Volatile Organic Compound Metabolism on Early Earth.

Biogenic volatile organic compounds (VOCs) constitute a significant portion of gas-phase metabolites in modern ecosystems and have unique roles in moderating atmospheric oxidative capacity, solar radiation balance, and aerosol formation. It has been theorized that VOCs may account for observed geological and evolutionary phenomena during the Archaean, but the direct contribution of biology to early non-methane VOC cycling remains unexplored. Here, we provide an assessment of all potential VOCs metabolized by the last universal common ancestor (LUCA). We identify enzyme functions linked to LUCA orthologous protein groups across eight literature sources and estimate the volatility of all associated substrates to identify ancient volatile metabolites. We hone in on volatile metabolites with confirmed modern emissions that exist in conserved metabolic pathways and produce a curated list of the most likely LUCA VOCs. We introduce volatile organic metabolites associated with early life and discuss their potential influence on early carbon cycling and atmospheric chemistry.

The Preservation and Spectral Detection of Historic Museum Specimen Microbial Mat Biosignatures Within Martian Dust: Lessons Learned for Mars Exploration and Sample Return.

The key building blocks for life on Mars could be preserved within potentially habitable paleo-depositional settings with their detection possible by utilizing mid-infrared spectroscopy; however, a definite identification and confirmation of organic or even biological origin will require the samples to be returned to Earth. In the present study, Fourier-transform infrared (FTIR) spectroscopic techniques were used to characterize both mineralogical and organic materials within Mars dust simulant JSC Mars-1 and ancient Antarctic cyanobacterial microbial mats from 1901 to 1904 Discovery Expedition. When FTIR spectroscopy is applied to cyanobacterial microbial mat communities, the resulting spectra will reflect the average biochemical composition of the mats rather than taxa-specific spectral patterns of the individual organisms and can thus be considered as a total chemical analysis of the mat colony. This study also highlights the potential difficulties in the detection of these communities on Mars and which spectral biosignatures will be most detectable within geological substrates. Through the creation and analysis of a suite of dried microbial mat material and Martian dust simulant mixtures, the spectral signatures and wavenumber positions of CHx aliphatic hydrocarbons and the C-O and O-H bands of polysaccharides remained detectable and may be detectable within sample mixtures obtained through Mars Sample Return activities.

A DNA Methylation Signature from Buccal Swabs to Identify Tuberculosis Infection.

BACKGROUND: Tuberculosis (TB) is amongst the largest infectious causes of death worldwide and there is a need for a time- and resource-effective diagnostic method. In this novel and exploratory study, we show the potential of using buccal swabs to collect human DNA and investigate the DNA methylation (DNAm) signatures as a diagnostic tool for TB. METHODS: Buccal swabs were collected from pulmonary TB patients (n= 7), TB exposed (n= 7), and controls (n= 9) in Sweden. Using Illumina MethylationEPIC array the DNAm status was determined. RESULTS: We identified 5644 significant differentially methylated CpG sites between the patients and controls. Performing the analysis on a validation cohort of samples collected in Kenya and Peru (patients, n=26; exposed, n=9; control, n=10) confirmed the DNAm signature. We identified a TB consensus disease module, significantly enriched in TB-associated genes. Lastly, we used machine learning to identify a panel of seven CpG sites discriminative for TB and developed a TB classifier. In the validation cohort the classifier performed with an AUC of 0.94, sensitivity of 0.92, and specificity of 1. CONCLUSION: In summary, the result from this study shows clinical implications of using DNAm signatures from buccal swabs to explore new diagnostic strategies for TB.

