Modeling of recovery efficiency of sampling devices used in planetary protection bioburden estimation.

IMPORTANCE: Planetary protection at the National Aeronautics and Space Administration (NASA) requires bioburden on certain spacecraft to be estimated via sampling in order to comply with biological cleanliness requirements. To achieve this, the recovery efficiency of devices used to sample the spacecraft pre-launch must be understood and their uncertainty quantified in order to produce the most reasonable estimates of bioburden. This study brings together experiments performed by NASA and the European Space Agency with approved swab and wipe sampling devices, inoculating steel coupons with laboratory strains of Bacillus spp. spores commonly recovered from spacecraft assembly clean rooms (B. atrophaeus, B. megaterium, B. safensis and B. thuringiensis), with a mathematical model of the assay process to assess recovery efficiency. The statistical treatment developed in this study allows comparison of bioburden estimates made from different devices processed by different methods. This study also gives stakeholders and practitioners a statistically rigorous approach to predict bioburden that can be folded into future modeling efforts.

Identification and Archive of Mars 2020 Spacecraft Microbial Isolates.

To support NASA's Mars 2020 mission, bioassays were performed to ensure the biological cleanliness of the spacecraft, instruments, and hardware assembly areas. Bioassays began in May 2014, as the first components were assembled, and continued until their launch in July 2020. Over this 6-year period, 1811 bioassay sampling sessions were conducted. To understand the nature of microbiological presence on and around the spacecraft, an archive of organisms resulting from the bioassays was assembled. This archive included 4232 microbial specimens preserved as frozen stocks. To date, more than 3489 microbial isolates have been tested by MALDI-TOF mass spectrometry analysis. Identifications were based on high confidence level matches to known microorganisms in the reference spectra database where 39 distinct genera were identified. Gram-positive bacteria were isolated almost exclusively. Most, but not all, were spore-forming genera. The most prevalent genera isolated in order of frequency were Bacillus, Priestia, Paenibacillus, Staphylococcus, Micrococcus, and Streptomyces. Within the largely represented Bacillus-like genera, the five most prevalent species were cereus, licheniformis, horneckiae, subtilis, and safensis.

Planetary Protection Implementation and Verification Approach for the Mars 2020 Mission.

The Mars 2020 Flight System comprises a Cruise Stage; Aeroshell; Entry, Descent, and Landing system; Perseverance rover; and the Ingenuity helicopter. The Perseverance rover was successfully delivered to Jezero Crater on February 18, 2021. Among its science objectives, Perseverance is meant to search for rocks that are capable of preserving chemical traces of ancient life, if it existed, and to core and cache rock and regolith samples. The Perseverance rover is gathering samples for potential return to Earth as part of a Mars Sample Return campaign. Thus, controlling the presence of Earth-sourced biological contamination is important to protect the integrity of the scientific results as well as to comply with international treaty and NASA requirements governing Planetary Protection prior to launch. An unprecedented campaign of sampling and environmental monitoring occurred, which resulted in over 16,000 biological samples collected throughout spacecraft assembly. Engineering design, microbial reduction measures, monitoring, and process controls enabled the mission to limit the total spore bioburden to 3.73 × 105 spores, which provided 25.4% margin against the required limit. Furthermore, the total spore bioburden of all landed hardware was 3.86 × 104, which provided 87% margin against the required limit. This manuscript outlines the Planetary Protection implementation approach and verification methodologies applied to the Mars 2020 flight system and its surrounding environments.

Applied Approach for Assessing Bioburden of the Mars 2020 Parachute Assembly.

The Mars 2020 mission delivered the Perseverance rover to the surface of Mars using a supersonic parachute manufactured at Airborne Systems, California. The Mars 2020 spacecraft, including the flight parachute, was subject to Planetary Protection spore bioburden compliance. Many previous missions with similar parachutes applied manufacturing specifications for calculating bioburden. Although the Mars 2020 parachute was manufactured in an uncontrolled environment, preliminary sampling of a flight-like parachute manufactured in the same facility suggested that the actual spore bioburden levels were potentially orders of magnitude lower than specification values for uncontrolled manufacturing (100,000 spores/m2). Several experiments were designed and carried out throughout the project timeline in an effort to estimate a representative bioburden for the flight parachute. Tests were performed on various parachute materials, including direct sampling and destructive assays of proxy materials. Different bioburden densities were applied to large continuous areas of the canopy, which experienced minimal handling, and seamed areas of the parachute that were likely to experience more handling during the stitching process. In addition, an approach to account for various thermal zones was developed and applied toward calculating log reduction for the parachute assembly. These various methods that were used toward different areas and materials of the Mars 2020 flight parachute provided a nuanced and data-backed estimate of spore bioburden density that can be adopted by future missions.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

