agrococcus, Arthrobacter, Bacillus, Kocuria, Oerskovia, Sphingomonas, Aerococcus, and Bosea represented a minor portion of cultivable diversity encountered. Members of Streptomyces and Agrobacterium were common to all samples. Although these microorganisms readily form colonies under laboratory conditions, they were unrelated to abundant environmental phylotypes recovered from same samples in a previous study. However, the comparative 16S rRNA analysis showed that microorganisms highly related to the ones obtained in this study were cultivated from other subterranean environments indicating that they might represent true microbial cave dwellers.

Microbial mats are a laminated organic-sedimentary ecosystem, found in a wide range of habitats. Fluctuating diel and seasonal physicochemical gradients characterize these ecosystems, resulting in both strata and microenvironments that harbor specific microbial communities. This study was undertaken to compare two types of microbial mats across seasons to further understand the structure of fungal communities in hypersaline microbial mats and their seasonal dynamics. The structure and diversity of fungal communities was documented in young transient and mature hypersaline microbial mats from a tropical region (Puerto Rico) using one culture-dependent and three culture-independent molecular techniques based on the internal transcribed spacer (ITS) region of ribosomal DNA: terminal restriction fragment length polymorphism (TRFLP), denaturing gradient gel electrophoresis (DGGE) and clone libraries. Two microbial mats (one young and transient, one mature) were sampled in Nov 2007 (wet season), Jan 2008 (intermediate season) and Mar 2008 (dry season) in the Cabo Rojo Solar Salterns on the southwestern coast of Puerto Rico. Traditional and molecular techniques revealed strong spatial and temporal heterogeneities in both microbial mats. Higher abundance of isolates and phylotypes were observed during the wet season, and diversity decreased from the top (oxic) to the bottom (anoxic) layers in both seasons. Some of the species isolated belong to the genera Aspergillus, Cladosporium, Hortaea, Pichia and Wallemia, which often are isolated from hypersaline environments. The most abundant clones belong to Acremonium strictum and Cladosporium halotolerans, which were not isolated in pure culture. The differences observed using culture-based and molecular techniques demonstrates the need of combining methods to study the diversity of fungi in a given substrate.

The crustacean genus Artemia naturally inhabits various saline and hypersaline environments and is the most frequently laboratory-hatched animal for live feed in mari- and aquaculture. Because of its high economic importance, Artemia–bacteria interactions were so far studied mostly in laboratory strains. In this study, we focused our attention on the Artemia-associated microbiota in its natural environment in the solar salterns of Eilat, Israel. We applied a culture-independent method (clone libraries) to investigate the bacterial community structure associated with Artemia in five evaporation ponds with salinities from slightly above seawater (5%) to the point of saturation (32%), in two different developmental stages: in nauplii and in the intestine of adult animals. Bacteria found in naupliar and adult stages were classified within the Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Cyanobacteria. The halophilic proteobacterial genera Halomonas spp. and Salinivibrio spp. dominated the Artemia microbiota in both stages in all ponds. We also analysed a clone library of entire adult animals, revealing a novel bacterial phylogenetic lineage. This is the first molecular study of bacteria associated with two developmental stages of Artemia along a salinity gradient.

The Secovlje saltern is one of the few remaining solar salterns for traditional, seasonal salt production. The bottom of the crystallizer ponds is covered with a microbial mat, known as the 'petola', that has continuously been cultivated from medieval times. Outside the salt production season, the petola is fertilized with anoxic marine mud and covered with saline water; during the season, it is covered by brine. Here, we have applied culture-independent techniques and microelectrode-based activity measurements to study the bacterial communities in three different layers of the petola during the peak of the harvesting season. For reference, we used nonactive petola that had been abandoned for several years. The upper 2 mm of the petola were dominated by the cyanobacterial species Coleofasciculus chthonoplastes and the Phormidium/Lyngbya group, and Gammaproteobacteria (Acinetobacter sp.), while the third anoxic layer was dominated by as yet uncultured phyla. The nonactive petola showed a higher biodiversity. Oxygen and sulfide concentrations differed between the mats studied, in terms of the depth of oxygen penetration and diel changes. This study provides the first molecular insight into the microbiology of the petola, and it represents an important contribution towards understanding the geomicrobiological cycles of the traditional Secovlje saltern.