Integrative proteome analysis of bone marrow interstitial fluid and serum reveals candidate signature for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer and clinically highly heterogeneous characterized by the accumulation of clonally proliferative immature precursors of myeloid lineage leading to bone marrow failure. Although, the current diagnostic methods for AML consist of cytogenetic and molecular assessment, there is a need for new markers that can serve as useful candidates in diagnosis, prognosis and understanding the pathophysiology of the disease. This study involves the investigation of alterations in the bone marrow interstitial fluid and serum proteome of AML patients compared to controls using label-free quantitative proteomic approach. A total of 201 differentially abundant proteins were identified in AML BMIF, while in the case of serum 123 differentially abundant proteins were identified. The bioinformatics analysis performed using IPA revealed several altered pathways including FAK signalling, IL-12 signalling and production of macrophages etc. Verification experiments were performed in a fresh independent cohort of samples using MRM assays led to the identification of a panel of three proteins viz., PPBP, APOH, ENOA which were further validated in a new cohort of serum samples by ELISA. The three-protein panel could be helpful in the diagnosis, prognosis and understanding of the pathophysiology of AML in the future. BIOLOGICAL SIGNIFICANCE: Acute Myeloid Leukemia (AML) is a type haematological malignancy which constitute one third of total leukemias and it is the most common acute leukemia in adults. In the current clinical practice, the evaluation of diagnosis and progression of AML is largely based on morphologic, immunophenotypic, cytogenetic and molecular assessment. There is a need for new markers/signatures which can serve as useful candidates in diagnosis and prognosis. The present study aims to identify and validate candidate biosignature for AML which can be useful in diagnosis, prognosis and understand the pathophysiology of the disease. Here, we identified 201 altered proteins in AML BMIF and 123 in serum. Among these altered proteins, a set of three proteins viz., pro-platelet basic protein (CXCL7), enolase 1 (ENO1) and beta-2-glycoprotein 1 (APOH) were significantly increased in AML BMIF and serum suggest that this panel of proteins could help in future AML disease management and thereby improving the survival expectancy of AML patients.

Quantifying the Potential for Nitrate-Dependent Iron Oxidation on Early Mars: Implications for the Interpretation of Gale Crater Organics.

Geological evidence and atmospheric and climate models suggest habitable conditions occurred on early Mars, including in a lake in Gale crater. Instruments aboard the Curiosity rover measured organic compounds of unknown provenance in sedimentary mudstones at Gale crater. Additionally, Curiosity measured nitrates in Gale crater sediments, which suggests that nitrate-dependent Fe2+ oxidation (NDFO) may have been a viable metabolism for putative martian life. Here, we perform the first quantitative assessment of an NDFO community that could have existed in an ancient Gale crater lake and quantify the long-term preservation of biological necromass in lakebed mudstones. We find that an NDFO community would have the capacity to produce cell concentrations of up to 106 cells mL-1, which is comparable to microbes in Earth's oceans. However, only a concentration of <104 cells mL-1, due to organisms that inefficiently consume less than 10% of precipitating nitrate, would be consistent with the abundance of organics found at Gale. We also find that meteoritic sources of organics would likely be insufficient as a sole source for the Gale crater organics, which would require a separate source, such as abiotic hydrothermal or atmospheric production or possibly biological production from a slowly turning over chemotrophic community.

Investigating Microbial Biosignatures in Aeolian Environments Using Micro-X-Ray: Simulation of PIXL Instrument Analyses at Jezero Crater Onboard the Perseverance Mars 2020 Rover.

