Medical Mycology and the Chemistry Classroom: Germinating

of a project-based Organic Chemistry laboratory environment: a phenomenographic approach. Nikita L. Burrows , Montana K. Nowak , Suazette R. Moori...
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Medical Mycology and the Chemistry Classroom: Germinating Student Interest in Organic Chemistry Joseph M. Bliss† and Christopher W. Reid*,‡ †

Department of Pediatrics, Women & Infants Hospital of Rhode Island, Warren Alpert Medical School of Brown University, Providence, Rhode Island 02905, United States ‡ Department of Science and Technology, Bryant University, Smithfield, Rhode Island 02917, United States S Supporting Information *

ABSTRACT: Efforts to provide active research context to introductory courses in basic sciences are likely to better engage learners and provide a framework for relevant concepts. A simple teaching and learning experiment was conducted to use concepts in organic chemistry to solve problems in the life sciences. Bryant University is a liberal arts university with new programs in biological and environmental sciences. This laboratory exercise was developed for introductory organic chemistry and designed to highlight its applicability to the biological sciences. Students participated in an ongoing project to identify antigens on the surface of Candida albicans hyphae by testing a variety of lipid extraction procedures and characterizing the fatty acid and carbohydrate composition of their extractions by thin-layer chromatography and gas chromatography−mass spectrometry. Student extracts were also tested in an immunoblot assay with a specific antibody fragment (scFv) that recognizes an unknown component of the cell surface of C. albicans hyphae. In order to build communication skills, students were required to prepare and present posters detailing the project and their findings. KEYWORDS: Second-Year Undergraduate, First-Year Undergraduate/General, Upper-Division Undergraduate, Organic Chemistry, Biochemistry, Interdisciplinary/Multidisciplinary, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Applications of Chemistry

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INTRODUCTION The application of active-learning and inquiry-driven pedagogies in science, technology, engineering, and mathematics (STEM) is motivated by the potential for greater retention of information and improved student attitudes.1 One approach involves the incorporation of faculty research into the undergraduate curriculum. These types of learning opportunities can build essential skills such as hypothesis testing, data analysis, and problem solving that can benefit all students, regardless of their ultimate career choices.2−5 There is an increasing awareness of the need for more interactive pedagogies in the classroom, with less emphasis on a rigid curriculum (teaching) and more focus on student understanding of the conveyed information (learning). Learning-focused STEM classrooms involve teachers guiding students in active and extended scientific study requiring the application of scientific knowledge and ideas, as well as analytical and critical thinking.6−8 The importance of a multidisciplinary approach to biology education that includes enhanced levels of mathematics and physical sciences is well understood.9−11 The increasing complexity of biological pathways, cellular communication, systems biology, and drug discovery demands a multidisciplinary approach that involves physicists, mathematicians, chemists, and biologists. As recipients of a multidisciplinary education, our undergraduates © 2013 American Chemical Society and Division of Chemical Education, Inc.

will be well prepared for future studies and for emergence into the evolving workforce. Bryant University, a primarily undergraduate liberal arts institution, has recently undergone a curriculum expansion to include degrees in environmental science and biology. Students in either of these majors have the option to take an introductory course in organic chemistry. While the course fulfills degree requirements for science majors, nonmajors with the required prerequisites may also take organic chemistry in fulfillment of their general education requirements. The primary challenges for the instructor are to actively engage the nonmajors in the study of organic chemistry and to demonstrate to the majors its applicability to environmental and biological sciences. The demographic of the organic chemistry course in question included four science majors (biology; environmental science) and one nonmajor (applied mathematics). Female students accounted for 80% of the class. Most of the students (four out of five) were not enrolled in the laboratory section, as it was not mandatory to fulfill degree requirements. As a result, these students lacked the opportunity to apply the concepts discussed in lecture. Here we report on a multiweek, activelearning project designed to foster student engagement, Published: September 18, 2013 1320

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during lectures included the biology and pathogenesis of C. albicans, general principles of immunochemistry, and the structure and function of antibodies and antibody fragments. Guided discussions were used to relay information pertaining to the project goals and experiments. Students were guided through discussions of biological (e.g., cell structure, membrane structure, membrane composition) and chemical (e.g., structure of biomolecules, chemical and physical properties of biomolecules) concepts and prompted to explore the relationship between the two.

