Quantitative evaluation of the feasibility of sampling the ice plumes at Enceladus for biomarkers of extraterrestrial life.

Enceladus, an icy moon of Saturn, is a compelling destination for a probe seeking biosignatures of extraterrestrial life because its subsurface ocean exhibits significant organic chemistry that is directly accessible by sampling cryovolcanic plumes. State-of-the-art organic chemical analysis instruments can perform valuable science measurements at Enceladus provided they receive sufficient plume material in a fly-by or orbiter plume transit. To explore the feasibility of plume sampling, we performed light gas gun experiments impacting micrometer-sized ice particles containing a fluorescent dye biosignature simulant into a variety of soft metal capture surfaces at velocities from 800 m ⋅ s-1 up to 3 km ⋅ s-1 Quantitative fluorescence microscopy of the capture surfaces demonstrates organic capture efficiencies of up to 80 to 90% for isolated impact craters and of at least 17% on average on indium and aluminum capture surfaces at velocities up to 2.2 km ⋅ s-1 Our results reveal the relationships between impact velocity, particle size, capture surface, and capture efficiency for a variety of possible plume transit scenarios. Combined with sensitive microfluidic chemical analysis instruments, we predict that our capture system can be used to detect organic molecules in Enceladus plume ice at the 1 nM level-a sensitivity thought to be meaningful and informative for probing habitability and biosignatures.

Optimization of Fluorescence Labeling of Trace Analytes: Application to Amino Acid Biosignature Detection with Pacific Blue.

Fluorescence labeling of biomolecules and fluorescence detection platforms provide a powerful approach to high-sensitivity bioanalysis. Reactive probes can be chosen to target specific functional groups to enable selective analysis of a chosen class of analytes. Particularly, when targeting trace levels of analytes, it is important to optimize the reaction chemistry to maximize the labeling efficiency and minimize the background. Here, we develop methods to optimize the labeling and detection of Pacific Blue (PB)-tagged amino acids. A model is developed to quantitate labeling kinetics and completeness in the circumstance where analyte labeling and reactive probe hydrolysis are in competition. The rates of PB hydrolysis and amino acid labeling are determined as a function of pH. Labeling kinetics and completeness as a function of PB concentration are found to depend on the ratio of the hydrolysis time to the initial labeling time, which depends on the initial PB concentration. Finally, the optimized labeling and detection conditions are used to perform capillary electrophoresis analysis demonstrating 100 pM sensitivities and high-efficiency separations of an 11 amino acid test set. This work provides a quantitative optimization model that is applicable to a variety of reactive probes and targets. Our approach is particularly useful for the analysis of trace amine and amino acid biosignatures in extraterrestrial samples. For illustration, our optimized conditions (reaction at 4 °C in a pH 8.5 buffer) are used to detect trace amino acid analytes at the 100 pM level in an Antarctic ice core sample.

Constraints on the formation environment of two chondrule-like igneous particles from Comet 81P/Wild 2.

Using chemical and petrologic evidence and modeling, we deduce that two chondrule-like particles named Iris and Callie, from Stardust cometary track C2052,12,74, formed in an environment very similar to that seen for type II chondrules in meteorites. Iris was heated near liquidus, equilibrated, and cooled at ≤ 100 °C/hr and within ≈ 2 log units of the IW buffer with a high partial pressure of Na such as would be present with dust enrichments of ≈ 103. There was no detectable metamorphic, nebular or aqueous alteration. In previous work Ogliore et al. (2012) reported that Iris formed late, > 3 Myr after CAIs, assuming 26Al was homogenously distributed, and was rich in heavy oxygen. Iris may be similar to assemblages found only in interplanetary dust particles and Stardust cometary samples called Kool particles. Callie is chemically and isotopically very similar but not identical to Iris.

Clinical and molecular characteristics of mitochondrial DNA depletion syndrome associated with neonatal cholestasis and liver failure.

OBJECTIVE: To determine the frequency of mitochondrial DNA depletion syndrome (MDS) in infants with cholestasis and liver failure and to further clarify the clinical, biochemical, radiologic, histopathologic, and molecular features associated with MDS due to deoxyguanosine kinase (DGUOK) and MPV17 gene mutations. STUDY DESIGN: We studied 20 infants with suspected hepatocerebral MDS referred to our tertiary care center between 2007 and 2013. Genomic DNA was isolated from blood leukocytes, liver, and/or skeletal muscle samples by standard methods. Mitochondrial DNA copy number relative to nuclear DNA levels was determined in muscle and/or liver DNA using real-time quantitative polymerase chain reaction and compared with age-matched controls. Nuclear candidate genes, including polymerase γ, MPV17, and DGUOK were sequenced using standard analyses. RESULTS: We identified pathogenic MPV17 and DGUOK mutations in 11 infants (6 females) representing 2.5% of the 450 cases of infantile cholestasis and 22% of the 50 cases of infantile liver failure referred to our center during the study period. All of the 11 patients manifested cholestasis that was followed by a rapidly progressive liver failure and death before 2 years of life. Mitochondrial DNA depletion was demonstrated in liver or muscle for 8 out of the 11 cases where tissue was available. Seven patients had mutations in the MPV17 gene (3 novel mutations), 4 patients had DGUOK mutations (of which 2 were novel mutations). CONCLUSION: Mutations in the MPV17 and DGUOK genes are present in a significant percentage of infants with liver failure and are associated with poor prognosis.