Assessing the past habitability of Mars and searching for evidence of ancient life at Jezero crater via the Perseverance rover are the key objectives of NASA's Mars 2020 mission. Onboard the rover, PIXL (Planetary Instrument for X-ray Lithochemistry) is one of the best suited instruments to search for microbial biosignatures due to its ability to characterize chemical composition of fine scale textures in geological targets using a nondestructive technique. PIXL is also the first micro-X-ray fluorescence (XRF) spectrometer onboard a Mars rover. Here, we present guidelines for identifying and investigating a microbial biosignature in an aeolian environment using PIXL-analogous micro-XRF (µXRF) analyses. We collected samples from a modern wet aeolian environment at Padre Island, Texas, that contain buried microbial mats, and we analyzed them using µXRF techniques analogous to how PIXL is being operated on Mars. We show via µXRF technique and microscope images the geochemical and textural variations from the surface to ∼40 cm depth. Microbial mats are associated with heavy-mineral lags and show specific textural and geochemical characteristics that make them a distinct biosignature for this environment. Upon burial, they acquire a diffuse texture due to the expansion and contraction of gas-filled voids, and they present a geochemical signature rich in iron and titanium, which is due to the trapping of heavy minerals. We show that these intrinsic characteristics can be detected via µXRF analyses, and that they are distinct from buried abiotic facies such as cross-stratification and adhesion ripple laminations. We also designed and conducted an interactive survey using the Padre Island µXRF data to explore how different users chose to investigate a biosignature-bearing dataset via PIXL-like sampling strategies. We show that investigating biosignatures via PIXL-like analyses is heavily influenced by technical constraints (e.g., the XRF measurement characteristics) and by the variety of approaches chosen by different scientists. Lessons learned for accurately identifying and characterizing this biosignature in the context of rover-mission constraints include defining relative priorities among measurements, favoring a multidisciplinary approach to the decision-making process of XRF measurements selection, and considering abiotic results to support or discard a biosignature interpretation. Our results provide guidelines for PIXL analyses of potential biosignature on Mars.

Hydrochemical and isotopic characteristics of water sources for biological activity across a massive evaporite basin on the Tibetan Plateau: Implications for aquatic environments on early Mars.

Covered by vast eolian landforms, gravel deposits, and playas, the worldwide typical evaporite deposit land, Qaidam Basin, in northwestern China is analogous to early Mars when the aridification process had lasted for millions of years since the end of a wetter climate. This study aims to investigate the chemical and isotopic characteristics of waters in an evaporite-rich environment, as well as the habitable conditions therein, that have undergone a transformation similar to early Mars. In May 2023, a total of 26 water samples were collected across the representative central axis of a longitudinal aridity gradient in the Qaidam Basin, including categories of meteoric water, freshwater, standing water accumulated after precipitation, salty lacustrine water, and hypersaline brines to inspect compounds made up of carbon, nitrogen, phosphorus, sulfur, halogen, and metallic elements. As evaporation intensified, the salt types transformed from HCO3-Ca·Na to Cl·SO4-Na or ClMg. The dominance of carbonate will gradually be replaced by sulfate and chloride, leaving much more dilute and less detectable contents. The presence of trace ClO4-, ClO3-, ClO2-, and BrO3- was confirmed in a few of the sampled Qaidam waters, indicating the preservation of oxyhalides in waters within an arid region and possibly the presence of relevant microbial enzymes. The isotopes of water, carbonaceous, and nitrogenous compounds provide valuable references for either abiogenic or biogenic signatures. With undetectable amount, phosphorus was found to be the limiting nutrient in evaporative aquatic environments but not necessarily antibiosignatures. Overall, these results suggest that the paleo-lacustrine environments on Mars are more likely to preserve biosignatures if they feature the dominance of carbonate minerals, bioavailable nitrate, phosphorus, and organic carbon, the presence of thermodynamically unstable oxyhalides, and isotope ratios that point to the involvement of biological activity.

