Initial Publication Date: July 10, 2022 | Reviewed: March 10, 2023

Molecular Parasitology

Swati Agrawal, University of Mary Washington

Location: Virginia 22407

Abstract

Embedding inquiry driven research in undergraduate courses allows integration of core concepts and competencies necessary for scientific thinking and become proficient in lab skills. These are critical skill for undergraduates to be successful in science careers and for admission into graduate school. However, there are only a handful of examples of collaborative CUREs in Biology where students have an opportunity to connect with a network of researchers outside their own institution, and none in the field of parasitology. In Spring 2021, we piloted a mini-CURE where student groups from University of Mary Washington and Georgia State University collaboratively completed research projects as part of a research-intensive course on Molecular Parasitology. The benefits of this approach were immediately obvious as students interacted across institutions, learned from each other's disciplinary expertise while informing their own research with data collected by their collaborators. It provided enrichment to the course by adding networking opportunities as well as cross-disciplinary knowledge sharing. We present here our CURE model as a way for other educators to design and implement similar cross-institutional inter-disciplinary CUREs that can be modified to align with their research expertise.

Student Goals

  1. Gain proficiency in modern molecular, cellular and biochemical lab techniques used in the field of Kinetoplastid research
  2. Independently design novel research projects and effectively contribute to Kinetoplastid research.
  3. Engage in a cross-institutional, cross-disciplinary research experience and gain proficiency in oral and written scientific communication.

Research Goals

  1. Understanding the biological relevance of programmed cell death in Kinetoplastid parasites.
  2. Identification and characterization of the genes in activation and regulation of apoptosis in Kinetoplastid parasites.

Context

University of Mary Washington is a small public liberal arts university. Molecular Parasitology course is one of the research intensive courses offered in the department to provide Biology students an opportunity to complete necessary research experience/capstone experience in the major. Students design projects based on published literature on cell death in Kinetoplastid (a very understudied area), and also data from previous semesters generated by molecular parasitology students at all three institutions. Students learn techniques for parasite culture, microscopy, biochemical assays like MTT and q-PCR techniques. This is a unique course where students get to collaborate with other students and faculty outside the institution and do collaborative research. Throughout the course several external parasitology experts are invited to give research talk, students from all three universities are invited to attend these talks over zoom, ask questions and discuss their research.

The course typically enrolls 16 students, all are senior Biology or Biomedical majors.

Target Audience: Major, Upper Division
CURE Duration: Full Term, Spring

Target Audience: Major, Upper Division
CURE Duration:A full term

CURE Design

The class Kinetoplastida is comprised of many pathogenic parasites, that cause serious human disease such as sleeping sickness (Trypanosoma brucei), Leishmaniasis (Leishmania donovani),  and Chagas disease(Trypanosoma cruzi  ). In 2016, these diseases affected up to 18 million individuals globally and resulted in over 35,000 fatalities. Current treatments for these diseases are not adequate due to the adverse side effects, costs associated with the treatment methods, and rising resistance to drugs. To provide insight into new and better treatments for these deadly diseases, it is essential to study the biology of Kinetoplastids. A non-disease causing Kinetoplastid parasite, Crithidia fasciculata, only infects mosquitoes. C. fasciculata is very similar in the cellular make-up and biochemical processes to the human disease causing Kinetoplastids mentioned above. These qualities of Crithidia fasciculata make it an excellent exploratory model organism that undergraduates can study safely to better understand Kinetoplastid physiology without the risk of becoming infected.

In animals (including humans) apoptosis (programmed cell death) is necessary for normal development, removal of damaged or non-functional cells . Apoptosis acts as a cellular defense mechanism to remove parasite infected or mutated cells such as in cancer. Apoptosis and cell cycle arrest in disease causing parasite (protists) have been proposed as mechanisms to facilitate differentiation in the insect vector and the infected organism (mammalian hosts) as well as control the inflammatory response in the mammalian host. Thus, apoptosis serves as an altruistic mechanism used by the parasite to survive in the host and ensures transmission from one host to the other. However molecular mechanisms underlying apoptosis in Kinetoplastids are poorly understood because they are highly divergent from other eukaryotes. Understanding this pathway can unravel many therapeutic targets for treatment.

