Minipcr Sickle Cell Genetics Lab: Diagnosing Baby Marie Answers

Adv Physiol Educ. 2016 Mar; 40(i): 110–115.

"Sickle prison cell anemia: tracking downwards a mutation": an interactive learning laboratory that communicates basic principles of genetics and cellular biology

Kevin Jarrett

¹Center for Community Outreach Development, The University of Alabama at Birmingham, Birmingham, Alabama;

Mary Williams

¹Center for Community Outreach Development, The University of Alabama at Birmingham, Birmingham, Alabama;

Spencer Horn

³Birmingham Metropolis Schools, Birmingham, Alabama

David Radford

^oneCenter for Community Outreach Evolution, The University of Alabama at Birmingham, Birmingham, Alabama;

J. Michael Wyss

¹Heart for Customs Outreach Development, The University of Alabama at Birmingham, Birmingham, Alabama;

ⁱⁱDepartments of Cell, Developmental and Integrative Biology and Medicine, The Academy of Alabama at Birmingham, Birmingham, Alabama; and

Received 2015 Sep 16; Accepted 2015 November iii.

Abstract

"Sickle cell anemia: tracking downward a mutation" is a full-24-hour interval, enquiry-based, biology experience for high school students enrolled in genetics or advanced biology courses. In the experience, students utilise restriction endonuclease digestion, cellulose acetate gel electrophoresis, and microscopy to notice which of three putative patients have the sickle prison cell genotype/phenotype using Dna and blood samples from wild-blazon and transgenic mice that deport a sickle cell mutation. The inquiry-based, problem-solving arroyo facilitates the students' understanding of the basic concepts of genetics and cellular and molecular biology and provides feel with contemporary tools of biotechnology. It also leads to students' appreciation of the causes and consequences of this genetic disease, which is relatively common in individuals of African descent, and increases their understanding of the starting time principles of genetics. This protocol provides optimal learning when led by well-trained facilitators (including the classroom instructor) and carried out in modest groups (6:1 student-to-teacher ratio). This high-quality feel can be offered to a large number of students at a relatively low cost, and it is especially effective in collaboration with a local scientific discipline museum and/or university. Over the past 15 yr, >12,000 students have completed this inquiry-based learning experience and demonstrated a consequent, substantial increase in their understanding of the disease and genetics in full general.

Keywords: sickle cell, restriction digest, cellulose acetate, genetics, inquiry-based learning

genetic variation and natural choice are known to lead to both benefits and deleterious effects on organisms. In this activity, students explore a genetic disease from the level of DNA to poly peptide to cell to phenotype using contemporary molecular biology tools and techniques such equally restriction endonucleases, DNA agarose electrophoresis, cellulose acetate electrophoresis, and microscopy. Combined with classroom discussion, this intensive experience provides students with an opportunity to integrate scientific discipline content noesis via a hands-on experience. This laboratory is one of four day-long laboratory experiences in molecular biology that are part of The University of Alabama at Birmingham Heart for Customs OutReach Development's GENEius program hosted at McWane Science Middle. As an out-of-form/in-research laboratory experience, students employ cutting-border biotechnology tools, gain an agreement of the concepts underlying the phenotypic expression of a genetic affliction, and take an opportunity to integrate their content knowledge with hands-on experience. Furthermore, the laboratory is modifiable, so that it can be offered in kit form and conducted in a schoolhouse laboratory.

Groundwork

The Us has a bang-up need for a science workforce for the 21st century, peculiarly in the area of biomedical sciences (12a, 12b); yet, relatively few students are strongly engaged in avant-garde scientific discipline courses in precollege. This lack of engagement is especially astute amongst minority students (9, 12a, 12b, 12c). Thus, many colleges are collaborating with precollege education to discover ways to connect these students with engaging experiences that teach the first principles of science in a fashion that excites them. Ane of the challenges in secondary science didactics is finding interactive lesson plans that teach important principles that are cost and fourth dimension effective and appoint students in the learning process. This sickle prison cell anemia laboratory has proven to be an engaging physiology lesson plan that fulfills all of these goals, especially for students of African descent, almost all of whom know someone who suffers from sickle cell anemia.

