The Science, Technology, Academics, and Research (STAR) program at Ochsner Medical Center was created to stimulate the interest of New Orleans-area high school students, particularly underrepresented minorities, in biomedical research and health-care, with the ultimate goal of encouraging further education in these areas and expanding the local pipeline to careers in these fields. Over the years, Ochsner has provided informal and individualized summer scientific training to high school students, many of whom have gone on to medical school and other areas of scientific education. The first formal internship program was developed through a collaboration between Edward Frohlich, MD, the Alton Ochsner Distinguished Scientist, and Barbara MacPhee, then the Principal of New Orleans Science and Mathematics High School (NOSMHS), and was launched in the summer of 2005. Six students from NOSMHS, under the guidance of Ochsner scientists and physicians, carried out individual research projects over a period of 6 weeks, culminating with a final presentation of their research results.
After a 1-year hiatus due to Hurricane Katrina, the program was reconstituted in summer 2007 as the Summer Science Youth Academy (SSYA), under the directorship of Kaela Barnett, MPA, of Ochsner's Academic Division. SSYA operated for a total of 10 weeks consisting of two overlapping 6-week sessions, each with classes of 5 or 6 students from NOSMHS or Patrick Taylor Science and Technology Academy, respectively. In addition to placements in the research laboratories, the SSYA curriculum was expanded to include lectures, workshops, and rotations focused on issues related to research and knowledge management (library resources, bioinformatics, scientific/medical writing and editing, grant applications, etc.), healthcare (regulatory compliance, patient privacy, ethics, clinical department rotations, and more), and professional development (professional attire, interview and presentation skills, teamwork, networking). Ochsner scientists, physicians, nurses, administrators, and staff served as instructors, mentors, and role models during these sessions.
While still under the administration of the Academic Division, in 2008 the program underwent another series of structural changes, including the acquisition of a new name—STAR—and a reversion to the single 6-week program. Significantly, the STAR program was opened to students from any local high school, and the capacity was increased to a total of 12 participants. Although the lectures, workshops, and rotations of the SSYA curriculum were largely retained, the scientific research portion of the internship was modified so that experiments and demonstrations were primarily carried out in a newly refurbished student learning laboratory, called iLab. The provisioning and operation of iLab was greatly facilitated this year by a generous grant-in-aid from Blue Cross Blue Shield of Louisiana.
Under the guidance of Ochsner scientists, the STAR students carried out a variety of medically relevant, scientific experiments employing cutting-edge technologies in the iLab. Pairs of students were assigned to one (or at most two) of the eight experiments/demonstrations in this year's curriculum and required to present their results in a poster format during the program-ending Culmination Ceremony. In addition, the students, with assistance from their faculty mentor, were encouraged to write and submit an abstract of their research to The Ochsner Journal. These abstracts follow and provide a good description of the type of hands-on, inquiry-based experimental activities in which the STAR students participated.
Alcohol Application For Decolonization of Nasal Carriers of Staphylococcus Aureus
Keywords: methicillin resistant S. aureus, nasal cultures
Ryan Gupta*; Erika Hotard†; George A Pankey, MD‡; Science, Technology, Academics, and Research Program; *Jesuit High School, New Orleans, LA; dagger;>Patrick Taylor Science and Technology Academy, Jefferson, LA; ‡Infectious Diseases Research Laboratory, Ochsner Clinic Foundation, New Orleans, LA
Background: Staphylococcus aureus is a bacteria commonly (30%) colonized in the noses of healthy people. Colonizers of S. aureus are more likely to have minor skin infections (pimples and boils). However, serious S. aureus infections (such as blood stream surgical wounds and pneumonia) may occur. A person colonized with S. aureus, especially methicillin-resistant S. aureus (MRSA), is more at risk of developing an infection if admitted to the hospital. We evaluated the use of alcohol application in the nose of colonizers to determine whether this inexpensive method would affect the presence of S. aureus.