A large number of students at the University of Mary Washington (UMW) express an interest in pursuing careers in medicine and/or biomedical research. It is imperative that these students have authentic hand-on research experience beyond typical lab courses that prepare them for career ahead. Working on an important problem using a surrogate for a pathogen allows the collaborative research team (students and professors) to safely explore the biology of these parasites and improve our knowledge to find a treatment for three important pathogenic organisms (C. fasciculata is non-pathogenic but biologically close representative of all pathogenic Kinetoplastids). This serves as a great hook to engage students in authentic research and increases opportunities to use inquiry driven teaching designed method. Students design original research projects in teams that aimed at investigating environmental triggers like nutrient, starvation, temperature changes, osmotic changes, and hypoxia to see which of these serve as signal for apoptosis using cellular, molecular and biochemical assays to better characterize the triggers of apoptosis and expand the current tool-kit for identifying morphological changes such as change in cell shape, motility and metabolic health. Students also analyze gene expression changes using q-PCR analysis in stressed vs non stressed parasites to understand molecular basis of cell death.

Working in teams, while having an opportunity to ask questions to not only instructors but also peers at Albright and Georgia State facilitates improvement of ideas and troubleshooting as well as improving communication skills. I routinely invite 5-6 expert parasitologist to come to class and talk about their research and their career path. These talks are a great opportunity for my students to hear about new and exciting research. They also provide networking opportunities and, in many cases, it helps them in graduate school and job applications. Albright, Georgia State, and University of Mary Washington students stay connected via slack channels and a common Crithidia CURE website.

Understanding the biological relevance of programmed cell death in Kinetoplastid parasites. It has been speculated in previous studies on Leishmania that the programmed cell death in the parasite is a mechanism to avoid hyper-activation of immune reaction from the host. Apoptosis of parasites in the insect vector creates a bottleneck so that the fittest parasites are selected for transmission into the human host. However, it is unclear as to what triggers apoptosis versus cell replication. Research findings and tools developed in this class will help the overall Kinetoplastid field, we expect that after few iterations of the class the data generated will be publishable with student co-authors. The data generated at the end of each semester is documented in the form of research papers written by students, which are saved and shared with students in the following semester across all three universities.

Core Competencies: Analyzing and interpreting data, Constructing explanations (for science) and designing solutions (for engineering), Developing and using models, Planning and carrying out investigations
Nature of Research: Applied Research, Basic Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Understanding the biological relevance of programmed cell death in Kinetoplastid parasites.
Research Goal 2: Identification and characterization of the genes in activation and regulation of apoptosis in Kinetoplastid parasites.


Student Goal 1: Gain proficiency in modern molecular, cellular and biochemical lab techniques used in the field of Kinetoplastid research

Students learn and get practice in tissue culture techniques to culture parasites Crithidia fasciculata

Students learn how to count parasites and generate OD based growth curves

Students practice designing experimental models for treatment of parasites with environmental stressors of choice. (We mostly use heat, PH, osmotic stress, oxidative stress)

Students learn how to use the colorimetric plate reader to do MTT assay to measure metabolic health of stressed vs unstressed parasites.

 

Review literature to gain an understanding of molecular mechanism of apoptosis in all eukaryotic systems.

Review research in other kinetoplastids systems for genes involved in apoptosis and prepare an annotated bibliography.

Generate a list of putative apoptosis pathway genes to be tested for q-PCR analysis.

Explain the principles of q-PCR

Design independent q-PCR experiments for analysis of differential gene expression post- treatment.

 



Student Goal 2: Independently design novel research projects and effectively contribute to Kinetoplastid research.

Read term-papers from previous cohort of students, to understand effect of different environmental stressors on C. fasciculata on ways to assess the morphological and metabolic defects.

Develop an outline of experimental design and hypothesis and represent it using Graphical abstract.

 

 

Navigate Tritryp DB database for bioinformatically curating the apoptosis gene candidates

Collect cellular localization predictions and domain predictions from TriTrypDB

Design SgRNA and constructs for CRISPR_cas9 mediated gene knock out of above genes

 



Student Goal 3: Engage in a cross-institutional, cross-disciplinary research experience and gain proficiency in oral and written scientific communication.

Attend common Journal clubs and external speaker presentations via zoom and engage in discussion sessions after the talks.

Communicate regularly with  partners at GSU and Albright students about new findings and results

Analyze and discuss data together with partners at GSU and Albright in context of each others experiments

 

Prepare and deliver oral or written presentation to  the whole class at UMW and to students and faculty at Albright and GSU.

