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Detecting biosignatures of life in extraterrestrial environments remains one of the primary objectives of scientific inquiry. Currently, both remote and direct detection methods are primarily aimed at identifying key molecular classes fundamental to terrestrial biology. However, a more universally applicable spectroscopic approach could involve searching for homochiral molecules. Thus, this perspective delves into the significance of homochirality as a critical factor in the origin of life. Without homochirality, the formation of self-recognizing and self-replicating complex molecules would be hindered. The various hypotheses concerning the origin of homochiral molecules have been explored and analyzed within this context. This perspective emphasizes the potential for discovering extraterrestrial microscopic life through the detection of homochiral molecules using chirodetecting methods such as chromatography and chiroptical spectroscopy or circular polarimetry as a promising remote technique. This discussion highlights the importance of homochirality in the broader search for life beyond Earth and underscores the need for innovative methodologies and instrumentation in astrobiological research. These techniques can be an effective method for detecting homochirality on future planetary missions.

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In this study, we conducted polymerase chain reaction (PCR) experiments using Escherichia coli (E. coli) and a Mars sand simulant (Mars Global Simulant MGS-1, Exolith Lab) to detect and analyze potential extraterrestrial life. The targeted DNA sequence is common among the bacterial kingdom on Earth. PCR experiments conducted after alkaline heat extraction, wherein samples with varying amounts of Mars sand simulant were compared, revealed that the simulant interfered with DNA detection. We then conducted PCR experiments following treatment with a sand DNA extraction kit on samples with various E. coli densities. DNA bands for a minimum E. coli density of 900 cells/(g sand) were confirmed, while no DNA bands were visible in the 90 cells/(g sand) sample with and without the Mars sand simulant. The total DNA mass contained in 900 cells was calculated to be 15.3 pg (i.e., 1.53 pg in 0.1 g sand sample we evaluated). We tested and compared the influence of the eluate of Mars sand simulant and DNA adsorption onto Mars sand simulant based on optical absorbance measurements. Our findings suggest that the mechanism by which the Mars sand simulant prevents PCR is through the adsorption of DNA onto the Mars sand simulant.

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Computation, if treated as a set of physical processes that act on information represented by states of matter, encompasses biological systems, digital systems, and other constructs and may be a fundamental measure of living systems. The opportunity for biological computation, represented in the propagation and selection-driven evolution of information-carrying organic molecular structures, has been partially characterized in terms of planetary habitable zones (HZs) based on primary conditions such as temperature and the presence of liquid water. A generalization of this concept to computational zones (CZs) is proposed, with constraints set by three principal characteristics: capacity (including computation rates), energy, and instantiation (or substrate, including spatial extent). CZs naturally combine traditional habitability factors, including those associated with biological function that incorporate the chemical milieu, constraints on nutrients and free energy, as well as element availability. Two example applications are presented by examining the fundamental thermodynamic work efficiency and Landauer limit of photon-driven biological computation on planetary surfaces and of generalized computation in stellar energy capture structures (a.k.a. Dyson structures). It is suggested that CZs that involve nested structures or substellar objects could manifest unique observational signatures as cool far-infrared emitters. While these latter scenarios are entirely hypothetical, they offer a useful, complementary introduction to the potential universality of CZs.

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Darwin's theory of evolution by natural selection was revolutionary because it provided a mechanism by which variation could be selected. This mechanism can only operate on living systems and thus cannot be applied to the origin of life. Here, we propose a viable alternative mechanism for prebiotic systems: autocatalytic selection, in which molecules catalyze reactions and processes that lead to increases in their concentration. Crucially, this provides a driver for increases in concentrations of molecules to a level that permits prebiotic metabolism. We show how this can produce high levels of amino acids, sugar phosphates, nucleotides and lipids and then lead on to polymers. Our outline is supported by a set of guidelines to support the identification of the most likely prebiotic routes. Most of the steps in this pathway are already supported by experimental results. These proposals generate a coherent and viable set of pathways that run from established Hadean geochemistry to the beginning of life.