Methods: Twelve high school students participated in this project.
Collecting specimens and inoculating media: Day 1: The left and right nostrils were swabbed separately. Each swab was inoculated onto a sheep blood agar plate and then streaked. A separate 70% isopropyl alcohol prep pad was applied in the left and right nostrils near the tip of the nose. After 12 hours, the nasal alcohol application was repeated. Day 2: The left and right nasal cultures were repeated, and the nasal alcohol application was repeated at 0 and 12 hours. Day 3: The left and right nasal cultures were repeated.
Reading plates/interpreting results: The blood agar plates were examined after 24 and 48 hours incubation. Any white or cream-colored colonies with a zone of beta-hemolysis on the blood agar were subcultured for further identification. The number of colonies was not quantitated. The Staphyloslide® Latex Test (BD, Franklin Lakes, NJ) was used to differentiate S. aureus that possessed clumping factor and/or Protein A from staphylococci that did not possess these properties. S. aureus isolates were then tested to identify MRSA using the Vitek (Biomerieux, Durham, NC) instrument.
Results: S. aureus was cultured from the noses of 5/12 (42%) of the students. In three instances, colonization occurred in only a single nostril; therefore, screening both nostrils is essential. No MRSA was isolated. After nasal alcohol applications were repeated every 12 hours for 2 days, S. aureus was not cultured from the nose of 3/5 (60%) of the students. Minor side effects from the alcohol application included slight burning, experienced by most students, but this did not require discontinuation of the applications. One known asthmatic student, who had an initial negative S. aureus culture, developed an asthma attack approximately 2 hours after the first alcohol application; further applications were discontinued in her case.
Conclusions: Further evaluation of the effect of nasal alcohol application on the colonization of S. aureus using additional subjects is needed to substantiate the encouraging (60%) positive result found with this project.
Aliens Among Us
Keywords: DNA, variable nucleotide tandem repeats
Geralynn Lane*; †Jihye Lim; Michelle Peters; Jawed Alam, PhD‡; Science, Technology, Academics, and Research Program; *Lusher Charter School, New Orleans, LA; †Patrick Taylor Science and Technology Academy, Jefferson, LA; ‡Laboratory of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, LA
Background: While camping in Tickfaw State Park in the summer of 1991, Katherine Naquin was abducted—by extraterrestrials, according to her father, Air Force Captain Jack Naquin. Despite the fact that nobody believed him, Naquin persisted with his story and continued to investigate his daughter's disappearance. His efforts were rewarded years later when the Pentagon confirmed the existence of a crashed alien spaceship to him. Working with a military colleague and data that were collected over several decades by the military, Naquin came to a startling conclusion: The aliens were using Katherine's DNA to mass produce human-like clones to secretly plant them on Earth, in hopes of eventually overtaking the planet and its inhabitants. Furthermore, he believed the aliens probably mutated the genes involved in outward appearance so that none of the clones would look exactly like Katherine and that they were also mixing DNA from a boy abducted at the same time to produce male clones as well. Because of the potential threat to all humankind, identifying the alien clones became a national imperative.1 After Katherine's period of absence was considered, it became apparent that some of the clones would be 16–17 years old. The STAR students were potential aliens and needed to be tested. Given a sample of Katherine's DNA that had been extracted from her baby teeth, the task for the STAR students was to find a safe way to extract their own DNA and then develop a test to determine if one or more of their fellow classmates might be aliens cloned from Katherine's DNA.
Methods: After much discussion and research, the students discovered that buccal cells can be safely and easily recovered from the cheek with saline rinses and enough DNA isolated for analysis. They also learned that human DNA contains regions called variable nucleotide tandem repeats (VNTRs) that are often used in forensic analysis because the length of VNTRs frequently varies among individuals. The students concluded that if a classmate's VNTRs were of the same size as Katherine's, then there was a high probability that that individual was a clone. Two VNTR loci were selected for analysis: DIS80 and D17S30. DNA from buccal cells was isolated by standard procedures. The VNTR loci were amplified by polymerase chain reaction (PCR) and analyzed by agarose gel electrophoresis.
