Structure and Function

TWiM #227: The Light and Dark Sides of the Fungal World

Podcast and Annotation Information

  • Annotation by Alice Lee, Jordyn Krinsky, Sydney Jennings, Hailey Kelley, Alex Bray, and Maggie Schlarmann
  • Podcast audio by TWiM: TWiM #227 Podcast Link
  • Podcast transcript by Otter.ai and edited by Isabelle Norris and Laurel Thompson: Access Podcast Transcripts
  • Papers Discussed:
    • Hermenau R, Kugel S, Komor AJ, Hertweck C. 2020. Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides. Proc Natl Acad Sci USA. 117(38):23802-23806. doi: 10.1073/pnas.2006109117.
    • Campuzano A, Castro-Lopez N, Martinez AJ, Olszewski MA, Ganguly A, Leopold Wager C, Hung CY, Wormley FL Jr. 2020. CARD9 Is Required for Classical Macrophage Activation and the Induction of Protective Immunity against Pulmonary Cryptococcosis. mBio. 11(1):e03005-19. doi: 10.1128/mbio.03005-19

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:20 minutes

The Most Interesting Things (according to students)

  • The mushrooms studied do not exist much in the field anymore.  Agaricus bisporus is no longer found in nature and is mainly cultivated.
  • Fungi  have their own bacterial pathogens and it displays how interconnected microbes are. For example, Mycetocola can protect mushrooms by neutralizing toxins and shows how bacteria can play a role in fungal health.

“The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.”(Hermenau et al. 2020)

1.2. Main paper; discussion starts at 17:14 minutes

The Most Interesting Things (according to students)

  •  Humans are very similar, biochemically, to the fungi since they are also eukaryotes. Because of this, we are having an increased threat from fungal diseases due to the fact that fungi are so similar to humans.
  • Global warming is making fungal infections more common. Rising temperatures are expanding the range of dangerous fungi, which shows the urgency of climate change.

“Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Consequently, clinical and animal studies indicate that CARD9 is an important regulator of protective immunity against fungal pathogens. Previous studies suggest that CARD9 is important for the induction of protection against Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system in immunocompromised patients. However, the effect of CARD9 deficiency on the induction of protective immune responses against C. neoformans is unknown. Immunization with a C. neoformans mutant that over expresses the transcription factor zinc finger 2, denoted LW10, results in protection against an otherwise lethal challenge with wild-type (WT) C. neoformans. Our results showed that CARD9 is essential for the induction of vaccine-mediated immunity against C. neoformans infection. We observed significant decreases in interleukin-17 (IL-17) production and significant increases in Th2-type cytokine (IL-4, IL-5, and IL-13) production in CARD9-deficient mice after inoculation with strain LW10. While leukocyte infiltration to the lungs of CARD9-deficient mice was similar in LW10 and WT C. neoformans-infected mice, macrophages derived from CARD9-deficient mice inherently skewed toward an M2 activation phenotype, were unable to contain the growth of LW10, and failed to produce nitric oxide in response to infection with LW10 or stimulation with lipopolysaccharide. These results suggest that CARD9-mediated signaling is required for M1 macrophage activation and fungicidal activity necessary for the induction of vaccine-mediated immunity against C. neoformans.” (Campuzano et al. 2020).

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 23 (ASM_23): The health of the environment and all organisms (microbes, plants, humans, other animals) are closely linked and interdependent, as described by the OneHealth paradigm.
  • Fundamental Statement 5 (ASM_5): The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analyses, molecular methods, and bioinformatic tools.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental Statement 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the bacterial pathogen responsible for brown blotch disease.
  • Recall the mechanism by which Mycetocola bacteria counteract the pathogen.
 S L
  • Propose an experiment to test the effectiveness of using Mycetocola as a biological control agent in preventing brown blotch disease in mushrooms.
 S H
  • Recall experimental methods used to assess fungal clearance in mice.
  • Identify the immune response to Cryptococcus in CARD9-deficient mice.
 M L
  • Predict how CARD9 deficiencies might affect susceptibility to other fungal infections beyond cryptococcosis.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • Proteomics Fractionation (7:53–8:19): This is a technique used to simplify complex protein mixtures by separating them into different groups, or fractions, based on their properties like size, charge, or solubility. The researchers fractionated the proteome where they identified important enzymes in toxin inactivation. This improved  the activity of Mycetocola to impair the destructive activity of the Pseudomonas as well as the dissemination.
  • Lipopeptide Cleavage (7:09–7:49): This is a method to break down of lipopeptide molecules, which are compounds containing both a peptide (protein-like) and a lipid (fat-like) component. Mycetocola protects the mushrooms by cleaving the lipopeptides. With this bacterium around, the bad toxins (virulence factors) of the Pseudomonas are neutralized.