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While Earth contains the only known example of life in the universe, it is possible that life elsewhere is fundamentally different from what we are familiar with. There is an increased recognition in the astrobiology community that the search for life should steer away from terran-specific biosignatures to those that are more inclusive to all life-forms. To start exploring the space of possibilities that life could occupy, we can try to dissociate life from the chemistry that composes it on Earth by envisioning how different life elsewhere could be in composition, lifestyle, medium, and form, and by exploring how the general principles that govern living systems on Earth might be found in different forms and environments across the Solar System. Exotic life-forms could exist on Mars or Venus, or icy moons like Europa and Enceladus, or even as a shadow biosphere on Earth. New perspectives on agnostic biosignature detection have also begun to emerge, allowing for a broader and more inclusive approach to seeking exotic life with unknown chemistry that is distinct from life as we know it on Earth.

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The search for life beyond Earth necessitates a rigorous and comprehensive examination of biosignatures, the types of observable imprints that life produces. These imprints and our ability to detect them with advanced instrumentation hold the key to our understanding of the presence and abundance of life in the universe. Biosignatures are the chemical or physical features associated with past or present life and may include the distribution of elements and molecules, alone or in combination, as well as changes in structural components or physical processes that would be distinct from an abiotic background. The scientific and technical strategies used to search for life on other planets include those that can be conducted in situ to planetary bodies and those that could be observed remotely. This chapter discusses numerous strategies that can be employed to look for biosignatures directly on other planetary bodies using robotic exploration including those that have been deployed to other planetary bodies, are currently being developed for flight, or will become a critical technology on future missions. Search strategies for remote observations using current and planned ground-based and space-based telescopes are also described. Evidence from spectral absorption, emission, or transmission features can be used to search for remote biosignatures and technosignatures. Improving our understanding of biosignatures, their production, transformation, and preservation on Earth can enhance our search efforts to detect life on other planets.

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What is life? Multiple definitions have been proposed to answer this question, but unfortunately, none of them has reached the consensus of the scientific community. Here, the strategy used to define what life is was based on first establishing which characteristics are common to all living systems (organic nature, entropy-producing system, self-organizing, reworkable pre-program, capacity to interact and adapt, reproduction and evolution) and from them constructing the definition taking into account that reproduction and evolution are not essential for life. On this basis, life is defined as an interactive process occurring in entropy-producing, adaptive, and informative (organic) systems. An unforeseen consequence of the inseparable duality between the system (living being) and the process (life) is the interchangeability of the elements of the definition to obtain other equally valid alternatives. In addition, in the light of this definition, cases of temporarily lifeless living systems (viruses, dormant seeds, and ultracold cells) are analyzed, as well as the status of artificial life entities and the hypothetical nature of extraterrestrial life. All living systems are perishable because the passage of time leads to increasing entropy. Life must create order by continuously producing disorder and exporting it to the environment and so we move and stay in the phase transition between order and chaos, far from equilibrium, thanks to the input of energy from the outside. However, the passage of time eventually leads us to an end in which life disappears and entropy increases.

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The concept of a biosignature is widely used in astrobiology to suggest a link between some observation and a biological cause, given some context. The term itself has been defined and used in several ways in different parts of the scientific community involved in the search for past or present life on Earth and beyond. With the ongoing acceleration in the search for life in distant time and/or deep space, there is a need for clarity and accuracy in the formulation and reporting of claims. Here, we critically review the biosignature concept(s) and the associated nomenclature in light of several problems and ambiguities emphasized by recent works. One worry is that these terms and concepts may imply greater certainty than is usually justified by a rational interpretation of the data. A related worry is that terms such as "biosignature" may be inherently misleading, for example, because the divide between life and non-life-and their observable effects-is fuzzy. Another worry is that different parts of the multidisciplinary community may use non-equivalent or conflicting definitions and conceptions, leading to avoidable confusion. This review leads us to identify a number of pitfalls and to suggest how they can be circumvented. In general, we conclude that astrobiologists should exercise particular caution in deciding whether and how to use the concept of biosignature when thinking and communicating about habitability or life. Concepts and terms should be selected carefully and defined explicitly where appropriate. This would improve clarity and accuracy in the formulation of claims and subsequent technical and public communication about some of the most profound and important questions in science and society. With this objective in mind, we provide a checklist of questions that scientists and other interested parties should ask when assessing any reported detection of a "biosignature" to better understand exactly what is being claimed.

