Physiological and Genetic Identification of Micrococcus luteus Isolated from the Hind Foot of a Chinchilla
Here is the link to my Lab Report. Thanks for a wonderful semester! Wishing everyone a great summer!
Here is the link to my Lab Report. Thanks for a wonderful semester! Wishing everyone a great summer!
Planococcus sp. Isolated from sub-artic decomposing wood.
Morgen Southwood April 26, 2017
Introduction
After learning about some extreme life styles that microbes could have, I was curious if I could find one in my own home. I sampled two places, a piece of wood from my fire wood stack (which is exposed to the extreme cold temperatures of Alaskan winters) and the ashes from my stove. If any microbes grew from the firewood it would mean that they were able to withstand temperatures below -50 degrees Celsius and be psycrophiles, if any microbes grew in my wood stove they would have had to be thermophiles. I was unable to grow any microbes from the wood stove but the growth from the previously frozen wood was abundant. Each of those colonies represented a microbe that had the physiological capability of surviving those conditions, and I was interested in observing them in the lab. The identification of this microbe would add to the depth of knowledge of what bacteria are and can be found on decomposing wood in the sub-artic.
The firewood pile was slowly decomposing, and it is likely that the microbe that was isolated from it was a part of the community that was utilizing the wood as an energy source. Decomposing communities frequently have a mix of fungi and bacteria. These communities emit extracellular enzymes to decay the wood, this results in an environment with high acidity and enzymes that are producing free radical oxygen species7. To thrive in this community it would be helpful to have mechanisms to process radical oxygen species. One such mechanism is the use of Catalase to catalyze the transformation of the reactive species hydrogen peroxide into O2. It is natural for bacteria in these community’s to be adapted to oxidative stress8
Throughout the course of this study I isolated one colony and performed many physiological and genomic tests to identify what species my isolate was. Considering the environment in which it was found, there were a few expected results: such as the microbe’s oxygen class and catalase test results. Once identified, I could then research the species and compare it’s physiology and genomic data to current literature to confirm the taxonomic assignment.
Methods
Collecting sample and isolation process
The sample was collected from a dry piece of wood. Sterile water was used to moisten the wood and a sterile swab was rolled along the wood’s surface. The sample was streaked using the quadratic method to grow pure isolated colonies onto a tryptic soy agar (TSA) plate. To control growth rates the plates were grown in incubators for 2-4 days at 37 degrees Celsius then moved to a refrigerator to ensure colonies did not grow large enough to touch. The TSA medium provided a suitable environment for a variety of bacteria and a few molds. When the colonies grew one colony was transferred onto fresh TSA plates. The quadratic streak was used 4 times to ensure a pure culture. At this time the culture was transferred into an agar slant. Tests were performed on the isolate by obtaining cells from either the TSA plates or from the slant agar.
Physiological Characteristics
The gram stain was performed to assess the thickness of the peptidoglycan wall. The sample was stained using the provided protocol, gram-positives and negative controls were compared to the isolate to determine its gram-state (lab 4). While the cells were prepped in slides some characteristics were observed with the microscope, such as cell alignment shape and length. Colony morphology including color, size, margin and elevation was observed from growth on the TSA agar.
Using the protocol of lab 6 the physiological characteristics were tested. A suspension of the microbe in broth was tested simultaneously in all 21 of the tests included in the API 20E test strip. Additionally the isolate underwent the thioglycolate test- for oxygen class, oxidase test- for the presence of cytochrome c-oxidase, and lastly the catalase test- for the presence of catalase enzyme.
The cells inoculated onto Eosin Methylene Blue (EMB) and MacConkey Agar (MAC) plates. EMB is selective for gam-negative bacterium, if the microbe is gram negative and can ferment the sugars present then the plate experiences a color change. MAC plates are also selective for Gram-negative microbes and differentiates between lactose fermenters (agar turns pink) and non-lactose-fermenters (agar turns colorless).
The susceptibility of the isolate to 8 different antibiotics was tested using the Kirby-Bauer Method agar and the disk diffusion test per the protocol of Lab 9. The antibiotics tested were Amikacin, Cefazolin, Cefoperazone, Gentamicin, Piperacillin, Tobramycin, Trimethoprim and Vancomycin. After a two-day waiting period while the microbe was given the opportunity to develop where it could, the diameter of prohibited growth was measured and compared to standard antibiotic specific diameters and then classified as either susceptible, intermediate or resistant.
