Multi-Technique Characterization of 3.45 Ga Microfossils on Earth: A Key Approach to Detect Possible Traces of Life in Returned Samples from Mars.

The NASA Mars 2020 Perseverance rover is actively exploring Jezero crater to conduct analyses on igneous and sedimentary rock targets from outcrops located on the crater floor (Máaz and Séítah formations) and from the delta deposits, respectively. The rock samples collected during this mission will be recovered during the Mars Sample Return mission, which plans to bring samples back to Earth in the 2030s to conduct in-depth studies using sophisticated laboratory instrumentation. Some of these samples may contain traces of ancient martian life that may be particularly difficult to detect and characterize because of their morphological simplicity and subtle biogeochemical expressions. Using the volcanic sediments of the 3.45 Ga Kitty's Gap Chert (Pilbara, Australia), containing putative early life forms (chemolithotrophs) and considered as astrobiological analogues for potential early Mars organisms, we document the steps required to demonstrate the syngenicity and biogenicity of such biosignatures using multiple complementary analytical techniques to provide information at different scales of observation. These include sedimentological, petrological, mineralogical, and geochemical analyses to demonstrate macro- to microscale habitability. New approaches, some unavailable at the time of the original description of these features, are used to verify the syngenicity and biogenicity of the purported fossil chemolithotrophs. The combination of elemental (proton-induced X-ray emission spectrometry) and molecular (deep-ultraviolet and Fourier transform infrared) analyses of rock slabs, thin sections, and focused ion beam sections reveals that the carbonaceous matter present in the samples is enriched in trace metals (e.g., V, Cr, Fe, Co) and is associated with aromatic and aliphatic molecules, which strongly support its biological origin. Transmission electron microscopy observations of the carbonaceous matter documented an amorphous nanostructure interpreted to correspond to the degraded remains of microorganisms and their by-products (extracellular polymeric substances, filaments…). Nevertheless, a small fraction of carbonaceous particles has signatures that are more metamorphosed. They probably represent either reworked detrital biological or abiotic fragments of mantle origin. This study serves as an example of the analytical protocol that would be needed to optimize the detection of fossil traces of life in martian rocks.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

In Situ Raman Spectroscopy Monitoring of Material Changes During Proton Irradiation.

Organic molecules are prime targets in the search for life on other planetary bodies in the Solar System. Understanding their preservation potential and detectability after ionic irradiation, with fluences potentially representing those received for several millions to billions of years at Mars or in interplanetary space, is a crucial goal for astrobiology research. In order to be able to perform in situ characterization of such organic molecules under ionic irradiation in the near future, a feasibility experiment was performed with polymer test samples to validate the optical configuration and the irradiation chamber geometry. We present here a Raman in situ investigation of the evolution of a series of polymers during proton irradiation. To achieve this goal, a new type of Raman optical probe was designed, which documented that proton irradiation (with a final fluence of 3.1014 at·cm-2) leads to an increase in the background level of the signal, potentially explained by the scission of the polymeric chains and by atom displacements creating defects in the materials. To improve the setup further, a micro-Raman probe and a temperature-controlled sample holder are under development to provide higher spectral and spatial resolutions (by reducing the depth of field and laser spot size), to permit Raman mapping as well as to avoid any thermal effects.

Equilibrium and non-equilibrium furanose selection in the ribose isomerisation network.

The exclusive presence of ß-D-ribofuranose in nucleic acids is still a conundrum in prebiotic chemistry, given that pyranose species are substantially more stable at equilibrium. However, a precise characterisation of the relative furanose/pyranose fraction at temperatures higher than about 50 °C is still lacking. Here, we employ a combination of NMR measurements and statistical mechanics modelling to predict a population inversion between furanose and pyranose at equilibrium at high temperatures. More importantly, we show that a steady temperature gradient may steer an open isomerisation network into a non-equilibrium steady state where furanose is boosted beyond the limits set by equilibrium thermodynamics. Moreover, we demonstrate that nonequilibrium selection of furanose is maximum at optimal dissipation, as gauged by the temperature gradient and energy barriers for isomerisation. The predicted optimum is compatible with temperature drops found in hydrothermal vents associated with extremely fresh lava flows on the seafloor.