Sickle cell anemia is a puzzler, a affliction that protects from 1 disease (malaria) but eventually kills the "saved" patient. Malaria is one of the near common infectious diseases worldwide, with ∼500,000 new cases each year (14). Nearly malaria infections occur in the equatorial regions of the world (primarily in Africa and Asia), simply it is rare in the United states, with only ∼1,000 new cases per year. Worldwide, malaria causes severe symptoms in many patients and leads to ∼1 expiry every xxx s (4). Plasmodium parasites (primarily P. falciparum just as well P. vivax, P. ovale, P. malariae, and P. knowlesi) cause malaria, which is transmitted to people by female Anopheles mosquitoes (the infectious vector, which is the only mosquito species that can transmit the disease). The musquito initially becomes infected by obtaining a claret repast containing microscopic malaria parasites from an infected person. When the mosquito accept its adjacent blood repast (∼1 wk afterward), the parasites are injected via the mosquito's saliva into some other person. These parasites multiply inside crimson blood cells, causing symptoms that include anemia, lightheadedness, shortness of breath, tachycardia, fever, chills, nausea, and, potentially, blackout and death. Malaria transmission tin be reduced by preventing mosquito bites using physical or chemical barriers or by decision-making mosquitoes via insecticide spraying and by draining continuing water where mosquitoes lay their eggs. Pharmaceutical treatments tin decrease the severity and frequency of malaria symptoms, but they do non prevent or cure the disease. In contrast, new genetic treatments may provide methods to prevent or even "cure" the affliction, a topic that may provide an excellent opportunity for extending the day-long sickle cell experience.

Hemoglobin A is the about common course of man hemoglobin. Sickle cell disease is a genetic disorder in which cherry blood cells incorporate hemoglobin S (Due south represents sickle). In contrast to hemoglobin A, hemoglobin S shortens the life of cherry blood cells (from ∼120 days to no more than than twenty days, ordinarily ∼xvi days) and causes the cells to become rigid and crescent shaped rather than round. The strong, sickle-shaped ruby-red blood cells block small blood vessels, leading to reduced blood flow and, ultimately, to damage of the tissue and organs normally perfused by that vessel. Individuals with the sickle cell trait produce both normal and abnormal hemoglobin. These individuals are heterozygous for the sickle cell cistron but practise non conduct as severe clinical symptoms as homozygous individuals with the sickle cell gene.

In contrast to its adverse effects on cardiovascular health, sickle cell disease has benign effects relative to another disease, malaria, an infection that significantly lowers cytoplasm pH (increasing acidity), causing hemoglobin to release oxygen and thereby slowing the transit of red blood cells through capillaries. Sickling of the red blood cells disrupts the proliferation of the Plasmodium parasites, in part by depleting cellular K⁺, which is required for the parasite to abound. This results in the rapid expiry of Plasmodium parasites. Fifty-fifty if non all parasites dice, sickling increases malaria resistance by reducing the rate of Plasmodium proliferation, thus providing the immune organization with time to mount a significant response to the parasite, greatly reducing the agin consequences of the infection (one, 13).

Sickle prison cell illness is an autosomal recessive disorder. Approximately 8.3% of African-Americans are heterozygous for the sickle cell gene (known every bit the sickle cell trait), only they practise not develop anemia or clinical signs of the disease. In dissimilarity, ∼0.1% of African-American newborns are homozygous for the sickle jail cell factor, leading to a high rate of morbidity and bloodshed (v). A unmarried base or signal mutation in the gene encoding hemoglobin leads to sickle jail cell disease. This affects the primary ruby claret jail cell protein that is necessary for the transport of oxygen from the lungs to tissues. The hemoglobin molecule is a tetramer equanimous of ii α-subunits and two β-subunits. A single Dna base change, from adenine to thymine, leads to the substitution of valine for glutamic acid in the sixth position on the β-subunits (globin chain), thus altering the net charge and conformation of the sickle hemoglobin protein, causing deoxygenated hemoglobin to class insoluble polymers. The polymerization of sickle jail cell hemoglobin is responsible for the feature distorted shape of sickle cells and the severe symptoms of the disease (ten).

While sickle cell disease protects against malaria, information technology did not develop as a directly response to malaria; information technology occurred by chance. People living in areas where malaria epidemics were common were at an increased risk of malaria, and with little or no medical intendance, the astringent fever, enlarged spleen, and anemia led to rapid expiry, particularly amongst children. The relatively minor number of individuals with homozygous sickle cell disease (SS) typically died early in life due to complications of sickle cell anemia. In contrast, fourscore times more individuals were heterozygous carriers of the disease (Every bit), and they were protected from malaria and, therefore, could pass the genes on to their children.