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We present a comparative study of the methods used in the search for extraterrestrial microorganism life, including a summary table where different life-detection techniques can be easily compared as an aid to mission and instrument design aimed at life detection. This is an extension of previous study, where detection techniques for a series of target characteristics and molecules that could constitute a positive life detection were evaluated. This comparison has been extended with a particular consideration to sources of false positives, the causes of negative detection, the results of detection techniques when presented regarding terrestrial life, and additional science objectives that could be achieved outside the primary aim of detecting life. These additions address both the scientific and programmatic side of exploration mission design, where a successful proposal must demonstrate probable outcomes and be able to return valuable results even if no life is found. The applicability of the life detection techniques is considered for Earth life, Earth-independent life (life emerging independently from that on Earth,) and Earth-kin life (sharing a common ancestor with life on Earth), and techniques effective in detecting Earth life should also be useful in the detection of Earth-kin life. However, their applicability is not guaranteed for Earth-independent life. As found in our previous study, there exists no realistic single detection method that can conclusively determine the discovery of extraterrestrial life, and no method is superior to all others. In this study, we further consider combinations of detection techniques and identify imaging as a valuable addition to molecule detection methods, even in cases where there is insufficient resolution to observe the detailed morphology of a microbial cell. The search for extraterrestrial life is further divided into a survey-and-detection and analysis-and-conclusion step. These steps benefit from different detection techniques, but imaging is necessary for both parts.

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It is not a stretch to say that the search for extraterrestrial life is possibly the biggest of the cosmic endeavors that humankind has embarked upon. With the continued discovery of several Earth-like exoplanets, the hope of detecting potential biosignatures is multiplying amongst researchers in the astrobiology community. However, to be able to discern these signatures as being truly of biological origin, we also need to consider their probable abiotic origin. The field of prebiotic chemistry, which is aimed at understanding enzyme-free chemical syntheses of biologically relevant molecules, could particularly aid in this regard. Specifically, certain peculiar characteristics of prebiotically pertinent messy chemical reactions, including diverse and racemic product yields and lower synthesis efficiencies, can be utilized in analyzing whether a perceived 'signature of life' could possibly have chemical origins. The knowledge gathered from understanding the transition from chemistry to biology during the origin of life could be used for creating a library of abiotically synthesized biologically relevant organic molecules. This can then be employed in designing, standardizing, and testing mission-specific instruments/analysis systems, while also enabling the effective targeting of exoplanets with potentially 'ongoing' molecular evolutionary processes for robust detection of life in future explorative endeavors.

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The discovery of mechanisms for the synthesis of homo-polymeric oligopeptides, such as polyglycine under conditions relevant to the astrophysical environment as well as in scenarios resembling primordial conditions that prevailed soon after Earth was formed, raises hopes in the search of extraterrestrial life. It also raises the possibility of extraterrestrial contribution to origin of life on Earth in the form of simple polypeptides. Bioinformatics analyses strongly predict such homo-polymeric peptides to be intrinsically disordered underscoring the potential involvement of IDPs in the origin of life which, even in its simplest form, could emerge spontaneously by autocatalysis of the primordial IDPs in self-organizing systems that evolved over time following natural selection.Communicated by Ramaswamy H. Sarma.