Draft Genome Sequences of Spacecraft-Associated Microbes Isolated from Six NASA Missions.

Whole-genome sequencing can be used to better understand and assess the functional abilities of microorganisms isolated from spacecraft hardware and associated surfaces for planetary protection (PP) purposes. We sequenced 191 isolates from 6 spaceflight missions with PP requirements and identified them using Illumina-based sequencing methods and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

Establishing Sterility Assurance for Bacillus canaveralius 29669 Spores Under High Heat Exposure.

The ever-increasing complexity in critical spacecraft hardware and materials has led to the development of new microbial reduction procedures as well as to changes in established processes such as heat microbial reduction (HMR). In the space biology field of Planetary Protection, 500°C for 0.5 s is the current HMR recommendation to reduce microorganisms from flight hardware. However, more studies are needed to effectively determine the microbial reduction capability of high-temperature (more than 200°C), short-duration (under 30 s) heat exposures. One of the many recent microbial reduction bioengineering research avenues harnesses electromagnetic energy for microbial reduction, with previous investigations demonstrating that infrared heaters are capable of the short temperature ramp time required for rapid heating investigations above 200°C. Therefore, this study employed a 6 kW infrared heater to determine the survivability of heat resistant Bacillus canaveralius 29669 to high-temperature, short-duration infrared temperatures. While B. canaveralius 29669 spores can survive microbial heat reduction processes above 200°C, we found evidence suggesting that the 500°C for 0.5 s temperature sterilization specification for Planetary Protection should be updated. This research presents spore survival data and a corresponding model pointing to a re-evaluation of the recommended HMR exposure of 500°C for 0.5 s, while simultaneously meeting requirements on the forward biological contamination of solar system bodies and opening up design possibilities for future spacecraft hardware.

Performance of Multiple Metagenomics Pipelines in Understanding Microbial Diversity of a Low-Biomass Spacecraft Assembly Facility.

NASA planetary protection (PP) requires an assessment of the biological contamination of the potential microbial burden on spacecraft destined to explore planetary bodies that may harbor signs of life, like Mars and Europa. To help meet these goals, the performance of multiple metagenomic pipelines were compared and assessed for their ability to detect microbial diversity of a low-biomass clean room environment used to build spacecraft destined to these planetary bodies. Four vendors were chosen to implement their own metagenomic analysis pipeline on the shotgun sequences retrieved from environmental surfaces in the relevant environments at NASA's Jet Propulsion Laboratory. None of the vendors showed the same microbial profile patterns when analyzing same raw dataset since each vendor used different pipelines, which begs the question of the validity of a single pipeline to be recommended for future NASA missions. All four vendors detected species of interest, including spore-forming and extremotolerant bacteria, that have the potential to hitch-hike on spacecraft and contaminate the planetary bodies explored. Some vendors demonstrated through functional analysis of the metagenomes that the molecular mechanisms for spore-formation and extremotolerance were represented in the data. However, relative abundances of these microorganisms varied drastically between vendor analyses, questioning the ability of these pipelines to quantify the number of PP-relevant microorganisms on a spacecraft surface. Metagenomics offers tantalizing access to the genetic and functional potential of a microbial community that may offer NASA a viable method for microbial burden assays for planetary protection purposes. However, future development of technologies such as streamlining the processing of shotgun metagenome sequence data, long read sequencing, and all-inclusive larger curated and annotated microbial genome databases will be required to validate and translate relative abundances into an actionable assessment of PP-related microbes of interest. Additionally, the future development of machine learning and artificial intelligence techniques could help enhance the quality of these metagenomic analyses by providing more accurate identification of the genetic and functional potential of a microbial community.