Learning Objectives

Afterward finishing the action and associated discussions, students should be able to:

• 1. Describe the basic genetic cause(s) of sickle cell disease, a disease that is relatively common.

• 2. Define the consequences of carrying a single or double copy of a recessive gene.

• 3. Develop an understanding of how a pocket-sized genetic alter results in a major alter in the phenotype of an private.

• 4. Employ biology, chemical science, and physics concepts to assess the crusade of a common disease.

• v. Assess the importance of natural selection in leading to gene changes that do good the organism in some environments only not in other environments.

Activity Level

This activity is suitable for loftier school and early college students who have a bones understanding of genetics. Because electrophoresis, brake digest, and pipetting techniques are novel for the target audition, additional time is needed to introduce these concepts. Teachers may find it helpful to carry out a micropipetting exercise exercise before the laboratory experience.

Prerequisite Student Knowledge or Skills

Before engaging in this action, students should have a basic understanding of the get-go principles of molecular biology, such as the primal dogma. Before the laboratory, a pipetting practice do and a basic understanding of the use of a microscope will prove helpful.

Time Required

The preassessment and lecture take ∼40 min. All components of the pupil laboratory experience accept 4–five h.

METHODS

Equipment and Supplies

The protocol requires filter purified h2o, 10× buffer E, restriction enzyme Bsu36I, BSA, and the advisable enzyme buffer. Reagents can exist purchased from Promega (www.promega.com). Cellulose acetate paper can be purchased from Sigma-Aldrich (ww.sigmaaldrich.com). SuperZ applicator and sample well plates can be ordered from Helena Laboratories (http://www.helena.com/catalog/sampleapplication.htm). six× Dna loading dye can be prepared using 50% glycerol and 0.4% bromophenol blue. 50× Tris-acetate-EDTA (TAE) and Tris-EDTA-borate (TEB) buffer tin be diluted to l× concentrations; 1.5% agarose gels for electrophoresis tin can be prepared by mixing agarose and l× TAE buffer and heating it in a microwave oven to melt the agarose. Ethidium bromide [EtBr; Fischer Scientific (www.fischersci.com)] stock solution (10 mg/ml) is added to melted agarose upon cooling (final: 0.01 mg/ml). The agarose-ethidium mix is poured into an agarose gel well tray. Finally, Deoxyribonucleic acid, whole blood, and hemolysates can be obtained from transgenic mice past contacting Dr. Tim Townes (ude.bau@senwott).

Instructions

The protocol for "Sickle cell anemia: tracking downward a mutation" has three chief steps (run into Supplemental Material, appendix 1): Deoxyribonucleic acid restriction digest, protein analysis, and microscopy. ¹

Deoxyribonucleic acid restriction digest.

Students working in small groups are provided with mock patient samples labeled Ten, Y, and Z, representing AA, AS, or SS genotypes; students are blinded to the identity of the samples. Each student squad assigns members to exist the control and experimental samples. The control educatee labels their microcentrifuge tubes by writing the letter of their patient and "u," significant uncut DNA, due east.g., Xu is uncut X. The experimental group, which will add the brake enzyme Bsu36I, writes their patient letter and "c," meaning that DNA is cut with the restriction enzyme, eastward.thou., Xc is cut 10. The brake assimilate solution is mixed into Xc, and an agarose gel is prepared. To begin digestion, 20 μl filter purified water, four μl of 10× buffer East, 4 μl BSA, 10 μl unknown DNA, and two μl Bsu36I should exist added to a microcentrifuge tube. The tube is placed in a microcentrifuge and pulsed for x s to mix the reagents. The tubes are incubated in a 37°C h2o bath for 2 h to allow digestion of the Deoxyribonucleic acid, and gel electrophoresis is and so performed. To view digested DNA by agarose gel electrophoresis, agarose gel electrophoresis of the digested Dna is carried out equally previously described (12). Four microliters of 6× loading dye are added to twenty μl of the restriction assimilate, and this is loaded onto a 1.5% agarose gel prepared using 1× TAE buffer. Because the agarose gel contains loftier concentrations of EtBr, a suspected carcinogen, protective gloves should exist worn and farthermost caution should be taken when handling the gel once electrophoresis is completed. The gel is place on an ultraviolet light source for visualization of the DNA bands, and photomicrographic images can exist taken of the gels to enable students to analyze the results and draw conclusions.

