RESUMO
Metformin has utility in cancer prevention and treatment, though the mechanisms for these effects remain elusive. Through genetic screening in C. elegans, we uncover two metformin response elements: the nuclear pore complex (NPC) and acyl-CoA dehydrogenase family member-10 (ACAD10). We demonstrate that biguanides inhibit growth by inhibiting mitochondrial respiratory capacity, which restrains transit of the RagA-RagC GTPase heterodimer through the NPC. Nuclear exclusion renders RagC incapable of gaining the GDP-bound state necessary to stimulate mTORC1. Biguanide-induced inactivation of mTORC1 subsequently inhibits growth through transcriptional induction of ACAD10. This ancient metformin response pathway is conserved from worms to humans. Both restricted nuclear pore transit and upregulation of ACAD10 are required for biguanides to reduce viability in melanoma and pancreatic cancer cells, and to extend C. elegans lifespan. This pathway provides a unified mechanism by which metformin kills cancer cells and extends lifespan, and illuminates potential cancer targets. PAPERCLIP.
Assuntos
Metformina/farmacologia , Acil-CoA Desidrogenase/genética , Envelhecimento , Animais , Tamanho Corporal , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Longevidade , Alvo Mecanístico do Complexo 1 de Rapamicina , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Neoplasias/tratamento farmacológico , Poro Nuclear/metabolismo , Fosforilação Oxidativa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.
Assuntos
Fosfatos de Dinucleosídeos/química , Técnicas Genéticas , RNA/química , Cinética , Polimerização , Moldes GenéticosRESUMO
Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA-Seq/métodos , Software , Primers do DNA/química , Primers do DNA/genética , Origem da Vida , RNA/química , RNA/genéticaRESUMO
Deep-sea hypersaline anoxic basins (DHABs) are uniquely stratified polyextreme environments generally found in enclosed seas. These environments select for elusive and widely uncharacterized microbes that may be living below the currently recognized window of life on Earth. Still, there is strong evidence of highly specialized active microbial communities in the Kryos, Discovery, and Hephaestus basins located in the Eastern Mediterranean Sea; the only known athalassohaline DHABs. Life is further constrained in these DHABs as near-saturated concentrations of magnesium chloride significantly reduces water activity (aw ) and exerts extreme chaotropic stress, the tendency of a solution to disorder biomolecules. In this review, we provide an overview of microbial adaptations to polyextremes focusing primarily on chaotropicity, summarize current evidence of microbial life within athalassohaline DHABs and describe the difficulties of life detection approaches and sampling within these environments. We also reveal inconsistent measurements of chaotropic activity in the literature highlighting the need for a new methodology. Finally, we generate recommendations for future investigations and discuss the importance of athalassohaline DHAB research to help inform extraterrestrial life detection missions.
Assuntos
Exobiologia , Microbiota , Cloreto de Magnésio , Mar Mediterrâneo , Água do MarRESUMO
Concurrent osmotic and chaotropic stress make MgCl2 -rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl2 brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl2 -rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl2 -saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.
Assuntos
Salinidade , Água do Mar , Oceanos e Mares , RNA Ribossômico 16S/genética , Cloreto de Sódio/análiseRESUMO
BACKGROUND: Long-read nanopore sequencing technology is of particular significance for taxonomic identification at or below the species level. For many environmental samples, the total extractable DNA is far below the current input requirements of nanopore sequencing, preventing "sample to sequence" metagenomics from low-biomass or recalcitrant samples. RESULTS: Here we address this problem by employing carrier sequencing, a method to sequence low-input DNA by preparing the target DNA with a genomic carrier to achieve ideal library preparation and sequencing stoichiometry without amplification. We then use CarrierSeq, a sequence analysis workflow to identify the low-input target reads from the genomic carrier. We tested CarrierSeq experimentally by sequencing from a combination of 0.2 ng Bacillus subtilis ATCC 6633 DNA in a background of 1000 ng Enterobacteria phage λ DNA. After filtering of carrier, low quality, and low complexity reads, we detected target reads (B. subtilis), contamination reads, and "high quality noise reads" (HQNRs) not mapping to the carrier, target or known lab contaminants. These reads appear to be artifacts of the nanopore sequencing process as they are associated with specific channels (pores). CONCLUSION: By treating sequencing as a Poisson arrival process, we implement a statistical test to reject data from channels dominated by HQNRs while retaining low-input target reads.