Transcriptomic and proteomic profiles of fetal versus adult mesenchymal stromal cells and mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) can regenerate tissues through engraftment and differentiation but also via paracrine signalling via extracellular vesicles (EVs). Fetal-derived MSCs (fMSCs) have been shown, both in vitro and in animal studies, to be more efficient than adult MSC (aMSCs) in generating bone and muscle but the underlying reason for this difference has not yet been clearly elucidated. In this study, we aimed to systematically investigate the differences between fetal and adult MSCs and MSC-derived EVs at the phenotypic, RNA, and protein levels. METHODS: We carried out a detailed and comparative characterization of culture-expanded fetal liver derived MSCs (fMSCs) and adult bone marrow derived MSCs (aMSCs) phenotypically, and the MSCs and MSC-derived EVs were analysed using transcriptomics and proteomics approaches with RNA Sequencing and Mass Spectrometry. RESULTS: Fetal MSCs were smaller, exhibited increased proliferation and colony-forming capacity, delayed onset of senescence, and demonstrated superior osteoblast differentiation capability compared to their adult counterparts. Gene Ontology analysis revealed that fMSCs displayed upregulated gene sets such as "Positive regulation of stem cell populations", "Maintenance of stemness" and "Muscle cell development/contraction/Myogenesis" in comparison to aMSCs. Conversely, aMSCs displayed upregulated gene sets such as "Complement cascade", "Adipogenesis", "Extracellular matrix glycoproteins" and "Cellular metabolism", and on the protein level, "Epithelial cell differentiation" pathways. Signalling entropy analysis suggested that fMSCs exhibit higher signalling promiscuity and hence, higher potency than aMSCs. Gene ontology comparisons revealed that fetal MSC-derived EVs (fEVs) were enriched for "Collagen fibril organization", "Protein folding", and "Response to transforming growth factor beta" compared to adult MSC-derived EVs (aEVs), whereas no significant difference in protein expression in aEVs compared to fEVs could be detected. CONCLUSIONS: This study provides detailed and systematic insight into the differences between fMSCs and aMSCs, and MSC-derived EVs. The key finding across phenotypic, transcriptomic and proteomic levels is that fMSCs exhibit higher potency than aMSCs, meaning they are in a more undifferentiated state. Additionally, fMSCs and fMSC-derived EVs may possess greater bone forming capacity compared to aMSCs. Therefore, using fMSCs may lead to better treatment efficacy, especially in musculoskeletal diseases.

Resolution and quantification of carbohydrates by enantioselective comprehensive two-dimensional gas chromatography.

Carbohydrates, in particular the d-enantiomers of ribose, 2-deoxyribose, and glucose, are essential to life's informational biopolymers (RNA/DNA) and for supplying energy to living cells through glycolysis. Considered to be potential biosignatures in the search of past or present life, our capacity to detect and quantify these essential sugars is crucial for future space missions to the Moon, Mars or Titan as well as for sample-return missions. However, the enantioselective analysis of carbohydrates is challenging and both research and routine applications, are lacking efficient methods that combine highly sensitive and reproducible detection with baseline enantioselective resolution and reliable enantiomeric excess (ee) measurements. Here, we present four different derivatization strategies in combination with multidimensional gas chromatography coupled to a reflectron time-of-flight mass spectrometer (GC×GC-TOF-MS) for the enantioselective resolution of C3 to C6 carbohydrates potentially suitable for sample-return analyses. Full mass spectral interpretation and calibration curves for one single-step (cyclic boronate derivatives) and three two-step derivatization protocols (aldononitrile-acetate, hemiacetalization-trifluoroacetylation, and hemiacetalization-permethylation) are presented for concentrations ranging from 1 to 50 pmol µL⁻1 with correlation coefficients R2 > 0.94. We compared several analytical parameters including reproducibility, sensitivity (LOD and LOQ), overall separation, chiral resolution (RS), mass spectrum selectivity, stability during long term storage, and reliability of ee measurements to guide the application-dependent selection of optimal separation and quantification performance.

Biosignature Detection and MinION Sequencing of Antarctic Cryptoendoliths After Exposure to Mars Simulation Conditions.

In the search for life in our Solar System, Mars remains a promising target based on its proximity and similarity to Earth. When Mars transitioned from a warmer, wetter climate to its current dry and freezing conditions, any putative extant life probably retreated into habitable refugia such as the subsurface or the interior of rocks. Terrestrial cryptoendolithic microorganisms (i.e., those inhabiting rock interiors) thus represent possible modern-day Mars analogs, particularly those from the hyperarid McMurdo Dry Valleys in Antarctica. As DNA is a strong definitive biosignature, given that there is no known abiotic chemistry that can polymerize nucleobases, we investigated DNA detection with MinION sequencing in Antarctic cryptoendoliths after an ∼58-sol exposure in MARTE, a Mars environmental chamber capable of simulating martian temperature, pressure, humidity, ultraviolet (UV) radiation, and atmospheric composition, in conjunction with protein and lipid detection. The MARTE conditions resulted in changes in community composition and DNA, proteins, and cell membrane-derived lipids remained detectable postexposure. Of the multitude of extreme environmental conditions on Mars, UV radiation (specifically UVC) is the most destructive to both cells and DNA. As such, we further investigated if a UVC exposure corresponding to ∼278 martian years would impede DNA detection via MinION sequencing. The MinION was able to successfully detect and sequence DNA after this UVC radiation exposure, suggesting its utility for life detection in future astrobiology missions focused on finding relatively recently exposed biomarkers inside possible martian refugia.