Hemoglobin protein analysis by cellulose acetate electrophoresis.

TEB buffer (75 ml) is poured in each side of the electrophoresis chamber, leaving the middle chamber empty, and 8.0 μl of each hemolysate sample is placed into the wells on the sample well plate. Hemolysates are blotted onto celluloses acetate paper and run at 200 V for 40 min. The gels are stained with amido black and decolorize with destaining solution (47.5% methanol and 5% acetic acid). Protocols for buffers, stains, and reagents tin can exist constitute at Lab-Manual.com (11).

Microscopic observation of blood-red blood cells.

Students should gear up a moisture mount using ane or two μl of transgenic (AA, AS, or SS) mouse claret (diluted 1:one with PBS) onto the center of the slide. A coverslip is placed on elevation of the blood. A tissue is then placed on top of the coverslip and pressed gently with a thumb. Using a ×100 oil-immersion lens, students can find the blood smear throughout the solar day to observe any changes in cell shape, size, or structure.

Troubleshooting

Because students work in minor groups with a facilitator, experimental results tend to be anticipated; however, students' lack of experience and the multistep protocols can event in less than optimal results. Common examples include the digested DNA sample appearing as a smear. This may result from the gel beingness punctured during loading or nuclease contamination. Another commonly obtained effect is the unexpected digestion of samples that should not be cutting, which is like due to cross contamination during the student'southward pipetting of the enzyme.

Safety Considerations

When using a microwave to liquefy gels, students should wear insulated gloves and let the gel cool (∼65°C) before adding EtBr. Only the instructors should handle EtBr while using advisable precautions. Students should wear eye protection and gloves when visualizing the agarose gels under ultraviolet light. The teacher may choose to apply culling dye for EtBr, eastward.g., alternative gel systems, such as the MiniOne Organisation (http://theminione.com/), which would allow students to runway their DNA bands migration, in real fourth dimension, without the use of EtBr or other chancy dyes. Upon completion of the protocol, gels and any other contaminated materials should exist properly tending every bit hazardous waste.

RESULTS

DNA digestion and electrophoresis demonstrate the phenotype of the DNA of patients Ten, Y, and Z, thereby allowing students to determine the correct genotypes for each mock patient sample. Typical results of the gel electrophoresis for each "patient's" Dna, both control (sample X uncut) and experimental (sample X cutting), are shown in Fig. 1. Students should be able to determine the right genotype because the restriction enzyme Bsu36I cuts normal DNA at the specific sequence CC$\widehat{T}$NAGG, resulting in 331- and 200-bp fragments of the 531-bp gene. Normal (A) DNA is cutting once by the restriction enzyme, but the mutated sickle (S) DNA is non cut. Thus, students will notice that the sample of patient X has two bands at 331 and 200 bp and that patient X has a normal homozygous AA genotype. Samples from patient Y yield iii bands at 331, 200, and 531 bp, suggesting that both normal and sickle Deoxyribonucleic acid are present. Students will conclude that patient Y is a heterozygous carrier of sickle cell anemia (genotype AS). A single ∼531-bp band volition exist apparent in the samples from patient Z, because the Deoxyribonucleic acid was non cut by the enzyme, indicating a homozygous genotype (SS). The success rate for this experiment is loftier, ∼80%, but the experiments that neglect can be as valuable since they can lead to a discussion focused on exploring why the failure occurred, thus potentially stimulating even deeper learning.

A: atypical agarose gel photo micrographic image of digested DNA from normal, homozygous, and heterozygous sickle cell samples. See B for farther details. Lane one, 100-bp DNA marking; lane 2, empty; lane iii, sample X uncut; lane iv, sample X cut; lane 5, sample Y uncut; lane 6, sample Y cut; lane 7, sample Z uncut; lane viii, sample Z cut. B: a sit-in mockup of ideal results of the sickle cell agarose gel electrophoresis in the experience. The green fill demonstrates the slow migration of the uncut Deoxyribonucleic acid. In control (unaffected) individuals, the DNA is cut by the restriction enzyme (Bsu36I) and splits into two bands (blue). The lack of an enzyme site in the homozygous sickle prison cell patient blocks the power of Bsu36I to cut the Dna, and, therefore, only one green band appears. In heterozygous carriers, both the uncut and cut band appear, indicating that the private has both unaffected (green) and affected (bluish) Dna. Lane one, 100-bp Deoxyribonucleic acid marker, lane 2, empty; lane 3, sample 10 uncut; lane 4, sample 10 cut; lane 5, sample Y uncut; lane vi, sample Y cut; lane 7, sample Z uncut; lane 8, sample Z cut.