4.2. Main Paper

  • Luminex Multiplex Assay (29:27–36:17):  This is a  bead-based immunoassay that allows for the simultaneous detection and quantification of multiple analytes in a single sample. This allowed the researchers to take small quantities of these lung homogenates and assay 21 different cytokines and chemokines from the same samples. They were able to take two different- infect either wild-type or mutant mice with either wild-type or mutant yeast, and look at two different times of infections and measure 21 different cytokines.
  • C. neoformans Strains (21:06–29:27): A strain is a particular version of a microbe.  LW10 is a C. neoformans mutant strain that over expresses the transcription factor zinc finger 2 (Znf2), resulting in filamentation and avirulence in mice. When infected with this strain of bacteria, the mice can develop an immunity to subsequent infection.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Cycliclipopeptides (3:20–8:59): This is a lipid chain which has a cyclic group and peptides. The main cycliopeptide, tolaasin, is a pore forming toxin. It makes holes in the cells of the mushroom and gets them to rot.
  • Pseudomonas tolaasii (3:20–8:59): Pseudomonas tolaasii is a bacterium that causes brown blotch disease. Once the mushroom, especially in the button stage, gets this disease they rot from the inside out.
  • Pseudodesmin (3:20–8:59): This is a biosurfactant that allows the bacteria to move around on the mushroom.

5.2. Main Paper

  • Fungal Pathogenesis (21:06–29:27): It is important that we better understand fungal pathogenesis, how fungi cause disease.  Right now we only have four classes of drugs to treat fungal infections and resistance is starting to emerge in some of these pathogenic fungi. Fungi are difficult to treat in humans in part because fungi and humans are both eukaryotes, so they share specific features of many biological processes.
  • Fungal Strain Differences (25:53–36:17): Different strains of a microbe have different features, usually due to genome or epigenetic difference.  When you infect mice with the LW10 strain of Cryptococcus neoformans the mice develop an immunity to subsequent infection. Mice that lack the CARD9 component die by a month after infection.

6. Podcast Questions

  1. Which bacterial species is responsible for brown blotch disease in cultivated mushrooms?
    1. Pseudomonas fluorescens
    2. Pseudomonas tolaasii
    3. Mycetocola spp.
    4. Bacillus subtilis
  2. What mechanism does Mycetocola use to control Pseudomonas tolaasii?
    1. It directly engulfs Pseudomonas tolaasii.
    2. It prevents Pseudomonas attachment.
    3. It breaks down the Pseudomonas toxins.
    4. It initiates the mushroom immune system.
  3. The podcasters mention possibly directly using a culture of Mycetocola to control brown blotch. Which description would be a good experiment for determining whether Mycetocola could be used as a biological control agent for brown blotch disease in mushroom cultivation?
    1. Cultivate 30 fields of mushrooms and spray all of them with a dilute inoculum of Mycetocola every other day and assay for brown blotch disease at day 10 and at day 50.
    2. Cultivate 30 fields of mushrooms, spray 15 with a combination of Mycetocola + Pseudomonas and 15 with Mycetocola alone as a control.  Assay for brown blotch at day 50.
    3. Cultivate 15 fields of mushrooms, spray 10 with a combination of Mycetocola + Pseudomonas and 15 with just sterile water as a control.  Assay for brown blotch at day 50.
    4. Cultivate 30 fields of mushrooms, spray 10 with Mycetocola + Pseudomonas, 10 with Pseudomonas alone, and 10 with Mycetocola alone.  Assay for brown blotch at day 50.
  4. What method did the researchers use to quantify the Cryptococcus fungal burden present in the lungs of infected wild-type and CARD9-deficient mice?
    1. Homogenized the mouse lungs, plated the inoculum, and looked for colonies.
    2. Performed fluorescent microscopy using fungi tagged with fluorescent proteins.
    3. Used visible range spectroscopy to quantify optical density of lung homogenates.
    4. Used mass spectroscopy of lung homogenates to identify  fungal metabolites.
  5. Which of the following best describes the immune response in CARD9-deficient mice infected with Cryptococcus?
    1. CARD9-deficient mice have an increased adaptive immune response.
    2. CARD9-deficient mice fail to recruit neutrophils and macrophages effectively.
    3. CARD9-deficient exhibit reduced inflammation and slower fungal clearance.
    4. CARD9-deficient mice show increased resistance to fungal infections.
  6. If a patient with a CARD9 mutation was to be exposed to an infectious dose of Candida albicans, what would be the most likely immune response outcome? What is your reasoning?
    1. Increased resistance to infection due to macrophages using alternative immune pathways
    2. Normal clearance of Candida albicans because Candida albicans is a very different fungus
    3. Impaired clearance of Candida albicans  because CARD9 is involved in fungal surveillance
    4. Enhanced activation of macrophages and improved fungal clearance due to high inflammation