Results and Conclusions: In the gel electrophoresis profile, no amplified DNA was detected for three students at the VNTR loci. This could have been caused by a failure at the DNA isolation step (the absence of DNA or the isolation of poor quality DNA) or a failure of the PCR amplification reaction. Four of the students matched the size and pattern of Katherine's DNA at one locus. Such a high percentage of positive results suggests a potential contamination of the students' DNA with Katherine's, as all students were also given a sample of Katherine's DNA as a control. None of the students had a perfect match with both of Katherine's VNTRs, making it unlikely that any of the STAR students was a clone. Conducting this experiment was an effective way of learning about DNA forensics, VNTRs, DNA isolation, PCR, and gel electrophoresis. It also demonstrated the many problems one can encounter during experimentation and the care needed to perform an experiment successfully.
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CELL SIGNALING AND IMMUNOBLOTTING
Keywords: immunoblotting, signal transduction, molecular weight
Ngoc Pham*; Kayla Rodney†; T Cooper Woods, PhD‡; Science, Technology, Academics, and Research Program; *Patrick Taylor Science and Technology Academy, Jefferson, LA; †Lusher Charter School, New Orleans, LA; ‡Laboratory of Molecular Cardiology, Ochsner Clinic Foundation, New Orleans, LA
Background: The overall goal of this project was to introduce the students to protein phosphorylation, Western blotting, and the use of antibodies in molecular biology through hands-on experimentation combined with in-lab lectures. Furthermore, the experiment was intended to help the students build a connection between changes in signal transduction and diseases such as diabetes. Protein phosphorylation is a signal transduction method that cells use to respond to stimuli. As an educational example, we measured the phosphorylation of extracellular signal-response kinase (ERK) 1/2, which controls cell proliferation in diseases, in two unknown samples using Western blotting. This method combines protein electrophoresis and antibodies to measure the levels of phosphorylated ERK as well as to determine the molecular weight of ERK.
Methods: The students first prepared a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoresed the unknown samples. The proteins were transferred onto a membrane and then incubated in blocking buffer, a primary antibody targeting phosphorylated ERK, and then an appropriate secondary antibody. Once the incubation period was completed, the membrane was soaked in chemiluminescent development solution and imaged with a ChemiDoc (Bio-Rad Laboratories, Hercules, CA) gel imager. Based on the in-lab lectures and the gel image, the students were asked to determine which sample they believed came from a diabetic patient and to estimate the molecular weight of ERK.
Results: The students' predictions of the molecular weight of ERK ranged from 10 to 140 kDa. Five students (42%) estimated the molecular weight within 10% of the actual value: 42 kDa. Tissue samples from diabetic patients previously have been shown to exhibit higher levels of ERK phosphorylation than those from nondiabetics. Based on knowledge of this and the fact that the band indicating phosphorylated ERK was clearly darker in sample A than that in sample B, 9 out of 12 students (75%) correctly predicted that sample A represented the diabetic patient.
Conclusion: As 75% of the students were able to correctly identify which sample was from the diabetic patient, we believe our goal of introducing the students to signal transduction was achieved. Though the predictions of molecular weight varied, almost half of the students predicted the value within a reasonable error range. Overall, this suggests that while the explanation of estimating molecular weight from a Western blot could be improved, this experiment successfully introduced the students to Western blotting and signal transduction.
