Spaceflight also significantly impacts the host microbiome, as evidenced by NASA’s study comparing the gut microbiomes of an astronaut and his Earthbound twin 14. The astronaut displayed notable changes, including an increased Firmicutes to Bacteroidetes ratio. Another study noted postflight microbiome differences among astronauts, with increased microbial similarity and reduced bacterial taxa 15. Whereas these studies were primarily concerned with long-term flights, there is a scarcity of work focused on changes to the microbiome upon stratosphere exposure. Only a few studies have been conducted under stratospheric balloon flight conditions, all showing significant challenges to bacterial survival. For example,
Our study investigated the impact of Faraday fabric-based payload shielding on
For the Faraday fabric-based payload-shielded setup, one sample box, initially secured in plastic biological shipping material (to comply with EH&S shipping regulations, aerospace flight standards for additional payload containment, and preventative risk mitigation, given the unpredictability of weather balloon recoveries and the potential risk to the public on unintended landing sites), was further enveloped in a triple-layered Faraday fabric (OUSEXI, a blend of polyester and high-conductivity metals like copper and nickel). The unshielded box was only wrapped in plastic biological shipping material. Within the orange payload box (Length: 33.4 cm, Width: 21.9 cm, and Height: 25.5 cm), there was a temperature and humidity sensor (Adafruit) and Shkalacar Assembled DIY Geiger Counter Kit, DIY Nuclear Radiation Detector Kit with a Miller Tube, Portable Geiger Counter, and an Assembled Radiation Detector System Experimental Module that were securely mounted above each sample box to collect a diverse range of data throughout the flight (columns AH-AR,
Summary of Recorded Flight Data. This table presents a summary of the flight data (recorded by the primary flight computer), which lasted for a total of 89 minutes from launch to landing. The table highlights the minimum and maximum values recorded for altitude, external temperature, pressure, and UV index during this flight mission (for the entire dataset see
Altitude | 0 km | 27 km |
External temperature | −57.8°C | 25.04°C |
External pressure | 1.83 kPa | 90.41 kPa |
External UV index | 1.44 | 19.13 |
The balloon was launched from Kennedy Space Center’s Educational Center on August 21, 2022, at 11:47 AM EST. The balloon’s total travel time was 1 hour and 29 minutes from take-off to landing, and it remained in the stratosphere for 1 hour and 2 minutes prior to its burst. The balloon reached a maximum altitude of 27 km before a parachute was deployed for the descent. Following the GPS tracking of the payload’s landing location, the payload was retrieved 1 hour and 28 minutes post-landing and moved to storage in dry ice, followed by long-term storage in a freezer at −80°C.
RAW 264.7 murine macrophage cells (ATCC# TIB-71, ATCC, USA) were grown and maintained in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 100 μg/mL Penicillin/Streptomycin (Life Technologies Inc., USA).
At 24 hours prior to infection, RAW 264.7 cells were seeded at approximately 3×104 cells/well in a 96-well tissue culture plate. One hour prior to infection, the cell medium was changed to incomplete DMEM (DMEM + 10% FBS). Weather balloon flight samples, both shielded and unshielded, containing
The TNF-α assay measures the release of tumor necrosis factor-alpha (TNF-α), a key inflammatory cytokine, from immune cells, indicating the cellular response to infection or stress. This assay is of interest because TNF-α plays a crucial role in immune regulation and its levels can provide insights into the potential inflammatory impact of
An equal amount of 25 µg protein per sample was utilized, and three replicates per sample type were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The entire gel lane corresponding to each sample was excised and diced into 1 mm2 cubes, followed by ingel trypsin digestion as we did previously 18,19. The resulting peptide samples were analyzed using a 250-mm Ultrahigh-Performance Liquid Chromatography (UHPLC) system coupled to an Orbitrap Fusion mass spectrometer (Thermo Scientific) similar to what was described previously 18,19. After the raw data were collected, tandem mass spectra were extracted, the charge state was deconvoluted, and the charge state was deisotoped using Proteome Discoverer (Thermo Scientific) version 2.1. Mascot (Matrix Science, London, UK; version 2.7.0) which was employed to analyze all MS/MS samples. The Mascot search was performed against the UniProt
To conduct the principal component analysis (PCA) of the differentially abundant proteins, we utilized Clustvis (version 2.00). Row-wise unit variance scaling was applied, and SVD with imputation was used to calculate the principal components. Prediction ellipses were generated to encompass new observations from the same group with a probability of 0.95, ensuring that they fall within the ellipse. Additionally, we performed a hierarchical clustering analysis. Both rows and columns were clustered using correlation distance, and average linkage was employed. Before clustering, rows were centered and unit variance scaling was applied to ensure accurate data representation.
