Capstone WEEK4: ANNOTATED BIBLIOGRAPHY 1) Ye, L., Chang, J. C., Lin, C., Sun, X., Yu, J., & Kan, Y. W. (2009). Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases. Proceedings of the National Academy of Sciences, 106(24), 9826-9830. Ye, Lin, studied about the prenatal diagnosis of hereditary diseases like sickle cell disease and their treat by inducing pluripotent stem cells and gene therapy. Their paper published in 2009 in Proceedings of the National Academy of Sciences. The development of reprogramming somatic cells to induced pluripotent stem cells gives a conceivable new way to treat sickle cell sickness. Induced pluripotent stem (iPS) cells can be produced using these patients' somatic cells and the change in the beta-globin gene rectified by gene targeting, and the cells developed into hematopoietic cells to be come back to the patient. 2) Pearson, E. G., & Flake, A. W. (2013, February). Stem cell and genetic therapies for the fetus. In Seminars in pediatric surgery (Vol. 22, No. 1, pp. 56-61). WB Saunders. Pearson, E. G., & Flake, A. W. describes about the stem cell and gene therapy for the fetus in their paper. Both of these technologies are very important for the treatment of sickle cell disease. The pre-birth determination and management of congenital sickness has gained huge ground over the earlier decade. At present, fetal treatment gives remedial choices to a scope of inborn disorders like sickle cell anemia; however, it is confined to the treatment of fetal pathophysiology. Betterment in pre-birth screening and the early finding of hereditary diseases like sickle cell anemia take into account preemptive treatment of foreseen postnatal infection by gene therapy and stem cell therapy. 3) Sebastiano, V., Maeder, M. L., Angstman, J. F., Haddad, B., Khayter, C., Yeo, D. T. & Joung, J. K. (2011). In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells, 29(11), 1717-1726. Sebastiano, V. explains about the benefits of zinc fingers in human gene correction in sickle cell disease. Zinc fingers are the modern technology used in the correction of defective gene in sickle cell disease. Recently, engineered zinc finger nucleases (ZFNs) have been appeared to generously expand HR frequencies in human iPSCs (induced pleuripotent stem cell), raising the possibility of utilizing this innovation to cure disease-causing mutations. Here, they depict the generation of iPSC lines (induced pleuripotent stem cell) from sickle cell anemia patients and in situ treatment of the disease bringing on change utilizing three ZFN pairs made by the freely accessible oligomerized pool engineering method. Posted on: Wednesday, November 4, 2015 12:20:59 PM PST These are the expectations for each week of the course from you. All MILESTONES are DUE the Thursday of the indicated week by 11:59pm. Late work will be penalized by 1 point per day until Sunday at 11:59pm then 5 points each day thereafter (in other words for Milestones 1-3 if it is later than Wednesday of the following week you will not receive any points - insert sad face) DUE Week 1 – No milestone - form groups, pick a topic, decide on research strategy and divide tasks among group members. (Experimental: Introduction, Methods, Results, and Discussion OR Grant Proposal: Introduction, Preliminary study, Specific Aim 1, and Specific Aim 2) DUE Week 2 – Milestone 1. A 1-page introduction of your topic and what you are going to research (problem statement ) in APA format, including dependent and independent variables, plus a bibliography, with each member contributing a minimum of 2 articles from each task for a total of (about) 8-10 references in APA. This is what I'm looking for: (1) Title page (with running head) and group names, (2) Introductory page(s), problem statement with variables, and (3) References all in APA. NOTE: EACH person MUST turn in a copy of the group work in order to receive a grade. DUE Week 3 – Milestone 2. (1) Outline of your research (e.g. a literature review) and (2) how the research you have uncovered will aid you in achieving your research goals. This should be individual per student per task. If your task is results, then focus on the results outline, if you are doing the introduction focus on that. Please continue to use your APA format paper from Milestone 1 for this outline. You MUST work together to form a cohesive outline (Milestone 4), but the effort must be individual. NOTE: EACH person MUST turn in individual work in order to receive a grade. DUE Week 4 – Milestone 3. Annotated bibliography as per the sample uploaded to doc sharing, and each group member must have a minimum of 3-5 references in their annotated bibliography. Again focus on your task and gear your research towards achieving your task goal. You MUST collaborate, but the uploaded work must be individual. NOTE: EACH person MUST turn in individual work in order to receive a grade. DUE Week 5 – Milestone 4. A DETAILED Outline of YOUR section of the paper (minimum of 4 pages) and will be turned in individually per student (and section). Please view the sample outline in doc sharing prior to turning in your document. Again it should be in your APA formatted paper from milestone 2. NOTE: EACH person MUST turn in individual work in order to receive a grade. DUE Week 6 – Milestone 5. Rough draft of sections (section papers will be individually written) in APA format. NOTE: EACH person MUST turn in individual work in order to receive a grade. DUE Week 7 – Milestone 6. Oral defense Rough Draft and sample presentation (in PPT done as a group) NOTE: EACH person MUST turn in a copy of the group work in order to receive a grade. Final Paper: DUE Week 8: Cohesive compilation of the paper (minimum 40 pages) in APA format printed out and bound (staple, folder, or wire-bound) NOTE: only 1 printed paper needed. Oral Defense: In class Week 9. WOOOOOOOOOOOOOOOOOOOO you are done! NOTE: EACH person MUST contribute to the oral defense and be able to answer questions in order to receive a passing grade. Topic: Stem cell gene therapy on fetal sickle disease. 