CELLULAR TRAFFICKING OF FLUORESCENT MARKER PROTEINS
Keywords: protein trafficking, fluorescent proteins, deconvolution microscopy
Ryan Gupta*; Erika Hotard†; Dawn DeHaro‡; Julia L Cook, PhD‡; Science, Technology, Academics, and Research Program; *Jesuit High School, New Orleans, LA; †Patrick Taylor Science and Technology Academy, Jefferson, LA; ‡Laboratory of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, LA
Background: The process of intracellular protein movement is essential to the livelihood of a cell and organism. In fact, certain diseases, such as cystic fibrosis and Alzheimer's disease, are caused by failures in this process. Thus, a thorough understanding of cellular compartmentalization and protein trafficking is essential to medical and scientific advancements. In this experiment, six expression vectors, targeted to unique cellular compartments, were provided to students blinded to the sample identities. Each of the six plasmid vectors encodes a fluorescent fusion protein that can be visualized microscopically. Based on the microscopic distribution of the fluorescent marker proteins, the identity of each protein was predicted.
Methods: Transfection: A T150 flask of Chinese hamster ovary (CHO)-K1 cells was trypsinized and replated onto six MatTek (Ashland, MA) dishes (glass bottom culture dishes, 1.5-C, poly-D-lysine coated). Working in pairs, the students performed cell counts and cell replating. Twenty-four hours later, cells were transfected using the commercial reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Imaging: Forty-eight hours post-transfection, students examined the cells using 3D deconvolution microscopy on an Axiovert 200M inverted microscope (Carl Zeiss, Jena, Germany). Images were captured using Slidebook 4.2 (Olympus, Center Valley, PA). Based on the distribution of the fluorescent proteins, groups rationally proposed a target organelle or cellular compartment to which each “unknown” protein was targeted.
Results: The data from the experiments clearly illustrate the wide range of areas to which proteins may traffic in cells, often dependent on small (≤20 amino acids) peptide domains within the larger protein. The region of the cell to which a protein travels is directly dependent on its unique characteristics and cellular function. This series of experiments demonstrated to the students proteins which traveled to the following regions: Golgi bodies, endosomes, microtubules, the endoplasmic reticulum, plasma membranes, and early endosomes.
Conclusions: These studies introduced the students to several concepts, including cell culture, transfection, cell structure and morphology, protein trafficking, fluorescence microscopy, and image capture.
CHEMOTHERAPY AND BONE LOSS
Keywords: cancer, chemotherapy, osteoporosis
Krystin Dees*; Taylor Davis†; Ty Nichols‡; Gregory Tobin§; Ranjitha Katikaneni‡; Tulasi Ponnapakkam, PhD‡; Robert Gensure, MD‡; Science, Technology, Academics, and Research Program; *Lusher Charter School, New Orleans, LA; †New Orleans Science and Mathematics High School, New Orleans, LA; ‡Pediatric Endocrinology Research Laboratory, Ochsner Clinic Foundation, New Orleans, LA; §Jesuit High School, New Orleans, LA
Background: Millions of Americans suffer from cancer, and chemotherapy is often used to kill the rapidly growing cells that cause it. During and after chemotherapy, patients often develop osteoporosis, a disease where there is an increased risk of fractures due to bone loss. In such cases, the osteoporosis could be caused by the chemotherapy treatment itself or by patients being ill and less active. Our hypothesis is that chemotherapy induces osteoporosis.
Methods: We investigated the effects of chemotherapy on the bones of mice for 4 weeks, using two sets of mice: one control group and one chemotherapy group. The chemotherapy group was injected with cyclophosphamide, and the control was injected with buffer every 2 weeks. After 4 weeks, the mice were scanned using dual-energy x-ray absorptiometry, and the scans were analyzed. The results for each group were averaged and tested for statistical significance.
Results: After 4 weeks the average bone mineral density (BMD) for the control group was 60.7 mg/cm2 and the chemotherapy group was 55.6 mg/cm2. This represented a 5.1 mg/cm2 (10%) decrease in BMD after only 4 weeks; this difference was statistically significant (p<0.05).
