Nanoscale 3D quantitative imaging of 1.88 Ga Gunflint microfossils reveals novel insights into taphonomic and biogenic characters.

Precambrian cellular remains frequently have simple morphologies, micrometric dimensions and are poorly preserved, imposing severe analytical and interpretational challenges, especially for irrefutable attestations of biogenicity. The 1.88 Ga Gunflint biota is a Precambrian microfossil assemblage with different types and qualities of preservation across its numerous geological localities and provides important insights into the Proterozoic biosphere and taphonomic processes. Here we use synchrotron-based ptychographic X-ray computed tomography to investigate well-preserved carbonaceous microfossils from the Schreiber Beach locality as well as poorly-preserved, iron-replaced fossil filaments from the Mink Mountain locality, Gunflint Formation. 3D nanoscale imaging with contrast based on electron density allowed us to assess the morphology and carbonaceous composition of different specimens and identify the minerals associated with their preservation based on retrieved mass densities. In the Mink Mountain filaments, the identification of mature kerogen and maghemite rather than the ubiquitously described hematite indicates an influence from biogenic organics on the local maturation of iron oxides through diagenesis. This non-destructive 3D approach to microfossil composition at the nanoscale within their geological context represents a powerful approach to assess the taphonomy and biogenicity of challenging or poorly preserved traces of early microbial life, and may be applied effectively to extraterrestrial samples returned from upcoming space missions.

A Hydrothermal-Sedimentary Context for the Origin of Life.

Critical to the origin of life are the ingredients of life, of course, but also the physical and chemical conditions in which prebiotic chemical reactions can take place. These factors place constraints on the types of Hadean environment in which life could have emerged. Many locations, ranging from hydrothermal vents and pumice rafts, through volcanic-hosted splash pools to continental springs and rivers, have been proposed for the emergence of life on Earth, each with respective advantages and certain disadvantages. However, there is another, hitherto unrecognized environment that, on the Hadean Earth (4.5-4.0 Ga), would have been more important than any other in terms of spatial and temporal scale: the sedimentary layer between oceanic crust and seawater. Using as an example sediments from the 3.5-3.33 Ga Barberton Greenstone Belt, South Africa, analogous at least on a local scale to those of the Hadean eon, we document constant permeation of the porous, carbonaceous, and reactive sedimentary layer by hydrothermal fluids emanating from the crust. This partially UV-protected, subaqueous sedimentary environment, characterized by physical and chemical gradients, represented a widespread system of miniature chemical reactors in which the production and complexification of prebiotic molecules could have led to the origin of life. Key Words: Origin of life-Hadean environment-Mineral surface reactions-Hydrothermal fluids-Archean volcanic sediments. Astrobiology 18, 259-293.

Mineralization and Preservation of an extremotolerant Bacterium Isolated from an Early Mars Analog Environment.

The artificial mineralization of a polyresistant bacterial strain isolated from an acidic, oligotrophic lake was carried out to better understand microbial (i) early mineralization and (ii) potential for further fossilisation. Mineralization was conducted in mineral matrixes commonly found on Mars and Early-Earth, silica and gypsum, for 6 months. Samples were analyzed using microbiological (survival rates), morphological (electron microscopy), biochemical (GC-MS, Microarray immunoassay, Rock-Eval) and spectroscopic (EDX, FTIR, RAMAN spectroscopy) methods. We also investigated the impact of physiological status on mineralization and long-term fossilisation by exposing cells or not to Mars-related stresses (desiccation and radiation). Bacterial populations remained viable after 6 months although the kinetics of mineralization and cell-mineral interactions depended on the nature of minerals. Detection of biosignatures strongly depended on analytical methods, successful with FTIR and EDX but not with RAMAN and immunoassays. Neither influence of stress exposure, nor qualitative and quantitative changes of detected molecules were observed as a function of mineralization time and matrix. Rock-Eval analysis suggests that potential for preservation on geological times may be possible only with moderate diagenetic and metamorphic conditions. The implications of our results for microfossil preservation in the geological record of Earth as well as on Mars are discussed.

Diagenesis and clay mineral formation at Gale Crater, Mars.