Assuntos
Nanoporos , Análise de Sequência de DNA/métodos , Software , Fluxo de Trabalho , Bacillus subtilis/genética , Curva ROCRESUMO
Reproductive senescence is a hallmark of aging. The molecular mechanisms regulating reproductive senescence and its association with the aging of somatic cells remain poorly understood. From a full genome RNA interference (RNAi) screen, we identified 32 Caenorhabditis elegans gene inactivations that delay reproductive senescence and extend reproductive lifespan. We found that many of these gene inactivations interact with insulin/IGF-1 and/or TGF-ß endocrine signaling pathways to regulate reproductive senescence, except nhx-2 and sgk-1 that modulate sodium reabsorption. Of these 32 gene inactivations, we also found that 19 increase reproductive lifespan through their effects on oocyte activities, 8 of them coordinate oocyte and sperm functions to extend reproductive lifespan, and 5 of them can induce sperm humoral response to promote reproductive longevity. Furthermore, we examined the effects of these reproductive aging regulators on somatic aging. We found that 5 of these gene inactivations prolong organismal lifespan, and 20 of them increase healthy life expectancy of an organism without altering total life span. These studies provide a systemic view on the genetic regulation of reproductive senescence and its intersection with organism longevity. The majority of these newly identified genes are conserved, and may provide new insights into age-associated reproductive senescence during human aging.
Assuntos
Envelhecimento/genética , Caenorhabditis elegans/genética , Redes Reguladoras de Genes , Longevidade/genética , Reprodução/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/fisiologia , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Estudo de Associação Genômica Ampla , Masculino , Interferência de RNA , Transdução de Sinais/genéticaRESUMO
Spinster (Spin) in Drosophila or Spinster homolog 1 (Spns1) in vertebrates is a putative lysosomal H+-carbohydrate transporter, which functions at a late stage of autophagy. The Spin/Spns1 defect induces aberrant autolysosome formation that leads to embryonic senescence and accelerated aging symptoms, but little is known about the mechanisms leading to the pathogenesis in vivo. Beclin 1 and p53 are two pivotal tumor suppressors that are critically involved in the autophagic process and its regulation. Using zebrafish as a genetic model, we show that Beclin 1 suppression ameliorates Spns1 loss-mediated senescence as well as autophagic impairment, whereas unexpectedly p53 deficit exacerbates both of these characteristics. We demonstrate that 'basal p53' activity plays a certain protective role(s) against the Spns1 defect-induced senescence via suppressing autophagy, lysosomal biogenesis, and subsequent autolysosomal formation and maturation, and that p53 loss can counteract the effect of Beclin 1 suppression to rescue the Spns1 defect. By contrast, in response to DNA damage, 'activated p53' showed an apparent enhancement of the Spns1-deficient phenotype, by inducing both autophagy and apoptosis. Moreover, we found that a chemical and genetic blockage of lysosomal acidification and biogenesis mediated by the vacuolar-type H+-ATPase, as well as of subsequent autophagosome-lysosome fusion, prevents the appearance of the hallmarks caused by the Spns1 deficiency, irrespective of the basal p53 state. Thus, these results provide evidence that Spns1 operates during autophagy and senescence differentially with Beclin 1 and p53.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteína Supressora de Tumor p53/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Envelhecimento/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Proteína Beclina-1 , Dano ao DNA/genética , Reparo do DNA/genética , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Lisossomos/genética , Macrolídeos/farmacologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Peixe-ZebraRESUMO
Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Transporte/metabolismo , Comportamento Alimentar/fisiologia , Metabolismo dos Lipídeos/fisiologia , Longevidade/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Tecido Adiposo/metabolismo , Animais , Compostos de Boro/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Dieta , Fixadores/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Mutação/genética , Proteína Oncogênica v-akt/metabolismo , Oxazinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Reprodução/fisiologia , Transdução de Sinais , Somatomedinas/metabolismoRESUMO
Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related decline. Lysosome related organelles (LROs) are specialized lysosomes found in organisms from humans to worms, and share many of the features of classic lysosomes. Defective LROs are associated with human immune disorders and neurological disease. Caenorhabditis elegans LROs are the site of concentration of vital dyes such as Nile red as well as age-associated autofluorescence. Even though certain short-lived mutants have high LRO Nile red and high autofluorescence, and other long-lived mutants have low LRO Nile red and low autofluorescence, these two biologies are distinct. We identified a genetic pathway that modulates aging-related LRO phenotypes via serotonin signaling and the gene kat-1, which encodes a mitochondrial ketothiolase. Regulation of LRO phenotypes by serotonin and kat-1 in turn depend on the proton-coupled, transmembrane transporter SKAT-1. skat-1 loss of function mutations strongly suppress the high LRO Nile red accumulation phenotype of kat-1 mutation. Using a systems approach, we further analyzed the role of 571 genes in LRO biology. These results highlight a gene network that modulates LRO biology in a manner dependent upon the conserved protein kinase TOR complex 2. The results implicate new genetic pathways involved in LRO biology, aging related physiology, and potentially human diseases of the LRO.