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 2ABD) and one from the main paper (Figure 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key aspects of experimental design, such as the controls in the experiments, as well as experimental setup and analysis features.
  • Analyze the data (images, HPLC-MS, MALDI-imaging) and draw conclusions about which bacterium neutralizes P. tolaasii’s virulence and how it/they do so.
  • Design an experiment to test Mycetocola spp. as a potential agent against fungal pathogens.

Experimental Background (Hermenau et al., Figure 2ABD)

When white button mushrooms are commercially cultivated these fungi are susceptible to brown blotch disease, which is caused by the bacterium Pseudomonas tolaasii. This pathogen secretes cyclic lipopeptide toxins, such as tolaasin I, which are responsible for damaging mushroom tissue. In this study, Hermenau et al. (2020) investigated the ability of other bacteria to inhibit P. tolaasii’s virulence by interfering with these toxins. They tested two Actinobacteria, Mycetocola tolaasinivorans and M. lacteus.  First, the ability to alter P. tolaasiI infection was tested by inoculating button mushroom or white potato tissue with the pathogen, pathogen plus the test bacteria, or NaCl (panel A).  Next, changes to the P. tolaasii-specific toxin, tolaasin I,  in the presence of each test bacterium were examined using high performance liquid chromatography-mass spectrometry (HPLC-MS; panel B).  To further characterize the physical interaction, bacteria and fungi were grown on solid medium next to each other with an overlapping section (panel D).  MALDI imaging was performed to map the spatial organization of the toxin and product relative to each species’ growth (first column in panel D).  The results are displayed as heatmaps, respectively, for toxin and product (panel D, center and right columns).

Several-part graphic of data related to the study, including photos, HPLC traces, chemical structures, and MALDI imaging.
Figure 2. “Helper bacteria inactivate the toxin produced by the mushroom pathogen. (A) In vivo infection assays with mushroom and potato tuber slices. (B) Extracted-ion chromatogram (EIC) traces of 1 (black, 994.1137 [M + 2H]2+) and 2 (red, 1003.1190 [M + 2H]2+) obtained from high-performance liquid chromatography (HPLC) analysis of culture extracts, isolated 1, and 1 hydrolyzed with 5 eq LiOH, 1 h, RT. (C) Comparison of MS/MS fragmentation patterns of 1 and 2 with highlighted key fragments. Gray bar indicates magnified area. c: calculated; o: observed masses. (D) MALDI imaging of solid cultures of P. tolaasii with M. lacteus, and M. tolaasinivorans, respectively, showing spatial distribution of 1 and 2. Black: colony of M. tolaasinivorans and M. lacteus, respectively; red: colony of P. tolaasii; green: mixed colonies.” (Hermenau et al. 2020)