Bioinformatics
Dna was extracted and purified from the isolates cells using protocol from lab 5. Cell lysis was accomplished through adding sodium dodecyl sulfate (SDS), which is a surfactant that chemically weakened the cell walls, and the use of PowerBeads and the centrifuge to mechanically break the cell walls. A series of patented solutions C1-4 were added following the protocol to purify and remove inhibitors and proteins until all that remained was a pure solution of DNA. The DNA was then sent to the UAF’s DNA Core Lab sequenced using the Illumina MiSeq DNA sequencer which utilized the next-generation sequencing methodology.
Using Illumina’s BaseSpace dashboard to handle the data, the isolate’s genome was analyzed and compared to an existing database to search for similar sequences of DNA. BaseSpace has many apps that provide different information about the genomic data. The SPAdes Genome Assembler provided the overall result of the genomes assembly. Of this information, only the number of contigs that were greater than 1000 base pairs long [# contigs (>=1000bp)], The total length of the assembled genome, the longest contig and the guanine and cytosine percentage in the genome (GC%) were observed. The next app that was used was the Kraken Metagenomics, which provided taxonomic assignment. This app provided sample information such as the total number of reads that were used for classification and the percent of reads that were classified. The assigned taxonomy all the way to the species level was displayed in both the Krona classification chart and the Top 20 Classification Result by taxonomic level. Both sources have specific classification percentages that confer certainty. The final app Prokka Genome Annotation assigned gene regions that likely code for either tRNAs, rRNAs, CRISPRs and coding genes (CDs) and the abundance of them. The APP provides an extensive list of regions that were specifically identified as the above elements.
Further analysis of the genomic information was necessary. The contig.fasta file from the SPAdes Genome Assembly produced by Illumina’s BaseSpace was analyzed by BLAST. BLAST had a different database and could possibly have the genome of the isolates species if base space did not. BLAST suggested Genus and species with associated query cover percentage and identity percentage.
Results
Physiological Characteristics
The Isolate appeared to be a pure culture by the third quadratic streak; a fourth was performed for certainty. The Microbes transfer to the agar slant was successful: Image 1, and was successfully re-cultured from the slant when necessary.
Image 1: Microbe growing on TSA agar
Image 2: Gram-stained microbe
The gram-stain was repeated three times before a successful stain was accomplished. The gram-stain revealed that the isolate was a gram-positive coccid: Image 2. While under the microscope it was observed that the cells could be aligned linearly, in pairs or quads but not in a longer strep chain. The purity of the isolate was confirmed with the microscopic observation of a mono-culture. The cells were measured to be 1 micrometer in diameter. Colony morphology on the TSA agar revealed a yellow colony that’s size increased with time, a smooth round margin and the colony was an elevated domed.
Image 3: API 20E test results
The API 20e tests had negative results for all for the 21 tests: Image 3. Considering that the API 20e test is designed for enteric gram negative microbes these results are confirmation that the microbe is gram-positive. The thioglycolate test had growth throughout the agar with thicker growth at the surface, which revealed that the microbe’s oxygen class was facultative aerobe. The catalase test produced bubbles and the oxidase test produced a blue color change on the strip indicating that the microbe was positive in both tests. These results reveal that the cells contained both cytochrome c-oxidase and the catalase enzyme respectively. The isolate failed to grow on EMB or MAC plates, considering that both plates are selective for gram negative, these results confirm the results of the gram positive test: Image 4.
Physiological Tests | Results |
Gram | Positive |
Cell morphology | 1 micrometer diameter, coccus, cells divide linearly |
Colony Morphology | Yellow colonies that’s size increases with time, a smooth round margin, elevated dome. |
API 20e | All negative results |
Fluid thyioglycolate | Facultative anaerobe |
Oxidase | Positive result |
Catalase | Positive result |
MAC | No growth |
EMB | No growth |
Table 1: Physiological Test Results
The microbe was susceptible to all eight antibiotics tested. The Inhibition Zones exceed the antibiotic specific threshold diameter for susceptibility see table 2 for details.