Integrated high performance microfluidic organic analysis instrument for planetary and space exploration.

The exploration of our solar system to characterize the molecular organic inventory will enable the identification of potentially habitable regions and initiate the search for biosignatures of extraterrestrial life. However, it is challenging to perform the required high-resolution, high-sensitivity chemical analyses in space and in planetary environments. To address this challenge, we have developed a microfluidic organic analyzer (MOA) instrument that consists of a multilayer programmable microfluidic analyzer (PMA) for fluidic processing at the microliter scale coupled with a microfabricated glass capillary electrophoresis (CE) wafer for separation and analysis of the sample components. Organic analytes are labeled with a functional group-specific (e.g. amine, organic acid, aldehyde) fluorescent dye, separated according to charge and hydrodynamic size by capillary electrophoresis (CE), and detected with picomolar limit of detection (LOD) using laser-induced fluorescence (LIF). Our goal is a sensitive automated instrument and autonomous process that enables sample-in to data-out performance in a flight capable format. We present here the design, fabrication, and operation of a technology development unit (TDU) that meets these design goals with a core mass of 3 kg and a volume of <5 L. MOA has a demonstrated resolution of 2 × 105 theoretical plates for relevant amino acids using a 15 cm long CE channel and 467 V cm-1. The LOD of LIF surpasses 100 pM (0.01 ppb), enabling biosignature detection in harsh environments on Earth. MOA is ideally suited for probing biosignatures in potentially habitable destinations on icy moons such as Europa and Enceladus, and on Mars.

Operation of a programmable microfluidic organic analyzer under microgravity conditions simulating space flight environments.

A programmable microfluidic organic analyzer was developed for detecting life signatures beyond Earth and clinical monitoring of astronaut health. Extensive environmental tests, including various gravitational environments, are required to confirm the functionality of this analyzer and advance its overall Technology Readiness Level. This work examines how the programmable microfluidic analyzer performed under simulated Lunar, Martian, zero, and hypergravity conditions during a parabolic flight. We confirmed that the functionality of the programmable microfluidic analyzer was minimally affected by the significant changes in the gravitational field, thus paving the way for its use in a variety of space mission opportunities.

Method for detecting and quantitating capture of organic molecules in hypervelocity impacts.

Enceladus is a prime candidate in the solar system for in-depth astrobiological studies searching for habitability and life because it has a liquid water ocean with significant organic content and ongoing cryovolcanic activity. The presence of ice plumes that jet up through fissures in the ice crust covering the sub-surface ocean, enables remote sampling and in-situ analysis via a fly-by mission. However, capture and transport of organic materials to organic analyzers presents distinctive challenges as it is unknown whether, and to what extent, organic molecules imbedded in ice particles can be captured and survive hypervelocity impacts. This manuscript provides a fluorescence microscopic method to parametrically determine the amount of an organic fluorescent tracer dye, Pacific Blue™ (PB) deposited on a metallic surface. This method can be used to measure the capture and survival outcomes of terrestrial hypervelocity impact experiments where an ice projectile labeled with Pacific Blue impacts a soft metal surface. This work is an important step in the advancement of instruments like the Enceladus Organic Analyzer for detecting biosignatures in an Enceladus plume fly-by mission. An apparatus consisting of a substrate humidification shroud coupled with an epifluorescence microscope with CCD detector is developed to measure the intensity of quantitatively deposited Pacific Blue droplets under controlled humidity. Calibration curves are produced that relate the integrated fluorescence intensity of humidified PB droplets on metal foils to the number of PB molecules deposited. To demonstrate the utility of this method, our calibrations are used to analyze and quantitate organic capture and survival (up to 11% capture efficiency) following ice particle impacts at a velocity of 1.7 km/s on an aluminum substrate.

Fabrication of high-quality glass microfluidic devices for bioanalytical and space flight applications.

Microfabricated glass microfluidic and Capillary Electrophoresis (CE) devices have been utilized in a wide variety of applications over the past thirty years. At the Berkeley Space Sciences Laboratory, we are working to further expand this technology by developing analytical instruments to chemically explore our solar system. This effort requires improving the quality and reliability of glass microfabrication through quality control procedures at every stage of design and manufacture. This manuscript provides detailed information on microfabrication technology for the production of high-quality glass microfluidic chips in compliance with industrial standards and space flight instrumentation quality control.•The methodological protocol provided in this paper includes the scope of each step of the manufacturing process, materials and technologies recommended and the specific challenges that often confront the process developer.•Types and sources of fabrication error at every stage have been identified and their solutions have been proposed and verified.•We present robust and rigorous manufacturing and quality control procedures that will assist other researchers in achieving the highest possible quality glass microdevices using the latest apparatus in a routine and reliable fashion.