For the protein analysis portion of the study, students use patient hemolysate samples (samples A–C) to make up one's mind the course of hemoglobin in these "patients." Cellulose acetate electrophoresis of lysates from red blood cells demonstrates that the glutamate to valine exchange, which occurs in sickle cell, changes the electrostatic charge of hemoglobin. Glutamate has a carboxyl (COOH) group that gives the normal β-globin poly peptide a negative charge. In contrast, valine has an isopropyl [CH(CH₃)₂] grouping, which carries a neutral charge. This difference can be detected on the cellulose acetate, since the protein containing the glutamate residues volition travel farther toward the positive pole of the gel than the protein containing the valine residues. The neutral or less negatively charged hemoglobin S does not drift a meaning distance away from the origin. Sample A will result in a single band close to the positive end, indicating that the sample contains normal β-globin protein and that the patient'due south genotype is AA. Sample B volition have two protein bands, indicating that the patient has an Equally genotype and is a carrier of the sickle cell factor. Sample C volition present a unmarried band that is relatively close to the negative pole (compared with sample A), indicating that this patient is homozygous for sickle jail cell disease (SS).

Misconceptions

Misconception ane.

Students ofttimes believe that sickle cell is a catching disease, because it has some relation to malaria. They larn in this exercise that sickle cell disease is a genetic disease, inherited from parents, and that is not transmitted from 1 organism to another.

Misconception 2.

Students also often believe that all sickle cell patients die in babyhood from organ failure, infection, and other complications. Participating in the sickle jail cell feel includes becoming informed well-nigh new treatments that allow sickle cell patients to alive into their 50s and beyond (7).

Misconception 3.

Students are often unaware that there are effective treatments and cures for genetic diseases like sickle jail cell affliction. They larn about new genetic approaches, combined with bone marrow transplantations, that have led to new opportunities to treat and even potentially cure sickle jail cell disease (8).

Misconception four.

Many call up that because they are not African-American, they do not need to worry about this disease. Sickle cell disease tends to preferentially affect certain ethnicities, considering being a carrier of unusual hemoglobin may help protect against malaria in childhood. Thus, in places where malaria has been widespread, the genes became more "advantageous." These areas include the Mediterranean region, sub-Saharan Africa, the Caribbean area, the Middle East, and Asia. Today, both migration and mixed parentage mean that nosotros alive in increasingly diverse communities, and, thus, almost anyone could be affected.

Misconception five.

Some students call back that if a person is a carrier, their condition is non something they need to know, peculiarly as a young person, east.k., because it complicates the way of childbearing. In this disease, like others, it is of import for parents and other relatives to exist open with each other and their children about their health status, including genetic disorders. Beingness a carrier of unusual hemoglobin does not require whatsoever treatment, only understanding the risks volition help individuals make informed choices.

Misconception 6.

Other students have the misconception that beingness a carrier means the private is fully protected from malaria, a cracking advantage in regions that have high malaria rates. Being a carrier of the sickle prison cell gene protects somewhat against malaria, especially during babyhood; notwithstanding, people who are carriers tin still contract malaria. Furthermore, the reward comes at a considerable potential cost for their children.

Assessment of Student Agreement

Pre- and posttests, using a clicker system (www.einstruction.com), are used to assess students' learning of the concepts underlying the sickle cell disease experience. The pretest is designed to assess students' prior knowledge of genetics, biotechnology, and the primal dogma of molecular biology (see Fig. 2 and Supplemental Material, appendix 2). After the preassessment, one of the GENEius plan facilitators presents a fifteen-min PowerPoint introduction to sickle cell disease. This presentation includes a review of the genetic and molecular ground for sickle prison cell illness and clinical features, prevalence, diagnosis, and treatment of sickle cell illness. The introduction also includes a description of the laboratory technique that the students will carry out in the laboratory besides equally descriptions/representative examples of predicted results and an interpretation and analysis of the results. PowerPoint (run into Supplemental Cloth, appendix 3) is used for the presentation since information technology easily provides animations, internet movies, and video clips, which capture and maintain student attention.