Deletion of Mocos Induces Xanthinuria with Obstructive Nephropathy and Major Metabolic Disorders in Mice.

Background: Xanthinuria type II is a rare autosomal purine disorder. This recessive defect of purine metabolism remains an under-recognized disorder. Methods: Mice with targeted disruption of the molybdenum cofactor sulfurase (Mocos) gene were generated to enable an integrated understanding of purine disorders and evaluate pathophysiologic functions of this gene which is found in a large number of pathways and is known to be associated with autism. Results: Mocos-deficient mice die with 4 weeks of age due to renal failure of distinct obstructive nephropathy with xanthinuria, xanthine deposits, cystic tubular dilation, Tamm-Horsfall (uromodulin) protein (THP) deposits, tubular cell necrosis with neutrophils, and occasionally hydronephrosis with urolithiasis. Obstructive nephropathy is associated with moderate interstitial inflammatory and fibrotic responses, anemia, reduced detoxification systems, and important alterations of the metabolism of purines, amino acids, and phospholipids. Conversely, heterozygous mice expressing reduced MOCOS protein are healthy with no apparent pathology. Conclusions: Mocos-deficient mice develop a lethal obstructive nephropathy associated with profound metabolic changes. Studying MOCOS functions may provide important clues about the underlying pathogenesis of xanthinuria and other diseases requiring early diagnosis.

Metallomics in deep time and the influence of ocean chemistry on the metabolic landscapes of Earth's earliest ecosystems.

Modern biological dependency on trace elements is proposed to be a consequence of their enrichment in the habitats of early life together with Earth's evolving physicochemical conditions; the resulting metallic biological complement is termed the metallome. Herein, we detail a protocol for describing metallomes in deep time, with applications to the earliest fossil record. Our approach extends the metallome record by more than 3 Ga and provides a novel, non-destructive method of estimating biogenicity in the absence of cellular preservation. Using microbeam particle-induced X-ray emission (µPIXE), we spatially quantify transition metals and metalloids within organic material from 3.33 billion-year-old cherts of the Barberton greenstone belt, and demonstrate that elements key to anaerobic prokaryotic molecular nanomachines, including Fe, V, Ni, As and Co, are enriched within carbonaceous material. Moreover, Mo and Zn, likely incorporated into enzymes only after the Great Oxygenation Event, are either absent or present at concentrations below the limit of detection of µPIXE, suggesting minor biological utilisation in this environmental setting. Scanning and transmission electron microscopy demonstrates that metal enrichments do not arise from accumulation in nanomineral phases and thus unambiguously reflect the primary composition of the carbonaceous material. This carbonaceous material also has δ13C between -41.3‰ and 0.03‰, dominantly -21.0‰ to -11.5‰, consistent with biological fractionation and mostly within a restricted range inconsistent with abiotic processes. Considering spatially quantified trace metal enrichments and negative δ13C fractionations together, we propose that, although lacking cellular preservation, this organic material has biological origins and, moreover, that its precursor metabolism may be estimated from the fossilised "palaeo-metallome". Enriched Fe, V, Ni and Co, together with petrographic context, suggests that this kerogen reflects the remnants of a lithotrophic or organotrophic consortium cycling methane or nitrogen. Palaeo-metallome compositions could be used to deduce the metabolic networks of Earth's earliest ecosystems and, potentially, as a biosignature for evaluating the origin of preserved organic materials found on Mars.

Modern Iron Ooids of Hydrothermal Origin as a Proxy for Ancient Deposits.

We constrained the origin and genetic environment of modern iron ooids (sand-sized grains with a core and external cortex of concentric laminae) providing new tools for the interpretation of their fossil counterparts as well as the analogous particles discovered on Mars. Here, we report an exceptional, unique finding of a still active deposit of submillimetric iron ooids, under formation at the seabed at a depth of 80 m over an area characterized by intense hydrothermal activity off Panarea, a volcanic island north of Sicily (Italy). An integrated analysis, carried out by X-ray Powder Diffraction, Environmental Scanning Electron Microscopy, X-ray Fluorescence and Raman spectroscopy reveals that Panarea ooids are deposited at the seafloor as concentric laminae of primary goethite around existing nuclei. The process is rapid, and driven by hydrothermal fluids as iron source. A sub-spherical, laminated structure resulted from constant agitation and by degassing of CO2-dominated fluids through seafloor sediments. Our investigations point the hydrothermal processes as responsible for the generation of the Panarea ooids, which are neither diagenetic nor reworked. The presence of ooids at the seawater-sediments interface, in fact, highlights how their development and growth is still ongoing. The proposed results show a new process responsible for ooids formation and gain a new insight into the genesis of iron ooids deposits that are distributed at global scale in both modern and past sediments.