Conclusions: These results after 4 weeks supported our hypothesis that chemotherapy induces osteoporosis. The study will continue for another 6 weeks, and we expect that BMD will decrease even further and will cause osteoporosis in the mice.
GENE CLONING AND EXPRESSION IN THE DEVELOPMENT OF PHARMACEUTICALS
Keywords: DNA, therapeutic cloning, pharmaceutical research
Ngoc Pham*; Kayla Rodney†; Michelle Peters‡; Jawed Alam, PhD‡; Science, Technology, Academics, and Research Program; *Patrick Taylor Science and Technology Academy, Jefferson, LA; †Lusher Charter School, New Orleans, LA; ‡Laboratory of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, LA
Background: DNA cloning is an important technology that has led to many medical discoveries and advances in medical treatment. For instance, before the development of recombinant DNA technology, insulin for diabetic patients was derived from pigs and cows. The use of nonhuman insulin has some potential disadvantages, e.g., an immune reaction to the animal insulin may cause side effects. Also, there is the possibility of contaminating proteins (such as the prion proteins that cause bovine spongiform encephalopathy, i.e., “mad cow disease”) finding their way into the insulin preparations. Fortunately, recombinant DNA technology has permitted the production of large quantities of human insulin in bacteria.
Objective: We sought to learn about recombinant DNA technology by performing the same type of experiments as those used by the pharmaceutical industry to produce drugs such as insulin.
Methods: Instead of insulin, a red fluorescent protein (RFP) was used in this study because it is visible during various stages of the production process. The purification of the RFP involved the following steps. First, the RFP gene was cut with restriction enzymes and ligated into an expression plasmid. The restriction and ligation reactions were then checked by gel electrophoresis. Next, the recombinant DNA was transformed into bacterial cells and selected by antibiotic resistance. The bacteria cells containing the RFP gene were grown. Finally, protein extracts of the bacterial cells were prepared and the RFP protein purified by column chromatography.1
Results: The restriction and ligation reactions were successful, as determined by the banding patterns of the DNA after gel electrophoresis. The desired recombinant clone was created, as evidenced by the presence of red colonies on the agar plate after transformation. These are the cells that produce the RFP protein. The RFP was successfully purified, as shown by the pink protein sample after chromatography. With the completion of DNA cloning and protein purification, the therapeutic protein of interest was produced.
Conclusions: DNA cloning is a powerful technology that can be used to make useful drugs for some human diseases. Conducting the actual experiments in a laboratory and seeing them succeed made it fun and easy for students to understand the different steps of this technology.
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“OF MICE AND MEN”: METHODS OF HYPERTENSION STUDY IN MICE
Keywords: hypertension, blood pressure, mice
Rachelle Bordlee*; Iesha Spencer†; Kevin Redding‡; Science, Technology, Academics, and Research Program; *St. Mary's Dominican High School, New Orleans, LA; †New Orleans Science and Mathematics High School, New Orleans, LA; ‡Laboratory of Molecular Genetics, Ochsner Clinic Founation, New Orleans, LA
Background: Hypertension, or high blood pressure, is a major problem that can lead to the number one cause of death in the United States: heart disease. Hypertension is an excess of pressure on the artery walls. The average healthy blood pressure for an adult is 120/80 mmHg. Hypertension is caused by many factors, such as obesity, stress, a high salt diet, a lack of exercise, smoking, age, ethnicity, and some medications. Researchers are currently tracing heart health problems in mice to improve treatment in humans.
Methods: The CODA 6 (Kent Scientific, Torrington, CT) is a noninvasive device to measure blood pressure on the tail of a mouse or rat, similar to the sphygmomanometer used on the arm of a human. It is very important that blood pressure is measured while the mouse is active; anesthesia will lower blood pressure, skewing readings. The mouse is restrained in a small cylinder, and a cuff is put on its tail. Wires connect the cuff to a computer, which calculates the systolic and diastolic blood pressure of the mouse. One mouse was measured by tail-cuff (CODA 6) for 3 acclimation cycles and 5 reading cycles that took about 15 minutes.