The Mars Science Laboratory rover Curiosity found host rocks of basaltic composition and alteration assemblages containing clay minerals at Yellowknife Bay, Gale Crater. On the basis of the observed host rock and alteration minerals, we present results of equilibrium thermochemical modeling of the Sheepbed mudstones of Yellowknife Bay in order to constrain the formation conditions of its secondary mineral assemblage. Building on conclusions from sedimentary observations by the Mars Science Laboratory team, we assume diagenetic, in situ alteration. The modeling shows that the mineral assemblage formed by the reaction of a CO2-poor and oxidizing, dilute aqueous solution (Gale Portage Water) in an open system with the Fe-rich basaltic-composition sedimentary rocks at 10-50°C and water/rock ratio (mass of rock reacted with the starting fluid) of 100-1000, pH of ∽7.5-12. Model alteration assemblages predominantly contain phyllosilicates (Fe-smectite, chlorite), the bulk composition of a mixture of which is close to that of saponite inferred from Chemistry and Mineralogy data and to that of saponite observed in the nakhlite Martian meteorites and terrestrial analogues. To match the observed clay mineral chemistry, inhomogeneous dissolution dominated by the amorphous phase and olivine is required. We therefore deduce a dissolving composition of approximately 70% amorphous material, with 20% olivine, and 10% whole rock component.

Dating carbonaceous matter in archean cherts by electron paramagnetic resonance.

Ancient geological materials are likely to be contaminated through geological times. Thus, establishing the syngeneity of the organic matter embedded in a mineral matrix is a crucial step in the study of very ancient rocks. This is particularly the case for Archean siliceous sedimentary rocks (cherts), which record the earliest traces of life. We used electron paramagnetic resonance (EPR) for assessing the syngeneity of organic matter in cherts that have a metamorphic grade no higher than greenschist. A correlation between the age of Precambrian samples and the shape of their EPR signal was established and statistically tested. As thermal treatments impact organic matter maturity, the effect of temperature on this syngeneity proxy was studied; cyanobacteria were submitted to cumulative short thermal treatment at high temperatures followed by an analysis of their EPR parameters. The resulting carbonaceous matter showed an evolution similar to that of a thermally treated young chert. Furthermore, the possible effect of metamorphism, which is a longer thermal event at lower temperatures, was ruled out for cherts older than 2 Gyr, based on the study of Silurian cherts of the same age and same precursors but various metamorphic grades. We determined that even the most metamorphosed sample did not exhibit the lineshape of an Archean sample. In the hope of detecting organic contamination in Archean cherts, a "contamination-like" mixture was prepared and studied by EPR. It resulted that the lineshape analysis alone does not allow contamination detection and that it must be performed along with cumulative thermal treatments. Such treatments were applied to three Archean chert samples, making dating of their carbonaceous matter possible. We concluded that EPR is a powerful tool to study primitive organic matter and could be used in further exobiology studies on low-metamorphic grade samples (from Mars for example).

Electron paramagnetic resonance study of a photosynthetic microbial mat and comparison with Archean cherts.

Organic radicals in artificially carbonized biomass dominated by oxygenic and non-oxygenic photosynthetic bacteria, Microcoleus chthonoplastes-like and Chloroflexus-like bacteria respectively, were studied by Electron Paramagnetic Resonance (EPR) spectroscopy. The two bacteria species were sampled in mats from a hypersaline lake. They underwent accelerated ageing by cumulative thermal treatments to induce progressive carbonization of the biological material, mimicking the natural maturation of carbonaceous material of Archean age. For thermal treatments at temperatures higher than 620 °C, a drastic increase in the EPR linewidth is observed in the carbonaceous matter from oxygenic photosynthetic bacteria and not anoxygenic photosynthetic bacteria. This selective EPR linewidth broadening reflects the presence of a catalytic element inducing formation of radical aggregates, without affecting the molecular structure or the microstructure of the organic matter, as shown by Raman spectroscopy and Transmission Electron Microscopy. For comparison, we carried out an EPR study of organic radicals in silicified carbonaceous rocks (cherts) from various localities, of different ages (0.42 to 3.5 Gyr) and having undergone various degrees of metamorphism, i.e. various degrees of natural carbonization. EPR linewidth dispersion for the most primitive samples was quite significant, pointing to a selective dipolar broadening similar to that observed for carbonized bacteria. This surprising result merits further evaluation in the light of its potential use as a marker of past bacterial metabolisms, in particular oxygenic photosynthesis, in Archean cherts.