Can Isotopologues Be Used as Biosignature Gases in Exoplanet Atmospheres?

Isotopologue ratios are anticipated to be one of the most promising signs of life that can be observed remotely. On Earth, carbon isotopes have been used for decades as evidence of modern and early metabolic processes. In fact, carbon isotopes may be the oldest evidence for life on Earth, though there are alternative geological processes that can lead to the same magnitude of fractionation. However, using isotopologues as biosignature gases in exoplanet atmospheres presents several challenges. Most significantly, we will only have limited knowledge of the underlying abiotic carbon reservoir of an exoplanet. Atmospheric carbon isotope ratios will thus have to be compared against the local interstellar medium or, better yet, their host star. A further substantial complication is the limited precision of remote atmospheric measurements using spectroscopy. The various metabolic processes that cause isotope fractionation cause less fractionation than anticipated measurement precision (biological fractionation is typically 2 to 7%). While this level of precision is easily reachable in the laboratory or with special in situ instruments, it is out of reach of current telescope technology to measure isotope ratios for terrestrial exoplanet atmospheres. Thus, gas isotopologues are poor biosignatures for exoplanets given our current and foreseeable technological limitations.

Is There Such a Thing as a Biosignature?

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.

A characteristic cerebellar biosignature for bipolar disorder, identified with fully automatic machine learning.

Background: Transcriptomic profile differences between patients with bipolar disorder and healthy controls can be identified using machine learning and can provide information about the potential role of the cerebellum in the pathogenesis of bipolar disorder.With this aim, user-friendly, fully automated machine learning algorithms can achieve extremely high classification scores and disease-related predictive biosignature identification, in short time frames and scaled down to small datasets. Method: A fully automated machine learning platform, based on the most suitable algorithm selection and relevant set of hyper-parameter values, was applied on a preprocessed transcriptomics dataset, in order to produce a model for biosignature selection and to classify subjects into groups of patients and controls. The parent GEO datasets were originally produced from the cerebellar and parietal lobe tissue of deceased bipolar patients and healthy controls, using Affymetrix Human Gene 1.0 ST Array. Results: Patients and controls were classified into two separate groups, with no close-to-the-boundary cases, and this classification was based on the cerebellar transcriptomic biosignature of 25 features (genes), with Area Under Curve 0.929 and Average Precision 0.955. The biosignature includes both genes connected before to bipolar disorder, depression, psychosis or epilepsy, as well as genes not linked before with any psychiatric disease. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed participation of 4 identified features in 6 pathways which have also been associated with bipolar disorder. Conclusion: Automated machine learning (AutoML) managed to identify accurately 25 genes that can jointly - in a multivariate-fashion - separate bipolar patients from healthy controls with high predictive power. The discovered features lead to new biological insights. Machine Learning (ML) analysis considers the features in combination (in contrast to standard differential expression analysis), removing both irrelevant as well as redundant markers, and thus, focusing to biological interpretation.

Field evaluation of a point-of-care triage test for active tuberculosis (TriageTB).