Assuntos
Envelhecimento/genética , Redes Reguladoras de Genes , Lisossomos/genética , Organelas/genética , Acetil-CoA C-Aciltransferase/genética , Envelhecimento/patologia , Animais , Caenorhabditis elegans , Regulação da Expressão Gênica , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Alvo Mecanístico do Complexo 2 de Rapamicina , Redes e Vias Metabólicas , Mitocôndrias/genética , Mitocôndrias/metabolismo , Complexos Multiproteicos/genética , Organelas/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
Many genetic and physiological treatments that extend lifespan also confer resistance to a variety of stressors, suggesting that cytoprotective mechanisms underpin the regulation of longevity. It has not been established, however, whether the induction of cytoprotective pathways is essential for lifespan extension or merely correlated. Using a panel of GFP-fused stress response genes, we identified the suites of cytoprotective pathways upregulated by 160 gene inactivations known to increase Caenorhabditis elegans longevity, including the mitochondrial UPR (hsp-6, hsp-60), the ER UPR (hsp-4), ROS response (sod-3, gst-4), and xenobiotic detoxification (gst-4). We then screened for other gene inactivations that disrupt the induction of these responses by xenobiotic or genetic triggers, identifying 29 gene inactivations required for cytoprotective gene expression. If cytoprotective responses contribute directly to lifespan extension, inactivation of these genes would be expected to compromise the extension of lifespan conferred by decreased insulin/IGF-1 signaling, caloric restriction, or the inhibition of mitochondrial function. We find that inactivation of 25 of 29 cytoprotection-regulatory genes shortens the extension of longevity normally induced by decreased insulin/IGF-1 signaling, disruption of mitochondrial function, or caloric restriction, without disrupting normal longevity nearly as dramatically. These data demonstrate that induction of cytoprotective pathways is central to longevity extension and identify a large set of new genetic components of the pathways that detect cellular damage and couple that detection to downstream cytoprotective effectors.
Assuntos
Caenorhabditis elegans , Longevidade , Mitocôndrias , Estresse Fisiológico/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Regulação da Expressão Gênica/fisiologia , Insulina/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Longevidade/genética , Longevidade/fisiologia , Redes e Vias Metabólicas/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Long-standing unexplained Venus atmosphere observations and chemical anomalies point to unknown chemistry but also leave room for the possibility of life. The unexplained observations include several gases out of thermodynamic equilibrium (e.g., tens of ppm O2, the possible presence of PH3 and NH3, SO2 and H2O vertical abundance profiles), an unknown composition of large, lower cloud particles, and the "unknown absorber(s)." Here we first review relevant properties of the venusian atmosphere and then describe the atmospheric chemical anomalies and how they motivate future astrobiology missions to Venus.
