Search

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.

Acinetobacter baumannii is a bacterial pathogen that is often multidrug-resistant (MDR) and causes a range of life-threatening illnesses, including pneumonia, septicemia, and wound infections. Some antibiotic treatments can reduce mortality if dosed early enough before an infection progresses, but there are few other treatment options when it comes to MDR-infection. Although several prophylactic strategies have been assessed, no vaccine candidates have advanced to clinical trials or have been approved. Herein, we rapidly produced protective whole-cell immunogens from planktonic and biofilm-like cultures of A. baumannii, strain AB5075 grown using a variety of methods. After selecting a panel of five cultures based on distinct protein profiles, replicative activity was extinguished by exposure to 10 kGy gamma radiation in the presence of a Deinococcus antioxidant complex composed of manganous (Mn2+) ions, a decapeptide, and orthophosphate. Mn2+ antioxidants prevent hydroxylation and carbonylation of irradiated proteins, but do not protect nucleic acids, yielding replication-deficient immunogenic A. baumannii vaccine candidates. Mice were immunized and boosted twice with 1.0 × 107 irradiated bacterial cells and then challenged intranasally with AB5075 using two mouse models. Planktonic cultures grown for 16 h in rich media and biofilm cultures grown in static cultures underneath minimal (M9) media stimulated immunity that led to 80–100% protection.

A concerted action on the part of international agencies and national governments has resulted in the near-eradication of poliomyelitis. However, both the oral polio vaccine (OPV) and the inactivated polio vaccine (IPV) have deficiencies which make them suboptimal for use after global eradication. OPV is composed of attenuated Sabin strains and stimulates robust immunity, but may revert to neurovirulent forms in the intestine which can be shed and infect susceptible contacts. The majority of IPV products are manufactured using pathogenic strains inactivated with formalin. Upon eradication, the production of large quantities of pathogenic virus will present an increased biosecurity hazard. A logical ideal endgame vaccine would be an inactivated form of an attenuated strain that could afford protective immunity while safely producing larger numbers of doses per unit of virus stock than current vaccines. We report here the development of an ionizing radiation (IR)-inactivated Sabin-based vaccine using a reconstituted Mn-decapeptide (MDP) antioxidant complex derived from the radioresistant bacterium Deinococcus radiodurans. In bacteria, Mn2+-peptide antioxidants protect proteins from oxidative damage caused by extreme radiation exposure. Here we show for the first time, that MDP can protect immunogenic neutralizing epitopes in picornaviruses. MDP protects epitopes in Polio Virus 1 and 2 Sabin strains (PV1-S and PV2-S, respectively), but viral genomic RNA is not protected during supralethal irradiation. IR-inactivated Sabin viruses stimulated equivalent or improved neutralizing antibody responses in Wistar rats compared to the commercially used IPV products. Our approach reduces the biosecurity risk of the current PV vaccine production method by utilizing the Sabin strains instead of the wild type neurovirulent strains. Additionally, the IR-inactivation approach could provide a simpler, faster and less costly process for producing a more immunogenic IPV. Gamma-irradiation is a well-known method of virus inactivation and this vaccine approach could be adapted to any pathogen of interest.

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.

Acinetobacter baumannii is a bacterial pathogen that is often multidrug-resistant (MDR) and causes a range of life-threatening illnesses, including pneumonia, septicemia, and wound infections. Some antibiotic treatments can reduce mortality if dosed early enough before an infection progresses, but there are few other treatment options when it comes to MDR-infection. Although several prophylactic strategies have been assessed, no vaccine candidates have advanced to clinical trials or have been approved. Herein, we rapidly produced protective whole-cell immunogens from planktonic and biofilm-like cultures of A. baumannii, strain AB5075 grown using a variety of methods. After selecting a panel of five cultures based on distinct protein profiles, replicative activity was extinguished by exposure to 10 kGy gamma radiation in the presence of a Deinococcus antioxidant complex composed of manganous (Mn2+) ions, a decapeptide, and orthophosphate. Mn2+ antioxidants prevent hydroxylation and carbonylation of irradiated proteins, but do not protect nucleic acids, yielding replication-deficient immunogenic A. baumannii vaccine candidates. Mice were immunized and boosted twice with 1.0 × 107 irradiated bacterial cells and then challenged intranasally with AB5075 using two mouse models. Planktonic cultures grown for 16 h in rich media and biofilm cultures grown in static cultures underneath minimal (M9) media stimulated immunity that led to 80–100% protection.

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.