Antibiotic Name | Minimum Inhibition zone for susceptibility (mm) | Microbe exceed inhibition zone
(yes/no) |
Amakicin | >=17 | Yes |
Cefazolin | >=18 | Yes |
Cefoperazone | >=21 | Yes |
Gentamicin | >=15 | Yes |
Piperacillin
Enterobacteriaeae S. pneumoniae |
>=21 >=18 |
Yes Yes |
Tobramycin | >=15 | Yes |
Trimethoprim | >=16 | Yes |
Vancomycin | >=17 | Yes |
Table 2: Antibiotic Susceptibility Results
Bionformatics
The analysis of the DNA with BaseSpace revealed unreliable data. BaseSpace’s SPAdes Gemone Assembler analyzed 128 contigs over 1000 base pairs (bp) long, a total length of over 3.7 million bp, the longest contig was 196 thousand bp, and the GC% was 44.4%. Kraken Metagenomics app was only able to classify 2.67% of reads, and of those reads only 17% of those reads identified the microbe as Lysinibacillus sphaericus. The Prokka Genome Annotation app identified 64 tRNAs, 0 rRNAs, 1 CRISPR, and 3739 CDS. Image 5 and 6 are from the Kraken app.
Image 5: Krona Classification Chart from BaseSpace displays the large portion of unclassified reads.
Image 6 To 20 Classification Results by Taxonomic Level from BaseSpace
A second analysis of the DNA was performed using the BLAST genomic database. BLAST classified the organism as Planoccus donghaensis with a query cover of 77% and a 78% identity. Several other species had similar query cover, including Planococcus antarticus: Image 7.
Image 7: BLAST Genomic Results
Discussion
The genotypic test results for this microbe were vague, the results of BaseSpace had a low number of reads that could be classified and of those reads there was little consensus. The results of the SPAdes Genome Assembler were above required values; # contigs >=1000 bp should be in the hundreds, total length should be at least a million bp and the longest contig should be at least 100000 bp. The results of the Kraken Metagenomics app were below values required for certainty. The percent of contigs read needed to be greater than 80% and the percent of those reads that needed to agree upon a classification to trust that classification to the genus level was 80% and to the genus level was 60%. The results did not meet these thresholds, and this is the reason that the BaseSpace indentified taxanomy of Lysinibacillus sphaericus was rejected.
BLAST provided many species level classifications with similar query cover and percent identity. The species classification with the highest query cover is poorly documented in literature. The first publication of a species under the genus Planococcus was P. citreus. It was identified in 1894, and was only approved in 19806. This genus is relatively young and is currently growing with many of the species being described within the last ten years. Of the few documents that relate to Planococcus donhaensis, there is one that notes the origin of the sample, the South Korean Sea. There are some qualities that are consistent between my sample and P. donghaensis, they are both, gram-positive, aerobic, coccus and oxidase positive 1. However these similarities are not enough to convince me that they are the same species. A hallmark of the genus Planococcus is that they are usually halo-tolerant gram-positive bacteria that frequently inhabit Antartica4. The isolates holotolerance was not tested but the similar cellular membrane composition and habitat conditions indicate that this genus is likely correct.
The negative results and lack of growth in the API 20e, MAC, and EMB are all consistent with the microbe’s gram-positive cell wall. These tests reveal nothing more than a conformation that it is indeed a gram-positive microbe. The oxygen class determined by the fluid thioglycolate test and the positive results of the Oxidase and Catalase test are both consistent with the oxygenic environment that the microbe was isolated from. These tests proved that the microbe could thrive in an oxygenated as well as an anoxic environment, that it contained cytochrome c oxidase which is a part of the electron transport chain found in microbes that utilize oxygen, and that it contained the catalase mechanism for dealing with oxidative damage, respectively.
At the genus level, morphology can vary greatly. Many of the morphological and physiological analyses made on the isolate are not held by every species in the Planoccocus genus. The morphological characteristics that are consistent with the isolate and across the genus are: gram-positive membranes, cocci cell shape, colonies are yellow orange in color, catalase positive and an aerobic oxygen class5. My isolate was not just aerobic but a facultative aerobe, which is inconsistent with two articles that state the genus is strictly aerobic5. The isolate was found to be susceptible to all antibiotics tested with the disk diffusion test. This is consistent with a study that found, to the level of the genus, that Planococcus’ were susceptible to all antibiotics tested3.