Summarize q-PCR experiments and bioinformatics findings in research paper for future molecular Parasitology CURE students at UMW and other universities

Suggest potential experimental modifications and troubleshooting methods for future students


Instructional Materials

Syllabus (Microsoft Word 2007 (.docx) 35kB Jul8 22)
Microscopy assignment (Microsoft Word 2007 (.docx) 1.8MB Jul8 22)
Crithidia fasciculata culture guide (Microsoft Word 2007 (.docx) 25kB Jul8 22)

Assessment

Graphical Abstract (Microsoft Word 2007 (.docx) 1.7MB Jul8 22)
Bioinformatics assignment (Microsoft Word 2007 (.docx) 2.6MB Jul8 22)
End of the semester Mini-Report (Microsoft Word 2007 (.docx) 14kB Jul8 22)

Instructional Staffing

I am the only instructor of this CURE. Sometimes I have undergraduate research interns already working on parasite research who help students sett up their experiments or train them on equipment.

Author Experience

Swati Agrawal, University of Mary Washington

Benefits of Course based undergraduate research (CURE) are numerous and well documented. For students at Primarily undergraduate institutions (PUI) these provide high-impact research experiences that can culminate in retention in STEM careers and motivation to pursue graduate level education. They provide opportunities for students to make discoveries, collaborate, engage in meaningful research and develop a sense of ownership of their lab work. For faculty, especially at PUI, these provide tractable models of modern, collaborative science and move toward the complex, interdisciplinary nature of scientific investigation as an effective platform for integrating missions in research and education. I wanted to design a CURE focused on Molecular Parasitology, because protozoan parasites are not widely used as model systems but they have very unique organelles and biology and tools for genetic manipulation are easily abundant.


Advice for Implementation

My goal in this CURE is to be able to provide all students extended opportunities to work in the lab,  so they can learn a technique, master it, and effectively use it to answer a research question. We typically have 6 hours in the week dedicated for this class. Most of the time we spend working in the lab. For four weeks spread through out the semester students have 36-48 hour growth curve experiments so I don't hold regular class during those weeks to compensate for their time spend outside the class.

I also want my students to be aware of common knowledge and concepts in molecular parasitology so I use a lot of iBiology lectures, I invite speakers  who are experts in the field through out the semester and we have journal clubs to get attuned with common ideas and paradigms in the field.

Lastly designing a CURE close to ones own research expertise has numerous benefits. A lot of students who take this class return to do extended research with me, oftentimes this culminates in their capstone experience. However for most students this research experience is meaningful in choosing future careers or jobs.

Iteration

This CURE is a semester long course. Students usually spend the first four week of class learning methods and tools necessary for parasite culture, microscopy, plate reader etc. They also design hypotheses and generate graphical abstracts for their experimental designs during this time, on which they get feedback from me and their peers. This provides an opportunity for them to reflect and modify anything necessary such as controls, concentrations, replicates, etc.

They run two mini-projects for two weeks each. They present their data after the first mini-project and get feedback from the instructor and peers to help them recognize any improvements they can make in the second mini-project. The most important goal for this CURE is for students to be able to design strong experiments with robust controls and replicates so data is reproducible. Usually by the second run of the experiments is stronger, students are more confident in their experimental design using all equipment and data analysis.

At the end of the semester students also reflect on future directions of the project, which is beneficial for the next cohort of students in the course.

Using CURE Data

We have compiled term papers from students and are in the process of uploading it to our website, which is a shared resource between Albright, Georgia State, and UMW. These are unpublished data that are not necessarily ready for submission to scientific journals. Several modifications and iterations of these experiments need to be run by multiple groups of students before we reach that stage. Nonetheless, students who have contributed an idea or data set will be co-authors on any publications.

Resources

1. Bell, Provost, & Bell. 2020. The Evolution of the Malate Dehydrogenase CUREs Community. The FASEB Journal P.G.E.Kennedy, Update on human African trypanosomiasis (sleeping sickness). J. Neurol. 266, 2334–2337 (2019).

2. Dicker, et al., Global, regional, and national age-sex-specific mortality and life expectancy, 1950–2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet 392, 1684–1735 (2018)N. L. Staub, et al., Scaling Up: Adapting a Phage-Hunting Course to Increase Participation of First-Year Students in Research. CBE Life Sci. Educ. 15, ar13 (2016).

3. Alvar, et al., Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLOS ONE 7, e35671 (2012).

4. Kaczanowski, Sajid, & Reece, Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites. Parasit. Vectors 4, 44 (2011).

5. Vision and Change in Undergraduate Biology Education » Final Report (October 10, 2021).T. Beneke, et al.,

6. A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R. Soc. Open Sci. 4, 170095 (2017).

7. Beneke, Gluenz, LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9. Methods Mol. Biol. Clifton NJ 1971, 189–210 (2019).




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