A comprehensive metagenomics framework to characterize organisms relevant for planetary protection.

BACKGROUND: Clean rooms of the Space Assembly Facility (SAF) at the Jet Propulsion Laboratory (JPL) at NASA are the final step of spacecraft cleaning and assembly before launching into space. Clean rooms have stringent methods of air-filtration and cleaning to minimize microbial contamination for exoplanetary research and minimize the risk of human pathogens, but they are not sterile. Clean rooms make a selective environment for microorganisms that tolerate such cleaning methods. Previous studies have attempted to characterize the microbial cargo through sequencing and culture-dependent protocols. However, there is not a standardized metagenomic workflow nor analysis pipeline for spaceflight hardware cleanroom samples to identify microbial contamination. Additionally, current identification methods fail to characterize and profile the risk of low-abundance microorganisms. RESULTS: A comprehensive metagenomic framework to characterize microorganisms relevant for planetary protection in multiple cleanroom classifications (from ISO-5 to ISO-8.5) and sample types (surface, filters, and debris collected via vacuum devices) was developed. Fifty-one metagenomic samples from SAF clean rooms were sequenced and analyzed to identify microbes that could potentially survive spaceflight based on their microbial features and whether the microbes expressed any metabolic activity or growth. Additionally, an auxiliary testing was performed to determine the repeatability of our techniques and validate our analyses. We find evidence that JPL clean rooms carry microbes with attributes that may be problematic in space missions for their documented ability to withstand extreme conditions, such as psychrophilia and ability to form biofilms, spore-forming capacity, radiation resistance, and desiccation resistance. Samples from ISO-5 standard had lower microbial diversity than those conforming to ISO-6 or higher filters but still carried a measurable microbial load. CONCLUSIONS: Although the extensive cleaning processes limit the number of microbes capable of withstanding clean room condition, it is important to quantify thresholds and detect organisms that can inform ongoing Planetary Protection goals, provide a biological baseline for assembly facilities, and guide future mission planning. Video Abstract.

Planetary Protection Implementation of the InSight Mission Launch Vehicle and Associated Ground Support Hardware.

The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) Mars mission launched from Vandenberg Air Force Base on an Atlas V 401 rocket on May 5, 2018. Prior to launch, the InSight spacecraft, associated launch vehicle hardware, and ground support equipment were required to satisfy Planetary Protection requirements to comply with international treaty obligations and demonstrate compliance with National Aeronautics and Space Administration (NASA) levied bioburden requirements. InSight was the first bioburden-controlled mission to launch from Vandenberg Air Force Base and required mission-unique policies and procedures to ensure Planetary Protection requirements were satisfied. All the launch vehicle hardware and associated ground support equipment with direct contact or line of sight to flight hardware were required to demonstrate a bioburden density of less than 1,000 spores/m2. Additionally, the environmental control system air ducts were required to demonstrate more stringent bioburden limits on internal duct surfaces (<100 spore/m2) and on air passing through the ducts (88 colony-forming units/m3). Although conservative approaches were used with the data analysis and launch recontamination analysis, InSight, the launch vehicle hardware, and ground support equipment were able to demonstrate compliance with the Planetary Protection requirements needed for launch approval. Here we detail the biological practices implemented on the launch vehicle hardware and ground support equipment that resulted in biologically clean hardware and the satisfaction of Planetary Protection.

Trophectoderm biopsy protocols can affect clinical outcomes: time to focus on the blastocyst biopsy technique.