The second most likely species suggested by was P. antarticus, at least this species shares a similar environment. P. antarticus thrived in an Antarctica, my sample would likely have similar mechanisms for surviving temperatures around -45 degrees Celsius in Faribanks winters. P. donghaensis may have had the ability to deal with these temperatures in its genome, but it is not certain since it’s environment doesn’t select for such characteristics.
The literature on the Planococcus species like donghaensis, kocurii, halocryophilus and antarticus often notes how closely related the species are and how further analysis like G-C content, fatty acid strains, DNA-DNA hybridization etc1,2 are needed to differentiate the species. To conclusively Identify the taxonomy of this isolated microbe, it would be advisable to repeat DNA isolation and genomic analysis that was preformed in this project, and to additionally perform other genotypic analyses like, DNA-DNA hybridization and 16s rRNA analysis and fatty acid identity. Considering the limit of species identified to this date, there is the potential that this microbe could be a new species.
Work Cited
5. Fackrell, H. (n.d.). Planococcus. Retrieved April 11, 2017, from Uwindsor.ca website: https://web2.uwindsor.ca/courses/biology/fackrell/Microbes/17375.htm
— Article and link: “Too Clean for Our Children’s Good? The Checkup’ by Perri Klass, MD, The New York Times, April 17, 2017.
— Summary: This article talks about the many various ways in which our children are protected from interaction with microbes, including giving birth by caesarian section, bottle-feeding, and possible exposure to antibiotics. Such protection on the one hand affords protection from disease but on the other hand offers greater risk that children may experience complications of the “built environment.’ It is a concern that living in such a clean, controlled environment could lead to an underdeveloped immune system and subsequent health problems which may have otherwise been avoidable had the body been exposed to a diverse array of microbes at a young age. In order to combat this problem, it is recommended that young children be introduced to these microbes in the outside environment through “controlled exposures’ in the form of either “natural exposure’ consisting of interaction with their environment or through a type of vaccine yet to be developed.
— Connections: This article include discussion of the development of the human microbiome, its importance in the overall health of an individual, the avenues by which children are typically first exposed to microbes, and also the concept of vaccination with microbes in order to improve health. All of these are topics which have been mentioned or discussed over the course of the semester.
— Critical analysis: I liked the contrast that the author provided between the microbes found outdoors as opposed to those found within the “built environment.’ While I had naturally assumed that the inside of a house or apartment may be “cleaner’ than the outside world, I had not given much thought to the members of the microbial populations to be found in each of the two environments; in reality, the inside of a dwelling is not necessarily any more microbe-free than the outside, it is instead simply inhabited by a different, and possibly narrower, variety of microbes. I did not detect anything scientifically inaccurate or confusing in this article, and think that it did perform an adequate job in relaying this information to the public. The author did not get too technical in any of their explanations, yet clearly stated the anticipated problem, reasons behind that belief, and also the possible solutions to the problem.
— Question: Are researchers suspecting that the health problems mentioned are primarily due to inadequate exposure to pathogenic bacteria? Or do interactions with the non-pathogenic bacteria also play a role in shaping the immune system of children? What kinds of “natural exposures’ are parents advised to pursue in order to assist their child’s immune system to develop properly?
In a Dragon’s Blood, Scientists Discover a Potential Antibiotic
Donald G. McNeil Jr., 4/17/17, New York Times
Summary: Scientists from George Mason University have found a substance in from the blood of a Komodo dragon that may be extremely useful with fighting germs. They recreated the substance in the lab and called it DRGN-1. Tests were done on mice with infected skin wounds for this study. This study showed the DRGN-1 could get through the membranes of bacteria, dissolve the biofilms that connect bacteria, and that it could speed up the process of wound healing.
Connections: Komodo dragons can be extremely dangerous because they kill with shock-inducing venom. However, in that same body lies a “rich source’ of potential antibiotics in their blood. This article explains that the chemical can break through the membranes of both gram-positive and gram-negative bacteria, which shows just how powerful it could be if used correctly.
Critical Analysis: It’s amazing to see that something so potentially dangerous can also be so helpful when it comes to medicine. I found this article to be a little too short and simple because it gave no evidence to how the chemical can help infections.
Question: How does the DRGN-1 chemical fight infections and does it have any other effect on the body after the infection has healed?
https://www.eurekalert.org/pub_releases/2017-04/cuot-fhe041917.php
Summary: This article explores recent research into the genome of 24 different fungal species in order to identify antibiotic and other bioactive compound production genes. This study has resulted in the discovery of over 1000 pathways for generation of bioactive compounds with pharmaceutical application.