The PROCESS experiment: exposure of amino acids in the EXPOSE-E experiment on the international space station and in laboratory simulations.

To understand the chemical behavior of organic molecules in the space environment, amino acids and a dipeptide in pure form and embedded in meteorite powder were exposed in the PROCESS experiment in the EXPOSE-E facility mounted on the European Technology Exposure Facility (EuTEF) platform on board the International Space Station (ISS). After exposure to space conditions for 18 months, the samples were returned to Earth and analyzed in the laboratory for reactions caused by solar UV and cosmic radiation. Chemical degradation and possible racemization and oligomerization, the main reactions caused by photochemistry in the vacuum ultraviolet domain (VUV, wavelength range 100-200 nm for photon energy from 6.2 to 12.4 eV) were examined in particular. The molecules were extracted and derivatized by silylation and analyzed by gas chromatograph coupled to a mass spectrometer (GC-MS) to quantify the rate of the degradation of the compounds. Laboratory exposure in several wavelength ranges from UV to VUV was carried out in parallel in the Cologne Deutsches Zentrum für Luft- und Raumfahrt (DLR) Center and Centre de biophysique moléculaire (CBM) laboratories. The results show that resistance to irradiation is a function of the chemical nature of the exposed molecules and the wavelengths of the UV light. The most altered compounds were the dipeptide, aspartic acid, and aminobutyric acid. The most resistant were alanine, valine, glycine, and aminoisobutyric acid. Our results also demonstrate the protective effect of meteorite powder, which reemphasizes the importance of exogenic contribution to the inventory of prebiotic organics on early Earth.

Experimental silicification of the extremophilic Archaea Pyrococcus abyssi and Methanocaldococcus jannaschii: applications in the search for evidence of life in early Earth and extraterrestrial rocks.

Hydrothermal activity was common on the early Earth and associated micro-organisms would most likely have included thermophilic to hyperthermophilic species. 3.5-3.3 billion-year-old, hydrothermally influenced rocks contain silicified microbial mats and colonies that must have been bathed in warm to hot hydrothermal emanations. Could they represent thermophilic or hyperthermophilic micro-organisms and if so, how were they preserved? We present the results of an experiment to silicify anaerobic, hyperthermophilic micro-organisms from the Archaea Domain Pyrococcus abyssi and Methanocaldococcus jannaschii, that could have lived on the early Earth. The micro-organisms were placed in a silica-saturated medium for periods up to 1 year. Pyrococcus abyssi cells were fossilized but the M. jannaschii cells lysed naturally after the exponential growth phase, apart from a few cells and cell remains, and were not silicified although their extracellular polymeric substances were. In this first simulated fossilization of archaeal strains, our results suggest that differences between species have a strong influence on the potential for different micro-organisms to be preserved by fossilization and that those found in the fossil record represent probably only a part of the original diversity. Our results have important consequences for biosignatures in hydrothermal or hydrothermally influenced deposits on Earth, as well as on early Mars, as environmental conditions were similar on the young terrestrial planets and traces of early Martian life may have been similarly preserved as silicified microfossils.

Darwin--a mission to detect and search for life on extrasolar planets.

The discovery of extrasolar planets is one of the greatest achievements of modern astronomy. The detection of planets that vary widely in mass demonstrates that extrasolar planets of low mass exist. In this paper, we describe a mission, called Darwin, whose primary goal is the search for, and characterization of, terrestrial extrasolar planets and the search for life. Accomplishing the mission objectives will require collaborative science across disciplines, including astrophysics, planetary sciences, chemistry, and microbiology. Darwin is designed to detect rocky planets similar to Earth and perform spectroscopic analysis at mid-infrared wavelengths (6-20 mum), where an advantageous contrast ratio between star and planet occurs. The baseline mission is projected to last 5 years and consists of approximately 200 individual target stars. Among these, 25-50 planetary systems can be studied spectroscopically, which will include the search for gases such as CO(2), H(2)O, CH(4), and O(3). Many of the key technologies required for the construction of Darwin have already been demonstrated, and the remainder are estimated to be mature in the near future. Darwin is a mission that will ignite intense interest in both the research community and the wider public.