develop analytical and critical thinking skills, and illustrate the importance of organic chemistry in the study of life sciences. Toward this end, students participated in a limited-scope project related to the authors’ research under the Rhode Island Research Alliance Collaborative Research Award to Women and Infants Hospital (Providence, RI) and Bryant University (Smithfield, RI), to identify the antigenic targets of specific antibody fragments12 directed against Candida albicans hyphae (Figure 1, panels A and B). This student research project was

Growth of C. albicans Hyphae

Prior to the start of the project, C. albicans (strain SC5314) was cultured overnight in YEPD (1% yeast extract, 2% peptone, 2% glucose) at 37 °C with vigorous aeration. The cells were collected by centrifugation (7000 rpm, 5 min), and the cell pellet was washed twice with sterile phosphate-buffered saline (PBS). The washed cell pellet was resuspended in 1 mL of sterile H2O and plated in fresh medium 199 in large Petri dishes at a final concentration of 5 × 105 cells/mL. Hyphae were grown for 24 h at 37 °C under static conditions. The resulting hyphae were collected and washed in PBS.12 Prior to use in the undergraduate classroom, hyphae were killed by incubation in 2% phenol for 4 h. Total Lipid Extraction

C. albicans hyphae (approximately 0.5 g wet weight) were resuspended in methanol and dried under a stream of N2. The dried cells were resuspended in 5 mL of the appropriate solvent system (Supporting Information Table 1) and incubated for 4 h with gentle mixing on a Glass-Col Rugged Rotator. After 4 h, the cell debris was removed by centrifugation (3000 rpm, 10 min). The solvent was placed in a clean test tube and the sample evaporated to dryness under a stream of N2. Thin-Layer Chromatography

Students resuspended their lipid extracts in approximately 0.5 mL of CHCl3. Lipid extracts were spotted onto aluminumbacked silica plates (4 × 7 cm) using 10 μL capillary tubes and developed using 65:35:8 (CHCl3:MeOH:H2O) as the mobile phase. Plates were developed until the solvent front reached 1 cm from the top of the thin-layer chromatography (TLC) plate. TLC plates were stained using I2(vapor), basic KMnO4, or 2,4dinitrophenylhydrazine (DNPH). Fatty Acid Methyl Ester (FAME) Preparation

A 0.25 mL sample of the lipid extract in CHCl3 was placed in a screw-top glass test tube and dried under a stream of N2. A 2 mL volume of 3 M methanolic HCl was added to the dried lipid sample and heated at 98 °C for 16 h. After cooling, the sample was dried under a stream of N2, resuspended in 2 mL of MeOH, and dried again. The dried sample was resuspended in dH2O (1 mL) and extracted twice with CHCl3 (2 × 1 mL). The CHCl3 phases were collected, pooled, dried over Na2SO4, and evaporated under a stream of N2. The dried samples were resuspended in 500 μL of CH2Cl2 and analyzed by GC−MS.

Figure 1. Project overview. (A) Immunofluorescence (left) and phasecontrast (right) photomicrographs showing C. albicans hyphae bound by the specific antibody fragments under investigation. (B) Structure of antibodies and antibody fragments under study. (C) Phosphomannan and (D) glycosyl-inositol phosphate (GPI), two possible fungal glycolipid antigens.

designed to be achieved during regular class hours, yet still have the potential to provide valuable information for the research program’s advancement.

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Alditol Acetate Preparation

The lipid extract (0.2 mL) was dried under a stream of N2 and hydrolyzed in approximately 1 mL of 2 M trifluoroacetic acid for 3 h at 98 °C. Once cooled, the hydrolyzed sample was dried under a stream of N2, the residue resuspended in approximately 1.5 mL of deionized H2O, and a drop of ammonium hydroxide added (28% stock solution). A small amount (approximately 20 mg) of NaBH4 was added to the solution, which was incubated

MATERIALS AND METHODS

Delivery of Relevant Background Information

The background information the students needed to understand the project was delivered using a combination of traditional lectures and guided discussions. Topics covered 1321

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Table 1. Curriculum for Introduction to Organic Chemistry and Correlation to Project Objectives Weeks

Research Activities

Lecture Topics

1−2 2−3

Structure and bonding Alkanes, alkenes, and alkynes

4−5 6−7

Aromatic compounds Stereochemistry

7−8

Nucleophilic substitutions and eliminations Chemistry of alcohols, phenols, and Analysis of glycolipids. Preparation of alditol acetates of lipid-associated carbohydrates. ethers Chemistry of carbonyl groups Preparation of fatty acid methyl esters. Derivatization of lipid extracts for analysis by GC−MS. Preparation of (aldehydes, ketones, carboxylic alditol acetates of lipid-linked carbohydrates. Reduction of aldehydes by NaBH4. Hemiacetals, hemiketals, and acids, and derivatives) their relationship to monosaccharide structure. Structural characterization A short tutorial on electron-impact mass spectrometry, its application to identifying fatty acids and sugars using student-generated data.