The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of γ-rays (12,000 Gy), 20 times greater than Escherichia coli , is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) “antioxidant” Mn 2+ –metabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)–scavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn 2+ speciation in D. radiodurans and E. coli cells and their responses to 10 kGy γ-irradiation. The Mn 2+ of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn 2+ of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn 2+ speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn 2+ complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn 2+ speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation-induced ROS because it lacks an adequate supply of LMW Mn antioxidants.

The radioprotective capacity of a rationally-designed Mn2+-decapeptide complex (MDP), based on Mn antioxidants in the bacterium Deinococcus radiodurans, was investigated in a mouse model of radiation injury. MDP was previously reported to be extraordinarily radioprotective of proteins in the setting of vaccine development. The peptide-component (DEHGTAVMLK) of MDP applied here was selected from a group of synthetic peptides screened in vitro for their ability to protect cultured human cells and purified enzymes from extreme damage caused by ionizing radiation (IR). We show that the peptides accumulated in Jurkat T-cells and protected them from 100 Gy. MDP preserved the activity of T4 DNA ligase exposed to 60,000 Gy. In vivo, MDP was nontoxic and protected B6D2F1/J (female) mice from acute radiation syndrome. All irradiated mice treated with MDP survived exposure to 9.5 Gy (LD70/30) in comparison to the untreated mice, which displayed 63% lethality after 30 days. Our results show that MDP provides early protection of white blood cells, and attenuates IR-induced damage to bone marrow and hematopoietic stem cells via G-CSF and GM-CSF modulation. Moreover, MDP mediated the immunomodulation of several cytokine concentrations in serum including G-CSF, GM-CSF, IL-3 and IL-10 during early recovery. Our results present the necessary prelude for future efforts towards clinical application of MDP as a promising IR countermeasure. Further investigation of MDP as a pre-exposure prophylactic and post-exposure therapeutic in radiotherapy and radiation emergencies is warranted.

Autosomal dominant mutations in BTB and Kelch domain containing 13 protein (KBTBD13) are associated with a new type of Nemaline Myopathy (NEM). NEM is a genetically heterogeneous group of muscle disorders. Mutations causing phenotypically distinct NEM variants have previously been identified in components of muscle thin filament. KBTBD13 is a muscle specific protein composed of an N terminal BTB domain and a C terminal Kelch-repeat domain. The function of this newly identified protein in muscle remained unknown. In this study, we show that KBTBD13 interacts with Cullin 3 (Cul3) and the BTB domain mediates this interaction. Using ubiquitination assays, we determined that KBTBD13 participates in the formation of a Cul3 based RING ubiquitin ligase (Cul3-RL) capable of ubiquitin conjugation. Confocal microscopy of transiently expressed KBTBD13 revealed its co-localization with ubiquitin. Taken together, our results demonstrate that KBTBD13 is a putative substrate adaptor for Cul3-RL that functions as a muscle specific ubiquitin ligase, and thereby implicate the ubiquitin proteasome pathway in the pathogenesis of KBTBD13-associated NEM.

Spores of Bacillus subtilis strains with (wild type) or without (α − β − ) most DNA-binding α/β-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl 2 concentrations of 0.3 μM to 1 mM. These haploid spores had Mn levels that varied up to 180-fold and Mn/Fe ratios that varied up to 300-fold. However, the resistance of these spores to desiccation, wet heat, dry heat, and in particular ionizing radiation was unaffected by their level of Mn or their Mn/Fe ratio; this was also the case for wild-type spore resistance to hydrogen peroxide (H 2 O 2 ). However, α − β − spores were more sensitive to H 2 O 2 when they had high Mn levels and a high Mn/Fe ratio. These results suggest that Mn levels alone are not essential for wild-type bacterial spores' extreme resistance properties, in particular ionizing radiation, although high Mn levels sensitize α − β − spores to H 2 O 2 , probably by repressing expression of the auxiliary DNA-protective protein MrgA. Notably, Mn 2+ complexed with the abundant spore molecule dipicolinic acid (DPA) with or without inorganic phosphate was very effective at protecting a restriction enzyme against ionizing radiation in vitro , and Ca 2+ complexed with DPA and phosphate was also very effective in this regard. These latter data suggest that protein protection in spores against treatments such as ionizing radiation that generate reactive oxygen species may be due in part to the spores' high levels of DPA conjugated to divalent metal ions, predominantly Ca 2+ , much like high levels of Mn 2+ complexed with small molecules protect the bacterium Deinococcus radiodurans against ionizing radiation.

We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.