OBJECTIVE: To compare two different blastocyst biopsy protocols. DESIGN: Retrospective single-center cohort study. SETTINGS: Private in vitro fertilization center. PATIENT(S): The study included 1,670 frozen-thawed embryo transfers (FETs) with preimplantation genetic testing for aneuploidy (PGT-A). INTERVENTION: None. MAIN OUTCOME MEASURE(S): Survival rate (SR) after thawing, clinical pregnancy rate (CPR), ongoing implantation rate (IR), and live birth rate (LBR). RESULT(S): Eight hundred thirty-five FETs with PGT-A cycles including only embryos biopsied in the sequential blastocyst hatching and biopsy protocol paired with the ablation of one-fourth of the zona pellucida (ZP) were matched with 835 FETs with PGT-A cycles including only embryos biopsied in the day 3 prehatching protocol by female age (±1 year), number of embryos transferred, use of gestational carrier or egg donor, and day of blastocyst transfer. Only FETs with euploid blastocysts graded no lower than 4BB were included, and cycles with fewer than five oocytes were excluded. SR after thawing, CPR, ongoing IR, and LBR were significantly higher in the FET cycles with the embryos biopsied in the sequential hatching and biopsy protocol. Four cases of monozygotic twin pregnancies were reported with the day 3 prehatching protocol and none with the sequential hatching and biopsy protocol. CONCLUSION(S): Our results show, for the first time, that using different blastocyst biopsy protocols can affect clinical outcomes. Because the study was retrospective, our findings should be validated in a prospective trial.

Transitory microbial habitat in the hyperarid Atacama Desert.

Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: (i) a physico-chemical characterization of the soil habitability after an exceptional rain event, (ii) identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], (iii) measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and (iv) microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today's extreme hyperaridity.

Prefrontal Theta Oscillations Promote Selective Encoding of Behaviorally Relevant Events.

The ability to capture the most relevant information from everyday experiences without constantly learning unimportant details is vital to survival and mental health. While decreased activity of the medial prefrontal cortex (mPFC) is associated with failed or inflexible encoding of relevant events in the hippocampus, mechanisms used by the mPFC to discern behavioral relevance of events are not clear. To address this question, we chemogenetically activated excitatory neurons in the mPFC of male rats and examined its impact on local network activity and differential associative learning dependent on the hippocampus. Rats were exposed to two neutral stimuli in two environments whose contingency with an aversive stimulus changed systematically across days. Over 2 weeks of differential and reversal learning, theta band activity began to ramp up toward the expected onset of the aversive stimulus, and this ramping activity tracked the subsequent shift of the set (stimulus modality to environment) predictive of the aversive stimulus. With chemogenetic mPFC activation, the ramping activity emerged within a few sessions of differential learning, which paralleled faster learning and stronger correlations between the ramping activity and conditioned responses. Chemogenetic mPFC activity, however, did not affect the adjustment of ramping activity or behavior during reversal learning or set-shifting, suggesting that the faster learning was not because of a general enhancement of attention, sensory, or motor processing. Thus, the dynamics of the mPFC network activation during events provide a relevance-signaling mechanism through which the mPFC exerts executive control over the encoding of those events in the hippocampus.

Regiocontrolled, palladium-catalyzed bisfunctionalization of allenyl esters. Multicomponent coupling approaches to highly substituted alpha,beta-unsaturated delta-lactones.

A palladium-catalyzed regioselective bisfunctionalization of allenyl esters with boronic acids (nucleophiles) and aldehydes (electrophiles) was demonstrated. The three-component coupling afforded alpha,beta-unsaturated delta-lactones under mild conditions and with excellent chemo-, regio-, and diastereoselectivity. Aromatic, heteroaromatic and vinylic boronic acids (R1B(OH)2) reacted with ethyl 2,3-butadienoate and benzaldehyde to afford the corresponding 4-R(1),6-Ph-disubstituted alpha,beta-unsaturated delta-lactones in 62-78% yields. Lactones derived from aromatic, heteroaromatic, and vinylic aldehydes were isolated in 51-58% yields, while aliphatic aldehydes were less reactive. The regiochemistry of bisfunctionalization of allenyl ester homologues remained controlled by the ester substituent, and the reactions afforded cis-4,5,6-trisubstituted alpha,beta-unsaturated delta-lactones and esters of (Z)-syn-3,4,5-trisubstituted-5-hydroxy-2-pentenoic acids in combined 47-65% yields. The superior performance of a pi-allylpalladium(II) dimer catalyst featuring an auxiliary allyl ligand derived from beta-pinene, among diverse palladium(II) catalysts, was demonstrated. A catalytic cycle involving an unsymmetrical bis-pi-allylpalladium complex as the key intermediate was proposed, and the communication highlights the synthetic potential of such intermediates. However, the efficiency of asymmetry transfer remained low (<20%).