9−11 12−14 15

Polarity and organic compounds: Choosing a solvent system for lipid extraction. Oxidation of alkenes: KMnO4 oxidation of alkenes. Analysis of lipid extracts by thin layer chromatography (oxidation of alkenes by KMnO4). E/Z isomerism and relation to unsaturated fatty acids. Sugars and epimerization of anomeric carbon and the effect on monosaccharide analysis by GC−MS.

to interpret the spectra that were generated from the lipid extracts they themselves produced. A detailed timeline of the experiments undertaken and a description of how these research activities are related to the curriculum for the introductory organic chemistry course offered at Bryant University are presented in Table 1.

overnight at room temperature. The reduced monosaccharides were treated with 5 drops of glacial acetic acid and evaporated to dryness. The residue was washed twice in 2% acetic acid in methanol (2 mL) and evaporated to dryness. The resulting residue was dissolved in 2 mL of acetic anhydride and placed at 98 °C for 3 h, cooled, and evaporated to dryness. The dried alditol acetate samples were dissolved in CH2Cl2 (0.25 mL), dried over Na2SO4, and analyzed by GC−MS.

Experimental Design and Data Generation

The overarching goal of the research project under study was to isolate and identify C. albicans antigens recognized by a subset of specific antibody fragments. Students worked with the underlying assumption that the antigenic targets were glycolipids. With this in mind, students were tasked with assessing the efficacy of different glycolipid extraction techniques for the isolation of antigens from the hyphal cell wall. Students were presented with examples of glycolipids produced by C. albicans (Figure 1, panels C and D). Discussions in class focused on the properties of these molecules, such as size and polarity, and how these properties might influence the extraction of glycolipids from the hyphal cell wall. Students then used this information to select several different solvent systems to test experimentally in their glycolipid extractions. Specifically, the students tested lipid extraction methods on C. albicans hyphae that varied in solvent polarity (Supporting Information Table 1). Students hypothesized that the 2:1 CHCl3:MeOH solvent system would be most likely to draw out glycolipids with predominantly nonpolar properties, while the Bligh−Dyer extraction might prove more efficient at extracting more polar glycolipids because of the twophase nature of the extraction and the ability to analyze both the polar and nonpolar phases. Each student was assigned one of the four solvent systems described in Supporting Information Table 1 for their experiment (with the exception of the nonscience major, who was paired with one of the science majors for this project). The students extracted glycolipids from the hyphal samples and characterized their extracts using TLC and GC−MS. TLC analysis was done by the students during class time, while a demonstration of the GC−MS and the principles of analyte separation was provided. The students’ FAME and alditol acetate samples were run outside of class and the resulting data provided in a subsequent lecture. Preliminary comparison of the various lipid extracts was performed during class time using TLC and staining with either I2(vapor) or basic KMnO4 (Figures 2 and 3A). Iodine vapor, a general staining technique, has a high affinity for unsaturated and aromatic compounds. Using I2(vapor) only a few differences were noted, particularly

GC−MS Analysis

Analysis of FAME and alditol acetate samples was performed on an Agilent 7890A with an Agilent 19091-J-433 (FAME) or DB-225MS (alditol acetate) column and equipped with a 5975C EI-MS detector. Oven temperature conditions can be found in Supporting Information. Immunoblot Analysis

Lipid extracts (10 μL in CHCl3) were spotted onto nitrocellulose membrane and allowed to air-dry completely. The membrane was blocked in 5% skim milk, and standard Western blot development was performed with C. albicansspecific scFv and appropriate secondary antibodies as previously described.12