Deinococcus radiodurans is extremely resistant to ionizing radiation. How this bacterium can grow under chronic γ radiation [50 grays (Gy) per hour] or recover from acute doses greater than 10 kGy is unknown. We show that D. radiodurans accumulates very high intracellular manganese and low iron levels compared with radiation-sensitive bacteria and that resistance exhibits a concentration-dependent response to manganous chloride [Mn(II)]. Among the most radiation-resistant bacterial groups reported, Deinococcus, Enterococcus, Lactobacillus , and cyanobacteria accumulate Mn(II). In contrast, Shewanella oneidensis and Pseudomonas putida have high iron but low intracellular manganese concentrations and are very sensitive. We propose that Mn(II) accumulation facilitates recovery from radiation injury.

Birt-Hogg-Dube (BHD) syndrome is a rare inherited genodermatosis characterized by hair follicle hamartomas, kidney tumors, and spontaneous pneumothorax. Recombination mapping in BHD families delineated the susceptibility locus to 700 kb on chromosome 17p11.2. Protein-truncating mutations were identified in a novel candidate gene in a panel of BHD families, with a 44% frequency of insertion/deletion mutations within a hypermutable C 8 tract. Tissue expression of the 3.8 kb transcript was widespread, including kidney, lung, and skin. The full-length BHD sequence predicted a novel protein, folliculin, that was highly conserved across species. Discovery of disease-causing mutations in BHD , a novel kidney cancer gene associated with renal oncocytoma or chromophobe renal cancer, will contribute to understanding the role of folliculin in pathways common to skin, lung, and kidney development.

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn(2+) and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn(2+)-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.

Fetal hypoxia in the II trimester of pregnancy caused immunodeficiency in newborn mice: inhibition of antibody production to sheep erythrocytes and disturbances in migration of early hemopoietic precursors from the bone marrow to the spleen.

For extremely ionizing radiation-resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the US Department of Energy's Hanford Site in Washington State. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of the genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms, which limit protein oxidation during dehydration.

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection., Author Summary One original goal of radiobiology was to explain why cells are so sensitive to ionizing radiation (IR). Early studies in bacteria incriminated DNA as the principal radiosensitive target, an assertion that remains central to modern radiation toxicity models. More recently, the emphasis has shifted to understanding why bacteria such as Deinococcus radiodurans are extremely resistant to IR, by focusing on DNA repair systems expressed during recovery from high doses of IR. Unfortunately, as key features of DNA-centric hypotheses of extreme resistance have grown weaker, the study of alternative cellular targets has lagged far behind, mostly because of their relative biological complexity. Recent studies have shown that extreme levels of bacterial IR resistance correlate with high intracellular Mn(II) concentrations, and resistant and sensitive bacteria are equally susceptible to IR-induced DNA damage. The current work establishes a mechanistic link between Mn(II) and protection of proteins from radiation damage. In contrast to resistant bacteria, naturally sensitive bacteria are highly susceptible to IR-induced protein oxidation. We propose that sensitive bacteria sustain lethal levels of protein damage at radiation doses that elicit relatively little DNA damage, and that extreme resistance in bacteria is dependent on protein protection., A high intracellular concentration of manganese inDeinococcus radiodurans protects proteins, but not DNA, from ionizing radiation-induced oxidative damage. Protein protection may be critical to the known radiation resistance of these bacteria.

BackgroundThermus thermophilusandDeinococcus radioduransbelong to a distinct bacterial clade but have remarkably different phenotypes.T. thermophilusis a thermophile, which is relatively sensitive to ionizing radiation and desiccation, whereasD. radioduransis a mesophile, which is highly radiation- and desiccation-resistant. Here we present an in-depth comparison of the genomes of these two related but differently adapted bacteria.ResultsBy reconstructing the evolution ofThermusandDeinococcusafter the divergence from their common ancestor, we demonstrate a high level of post-divergence gene flux in both lineages. Various aspects of the adaptation to high temperature inThermuscan be attributed to horizontal gene transfer from archaea and thermophilic bacteria; many of the horizontally transferred genes are located on the single megaplasmid ofThermus. In addition, theThermuslineage has lost a set of genes that are still present inDeinococcusand many other mesophilic bacteria but are not common among thermophiles. By contrast,Deinococcusseems to have acquired numerous genes related to stress response systems from various bacteria. A comparison of the distribution of orthologous genes among the four partitions of theDeinococcusgenome and the two partitions of theThermusgenome reveals homology between theThermusmegaplasmid (pTT27) andDeinococcusmegaplasmid (DR177).ConclusionAfter the radiation from their common ancestor, theThermusandDeinococcuslineages have taken divergent paths toward their distinct lifestyles. In addition to extensive gene loss,Thermusseems to have acquired numerous genes from thermophiles, which likely was the decisive contribution to its thermophilic adaptation. By contrast,Deinococcuslost few genes but seems to have acquired many bacterial genes that apparently enhanced its ability to survive different kinds of environmental stresses. Notwithstanding the accumulation of horizontally transferred genes, we also show that the single megaplasmid ofThermusand the DR177 megaplasmid ofDeinococcusare homologous and probably were inherited from the common ancestor of these bacteria.