Mammalian systems can be a subject of cellular engineering in a similar way to bacterial cells. Our team decided to modify the existing mammalian cell signalling network of the innate immune response to bacterial infection. Binding of bacterial components (pathogen associated molecular patterns, or PAMPs) to a family of Toll-like receptors (TLRs) activates the cells of the immune system but an exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signalling cascade at the weak spot – protein MyD88 – which is the consensus adaptor protein of the surface-expressed TLRs. A mathematical model of cell activation with an engineered feedback loop predicts a decrease of cellular activation after repeated stimulation of TLR. We have prepared 26 BioBricks for mammalian systems and deposited them into the BioBrick Registry at MIT. Mammalian cells transfected with the feedback loop construct performed as designed with a decrease in the cellular response upon repeated stimulation with PAMPs. Cell activation decreased without completely abolishing the responsiveness to the bacterial stimulus, therefore our engineered system represents an artificial type of immunotolerance. The Slovenian team was composed of seven undergraduate students of microbiology and biochemistry and five mentors from the National Institute of Chemistry and University of Ljubljana Faculty of Chemistry and Chemical Technology.

Background: Targeting cell metabolism has emerged as a therapeutic strategy for the treatment of cancer. Metabolically active thyroid cancers are resistant to treatment with radioactive iodine. Aberrant expression of genes controlling glycolysis was demonstrated in thyroid cancer cells, but little is known regarding the role of mitochondrial proteins in thyroid carcinogenesis. Cytochrome c oxidase 4 (COX4) plays pivotal roles in oxidative phosphorylation and the cellular response to oxidative stress. COX4 may thus represent a promising therapeutic target. Objectives: We examined expression of COX4 in human thyroid tumors and performed functional studies using thyroid cancer cell lines. Material and Methods Expression of COX4 was examined by immunostaining in 25 follicular adenomas (FAs), 22 follicular cancers (FTCs), 90 papillary cancers (PTCs) and in 48 samples from normal thyroid tissue. FTC-derived (FTC133) and PTC-derived (BCPAP) cell lines were used to create COX4-deficient cells by lentiviral transfection. The efficiency of COX4 inhibition was examined. Mitochondrial membrane potential was examined by JC-1 staining. DNA-damage signaling was examined after cell exposure to γ radiation (6-18 Gy). We also determined the effects of 2-deoxyglucose (2DG) on viability of thyroid cancer cells with compromised COX4. Caspase-3 and PARP cleavage assays were performed to measure apoptosis. Results Positive immunostaining with anti-COX4 was detected in 6/48 (12.5%) normal tissue, 5/20 (25%) FAs, 7/22 (31%) FTCs, and 51/90 (56%) PTCs. The intensity of COX4 immunostaining was significantly higher in thyroid cancers than in either normal thyroid (p = 0.0001) or benign FAs (0.001). COX4 expression was more frequently detected in PTCs than in FTCs (p = 0.03). The mRNA level of COX4 was higher in BCPAP and FTC133 cells compared to normal thyroid. In both cell lines, silencing of COX4 altered intra-cellular distribution of JC-1 staining. In control cells, JC-1 staining was perinuclear, but in COX4-deficient cells it became diffusely cytoplasmic. COX4 silencing affected cell growth and response to γradiation in a cell type specific manner. In BCPAP cells, downregulation of COX4 was associated with inhibition of cell growth, block in G1 phase and inhibition of Cyclin D1. In BCPAP cells, COX4 silencing activated DNA-damage signaling and increased sensitivity to γ radiation. Inhibitor of glycolysis (2DG) was more efficient against COX4-deficient than COX4-expressing BCPAP cells. In FTC133 cells, silencing of COX4 increased the rate of growth and induced expression of Cyclin D1. COX4 silencing did not increase FTC133 cell sensitivity to γradiation nor to treatment with 2DG. Conclusion COX4 is implicated in regulation of thyroid cancer cell growth and response to DNA damaging or metabolic treatments. These data suggest that evaluation of COX4 in thyroid cancer could serve as a biomarker of response to treatment with metabolic agents. Citation Format: Vasyl V. Vasko, Athanasios Bikas, Aneeta Patel, John Costello, Rok Tkavc, Kenneth D. Burman, Kirk Jensen. Expression of cytochrome C oxidase 4 (COX4) in thyroid cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1001.

The crustacean genus Artemia naturally inhabits various saline and hypersaline environments and is the most frequently laboratory-hatched animal for live feed in mari- and aquaculture. Because of its high economic importance, Artemia-bacteria interactions were so far studied mostly in laboratory strains. In this study, we focused our attention on the Artemia-associated microbiota in its natural environment in the solar salterns of Eilat, Israel. We applied a culture-independent method (clone libraries) to investigate the bacterial community structure associated with Artemia in five evaporation ponds with salinities from slightly above seawater (5%) to the point of saturation (32%), in two different developmental stages: in nauplii and in the intestine of adult animals. Bacteria found in naupliar and adult stages were classified within the Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Cyanobacteria. The halophilic proteobacterial genera Halomonas spp. and Salinivibrio spp. dominated the Artemia microbiota in both stages in all ponds. We also analysed a clone library of entire adult animals, revealing a novel bacterial phylogenetic lineage. This is the first molecular study of bacteria associated with two developmental stages of Artemia along a salinity gradient.