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RESULTS AND DISCUSSION

Integration of Research Activities in the Chemistry Classroom

The research project was initiated during the first week of classes and lasted most of the semester. In general, a 30−40 min segment of each week’s lecture schedule was set aside for developing methodology, conducting experiments, or discussing results. Research activities for a given week were closely tied to the concepts being delivered in lectures at that time, in order to more effectively convey inherently challenging concepts in chemistry. As the course material covered the properties of organic molecules, in particular hydrophobicity and polarity, students applied this knowledge to developing procedures for lipid extraction. Class discussions on the reactivity of functional groups (alkenes, alcohols, and carbonyl groups) were supported by the analysis of the students’ lipid extracts via TLC and the derivatization of lipids and sugars for GC−MS analysis. Lectures also covered the characterization of organic molecules using methods such as GC−MS, and the identification of fatty acid chemical structures by electronimpact mass spectrometry. Instead of being given random GC− MS and electron-impact spectra to analyze, the students learned 1322

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which looked the most complex by TLC, did not have the diversity of fatty acids found in the Bligh−Dyer and 2:1 CHCl3:MeOH. This led to a class discussion regarding what biomolecules could be oxidized by KMnO4. Students further characterized their extractions by analyzing for sugar content via alditol acetate analysis (data not shown). Finally, the students tested their lipid extracts for reactivity with the C. albicans-specific antibody fragments using Western blots (data not shown). Students blotted their samples on a nitrocellulose membrane, and the resulting development of the membrane was completed outside of class. While extraction and identification of the specific antigen(s) recognized by these antibody fragments was beyond the scope of the students’ research objectives, it was nonetheless possible that the antigenic target of interest was present in one or more of the lipid extracts generated. Results of the immune blot indicated that only the MTBE extraction contained antigen.

Figure 2. Schematic diagram illustrating some of the chemistry behind the TLC stains used in this project. The detection of olefins via potassium permangenate oxidation (top reaction) and the detection of carbonyl groups by dinitrophenyl hydrazine (bottom reaction) are shown.

Interpretation of Data

The students used the data from the TLC and GC−MS analyses to evaluate the ability of their respective methodologies to provide a comprehensive representation of the glycolipids present in the hyphal cell wall. Each student shared his or her data with the class, which allowed everyone to draw conclusions regarding how their method compared to the other methods tested, and to determine which yielded the most comprehensive lipid extraction. Class discussions regarding the data were used to encourage analytical thinking and have the students draw conclusions regarding their own and class data. While the 2:1 CHCl3:MeOH and 10:10:3 CHCl3:MeOH:H2O contained the broadest range of fatty acids, only the MBTE extraction tested positive for the antigen in immune-blot analysis. Students were informed that the work they completed during the semester would be followed up during the summer and the reactive antigen present in the MBTE sample would be further purified and identified.

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ASSESSMENT Students were required to maintain a laboratory notebook dedicated to their project. Students were instructed to make note of all relevant details, including hypotheses, experimental design, experimental protocols, and data and their interpretation. At the end of the semester, students submitted the notebook for grading. Students were given marks for the completeness and accuracy of their data recording. An emphasis was placed on their ability to accurately describe each experiment and the rationale for conducting it. Students were required to clearly state experimental results and draw conclusions based on their own and their classmates’ results. The project culminated with each student preparing a poster presentation detailing the project objectives, experimental methods, results, and conclusions. The students presented their posters at Bryant’s inaugural Research and Engagement Day, an initiative the university launched to promote the research activities of faculty and students among the Bryant community. Students were assessed on their ability to write a concise yet accurate abstract describing the project, as well as on their ability to effectively convey their findings and the underlying chemical concepts during the poster presentation. The rubric used for evaluation of the presentation is included in the Supporting Information.

Figure 3. Representative data generated by students upon analysis of their respective lipid extractions. Panel A: TLC analysis of crude lipid extracts stained with either I2(vapor)(left) or KMnO4 (right); 1, Bligh−Dyer extraction (organic phase); 2, Bligh−Dyer extraction (aqueous phase); 3, CHCl 3 :MeOH (2:1) extraction; 4, CHCl3:MeOH:H2O (10:10:3) extraction. Panel B: representative FAME analysis of crude lipid extracts by GC−MS.

in the Bligh−Dyer aqueous phase (Figure 3A). When the students used basic KMnO4 as their stain, differences between the extraction methods were more evident (Figure 3A). A discussion of the reaction of KMnO4 with alkenes and polyols ensued with the students. Based on the results of KMnO4 staining, the students determined that the single phase 10:10:3 (CHCl3:MeOH:H2O) contained the more complex mixture of the hyphal cell surface. FAME analysis of the student lipid extractions are presented in Figure 3B and Supporting Information Table 2. The results generated by the group showed that both the classical two-phase Bligh−Dyer and the single phase 2:1 CHCl3:MeOH extractions provided similar lipid profiles. The 10:10:3 (CHCl3:MeOH:H2O) extraction, 1323