We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.

The fate of hematopoietic stem cells (HSCs) is determined by microenvironmental niches, but the molecular structure of these local networks is not yet completely characterized. Our recent observation that glycosaminoglycan hyaluronic acid (HA), a major component of the bone marrow extracellular matrix, is required for in vitro hematopoiesis led us to suggest a role for HA in structuring the hematopoietic niche. Accordingly, HA deprivation induced by various treatments might lead to an imbalance of normal HSC homeostasis. Since 5-fluorouracil (5-FU) administration sharply decreases the amount of cell surface-associated HA in bone marrow, we examined whether the administration of exogenous HA enhances suppressed hematopoiesis in 5-FU-treated mice. HA administered to mice following 5-FU infusion facilitated the recovery of leukocytes and thrombocytes in the peripheral blood. Intravenously infused HA was found in the bone marrow, where it bound endothelial cells and resident macrophages and increased expression of the hematopoiesis-supportive cytokines interleukin-1 and interleukin-6. In agreement with these observations, enhanced hematopoietic activity was detected in the bone marrow, as measured by elevated counts of long-term culture-initiating cells (LTC-ICs), committed progenitors, and the total number of mature bone marrow cells. Overall, our results suggest that HA is required for regulation of the hematopoiesis-supportive function of bone marrow accessory cells and, therefore, participates in hematopoietic niche assembly.

We report the use of spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to describe the numerous genomic imbalances characteristic of stage IV clear cell renal cell carcinoma (CCRCC). SKY and CGH were performed on 10 cell lines established from nephrectomy specimens, and CGH on uncultured material from five of the primary renal tumors. The mutational status of VHL (3p25) and MET (7q31), genes implicated in renal carcinogenesis, were determined for each case. Each case showed marked aneuploidy, with an average number of copy alterations of 14.6 (+/-2.7) in the primary tumors and 19.3 (+/-4.6) in the cell lines. Both whole-chromosome and chromosome-segment imbalances were noted by CGH: consistent losses or gains included +5q23-->ter (100%), -3p14-->ter (80%), and +7 (70%). All VHL mutations and 83% of the genomic imbalances found in the primary tumors were also found in the cell lines derived from them. SKY showed many complex structural rearrangements that were undetected by conventional banding analysis in these solid tumors. All cases with VHL inactivation had 3p loss and 5q gain related primarily to unbalanced translocations between 3p and 5q. In contrast, gains of chromosome 7 resulted primarily from whole-chromosome gains and were not associated with mutations of MET. SKY and CGH demonstrated that genomic imbalances in advanced RCC were the result of either segregation errors [i.e., whole chromosomal gains and losses (7.8/case)] or chromosomal rearrangements (10.7/case), of which the majority were unbalanced translocations.

We have investigated the role of endogenous retroviral mink cell focus-forming (MCF) genes in the regulation of mouse bone marrow hemopoietic progenitor cell proliferation. The p15E protein coded by the MCF env gene is expressed by early hemopoietic progenitors, mostly on spleen colony forming units (CFUs-12) and on erythropoietin-independent erythroid progenitors. Stimulation of cell proliferation in hemopoietic precursors by steroid hormone (testosterone propionate) treatment resulted in upregulation of the expression of the endogenous p15E protein on bone marrow cells. Blocking of endogenous retroviral MCF env gene expression on the activated hemopoietic progenitors by antisense oligonucleotides inhibited their proliferation. These data suggest that the endogenous MCF env genes act as growth-regulators for pluripotent hemopoietic progenitors.

This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow–derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow–derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.

Birt-Hogg-Dube syndrome (BHD), an inherited autosomal genodermatosis characterized by benign tumors of the hair follicle, has been associated with renal neoplasia, lung cysts, and spontaneous pneumothorax. To identify the BHD locus, we recruited families with cutaneous lesions and associated phenotypic features of the BHD syndrome. We performed a genomewide scan in one large kindred with BHD and, by linkage analysis, localized the gene locus to the pericentromeric region of chromosome 17p, with a LOD score of 4.98 at D17S740 (recombination fraction 0). Two-point linkage analysis of eight additional families with BHD produced a maximum LOD score of 16.06 at D17S2196. Haplotype analysis identified critical recombinants and defined the minimal region of nonrecombination as being within a