Metagenome analysis of ISS cargo resupply vehicles (CRV)

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective xce xb22 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return Spaceflight [LAR FLT]) or >50 days (N=20 ISS Terminal Spaceflight [ISS-T FLT]). After 29 days the 20 LAR FLT animals were returned live to back to Earth on January 13 2018. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Basal (BSL) groups of animals sacrificed (LAR BSL & ISS-T BSL; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). LAR BSL animals were dissected and samples were collected upon euthanasia. A Ground Control (GC) group LAR GC mimicked the LAR FLT group which was housed at KSC then shipped alive to Novartis xe2 x80 x99s Facilities where both the LAR FLT and LAR GC groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS-T FLT mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR groups. Following blood draw and hind limb dissection the ISS-T FLT animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80 xcb x9aC or colder until return. The ISS-T Ground Control (ISS-T GC) (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-T FLT frozen ISS-T GC and frozen 0-day ISS-T BSL animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received feces from only sham treated animals (no drug treated animals) from the following groups. FLT: LAR (n=9) ISS-T (n=7); GC: LAR (N=7) ISS-T (N=9); BSL: LAR (n=7) ISS-T (n=9). DNA was extracted and analyzed by sequencing using a variety of different targeted and un-targeted metagenome profiling assays.

The objective of the Rodent Research-9 (RR-9) mission was to use mice to understand the molecular basis of phenomena that affect astronauts during long-duration spaceflight particularly visual impairment and joint tissue degradation. To this end a flight group (FLT) of 10-week-old male C57BL/6J mice was launched from Kennedy Space Center (KSC) on 8/14/2017 and housed in Rodent Habitats on the ISS for 33 days before being returned alive to Earth. After splashdown in the Pacific Ocean the animals were transported to Loma Linda University (LLU) for testing euthanasia and dissection on 9/18/2018. A Basal Control (BSL) was housed in standard cages at Kennedy Space Center (KSC) and euthanized one day after launch of the FLT animals (8/15/2017). Ground Control (GC) and Vivarium Control (VIV) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However all the GC and VIV mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC and VIV studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018 while the VIV was euthanized and dissected 6/22/2018 - 6/23/2018. Because this resulted in a different cohort of mice being used for the GC and VIV controls as compared to the flight (FLT) and basal (BSL) groups two cohort controls were included in the study. The first Cohort Control 1 (CC_C1) was from the same cohort as the FLT and BSL animals and was sacrificed and dissected 4 days after the FLT group (9/22/2017). The second Cohort Control 2 (CC_C2) was from the same cohort as the GC and VIV animals and was sacrificed and dissected 2-8 days after the GC and VIV groups (6/24/2018 - 6/26/2018). The CC_C1 and CC_C2 groups were housed in standard cages and fed standard chow in contrast to all other groups which received Rodent Foodbars. To clarify the connections between treatment groups and animal cohorts the following group abbreviations are used in the sample metadata: Flight (FLT_C1); Basal (BSL_C1); Ground Control (GC_C2); Vivarium Control (VIV_C2) Cohort Control 1 (CC_C1); Cohort Control 2 (CC_C2). Fecal pellets were isolated directly from mice during dissection and preserved by flash freezing in liquid nitrogen before stored at -80 C. DNA was then extracted shotgun metagenomic libraries generated and libraries sequenced (target 10 M clusters at PE 250 bp). Metagenomic data was generated from the following groups: Basal Control (n=5) Ground Control (n=5) Vivarium Control (n=5) Cohort Control 1 (n=5) Cohort Control 2 (n=5) Flight (n=5).

Several studies have reported that microorganisms are co-transported with globally dispersed aerosols and that microbial DNA and viable cells have been detected at both low and high altitudes. In an effort to investigate how microbial patterns change with altitude two research flights were flown using NASA s C-20A Gulfstream III aircraft over the Sierra mountains in the Southern California/Nevada region over two consecutive days. During each flight the aircraft reached 40,000 ft and lowered to 30,000 ft 20,000 ft and 10,000 ft in a stair-step pattern. Bioaerosol samples were collected at each altitude for 30 minutes on gelatinous filters using the Aircraft Bioaerosol Collector (ABC). DNA was extracted from the bioaerosol sample. Whole genome sequencing was performed on the extracted DNA using an Illumina NextSeq 500.

The microbial composition of the International Space Station (ISS) environment is of critical interest due to potential impact of its constituents on human health and operational mission success. This study examined the whole metagenome of ISS microbes at both a species- and gene-level resolution. Air filter and dust samples from the ISS were analyzed and compared to samples collected in a terrestrial cleanroom environment. Samples were collected from ISS and cleanroom environments and treated to examine DNA from total versus viable populations. Microbial genes relevant to human health such as antimicrobial resistance and virulence genes were quantified.