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The reliable identification of biosignatures is key to the search for life elsewhere. On ocean worlds like Enceladus or Europa, this can be achieved by impact ionization mass spectrometers, such as the SUrface Dust Analyzer (SUDA) on board NASA's upcoming Europa Clipper mission. During spacecraft flybys, these instruments can sample ice grains formed from subsurface water and emitted by these moons. Previous laboratory analog experiments have demonstrated that SUDA-type instruments could identify amino acids, fatty acids, and peptides in ice grains and discriminate between their abiotic and biotic origins. Here, we report experiments simulating impact ionization mass spectra of ice grains containing DNA, lipids, and metabolic intermediates extracted from two bacterial cultures: Escherichia coli and Sphingopyxis alaskensis. Salty Enceladan or Europan ocean waters were simulated using matrices with different NaCl concentrations. Characteristic mass spectral signals, such as DNA nucleobases, are clearly identifiable at part-per-million-level concentrations. Mass spectra of all substances exhibit unambiguous biogenic patterns, which in some cases show significant differences between the two bacterial species. Sensitivity to the biosignatures decreases with increasing matrix salinity. The experimental parameters indicate that future impact ionization mass spectrometers will be most sensitive to the investigated biosignatures for ice grain encounter speeds of 4-6 km/s.

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The detection and analysis of extraterrestrial life are important issues of space science. Mars is among the most important planets to explore for extraterrestrial life, owing both to its physical properties and to its ancient and present environments as revealed by previous exploration missions. In this paper, we present a comparative study of methods for detecting extraterrestrial life and life-related substances. To this end, we have classified and summarized the characteristics targeted for the detection of extraterrestrial life in solar system exploration mission and the methods used to evaluate them. A summary table is presented. We conclude that at this moment (i) there is no realistic single detection method capable of concluding the discovery of extraterrestrial life, (ii) no single method has an advantage over the others in all respects, and (iii) there is no single method capable of distinguishing extraterrestrial life from terrestrial life. Therefore, a combination of complementary methods is essential. We emphasize the importance of endeavoring to detect extraterrestrial life without overlooking possible alien life forms, even at the cost of tolerating false positives. Summaries of both the targets and the detection methods should be updated continuously, and comparative studies of both should be pursued. Although this study assumes Mars to be a model site for the primary environment for life searches, both the targets and detection methods described herein will also be useful for searching for extraterrestrial life in any celestial environment and for the initial inspection of returned samples.

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We present a laboratory experiment of ATP measurement using Mars soil simulant and Escherichia coli (E. coli) with a point of view for future application to searching extraterrestrial life. We used Mars Global Simulant MGS-1 (Exolith Lab) as soil simulant, added E. coli suspension to it, then the soil simulant with E. coli was dried. Various configurations of samples with different E. coli density, 1.75 × 102, 1.75 × 103, 1.75 × 104, 1.75 × 105, and 1.75 × 106 cells (g soil)-1, were prepared together with controls. ATP extraction reagent and luminescence reagent were added to the sample, and bioluminescence measurement was performed. The result suggests significant detection of ATP for samples with E. coli density used in this work. Similar experiments but without the soil simulant were carried out, and results with and without the soil simulant are compared. Based on the ATP measurement studied in this work, we discussed extraterrestrial life search, planetary protection, relation with the panspermia hypothesis, and also other applications.

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How do scientists maintain their research programs in the face of not finding anything? Continual failure to produce results can result in declining support, scientific controversy and credibility challenges. We elaborate on a crucial mechanism for sustaining the credibility of research programs through periods of non-detection: the maintenance of ambiguity. By this, we refer to scientific strategies that resist closure or an experiment's premature end by creating doubt in negative findings and fostering hope for future positive results. To illustrate this concept, we draw upon the recent history of Martian exobiology. Since the 1960s, planetary scientists have continually tried and failed to find evidence of life on Mars. And yet, interest in extraterrestrial life detection remains high, with more missions to Mars underway. Through three destabilizing events of non-detection, we show how exobiologists sustained the search for Martian life by casting doubt on negative findings, pointing to other possible unexplored routes to success, and finally reconfiguring operations around new methods or goals. New approaches may take the form of shifts in scale, method and object of interest. By pivoting to a different scale, method or object, exobiologists have continued to study a subject continually lacking proof of existence and made important discoveries about life on Earth.