7.1.2. Questions

  1. Mushroom and potato slices were treated with the pathogenic bacterium (Pseudomonas tolaasii), one of two potential helper bacteria with P. tolaasii, and table salt (NaCl) to determine if either of these bacteria could defend against brown blotch disease.  In this experiment, ______ is the positive control and ______ is the negative control.
    1. NaCl; P. tolaasii + M. lacteus
    2. P. tolaasii alone; NaCl treated
    3. P. tolaasii + M. lacteus; NaCl
    4. No positive control; potato
  2. Which bacterium/a is/are most effective at preventing brown blotch disease in mushrooms caused by P. tolaasii (panel A)? What is your evidence?
    1. Mycetocola lacteus; the tissue slices show extensive infection
    2. Mycetocola lacteus; the tissue slices do not show infection
    3. Mycetola  tolaasinivorans; the tissue slices show much infection
    4. Mycetola  tolaasinivorans; the tissue slices do not show infection
  3. The researchers identified the active bacterial component through high pressure liquid chromatography-mass spectrometry (HPLC-MS) by comparing metabolic profiles of bacterial co-cultures to a mixture of known toxins, tolaasins (panel B). In this experiment, ______ is/are the positive control and ______ is/are the negative control.
    1. Solvent; both 1 and 1 + LiOH
    2. No positive control; 1 + LiOH only
    3. 1 and 1 + LiOH; no negative control
    4. Both solvent and 1; 1 + LiOH
  4. Based on the HPLC-MS data shown in panel B, which bacterium(a) affect(s) the specific P. tolaasii toxin known as tolaasin? What is your evidence?
    1. Both; The “1” retention time of the P. tolaasii “only” culture is shifted to the “2” retention time when either bacteria are included in the culture.
    2. M. lacteus; The “1” retention time of the P. tolaasii “only” culture is shifted to the “2” retention time only when M. lacteus is included in the culture.
    3. M. tolaasinivorans; The “1” retention time of the P. tolaasii “only” culture is shifted to the “2” retention time when M. tolaasinivorans is in the culture.
    4. Neither; The “1” retention time of the P. tolaasii “only” culture is not shifted to the “2” retention time when either bacteria are included in the culture.
  5. Which color outlines the location of the pathogen in the co-culture (panel D, left panels) and what color in the heat maps (panel D, center and right panels) represents the highest concentration of the compound that is being monitored?
    1. Black; blue
    2. Red; blue
    3. Black; red
    4. Red; red
  6. Does the distribution of tolaasin (1) and the tolassin cleavage product (2) support or conflict with the data in panels A and B? What is your evidence?
    1. Support; 1 is found in pathogen only areas and 2 is primarily found in interaction zones.
    2. Conflict; Both 1 and 2 are found in the pathogen only, 2 is concentrated in the Mycetocola.
    3. Support; 1 is found in pathogen areas and 2 is found at the outer edges in the Mycetocola.
    4. Conflict; Both 1 and 2 are found equally distributed throughout the co-culture of bacteria.
  7. Which description would be a good experiment for determining whether Mycetocola could be used as a biological control agent for brown blotch disease in mushroom cultivation?
    1. Cultivate 30 fields of mushrooms and spray all of them with a dilute inoculum of Mycetocola every other day and assay for brown blotch disease at day 10 and at day 50.
    2. Cultivate 30 fields of mushrooms, spray 15 with a combination of Mycetocola + Pseudomonas and 15 with Mycetocola alone as a control.  Assay for brown blotch at day 50.
    3. Cultivate 15 fields of mushrooms, spray 10 with a combination of Mycetocola + Pseudomonas and 15 with just sterile water as a control.  Assay for brown blotch at day 50.
    4. Cultivate 30 fields of mushrooms, spray 10 with Mycetocola + Pseudomonas, 10 with Pseudomonas alone, and 10 with Mycetocola alone.  Assay for brown blotch at day 50.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in bar plots and survival line plots for these experiments.
  • Identify key features of experimental design, including controls and variable types in these experiments.
  • Analyze the data to make conclusions about the role of CARD9 in survival and fungal burden in mice infected with the LW10 strain of Cryptococcus neoformans.
  • Predict which tissues are most likely to be at risk for CARD9-deficient patients infected with C. neoformans based on these results.

Experimental Background (Campuzano et al. et al., Figure 2)

Cryptococcus neoformans is a fungal pathogen that poses a serious threat to immunocompromised individuals. C. neoformans is usually inhaled and can disseminate to a number of tissues, including the brain causing fungal meningitis. In this study, Campuzano et al. (2020) explore the role of CARD9 in immunity to this pathogen.  CARD9 is a key adaptor protein that transduces signals from C-type lectin receptors (CLRs) on innate immune cells in response to fungal carbohydrates.  This makes CARD9 a critical mediator of antifungal immunity, but the effect of CARD9 deficiency on immunity to this pathogen is unknown. The researchers investigated this by infecting either wild-type mice (WT) or mice deficient in CARD9 (CARD9 KO) with a strain of C. neoformans (strain LW10.) that induces immunity rather than disease in wild-type mice.  They quantified mouse survival (panel A) as well as C. neoformans dissemination to the lungs (panel B), brain (panel C), and spleen (panel D).  Dissemination data, also known as fungal burden, was quantified by finding how many living fungi can be cultivated from homogenized tissue, and is reported as colony forming units (CFU).