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports-among others-the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.

Potential Role of Inorganic Confined Environments in Prebiotic Phosphorylation.

A concise outlook on the potential role of confinement in phosphorylation and phosphate condensation pertaining to prebiotic chemistry is presented. Inorganic confinement is a relatively uncharted domain in studies concerning prebiotic chemistry, and even more so in terms of experimentation. However, molecular crowding within confined dimensions is central to the functioning of contemporary biology. There are numerous advantages to confined environments and an attempt to highlight this fact, within this article, has been undertaken, keeping in context the limitations of aqueous phase chemistry in phosphorylation and, to a certain extent, traditional approaches in prebiotic chemistry.

A Statistical Approach to Illustrate the Challenge of Astrobiology for Public Outreach.

In this study, we attempt to illustrate the competition that constitutes the main challenge of astrobiology, namely the competition between the probability of extraterrestrial life and its detectability. To illustrate this fact, we propose a simple statistical approach based on our knowledge of the Universe and the Milky Way, the Solar System, and the evolution of life on Earth permitting us to obtain the order of magnitude of the distance between Earth and bodies inhabited by more or less evolved past or present life forms, and the consequences of this probability for the detection of associated biosignatures. We thus show that the probability of the existence of evolved extraterrestrial forms of life increases with distance from the Earth while, at the same time, the number of detectable biosignatures decreases due to technical and physical limitations. This approach allows us to easily explain to the general public why it is very improbable to detect a signal of extraterrestrial intelligence while it is justified to launch space probes dedicated to the search for microbial life in the Solar System.

Near-infrared luminescent metallacrowns for combined in vitro cell fixation and counter staining.

Cell fixation is an essential approach for preserving cell morphology, allowing the targeting and labelling of biomolecules with fluorescent probes. One of the key requirements for more efficient fluorescent labelling is the preservation of cell morphology, which usually requires a combination of several fixation techniques. In addition, the use of a counter stain is often essential to improve the contrast of the fluorescent probes. Current agents possess significant limitations, such as low resistance toward photobleaching and sensitivity to changes in the microenvironment. Luminescent Ln3+ 'encapsulated sandwich' metallacrowns (MCs) overcome these drawbacks and offer complementary advantages. In particular, they emit sharp emission bands, possess a large difference between excitation and emission wavelengths and do not photobleach. Herein, MCs formed with pyrazinehydroxamic acid (Ln3+[Zn(ii)MCpyzHA], Ln3+ = Yb, Nd) were used, combined with near-infrared (NIR) counter staining and fixation agents for HeLa cells upon an initial five minute exposure to UV-A light. The validity and quality of the cell fixation were assessed with Raman spectroscopy. Analysis of the NIR luminescence properties of these MCs was performed under different experimental conditions, including in a suspension of stained cells. Moreover, the high emission intensity of Ln3+[Zn(ii)MCpyzHA] in the NIR region allows these MCs to be used for imaging with standard CCD cameras installed on routine fluorescence microscopes. Finally, the NIR-emitting Ln3+[Zn(ii)MCpyzHA] compounds combine, within a single molecule, features such as cell fixation and staining abilities, good photostability and minimal sensitivity of the emission bands to the local microenvironment, and they are highly promising for establishing the next generation of imaging agents with a single biodistribution.

The Close-Up Imager Onboard the ESA ExoMars Rover: Objectives, Description, Operations, and Science Validation Activities.