BACKGROUND: To improve tuberculosis (TB) diagnosis, the World Health Organisation (WHO) has called for a non-sputum based triage test to focus TB testing on people with a high likelihood of having active pulmonary tuberculosis (TB). Various host or pathogen biomarker-based testing devices are in design stage and require validity assessment. Host biomarkers have shown promise to accurately rule out active TB, but further research is required to determine generalisability. The TriageTB diagnostic test study aims to assess the accuracy of diagnostic test candidates, as well as field-test, finalise the design and biomarker signature, and validate a point-of-care multi-biomarker test (MBT). METHODS: This observational diagnostic study will evaluate sensitivity and specificity of biomarker-based diagnostic candidates including the MBT and Xpert® TB Fingerstick cartridge compared with a gold-standard composite TB outcome classification defined by symptoms, sputum GeneXpert® Ultra, smear and culture, radiological features, response to TB therapy and presence of an alternative diagnosis. The study will be conducted in research sites in South Africa, Uganda, The Gambia and Vietnam which all have high TB prevalence. The two-phase design allows for finalisation of the MBT in Phase 1 in which candidate host proteins will be evaluated on stored serum from Asia, South Africa and South America and on fingerstick blood from 50 newly recruited participants per site. The MBT test will then be locked down and validated in Phase 2 on 250 participants per site. DISCUSSION: By targeting confirmatory TB testing to those with a positive triage test, 75% of negative GXPU may be avoided, thereby reducing diagnostic costs and patient losses during the care cascade. This study builds on previous biomarker research and aims to identify a point-of-care test meeting or exceeding the minimum World Health Organisation target product profile of a 90% sensitivity and 70% specificity. Streamlining TB testing by identifying individuals with a high likelihood of TB should improve TB resources use and, in so doing, improve TB care. TRIAL REGISTRATION: NCT04232618 (clinicaltrials.gov) Date of registration: 16 January 2020.

The Hypothesis of a "Living Pulse" in Cells.

Motility is a great biosignature and its pattern is characteristic for specific microbes. However, motion does also occur within the cell by the myriads of ongoing processes within the cell and the exchange of gases and nutrients with the outside environment. Here, we propose that the sum of these processes in a microbial cell is equivalent to a pulse in complex organisms and suggest a first approach to measure the "living pulse" in microorganisms. We emphasize that if a "living pulse" can be shown to exist, it would have far-reaching applications, such as for finding life in extreme environments on Earth and in extraterrestrial locations, as well as making sure that life is not present where it should not be, such as during medical procedures and in the food processing industry.

Organic Biosignature Degradation in Hydrothermal and Serpentinizing Environments: Implications for Life Detection on Icy Moons and Mars.

Evidence of liquid water is a primary indicator of habitability on the icy moons in our outer solar system as well as on terrestrial planets such as Mars. If liquid water-containing environments host life, some of its organic remains can be fossilized and preserved as organic biosignatures. However, inorganic materials may also be present and water-assisted organic-inorganic reactions can transform the organic architecture of biological remains. Our understanding of the fate of these organic remains can be assisted by experimental simulations that monitor the chemical changes that occur in microbial organic matter due to the presence of water and minerals. We performed hydrothermal experiments at temperatures between 100°C and 300°C involving lipid-rich microbes and natural serpentinite mineral mixtures generated by the subaqueous hydrothermal alteration of ultramafic rock. The products reveal what the signals of life may look like when subjected to water-organic-inorganic reactions. Straight- and branched-chain lipids in unaltered samples are joined by cyclization and aromatization products in hydrothermally altered samples. Hydrothermal reactions produce distinct products that are not present in the starting materials, including small, single-ring, heteroatomic, and aromatic compounds such as indoles and phenols. Hydrothermal reactions in the presence of serpentinite minerals lead to significant reduction of these organic structures and their replacement by diketopiperazines (DKPs) and dihydropyrazines (DHPs), which may be compounds that are distinct to organic-inorganic reactions. Given that the precursors of DKPs and DHPs are normally lost during early diagenesis, the presence of these compounds can be an indicator of coexisting recent life and hydrothermal processing in the presence of minerals. However, laboratory experiments reveal that the formation and preservation of these compounds can only occur within a distinct temperature window. Our findings are relevant to life detection missions that aim to access hydrothermal and serpentinizing environments in the subsurfaces of icy moons and Mars.

Are Large Sulfur Isotope Variations Biosignatures in an Ancient, Impact-Induced Hydrothermal Mars Analog?