Pre- and posttest scores (percent right) for high school students taking this module of GENEius during the 2004–2011 school years (n = 18,128). See the test instrument in the Supplemental Fabric appendix 2.

After the introduction, each group of students (maximum of 6 students) is assigned a facilitator (typically an undergraduate, graduate, or postdoctoral trainee; however, peer students who accept previously conducted the laboratory can besides exist outstanding facilitators, especially if the project is done in a school setting). The facilitators assist the students in conducting the experimental procedure and help them to understand the scientific concepts underlying the feel.

At the end of the experiments, students are given a posttest (Supplemental Material, appendix 2) to evaluate their understanding and knowledge gained from the laboratory experience. Students answer to basic questions regarding the experiment and the biotechnology used. Later the posttest, students appoint in a competitive and interactive "game" in which questions are asked well-nigh all aspects of the feel. Students savor the game, which challenges their noesis and understanding of concepts related to genetics in general and more specifically to the techniques with which they have simply gained experience. For this action, students are divided into groups of six, with each grouping having a spokesperson that provides the answer that the group decides on. Scores are kept, and, at the conclusion of the game, the group with the near points wins. In the instance of a tie, students must name and describe the expected results for the two types of electrophoresis used in the laboratory experiment.

Inquiry Applications

Enquiry application ane.

While this action could be carried out in a cookbook fashion, the feel is enhanced by the teacher'due south encouraging students to conduct out discovery-based learning and move beyond the experiment. It is possible that some groups' experiments will neglect to yield the intended results. From these failures, students are encouraged to learn the basis for the failure, exercising their troubleshooting and critical thinking skills. From these "failures," they may realize that the deeper interrogation, used to account for the failure, may lead to a more thorough understanding of a technique, a biological process, and a disease.

Enquiry application two.

Involvement generated by the feel will probable pb to the students' desire to learn more than most sickle cell. Teachers may encourage students to research journal articles about sickle prison cell illness, thereby realizing the potential of recently adult therapies to alter the form of the illness. Their literature exploration should be discussed inside working groups that somewhen report their findings to the unabridged form, thus promoting team and presentation skills.

Wider Educational Applications

In that location are many concepts central to the sickle jail cell disease experience that may exist extended and transferred to other related topics, for example:

• 1. Students may be encouraged to compare and dissimilarity the consequences of genetic mutations in sickle prison cell disease with those from other mutual genetic diseases.

• 2. Students may be encouraged to consider if a genetic variant always carries some benefit to the organism begetting it. For example, is at that place a benefit to having one copy of the cystic fibrosis gene or having Huntington's disease? Many other examples may be probed.

• 3. How might heart colour or mitochondrial differences convey advantages to some bearing those genetic traits?

• four. Students may also explore the ethical considerations related to how and when genetic information should be shared with individuals, in particular, with children. What is the consequence of non conveying this information to children, and why is information technology beneficial to acquire more than virtually one's genetic makeup?

• five. The progress in genetic treatments for sickle prison cell affliction is moving very rapidly and, in many means, more than apace than in other areas of genetic affliction prevention. This is largely due to the availability of fauna models of the disease; students may be reminded that they used blood obtained from animal models in their experiments. This may lead to a discussion of the use of animals in research, peculiarly for genetic diseases and other biomedical studies.

GRANTS

This piece of work was funded in part past National Institutes of Health Scientific discipline Teaching Partnership Honor Grants RR-022745 and OD-016490 and a grant from the United States Department of Education through the Alabama Commission on Higher Education.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: K.J., M.W., D.R., and J.M.W. formulation and design of research; 1000.J., M.W., S.H., and D.R. performed experiments; K.J. analyzed data; Chiliad.J. interpreted results of experiments; Thousand.J. prepared figures; Thousand.J., South.H., and J.Yard.W. drafted manuscript; K.J., M.W., D.R., and J.Chiliad.W. edited and revised manuscript; Grand.J., M.W., S.H., and J.M.W. approved terminal version of manuscript.

Supplementary Material

ACKNOWLEDGMENTS

The authors thank the McWane Scientific discipline Heart for the generous support for, and collaboration in, these outreach activities.

Footnotes

^aneSupplemental Material for this commodity is available at the Advances in Physiology Education website.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888518/

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