The Close-Up Imager (CLUPI) onboard the ESA ExoMars Rover is a powerful high-resolution color camera specifically designed for close-up observations. Its accommodation on the movable drill allows multiple positioning. The science objectives of the instrument are geological characterization of rocks in terms of texture, structure, and color and the search for potential morphological biosignatures. We present the CLUPI science objectives, performance, and technical description, followed by a description of the instrument's planned operations strategy during the mission on Mars. CLUPI will contribute to the rover mission by surveying the geological environment, acquiring close-up images of outcrops, observing the drilling area, inspecting the top portion of the drill borehole (and deposited fines), monitoring drilling operations, and imaging samples collected by the drill. A status of the current development and planned science validation activities is also given. Key Words: Mars-Biosignatures-Planetary Instrumentation. Astrobiology 17, 595-611.

Biosignatures on Mars: What, Where, and How? Implications for the Search for Martian Life.

UNLABELLED: The search for traces of life is one of the principal objectives of Mars exploration. Central to this objective is the concept of habitability, the set of conditions that allows the appearance of life and successful establishment of microorganisms in any one location. While environmental conditions may have been conducive to the appearance of life early in martian history, habitable conditions were always heterogeneous on a spatial scale and in a geological time frame. This "punctuated" scenario of habitability would have had important consequences for the evolution of martian life, as well as for the presence and preservation of traces of life at a specific landing site. We hypothesize that, given the lack of long-term, continuous habitability, if martian life developed, it was (and may still be) chemotrophic and anaerobic. Obtaining nutrition from the same kinds of sources as early terrestrial chemotrophic life and living in the same kinds of environments, the fossilized traces of the latter serve as useful proxies for understanding the potential distribution of martian chemotrophs and their fossilized traces. Thus, comparison with analog, anaerobic, volcanic terrestrial environments (Early Archean >3.5-3.33 Ga) shows that the fossil remains of chemotrophs in such environments were common, although sparsely distributed, except in the vicinity of hydrothermal activity where nutrients were readily available. Moreover, the traces of these kinds of microorganisms can be well preserved, provided that they are rapidly mineralized and that the sediments in which they occur are rapidly cemented. We evaluate the biogenicity of these signatures by comparing them to possible abiotic features. Finally, we discuss the implications of different scenarios for life on Mars for detection by in situ exploration, ranging from its non-appearance, through preserved traces of life, to the presence of living microorganisms. KEY WORDS: Mars-Early Earth-Anaerobic chemotrophs-Biosignatures-Astrobiology missions to Mars.

Tracing Biosignature Preservation of Geothermally Silicified Microbial Textures into the Geological Record.

New Zealand and Argentine (Late Jurassic-Recent) siliceous hot-spring deposits (sinter) reveal preservation pathways of environmentally controlled, microbe-dominated sedimentary facies over geological time scales. Texturally distinctive, laminated to thinly layered, dense and vertically oriented, microtubular "palisade" fabric is common in low-temperature (<40°C) sinter-apron terraces. In modern hot springs, the dark green to brown, sheathed, photosynthetic cyanobacterium Calothrix spp. (family Rivulariaceae) constructs felted palisade mats in shallow terrace(tte) pools actively accreting opaline silica. The resulting stacked layers of silicified coarse filaments-a stromatolite-are highly porous and readily modified by postdepositional environmental perturbations, secondary silica infill, and diagenetic silica phase mineral transformations (opal-A to quartz). Fossil preservation quality is affected by relative timing of silicification, and later environmental and geological events. A systematic approach was used to characterize palisade fabric in sinters of different ages to refine tools for recognizing biosignatures in extreme environments and to track their long-term preservation pathways into the geological record. Molecular techniques, scanning electron microscopy, Raman spectrometry, X-ray powder diffraction, petrography, and lipid biomarker analyses were applied. Results indicate that microbial communities vary at the micron scale and that early and rapid silicification is paramount to long-term preservation, especially where minimal postdepositional disturbance follows fossilization. Overall, it appears that the most robust biomarkers of fossil microbial activity in hot-spring deposits are their characteristic macro- and microtextures and laser micro-Raman identified carbon. Studies of Phanerozoic geothermal deposits with mineralized microbial components are relevant analogs for Precambrian geobiology because early life is commonly preserved as microbial microfossils and biofilms in silica, some of it hydrothermal in origin. Yet the diagenetic "movie" has already been run. Hence, studying younger sinters of a range of ages provides an opportunity to "play it again" and follow the varied influences on biosignatures into the deep-time geological record.