Discrepancies have emerged concerning the application of sulfur stable isotope ratios as a biosignature in impact crater paleolakes. The first in situ δ34S data from Mars at Gale crater display a ∼75‰ range that has been attributed to an abiotic mechanism. Yet biogeochemical studies of ancient environments on Earth generally interpret δ34S fractionations >21‰ as indicative of a biological origin, and studies of δ34S at analog impact crater lakes on Earth have followed the same approach. We performed analyses (including δ34S, total organic carbon wt%, and scanning electron microscope imaging) on multiple lithologies from the Nördlinger Ries impact crater, focusing on hydrothermally altered impact breccias and associated sedimentary lake-fill sequences to determine whether the δ34S properties define a biosignature. The differences in δ34S between the host lithologies may have resulted from thermochemical sulfate reduction, microbial sulfate reduction, hydrothermal equilibrium fractionation, or any combination thereof. Despite abundant samples and instrumental precision currently exclusive to Earth-bound analyses, assertions of biogenicity from δ34S variations >21‰ at the Miocene Ries impact crater are tenuous. This discourages the use of δ34S as a biosignature in similar environments without independent checks that include the full geologic, biogeochemical, and textural context, as well as a comprehensive acknowledgment of alternative hypotheses.

Fungal-induced fossil biomineralization.

Exceptional preservation of fossils has often been attributed to the actions of bacteria that aid in the preservation of soft tissues that normally decay rapidly. However, it is well known that fungi play a major role in organic matter decomposition, biogeochemical cycling of elements, and metal-mineral transformations in modern ecosystems. Although the fungal fossil record can be traced back over a billion years, there are only a few recorded examples of fungal roles in fossilization. In this research, we have carried out a detailed geobiological investigation on early Pleistocene hyena coprolites (fossilized dung) in an attempt to ascertain possible fungal involvement in their formation. Using an advanced microscopic and mineralogical approach, we found that numerous hydroxyapatite nanofibers (25-34 nm on average), interwoven to form spheroidal structures, constituted the matrix of the coprolites in addition to food remains. These structures were found to be extremely similar in texture and mineral composition to biominerals produced during laboratory culture of a common saprophytic and geoactive fungus, Aspergillus niger, in the presence of a solid source of calcium (Ca) and phosphorus (P). This observation, and our other data obtained, strongly suggests that fungal metabolism can provide a mechanism that can result in fossil biomineralization, and we hypothesize, therefore, that this may have contributed to the formation of well-preserved fossils (Lagerstätten) in the geological record. The characteristic polycrystalline nanofibers may also have served as a potential biosignature for fungal life in early Earth and extraterrestrial environments.

Expanding the taxonomic and environmental extent of an underexplored carbon metabolism-oxalotrophy.

Oxalate serves various functions in the biological processes of plants, fungi, bacteria, and animals. It occurs naturally in the minerals weddellite and whewellite (calcium oxalates) or as oxalic acid. The environmental accumulation of oxalate is disproportionately low compared to the prevalence of highly productive oxalogens, namely plants. It is hypothesized that oxalotrophic microbes limit oxalate accumulation by degrading oxalate minerals to carbonates via an under-explored biogeochemical cycle known as the oxalate-carbonate pathway (OCP). Neither the diversity nor the ecology of oxalotrophic bacteria is fully understood. This research investigated the phylogenetic relationships of the bacterial genes oxc, frc, oxdC, and oxlT, which encode key enzymes for oxalotrophy, using bioinformatic approaches and publicly available omics datasets. Phylogenetic trees of oxc and oxdC genes demonstrated grouping by both source environment and taxonomy. All four trees included genes from metagenome-assembled genomes (MAGs) that contained novel lineages and environments for oxalotrophs. In particular, sequences of each gene were recovered from marine environments. These results were supported with marine transcriptome sequences and description of key amino acid residue conservation. Additionally, we investigated the theoretical energy yield from oxalotrophy across marine-relevant pressure and temperature conditions and found similar standard state Gibbs free energy to "low energy" marine sediment metabolisms, such as anaerobic oxidation of methane coupled to sulfate reduction. These findings suggest further need to understand the role of bacterial oxalotrophy in the OCP, particularly in marine environments, and its contribution to global carbon cycling.