For functional analysis of differentially abundant proteins, we utilized ShinyGO (version 0.80), using
This study was part of the 2022 Florida Space Trek Academy, organized by Atlantis Educational Services and sponsored by NASA’s Florida Space Grant Consortium. Undergraduate and graduate university student team members underwent preparatory meetings and submitted written proposals seven weeks before the stratospheric balloon flight. Daily debriefings and support sessions ensured progress and cohesion. The program involved weather balloon deployment discussions, role assignments, and weather analysis for suitable launch sites. Activities included parachute creation, sensor component setup, and students’ payload preparation, launch, and retrieval efforts followed by payload release.
A payload of frozen mid-exponential phase
An overview of the experimental design. (A) Timeline of the experiment. (B). Overall experimental plan.
Payload configuration. A: Shielded (left) and unshielded (right) sample boxes.
On the flight day, the weather balloon containing the
Readings of temperature, pressure, and UV index from during the ascent and descent of the balloon flight.
First, we evaluated the effect of the flight in the presence or absence of shielding on
Next, we performed a gentamicin protection assay in RAW 264.7 murine macrophages to evaluate whether Faraday fabric-based payload shielding during the weather balloon flight affects the ability of
The intracellular survival of
To complement these findings, we executed a ground control (GC) experiment designed to replicate the ambient temperature conditions encountered during the flight. Here, frozen bacterial samples were subjected to a 2-hour and 57-minute incubation at room temperature within a styrofoam cooler, mirroring the complete duration of the balloon flight experiment. Post incubation, these samples were swiftly returned to −80°C storage pending further analysis. Analysis revealed that the ground control samples exhibited lower overall viability than the flight samples (
We subsequently assessed the release of tumor necrosis factor-alpha (TNF-α) from murine macrophages after exposure to
The effect of
Next, the impact of payload shielding on the proteome of
In our study, 1355
Proteomic analysis of the
To explore the relationship between the proteins with altered abundance in shielded and unshielded samples, we performed principal component analysis (PCA), which revealed a distinct grouping of the samples based on their proteomic profiles (
To gain insights into the biological functions of the differentially abundant proteins in
Bioinformatic analysis of differentially abundant proteins in
Furthermore, through manual inspection of the differentially abundant proteins in shielded versus unshielded conditions, we observed a decrease in chemotaxis proteins in the shielded samples compared to the unshielded samples (
In addition, decreased abundance of proteins related to peptidoglycan synthesis, turnover, cell division, and other cell wall processes were observed in
Finally, the largest group of differentially abundant proteins was associated with various metabolic processes. Notably, tricarboxylic acid cycle proteins SucD (A0A0H3CPV5, +1.60) and Mdh (A0A0H3CQC4, +1.60) exhibited increased abundance in shielded conditions, as did several proteins involved in fatty acid biosynthesis and amino acid metabolism (
This study was conducted during the summer of 2022 at Florida Space Trek Academy, where university student teams conducted experiments on small, high-altitude weather balloons. Our study demonstrates that student-driven research experiences can be harnessed to generate proof-of-principle feasibility data using short-duration flight experiments (1–4 hours) with small high-altitude weather balloons. Specifically, our study assessed the impact of weather balloon flight conditions and Faraday fabric-based payload shielding of samples of
Based on previously published research, we expected that
We also examined the intracellular survival of Faraday fabric-shielded and unshielded
Our study showed that while Faraday fabric shielding used during the stratospheric flight did not impact bacterial viability, it significantly enhanced the intracellular survival of
Moreover, the release of TNF-α from macrophages was significantly decreased when
We performed a proteomic investigation of changes in bacterial protein abundance between these two weather balloon flight samples to gain more insight into the reason behind improved intracellular cellular survival of Faraday fabric-shielded
Our study offers insights into the resilience of