Assuntos
Vênus , Exobiologia , Meio Ambiente Extraterreno , Gases/química , Atmosfera/químicaRESUMO
Gloeocapsopsis dulcis strain AAB1 is an extremely xerotolerant cyanobacterium isolated from the Atacama Desert (i.e., the driest and oldest desert on Earth) that holds astrobiological significance due to its ability to biosynthesize compatible solutes at ultra-low water activities. We sequenced and assembled the G. dulcis genome de novo using a combination of long- and short-read sequencing, which resulted in high-quality consensus sequences of the chromosome and two plasmids. We leveraged the G. dulcis genome to generate a genome-scale metabolic model (iGd895) to simulate growth in silico. iGd895 represents, to our knowledge, the first genome-scale metabolic reconstruction developed for an extremely xerotolerant cyanobacterium. The model's predictive capability was assessed by comparing the in silico growth rate with in vitro growth rates of G. dulcis, in addition to the synthesis of trehalose. iGd895 allowed us to explore simulations of key metabolic processes such as essential pathways for water-stress tolerance, and significant alterations to reaction flux distribution and metabolic network reorganization resulting from water limitation. Our study provides insights into the potential metabolic strategies employed by G. dulcis, emphasizing the crucial roles of compatible solutes, metabolic water, energy conservation, and the precise regulation of reaction rates in their adaptation to water stress.
Assuntos
Brassicaceae , Cianobactérias , Dessecação , Cianobactérias/genética , Redes e Vias Metabólicas , Sequência Consenso , DesidrataçãoRESUMO
Growing evidence of the potential habitability of Ocean Worlds across our solar system is motivating the advancement of technologies capable of detecting life as we know it-sharing a common ancestry or physicochemical origin with life on Earth-or don't know it, representing a distinct emergence of life different than our one known example. Here, we propose the Electronic Life-detection Instrument for Enceladus/Europa (ELIE), a solid-state single-molecule instrument payload that aims to search for life based on the detection of amino acids and informational polymers (IPs) at the parts per billion to trillion level. As a first proof-of-principle in a laboratory environment, we demonstrate the single-molecule detection of the amino acid L-proline at a 10 µM concentration in a compact system. Based on ELIE's solid-state quantum electronic tunneling sensing mechanism, we further propose the quantum property of the HOMO-LUMO gap (energy difference between a molecule's highest energy-occupied molecular orbital and lowest energy-unoccupied molecular orbital) as a novel metric to assess amino acid complexity. Finally, we assess the potential of ELIE to discriminate between abiotically and biotically derived α-amino acid abundance distributions to reduce the false positive risk for life detection. Nanogap technology can also be applied to the detection of nucleobases and short sequences of IPs such as, but not limited to, RNA and DNA. Future missions may utilize ELIE to target preserved biosignatures on the surface of Mars, extant life in its deep subsurface, or life or its biosignatures in a plume, surface, or subsurface of ice moons such as Enceladus or Europa. One-Sentence Summary: A solid-state nanogap can determine the abundance distribution of amino acids, detect nucleic acids, and shows potential for detecting life as we know it and life as we don't know it.
Assuntos
Júpiter , Ácidos Nucleicos , Exobiologia , Planeta Terra , Aminoácidos , Meio Ambiente Extraterreno/químicaRESUMO
An origin of Earth life on Mars would resolve significant inconsistencies between the inferred history of life and Earth's geologic history. Life as we know it utilizes amino acids, nucleic acids, and lipids for the metabolic, informational, and compartment-forming subsystems of a cell. Such building blocks may have formed simultaneously from cyanosulfidic chemical precursors in a planetary surface scenario involving ultraviolet light, wet-dry cycling, and volcanism. On the inferred water world of early Earth, such an origin would have been limited to volcanic island hotspots. A cyanosulfidic origin of life could have taken place on Mars via photoredox chemistry, facilitated by orders-of-magnitude more sub-aerial crust than early Earth, and an earlier transition to oxidative conditions that could have been involved in final fixation of the genetic code. Meteoritic bombardment may have generated transient habitable environments and ejected and transferred life to Earth. Ongoing and future missions to Mars offer an unprecedented opportunity to confirm or refute evidence consistent with a cyanosulfidic origin of life on Mars, search for evidence of ancient life, and constrain the evolution of Mars' oxidation state over time. We should seek to prove or refute a martian origin for life on Earth alongside other possibilities.