line and bar chars showing LW10, WT and CARD9 KO levels post-inoculation in mice.
Figure 2. “CARD9 is required for the induction of protection against cryptococcosis. (A to D) C57BL/6 and CARD9-deficient mice were given an intranasal inoculation with C. neoformans-derived strain LW10. (A) Mice were observed for up to 45 days for survival analysis. (B to D) Next, pulmonary fungal burden was determined on day 7 or 14 postinoculation (B), or fungal burden was determined in the brains (C) and spleens (D) of mice on day 14 post inoculation. Survival data are from one experiment using 10 mice per group. Fungal burden data are cumulative results for three experiments using five mice per group per time point. Values that are significantly different from the values for CARD9-deficient mice by the (A) log rank test or (B to D) an unpaired Student’s t test (two-tailed test) are indicated by asterisks as follows: *, P  0.05; ****, P  0.0001.” (Campuzano et al. 2020).

7.2.2. Questions

  1. Wild-type or mice deficient in CARD9 were infected with an engineered strain of C. neoformans (LW10) and survival followed for 45 days (panel A).  In this experiment, ______ is the positive control and ______ is the negative control.
    1. Infection; no infection
    2. No positive control; wild-type
    3. Infection; wild-type mice
    4. Wild-type; no negative control
  2. Which of the following best describes the survival outcome differences of wild-type and CARD9-deficient mice infected with the LW10 strain of Cryptococcus neoformans?
    1. CARD9-deficient mice survived considerably longer than wild-type mice.
    2. CARD9-deficient mice had similar survival rates to that of wild-type mice.
    3. CARD9-deficient mice had reduced survival compared to wild-type mice.
    4. CARD9-deficient mice had improved survival when treated with drugs.
  3. Wild-type or mice deficient in CARD9 were infected with an engineered strain of C. neoformans (LW10) and fungal burden in the lung quantified at 14 days post-infection (panel B, second two bars) .  In this experiment, ______ is the dependent variable and ______ is the independent variable.
    1. Mouse genotype; fungal strain
    2. Fungal strain; mouse genotype
    3. Fungal burden; mouse genotype
    4. Fungal burden; fungal strain
  4. What notation indicates statistically significant differences in lung fungal burden (panel B) and which data are statistically significant, if any?
    1. Bar heights; wild-type and CARD9 KO mice show significant differences at day 7 and 14.
    2. Asterisks; wild-type and CARD9 KO mice show significant differences at day 7 and 14.
    3. Bar heights; wild-type and CARD9 KO mice show significant differences only at day 14.
    4. Asterisks; wild-type and CARD9 KO mice show significant differences only at day 14.
  5. How does the pulmonary fungal burden in CARD9-deficient mice compare to that in wild-type mice over time?
    1. It was not significantly different between the mouse genotypes at day 7, but was statistically significantly different (lower) at day 14.
    2. It was not significantly different between the mouse genotypes at day 7, but was statistically significantly different (higher) at day 14.
    3. It was significantly different between the mouse genotypes at day 7 and day 14, but the difference was much greater at day 14.
    4. It was significantly different between the mouse genotypes at day 7, but the difference was less and not significant at day 14.
  6. Based on the data presented in this figure, which organs are most at risk of having a high fungal burden in patients with CARD9 deficiencies?
    1. Only the lungs
    2. Brain, lungs, and spleen
    3. Only the brain
    4. Lungs and spleen

8. Paper Information and Licensing

8.1. Snippet paper

  • Hermenau R, Kugel S, Komor AJ, Hertweck C. 2020. Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides. Proc Natl Acad Sci USA. 117(38):23802-23806. doi: 10.1073/pnas.2006109117.
  • This article is licensed for Creative Commons use using CC BY-NC-ND 4.0, which allows non-commercial re-use with proper attribution and no changes. See the article’s information on its web page.

8.2. Main paper

  • Campuzano A, Castro-Lopez N, Martinez AJ, Olszewski MA, Ganguly A, Leopold Wager C, Hung CY, Wormley FL Jr. 2020. CARD9 Is Required for Classical Macrophage Activation and the Induction of Protective Immunity against Pulmonary Cryptococcosis. mBio. 11(1):e03005-19. doi: 10.1128/mbio.03005-19.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s information on its web page.

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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