Importance of a martian hematite site for astrobiology.

Defining locations where conditions may have been favorable for life is a key objective for the exploration of Mars. Of prime importance are sites where conditions may have been favorable for the preservation of evidence of prebiotic or biotic processes. Areas displaying significant concentrations of the mineral hematite (alpha-Fe2O3), recently identified by thermal emission spectrometry, may have significance in the search for evidence of extraterrestrial life. Since iron oxides can form as aqueous mineral precipitates, the potential exists to preserve microscopic evidence of life in iron oxide-depositing ecosystems. Terrestrial hematite deposits proposed as possible analogs for hematite deposits on Mars include massive (banded) iron formations, iron oxide hydrothermal deposits, iron-rich laterites and ferricrete soils, and rock varnish. We report the potential for long-term preservation of microfossils by iron oxide mineralization in specimens of the approximately 2,100-Ma banded iron deposit of the Gunflint Formation, Canada. Scanning and analytical electron microscopy reveals micrometer-scale rods, spheres, and filaments consisting predominantly of iron and oxygen with minor carbon. We interpret these objects as microbial cells permineralized by an iron oxide, presumably hematite. The confirmation of ancient martian microbial life in hematite deposits will require the return of samples to terrestrial laboratories. A hematite-rich deposit composed of aqueous iron oxide precipitates may thus prove to be a prime site for future sample return.

Microscopic physical biomarkers in carbonate hot springs: implications in the search for life on Mars.

Physical evidence of life (physical biomarkers) from the deposits of carbonate hot springs were documented at the scale of microorganisms--submillimeter to submicrometer. The four moderate-temperature (57 to 72 degrees C), neutral pH springs reported on in this study, support diverse communities of bacteria adapted to specific physical and chemical conditions. Some of the microbes coexist with travertine deposits in endolithic communities. In other cases, the microbes are rapidly coated and destroyed by precipitates but leave distinctive mineral fabrics. Some microbes adapted to carbonate hot springs produce an extracellular polymeric substance which forms a three-dimensional matrix with living cells and cell remains, known as a biofilm. Silicon and iron oxides often coat the biofilm, leading to long-term preservation. Submicrometer mineralized spheres composed of calcium fluoride or silica are common in carbonate hot spring deposits. Sphere formation is biologically mediated, but the spheres themselves are apparently not fossils or microbes. Additionally, some microbes selectively weather mineral surfaces in distinctive patterns. Hot spring deposits have been cited as prime locations for exobiological exploration of Mars. The presence of preserved microscopic physical biomarkers at all four sites supports a strategy of searching for evidence of life in hot spring deposits on Mars.

Search for life on Mars.

A multi-user integrated suite of instruments designed to optimize the search for evidence of life on Mars is described. The package includes: -Surface inspection and surface environment analysis to identify the potential Mars landing sites, to inspect the surface geology and mineralogy, to search for visible surficial microbial macrofossils, to study the surface radiation budget and surface oxidation processes, to search for niches for extant life. -Subsurface sample acquisition by core drilling -Analysis of surface and subsurface minerals and organics to characterize the surface mineralogy, to analyse the surface and subsurface oxidants, to analyse the mineralogy of subsurface aliquots, to analyse the organics present in the subsurface aliquots (elemental and molecular composition, isotopes, chirality). -Macroscopic and microscopic inspection of subsurface aliquots to search for life's indicators (paleontological, biological, mineralogical) and to characterize the mineralogy of the subsurface aliquots. The study is led by ESA Manned Spaceflight and Microgravity Directorate.

The flocculation of wine yeasts: biochemical and morphological characteristics in Kloeckera apiculata.

The floc-forming ability of flocculent strains of Kloeckera apiculata, isolated from musts, was tested for susceptibility to proteinase and sugar treatments. Three different flocculation phenotypes were discriminated by protease digestion, whereas the inhibition of flocculation by sugars distinguished two definite patterns: one mechanism of flocculation involved a galactose-specific protein and the other a broad-specificity lectin. SEM and TEM observation of the cell surface of two different Kloeckera strains revealed fine fibrils and a diffuse structure at the point of contact in one strain, and thick masses of mucus on the cell wall of the other strain.