Connection: The article could be characterized as part history of the use of antibiotics and the rise of antibiotic resistance. We have at length discussed the prevalence and mechanisms for bacterial antibiotic resistance as well as the known pathways for antibiotic production in microbes like fungi.
Critical Analysis: The studied referenced in the article shows the promise of new antibiotic and even anti-cancer medications as a result of identifying these genomic pathways in fungi. The researchers believe that the knowledge gained from these sequences will also improve the efficiency of production and efficacy of existing antibiotics. At one point in the article, they refer to the predictive capability of the researchers experiments with the new sequencing data, claiming that not only could they predict the chemicals these fungi were capable of making, but identifying new versions of the same antibiotic chemicals. The reader must infer from the phrasing of this part that the researchers were able to trace the gene and find fungi that were previously unknown to have the ability to produce that particular antibiotic. The implications of information like that open the door wide to not only new means of production, but new variants of chemicals that have otherwise been fighting an uphill battle against antibiotic resistance.
Question: If it is true that the researchers found antibiotic production previously undiscovered in some fungi, they use the example of the chemical yanuthone, are these inactive genes that must be activated, and how are they accurately and consistently activating these genes to produce this chemical?
Article Title: Fungal duo isolated from toxic lake produce novel antibiotic
Source: C&En
Date: 4-19-17
Link: https://cen.acs.org/articles/95/i17/Fungal-duo-isolated-toxic-lake-produce-novel-antibiotic.html
Summary: In Montana there is an abandoned mining pit called Berkeley Pit. Since it was abandoned in 1983 water has leaked in and make it into a toxic pool with a pH of 2.5. It is so toxic that thousands of snow geese died last winter after they landed in it. However microbes love the pit. Two scientist from the University of Montana Andrea A. Stierleand and Donald B. Sterile who have been studying the fungus in the lake have found that two Penicillium fungus together make a new antibiotic. The antibiotic isn’t really a super new shape but it seems to act differently from know antibiotics.
Connection: Antibiotics we learned we discovered from a penicillin fungus by Alexander Fleming, so I thought it was really cool that we still find antibacterials like that. Could it be that fungus are adapting their antibacterials to fight resistant bacteria? This article also connects to the section on what bacteria use as energy sources, as finding life in an inhospitable place like an abandoned mining pit with pH2.5 is incredible and show how microbes can adapt to use almost anything.
Critical Analysis: The article doesn’t give much on the antibacterial agent it’s self, however it does provide us with a picture of the its chemical structure and a link to the article that the scientists published. The purpose of the post must have been to inform the public of a new discovery in science and I think it does this very well. How the article starts by describing the location of the discovery really draws readers and helps the mission of the article.
Question: We have learned a bit about how bacteria survive in inhospitable places like this pit, but how do fungus do it? How do fungus deal with low pH and high concentrations of heavy metals? Also what do the fungus use as an energy source?
Link: https://www.cnn.com/2017/04/18/health/frog-flu-virus-study/index.html
Summary: The frog species Hydrophylax bhuvistaraa secretes slime that contains a peptide that targets human H1 flu virus. Urumin, the peptide targets viruses without being toxic and harmful to human cells. This could present new ways to fight influenza in humans. Urumin targets hemagglutinin, completely denaturing the virus after exposure.
Connections: We learned about the antibiotic penicillin in class. Penicillin is made by a fungi in order to kill bacteria it might be in competition with for nutrients. Urumin, which is secreted by the frogs, is also for the frogs own benefit. Just in the way that we sued penicillin, a naturally produced antibiotic to our advantage, we hope to use Urumin to treat influenza.
Critical Analysis: This article seems very scientifically accurate. However, I do think mainstream media tends to sensationalize these kinds of discoveries. They had quotes from experts backing up their claims, but it would likely be a long time before Urumin would ever be able to actually be used in humans.
Question: What exactly is hemagglutinin in the H1 virus, what makes it unique to the virus?
Date: April 12, 2017
Source: Nova Southeastern University
URL: https://www.sciencedaily.com/releases/2017/04/170412115752.htm
Summary: Researchers at Nova Southeastern University (NSU) are looking for ways on how to treat infections without depending too much on antibiotics, since microbe’s resistance to antibiotics are alarmingly rising.