Feasibility of Detecting Bioorganic Compounds in Enceladus Plumes with the Enceladus Organic Analyzer.

Enceladus presents an excellent opportunity to detect organic molecules that are relevant for habitability as well as bioorganic molecules that provide evidence for extraterrestrial life because Enceladus' plume is composed of material from the subsurface ocean that has a high habitability potential and significant organic content. A primary challenge is to send instruments to Enceladus that can efficiently sample organic molecules in the plume and analyze for the most relevant molecules with the necessary detection limits. To this end, we present the scientific feasibility and engineering design of the Enceladus Organic Analyzer (EOA) that uses a microfluidic capillary electrophoresis system to provide sensitive detection of a wide range of relevant organic molecules, including amines, amino acids, and carboxylic acids, with ppm plume-detection limits (100 pM limits of detection). Importantly, the design of a capture plate that effectively gathers plume ice particles at encounter velocities from 200 m/s to 5 km/s is described, and the ice particle impact is modeled to demonstrate that material will be efficiently captured without organic decomposition. While the EOA can also operate on a landed mission, the relative technical ease of a fly-by mission to Enceladus, the possibility to nondestructively capture pristine samples from deep within the Enceladus ocean, plus the high sensitivity of the EOA instrument for molecules of bioorganic relevance for life detection argue for the inclusion of EOA on Enceladus missions. Key Words: Lab-on-a-chip-Organic biomarkers-Life detection-Planetary exploration. Astrobiology 17, 902-912.

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene.

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

Interstellar dust. Evidence for interstellar origin of seven dust particles collected by the Stardust spacecraft.

Seven particles captured by the Stardust Interstellar Dust Collector and returned to Earth for laboratory analysis have features consistent with an origin in the contemporary interstellar dust stream. More than 50 spacecraft debris particles were also identified. The interstellar dust candidates are readily distinguished from debris impacts on the basis of elemental composition and/or impact trajectory. The seven candidate interstellar particles are diverse in elemental composition, crystal structure, and size. The presence of crystalline grains and multiple iron-bearing phases, including sulfide, in some particles indicates that individual interstellar particles diverge from any one representative model of interstellar dust inferred from astronomical observations and theory.

MPV17 mutation causes neuropathy and leukoencephalopathy with multiple mtDNA deletions in muscle.

Disorders of mitochondrial DNA (mtDNA) maintenance are clinically and genetically heterogeneous, embracing recessive mtDNA depletion syndromes affecting children and adult-onset multiple mtDNA deletion disorders. Here we show that mutation of MPV17 - a gene implicated in severe, infantile hepatocerebral mtDNA depletion disorders characterised by a loss of mtDNA copies - can also cause clonally-expanded mtDNA deletion and focal cytochrome c oxidase (COX) deficiency in skeletal muscle associated with an adult presentation of neuropathy and leukoencephalopathy. The mpv17 protein is therefore intimately involved in both the mtDNA replication and repair processes and associated with both quantitative and qualitative mtDNA abnormalities.

Isotopic compositions of cometary matter returned by Stardust.

Hydrogen, carbon, nitrogen, and oxygen isotopic compositions are heterogeneous among comet 81P/Wild 2 particle fragments; however, extreme isotopic anomalies are rare, indicating that the comet is not a pristine aggregate of presolar materials. Nonterrestrial nitrogen and neon isotope ratios suggest that indigenous organic matter and highly volatile materials were successfully collected. Except for a single (17)O-enriched circumstellar stardust grain, silicate and oxide minerals have oxygen isotopic compositions consistent with solar system origin. One refractory grain is (16)O-enriched, like refractory inclusions in meteorites, suggesting that Wild 2 contains material formed at high temperature in the inner solar system and transported to the Kuiper belt before comet accretion.

Comet 81P/Wild 2 under a microscope.

The Stardust spacecraft collected thousands of particles from comet 81P/Wild 2 and returned them to Earth for laboratory study. The preliminary examination of these samples shows that the nonvolatile portion of the comet is an unequilibrated assortment of materials that have both presolar and solar system origin. The comet contains an abundance of silicate grains that are much larger than predictions of interstellar grain models, and many of these are high-temperature minerals that appear to have formed in the inner regions of the solar nebula. Their presence in a comet proves that the formation of the solar system included mixing on the grandest scales.

Organics captured from comet 81P/Wild 2 by the Stardust spacecraft.

Organics found in comet 81P/Wild 2 samples show a heterogeneous and unequilibrated distribution in abundance and composition. Some organics are similar, but not identical, to those in interplanetary dust particles and carbonaceous meteorites. A class of aromatic-poor organic material is also present. The organics are rich in oxygen and nitrogen compared with meteoritic organics. Aromatic compounds are present, but the samples tend to be relatively poorer in aromatics than are meteorites and interplanetary dust particles. The presence of deuterium and nitrogen-15 excesses suggest that some organics have an interstellar/protostellar heritage. Although the variable extent of modification of these materials by impact capture is not yet fully constrained, a diverse suite of organic compounds is present and identifiable within the returned samples.