The telemetry system from Data Sciences International (St. Paul, MN) is a more accurate, yet more costly device. The telemeter consists of a small tube connected to a radio-transmitter device. The tube is surgically inserted into the carotid artery of the mouse, and the transmitter is inserted under the skin. When activated, the radio transmission is picked up and recorded by a computer. The computer records blood pressure at any given interval without human supervision. One mouse measured by telemetry was implanted a week before measurement. The readings were taken for 30 seconds every half hour for 3 days; 2 readings were witnessed.
Results: Blood pressure readings for the CODA 6 were unreadable because the mouse was very anxious. Blood pressure readings for the radio-telemeter were very accurate.
Conclusions: The CODA 6 is noninvasive and less costly than the telemeter, but it cannot guarantee accurate results. Radio-telemeters provide accurate results but are more costly and require a delicate surgery. The choice of a method varies on user preference. To optimize accurate results for both methods, the surrounding atmosphere must be controlled to calm the mouse. For the CODA 6, the mouse is calmed by placing the cylinder on a heated pad, covering the cylinder with a cloth for darkness, and keeping the room still and quiet. A radio-telemeter allows the mouse freedom of movement around its cage, therefore lowering anxiety. These two methods of recording blood pressure in mice are furthering research on hypertension, heart disease, and related disorders.
TESTING FOR GENETICALLY MODIFIED FOODS
Keywords: genetically modified organisms
Sobia Arshad*; Erik Olivo†; Jawed Alam, PhD‡; *Patrick Taylor Science and Technology Academy, Jefferson, LA; †Benjamin Franklin High School, New Orleans, LA; ‡Laboratory of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, LA
Background: There has been a rapid growth in the number of food products in the global market that are derived from genetically modified (GM) crops. As scientists create new technologies and experiment with genetic engineering, for instance, to make crops resistant to disease, temperature extremes, or chemicals such as herbicides, the amount of GM food in our markets will continue to increase. Although GM foods could be one solution to world hunger, there may be health concerns—such as allergies— associated with some GM products. Also, it seems reasonable that the public should know what they are eating and from where it comes. Unfortunately, many people have not been well educated on this subject and, therefore, are not able to make informed decisions regarding their food consumption.
Objective: We sought to learn how to test for the presence of DNA indicative of genetic modification and to conduct this test with common food products. We hypothesized that most of our foods would be derived in part from genetically modified crops.
Methods: The food product was crushed to very small pieces in a plastic bag, and a small sample was transferred to a microcentrifuge tube. The food was dissolved in Edward's buffer, and the cells were lysed by boiling them for 10 minutes. The cell lysate was collected by centrifugation and extracted with a phenol/chloroform mixture. The DNA was precipitated with two volumes of ethanol, collected by centrifugation, and dissolved in water. An aliquot of the DNA samples, along with positive and negative controls, was mixed with reaction buffer and primers in a polymerase chain reaction (PCR) strip. The PCR strip was placed in a thermal cycler, and the DNA was amplified for 32 cycles. The PCR reaction products were analyzed by electrophoresis through a 2% agarose gel.
Results: Of the 10 products tested, four (corn, corn meal, Doritos®, and Fritos®) included GM ingredients, five (corn chips, corn flakes, crackers, popcorn, and sugar) did not, and the test of the tortilla chips was inconclusive.
Conclusions: Genetic engineering allows farmers to mass produce crops that will last longer, grow faster, and so forth, meaning they might someday become a viable solution to ending world hunger. At the same time, some GM foods may lead to health problems. This experiment was conducted to determine whether everyday food items included GM crops. For the most part, the lab experiment went well, but some groups encountered problems that resulted in inconclusive data. Knowledge of the GM status of several common foods will allow these students to make more informed eating decisions.
- Academic Division of Ochsner Clinic Foundation