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CONCLUSIONS Many students find organic chemistry to be a challenging subject matter. Students who are not pursuing a major in chemistry often fail to see the relevance of the topics covered in this course to their overall education. Through the use of hands-on experiments to illustrate textbook concepts, these difficult topics no longer come across as vague or unimportant to the biology and environmental science students, but instead frame them in the context of “real-life” problems. In this particular instance, students applied principles of polarity toward the extraction and analysis of important cell-surface components of C. albicans, a medically important fungal pathogen. For many of the topics covered in lecture, students had a hands-on frame of reference they could turn to. Rather than try to recall random textbook examples that illustrate a particular concept, students could recall an experiment they actually performed, or a set of data they actually generated and interpreted. This undoubtedly improves retention of information and helps students feel engaged and invested in the subject matter. Hands-on learning could arguably have been achieved by incorporating experiments designed for undergraduate laboratory courses into a lecture-based course. However, by bringing faculty research into the classroom, the students benefited by being involved in an active original research project in which they participated in the processes of scientific inquiry, experimental design, data collection, and analysis. By involving the students in the choice and development of methodologies, the students were better poised to understand what was occurring on a molecular level during each step of the experimental protocol. Because the students were carrying out original experiments for which the outcome was not already known, they were exposed to the process of troubleshooting experiments and learned to interpret real-world data, which are often more complex than those obtained from standard laboratory manual experiments. The incorporation of poster presentations helped to cement the students’ understanding of the underlying concepts covered by the experiments and showcase their work to the broader university community. Through the presentations, they learned how to effectively communicate ideas to a diverse audience and to field questions that go beyond the material captured on the poster. These abilities are a valuable asset to all students, regardless of their future career paths.

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thank A. Reid and S. Grabelle for critical review of the manuscript.

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REFERENCES

(1) National Research Council. National Science Education Standards; The National Academies Press: Washington, DC, 1996. (2) Major, C. H. Connecting What We Know and What We Do through Problem-Based Learning. AAHE Bull. 1998, 51, 7. (3) Burke, B. A.; Walton, E. Modeling Effective Teaching and Learning in Chemistry. J. Chem. Educ. 2002, 79, 155. (4) Cummins, R. H.; Green, W. J.; Elliott, C. “Prompted” InquiryBased Learning in the Introductory Chemistry Laboratory. J. Chem. Educ. 2004, 81, 239. (5) Yang, S.-P.; Li, C.-C. Using Student-Developed, Inquiry-Based Experiments To Investigate the Contributions of Ca and Mg to Water Hardness. J. Chem. Educ. 2009, 86, 506. (6) Lucas, T.; Rowley, N. M. Enquiry-Based Learning: Experiences of First-Year Chemistry Students Learning Spectroscopy. Chem. Educ. Res. Pract. 2011, 12, 478. (7) Prince, M. J. Does Active Learning Work? A Review of the Research. J. Eng. Educ. 2004, 93 (3), 223. (8) Spencer, J. N. New Approaches to Chemistry Teaching: 2005 George C. Pimentel Award. J. Chem. Educ. 2006, 83, 528. (9) Pursell, D. P. Enhancing Interdisciplinary, Mathematics, and Physical Science in an Undergraduate Life Science Program through Physical Chemistry. CBELife Sci. Educ. 2009, 8, 15. (10) Brenner, K. Fueling Educational Reform: Bio2010Biology for the Future. Cell Biol. Educ. 2003, 2, 85. (11) National Research Council. BIO2010: Transforming Undergraduate Education for Future Research Biologists; The National Academies Press: Washington, DC, 2003. (12) Haidaris, C. G.; Malone, J.; Sherrill, L. A.; Bliss, J. M.; Gaspari, A. A.; Insel, R. A.; Sullivan, M. A. Recombinant Human Antibody Single Chain Variable Fragments Reactive with Candida albicans Surface Antigens. J. Immunol. Methods 2001, 257, 185.

ASSOCIATED CONTENT

* Supporting Information S

Details regarding GC−MS conditions and lipid extraction techniques; results of FAME analysis; example rubric for grading project presentations. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by a Rhode Island Research Alliance Collaborative Grant Award to J.B. and C.R. We would like to 1324

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