Suppression of squamous cell carcinoma in hairless mice by dietary nutrient variation.

In experiments involving the induction of squamous cell carcinoma in 1846 hairless mice that were maintained on a wide variety of diets, it was found that those diets with the least optimum balance of nutrients had the greatest inhibitory effect on growth of cancer. Rate of onset and severity of tumors was caused to vary over a 20-fold range by means of dietary balance alone. These experiments suggest that dietary variation in general and intentional malnutrition in particular should be given special attention in the control of existing cancer in humans.

Serotonin binding sites. II. Muramyl dipeptide binds to serotonin binding sites on myelin basic protein, LHRH, and MSH-ACTH 4-10.

Previously, we reported the existence of structurally similar serotonin binding sites on myelin basic protein, LHRH, and MSH-ACTH 4-10. We now report that the adjuvant peptide, muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln) also binds to these sites. This observation may help to explain previous observations of serotonin-like activity by muramyl peptides, including the promotion of slow-wave sleep and fever induction. The observation may also provide an important link between the immune system and the nervous system that may explain the role of muramyl dipeptide adjuvants in causing autoimmune diseases to serotonin-regulated proteins and their receptors, as well as the alterations in serotonin levels that are often observed in autoimmune diseases. The observation provides concrete evidence for a dual-antigen hypothesis for the induction of autoimmune diseases by an adjuvant-peptide complex. Application of such a mechanism for induction of autoimmunity may be of importance in understanding a number of postinfectious and postvaccinal neuropathies, and suggests a possible etiology for autism, in which many patients have high blood serotonin levels, autoimmune reactions to myelin basic protein, and antibodies to serotonin binding sites. Finally, the observation suggests that glycopeptides may act as neurotransmitters.

Bovine pineal antireproductive tripeptide binds to luteinizing hormone-releasing hormone: a model for peptide modulation by sequence specific peptide interactions?

We report results of chromatographic, pH titration and nuclear magnetic resonance (NMR) spectroscopy studies demonstrating that the bovine pineal antireproductive tripeptide, Thr-Ser-Lys (BPART), binds to luteinizing hormone-releasing hormone (LHRH) at a site comprised of LHRH 2-5 (His-Trp-Ser-Tyr). BPART and LHRH have been shown to be antagonists in vitro. The binding constant is ca. 2 X 10(3)/mole. An NMR study of fifty other peptide pairs demonstrates that the binding is sequence and residue specific. The binding provides evidence of the amino acid pairing hypothesis, and suggests the possibility of modulation of one peptide by directly binding with another peptide.

Clinical suppression of experimental allergic encephalomyelitis by muramyl dipeptide "adjuvant".

Experimental allergic encephalomyelitis (EAE) is a model for several human diseases including multiple sclerosis and post-vaccinal encephalopathies. EAE is generally thought to be an autoimmune response to the antigen myelin basic protein (MBP). Oddly, MBP can also suppress EAE, and many observations suggest that an independent immune response to so-called "adjuvant" material is also necessary to EAE induction. Thus, EAE may be a result of a pair of interactive immune responses, one against MBP, and one against adjuvant. If so, the adjuvant should, like MBP, suppress EAE. We present data from experiments on strain 13 guinea pigs demonstrating EAE suppression by muramyl dipeptide, an active component of complete Freund's adjuvant. These results are striking because classically adjuvants are defined as immunopotentiators, not immunosuppressants. Our results, therefore, suggest that a revaluation of the role of adjuvants in inducing autoimmune diseases may be necessary.

Covalent linkage of phosphoinositides to myelin basic protein: in vivo occurrence and in vitro studies with experimental allergic encephalomyelitis.

We have previously reported the covalent attachment of phosphoinositides to myelin basic protein (MBP) in vitro. In this study, in vivo phosphoinositidation of MBP was observed to occur after intracerebral injection with [32p]HPO4(2-). In the in vitro study of experimental allergic encephalomyelitis, a four-fold increase in phosphoinositidation of MBP was observed in the myelin from the guinea pigs sacrificed during the development of motor dysfunction, as compared to the control group. A decrease (40%) was observed among most of the animals sacrificed before the onset of motor dysfunction.