Assuntos
Marte , Meteoroides , Planeta Terra , Exobiologia , Meio Ambiente Extraterreno/química , GeologiaRESUMO
Enterococcus faecalis is a multidrug resistant, opportunistic human pathogen and a leading cause of hospital acquired infections. Recently, isolates have been recovered from the air and surfaces onboard the International Space Station (ISS). Pangenomic and functional analyses were carried out to assess their potential impact on astronaut health. Genomes of each ISS isolate, and both clinical and commensal reference strains, were evaluated for their core and unique gene content, acquired antibiotic resistance genes, phage, plasmid content, and virulence traits. In order to determine their potential survival when outside of the human host, isolates were also challenged with three weeks of desiccation at 30% relative humidity. Finally, pathogenicity of the ISS strains was evaluated in the model organism Caenorhabditis elegans. At the culmination of this study, there were no defining signatures that separated known pathogenic strains from the more commensal phenotypes using the currently available resources. As a result, the current reliance on database information alone must be shifted to experimentally evaluated genotypic and phenotypic characteristics of clinically relevant microorganisms.
RESUMO
Nanopore sequencing, as represented by Oxford Nanopore Technologies' MinION, is a promising technology for in situ life detection and for microbial monitoring including in support of human space exploration, due to its small size, low mass (~100 g) and low power (~1 W). Now ubiquitous on Earth and previously demonstrated on the International Space Station (ISS), nanopore sequencing involves translocation of DNA through a biological nanopore on timescales of milliseconds per base. Nanopore sequencing is now being done in both controlled lab settings as well as in diverse environments that include ground, air, and space vehicles. Future space missions may also utilize nanopore sequencing in reduced gravity environments, such as in the search for life on Mars (Earth-relative gravito-inertial acceleration (GIA) g = 0.378), or at icy moons such as Europa (g = 0.134) or Enceladus (g = 0.012). We confirm the ability to sequence at Mars as well as near Europa or Lunar (g = 0.166) and lower g levels, demonstrate the functionality of updated chemistry and sequencing protocols under parabolic flight, and reveal consistent performance across g level, during dynamic accelerations, and despite vibrations with significant power at translocation-relevant frequencies. Our work strengthens the use case for nanopore sequencing in dynamic environments on Earth and in space, including as part of the search for nucleic-acid based life beyond Earth.
RESUMO
Recent studies regarding the origins of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly related to life on Earth, indicating a shared ancestry due to lithological exchange, or (2) is unrelated to life on Earth, suggesting convergent origins of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations (i.e., 104 cells in 50 mg of soil) observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results from 2 pg of purified Bacillus subtilis spore DNA simulating ideal extraction yields equivalent to 1 ppb life-detection sensitivity. We achieve this by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier. After filtering of carrier, low-quality, and low-complexity reads we detected 5 B. subtilis reads, 18 contamination reads (including Homo sapiens), and 6 high-quality noise reads believed to be sequencing artifacts.
Assuntos
Biomassa , Exobiologia/métodos , Marte , Ácidos Nucleicos/isolamento & purificação , Análise de Sequência de DNA , Solo/química , DNA/análise , DNA/isolamento & purificação , Humanos , Sequenciamento por Nanoporos , Esporos Bacterianos/genética , Água/químicaRESUMO
The detection of extant life is a major focus of many planned future planetary missions, a current challenge of which is the ability to target biomarkers capable of providing unambiguous evidence of life. DNA sequencing is increasingly recognized as a powerful tool for life detection for planetary exploration missions; beyond use of sequence information to determine the origins of the sample (e.g., extant life or forward contamination), recent advances in the field have enabled interrogation of single molecules, with or without amplification. The focus of this work is on failure modes, specifically the issues encountered when there is no-to-low input DNA into a sequencing device, and the potential for the generation of sequencing artifacts that could be interpreted as a false positive. Using Oxford Nanopore Technologies (ONT) MinION, we assess whether single molecule sequencing, involving no amplification, generates noise signals that could be misinterpreted in the context of a planetary exploration mission, and also whether the ability of the instrument to handle these types of situations could make it feasible for clean room monitoring. Utilizing quality score filtering techniques in place at the time of this experiment, runs containing only initial flowcell chemistry and/or library reagents generated 5 passing reads out of a total of 3568 measured reads, and contained estimated sequences with low complexity that did not map to the NCBI database. The noise characteristics in all instances suggest that quality thresholds were appropriately chosen by ONT: new chemistry and basecalling workflows have shown further suppression of noise sources, which completely mitigate the generation of spurious reads.