Connection: In class, we talked about how different kinds of bacteria can be susceptible or resistant to antibiotics.
Critical Analysis: This article is pretty short, and I think the researcher’s result could be elaborated more. What I learned from this article is that the researchers from NSU wanted to find a different way of treating infections with minimal use of antibiotics. I think that it’s interesting that they want to find a different approach to treating infections. However, this is going to be extremely hard because humans are still dependent to antibiotics, even though bacteria’s resistance to antibiotics are increasing. With this in mind, researchers from NSU discovered, with the help from University of Minnesota and Duke university, that by shaking the biofilm that bacteria made, bacteria’s ability to communicate with each other was negatively affected. I, also, learned that by applying a certain amount of frequency into the bacteria will cause them to be confuse in a way that it affects their growth and cooperation.
Question: Do you think that this method of shaking the bacteria in a perfect frequency will be efficient in treating bacterial infections in the future? Why or why not?
Article:Researchers Discover Antifungal Agent from Pathogen Box Project
Source: American Society for Microbiology
https://www.asm.org/index.php/mbiosphere/item/6334-scientists-identify-promising-new-compound-for-deadly-fungal-pathogens
Summary:
The pathogen box, which is an open-source drug discovery project, is seeking to find/create solutions to under-researched/neglected diseases. Researchers can receive this pathogen box, which is composed of 96 well plates with different compounds (thought to be anti-microbial agents, or known to have certain effects on microbes), for free as long as they report any findings within 2 years. In recent tests, a compound targeting cell walls and membranes in fungi (with low toxicity to humans) could potentially be used to treat common fungal infections Cryptococcus neoformans and Candida albicans.
Connections:
In class we have discussed finding different antibiotic targets for fungi, and the difficulty with doing so due to the physiological similarities between humans and fungi.
Critical Analysis:
I found it interesting that there is an ongoing project like this, with seemingly high potential, that is actually providing a vast amount of knowledge on antibacterial targets. This is actually a really great idea, the fact that the boxes are free to researchers as long as data is shared in order to add to a database is really creative and cool. I feel like techniques like this could really be used to stimulate interest and action in certain subfields and topics. This article was well written, interesting and pertinent to bio students, but simple enough for non-bio folk to have no problems reading and comprehending.
Question:
I would like to know how popular the pathogen box is (as far as how many people are using it) and what data they have gathered thus far (since its start in 2015).
Date Published: April 17, 2017
Source: https://schaechter.asmblog.org/schaechter/2017/04/staying-safe-in-space.html
Author: Jennifer Tsang
Summary:
Jennifer Tsang wrote mainly about safeguards with future microbial interactions in outer space. She touched upon safeguards against interplanetary contamination, about how NASA is preparing a lander destined for further investigation of Europa’s saltwater ocean underneath its icy surface (to look for extraterrestrial life, no less), and about their methods on how to handle any possible contaminants on the lander’s outer walls once it comes back from its long voyage.
Human gut microbiota from outer space, according to Jennifer’s research, decrease in diversity and compromise the immune system, which opportunistic pathogens may take advantage of. Bacteria also become more virulent and more resistant to antibiotics while exposed to increased radiation levels and microgravity.
Connections:
The author actually mentioned L.G. Baas Becking’s Principle of Ubiquity, which states that we can find microbial life everywhere on Earth, in every environment, in every biome, but that certain microorganisms exist only in a particular habitat–“…The environment selects.”
Antibiotic resistance was also touched upon by the author, how in space bacteria actually experience an enhanced resistance against them due to conditions in the environment.
Lastly, this article goes well under the astrobiology category of our curriculum.
Critical Analysis:
This article was interesting to me because of the astrobiological implications of the topics Jennifer Tsang has discussed. I learned that in space, the microbial content in our bodies gets significantly altered in a way that could mean harm to us in the future and may pose a huge risk for future space endeavors, especially for the astronauts involved, who are directly handling the missions.
The author appeared more credible in my eyes once she started putting links to her sources throughout the article.
Question(s):
How do we prevent our gut microbe diversity from decreasing so much that our immune functions gets compromised while in a zero-gravity environment? Is there a way for us to retain them, using our knowledge right now, in order to help our astronauts cope in space?