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A major objective in the exploration of Mars is to test the hypothesis that the planet has ever hosted life. Biogenic compounds, especially biominerals, are believed to serve as biomarkers in Raman-assisted remote sensing missions. However, the prerequisite for the development of these minerals as biomarkers is the uniqueness of their biogenesis. Herein, tetragonal bipyramidal weddellite, a type of calcium oxalate, is successfully achieved by UV-photolyzing pyruvic acid (PA). The as-prepared products are identified and characterized by micro-Raman spectroscopy and field emission scanning electron microscopy. Persistent mineralization of weddellite is observed with altering key experimental parameters, including pH, Ca2+ and PA concentrations. In particular, the initial concentration of PA can significantly influence the morphology of weddellite crystal. Oxalate acid is commonly of biological origin; thus calcium oxalate is considered to be a biomarker. However, our results reveal that calcium oxalate can be harvested by a UV photolysis pathway. Moreover, prebiotic sources of organics (e.g., PA, glycine, alanine, and aspartic acid) have been proven to be available through abiotic pathways. Therefore, our results may provide a new abiotic pathway of calcium oxalate formation. Considering that calcium oxalate minerals have been taken as biosignatures for the origin and early evolution of life on Earth and astrobiological investigations, its formation and accumulation by the photolysis of abiological organic compounds should be taken into account.

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Spectroscopic instruments are increasingly being implemented in the search for extraterrestrial life. However, microstructural spectral analyses of alien environments could prove difficult without knowledge on the molecular identification of individual spectral signatures. To bridge this gap, we introduce unsupervised K-means clustering as a statistical approach to discern spectral patterns of biosignatures without prior knowledge of spectral regions of biomolecules. Spectral profiles of bacterial isolates from analogous polar ice sheets were measured with Raman spectroscopy. Raman analysis identified carotenoid and violacein pigments, and key cellular features including saturated and unsaturated fats, triacylglycerols, and proteins. Principal component analysis and targeted spectra integration biplot analysis revealed that the clustering of bacterial isolates was attributed to spectral biosignatures influenced by carotenoid pigments and ratio of unsaturated/saturated fat peaks. Unsupervised K-means clustering highlighted the prevalence of the corresponding spectral peaks, while subsequent supervised permutational multivariate analysis of variance provided statistical validation for spectral differences associated with the identified cellular features. Establishing a validated catalog of spectral signatures of analogous biotic and abiotic materials, in combination with targeted supervised tools, could prove effective at identifying extant biosignatures.

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As we expand the search for life beyond Earth, a water-dominated planet, we turn our eyes to other aquatic worlds. Microbial life found in Earth's many extreme habitats are considered useful analogs to life forms we are likely to find in extraterrestrial bodies of water. Modern-day benthic microbial mats inhabiting the low-oxygen, high-sulfur submerged sinkholes of temperate Lake Huron (Michigan, USA) and microbialites inhabiting the shallow, high-carbonate waters of subtropical Laguna Bacalar (Yucatan Peninsula, Mexico) serve as potential working models for exploration of extraterrestrial life. In Lake Huron, delicate mats comprising motile filaments of purple-pigmented cyanobacteria capable of oxygenic and anoxygenic photosynthesis and pigment-free chemosynthetic sulfur-oxidizing bacteria lie atop soft, organic-rich sediments. In Laguna Bacalar, lithification by cyanobacteria forms massive carbonate reef structures along the shoreline. Herein, we document studies of these two distinct earthly microbial mat ecosystems and ponder how similar or modified methods of study (e.g., robotics) would be applicable to prospective mat worlds in other planets and their moons (e.g., subsurface Mars and under-ice oceans of Europa). Further studies of modern-day microbial mat and microbialite ecosystems can add to the knowledge of Earth's biodiversity and guide the search for life in extraterrestrial hydrospheres.