Bioreducible poly(2-ethyl-2-oxazoline)-PLA-PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and Doxorubicin.

The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)-poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz-PLA-g-PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8days following mcDNA-micelles intratumoral administration. Furthermore, drug-gene co-delivery in PEOz-PLA-g-PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.

Habitability on Mars from a microbial point of view.

Extraterrestrial habitability is a complex notion. We briefly review what is known about the origin of life on Earth, that is, life based on carbon chemistry and water. We then discuss habitable conditions (past and present) for established life and for the survival of microorganisms. Based on these elements, we propose to use the term habitable only for conditions necessary for the origin of life, the proliferation of life, and the survival of life. Not covered by this term would be conditions necessary for prebiotic chemistry and conditions that would allow the recognition of extinct or hibernating life. Finally, we apply this concept to the potential emergence of life on Mars where suitable conditions for life to start, proliferate, and survive have been heterogeneous throughout its history. These considerations have a profound impact on the nature and distribution of eventual traces of martian life, or any precursor, and must therefore inform our search-for-life strategies.

Raman imaging of metastable opal in carbonaceous microfossils of the 700-800 ma old Draken Formation.

Opaline silica was detected, with Raman spectroscopy, in carbonaceous microfossils (especially Myxococcoides) in silicified filamentous microbial mats within dolomitized conglomerates of the Draken Formation (-800 to -700 Ma). High-resolution electron microscopy (HRTEM) and microprobe analyses were used to confirm the nature of this phase in the quartz matrix of the microbial mats. The silica likely precipitated in a microcrystalline form onto the organic macromolecules around, and within, the degrading microorganisms and preserved them by inhibiting the natural phase change to quartz. The Raman signal of opaline silica associated with carbonaceous matter and other biosignatures could be a potential indicator of biogenicity. This kind of association could be very useful during the future ExoMars mission (ESA/Roscosmos, 2018) that will search for traces of past life on Mars.

Potential fossil endoliths in vesicular pillow basalt, Coral Patch Seamount, eastern North Atlantic Ocean.

The chilled rinds of pillow basalt from the Ampère-Coral Patch Seamounts in the eastern North Atlantic were studied as a potential habitat of microbial life. A variety of putative biogenic structures, which include filamentous and spherical microfossil-like structures, were detected in K-phillipsite-filled amygdules within the chilled rinds. The filamentous structures (∼2.5 µm in diameter) occur as K-phillipsite tubules surrounded by an Fe-oxyhydroxide (lepidocrocite) rich membranous structure, whereas the spherical structures (from 4 to 2 µm in diameter) are associated with Ti oxide (anatase) and carbonaceous matter. Several lines of evidence indicate that the microfossil-like structures in the pillow basalt are the fossilized remains of microorganisms. Possible biosignatures include the carbonaceous nature of the spherical structures, their size distributions and morphology, the presence and distribution of native fluorescence, mineralogical and chemical composition, and environmental context. When taken together, the suite of possible biosignatures supports the hypothesis that the fossil-like structures are of biological origin. The vesicular microhabitat of the rock matrix is likely to have hosted a cryptoendolithic microbial community. This study documents a variety of evidence for past microbial life in a hitherto poorly investigated and underestimated microenvironment, as represented by the amygdules in the chilled pillow basalt rinds. This kind of endolithic volcanic habitat would have been common on the early rocky planets in our Solar System, such as Earth and Mars. This study provides a framework for evaluating traces of past life in vesicular pillow basalts, regardless of whether they occur on early Earth or Mars.

How does crystalline substrate plasticity modify thin film buckling?

We report experimental atomic force microscopy observations and analytical modeling of buckling structures of thin films deposited on single crystal substrates. The formation of straight-sided blisters just above the step structures resulting from the dislocations emergence has been observed and explained in the framework of the Föppl-von Karman theory of thin plates. A critical step height above which the buckling may occur has been determined and the asymmetry of the resulting blisters has been explained. Finally, the new buckling criterion has been compared with the classical one in the plane case and allows us to explain the blisters localization on step structures.