1 page: Outline 2 Page: bibliography – a summary of what the paper is about. Papers that are talking about material and methods. (do research on it). Materials and methods: (zinc fingers) Explain the process, how its done. Step by step. (Break it down) The stages of the fetus: which one would be the best or least dangerous for the procedure to be done. What months for example, 3-5 months??? . You maybe need to also get done an ultrasound to see the baby and if it moves. To be able to inject the baby with zinc fingers to prevent sickle cell. Sickle cell used with zinc fingers. The risk on procedure. Pros and cons on zinc fingers Parents need to sign consent to approve this study. Test it on about 10-20 people, why so low the population because it a new study! ☺ How we test for the parents. Randomly pick people. The whole needle procedure! Graph/pictures. I also added this YouTube video: https://www.youtube.com/watch?v=f3pjyQvBdz0 Capstone 5/8/15 Methods: Detailed Outline Fucoidan consumption associated with slowing the progression of breast cancer I. Participants A. Clinical single blinded study was approved to study the effects of seaweed consumption on breast cancer patients i. The Human Subjects Research and Review Board of West Coast University and Material Command (Los Angeles, California) ii. The Jackson Memorial Hospital (Miami, Florida) iii. West Coast University Institutional Review Boards iv. IRB number, 91-027 v. Clinical registration number WCU4166852 B. 18 diagnosed female breast cancer patients recently diagnosed in stages 3 and 4 referred to our clinical trial i. 9/18 had stage 3 breast cancer ii. 9/18 had stage 4 breast cancer II. C. Control group consisted of 10 female breast cancer patients in any stage of cancer D. Inclusion Criteria i. Breast cancer patients between the ages of 30-70 years old ii. Participants must have no allergies to seaweed, shellfish, iodine, or soy iii. Participants must not have been on hormone replacement therapy, radiation therapy, or chemotherapy within the previous six months of the study and no history of cancer (other than breast cancer) within the past twenty years iv. Participants must not have diabetes or gastrointestinal disorders v. Alcohol intake limited to less than one per week vi. No oral antibiotics taken within the last three months vii. Women agree to consume their normal diet, avoiding seaweeds and to continue their habitual use of medications, chemotherapy, vitamins and supplements during the study Description of breast cancer stage under investigation A. Stage 3 Breast Cancer i. Cancer extends beyond immediate region of the tumor ii. Invades nearby lymph nodes and muscles but has not spread to vital organs iii. Divided into three groups based on the size of the tumor: Stage 3A, 3B, and 3C iv. Stage 3A falls under three distinct descriptions 1 v. vi. B. III. a. Tumor is less than 2cm and has spread to 4-9 lymph nodes b. Tumor is larger than 5cm and cancer clusters found in lymph nodes c. Tumor is larger than 5cm and has spread to lymph nodes near the breastbone or underarm Stage 3B description a. Tumor size is variable and can be any size b. Cancer has spread to the chest wall or breast skin c. Cancer has spread to lymph nodes d. Evidence of swelling, inflammation or ulcers is visible e. Cancer has invaded at least 9 lymph nodes Stage 3C description a. No tumor or a tumor of variable size and cancer has invaded at least 10 or more lymph nodes b. No tumor or a tumor of variable size and cancer has spread to lymph nodes near collar bone c. No tumor or tumor of variable size and cancer has spread to lymph nodes near underarm or breastbone Stage 4 i. Cancer spread to other parts of the body, such as the brain, bones, lung and liver ii. This stage is considered incurable Study Design A. Clinical study spanned a total of 5 years from April 2015-April 2020 B. The ten control group breast cancer participants ingested a sugar placebo pill as their medication C. The remaining 18 breast cancer patients ingested 5 grams per day for a total of 10 500 mg capsules i. Seaweed capsules were obtained from a manufacturer based in Okinawa, Japan IV. Health and Mortality Surveillance A. Pre-evaluation tests were taken prior to the start of treatment to establish each participant’s baseline values i. Colony formation, MTT assay and xenograft to determine breast cancer cell numbers (EXPLAIN) ii. Blood tests- prior confirmatory results that diagnosed breast cancer patients a. Hormone receptor test- tests if the breast cancer cells have receptors for the hormone estrogen and progesterone b. HER2/neu test (ERBB2 test)- known as the human epidermal growth factor receptor 2 proto-oncogene. This is usually overexpressed and amplified in invasive breast tumor tissue 2 iii. Mammogram a. X-ray picture of the breast b. Used to check breast cancer who show no signs or symptoms of the disease c. Used to check for breast cancer progression d. Description of Procedure- breasts are compressed between two firm surfaces to spread out the breast tissue. The x-ray takes black and white images of the breast B. Daily seaweed capsule ingestion at 5 grams per day (ten 500 mg capsules per day) C. Every 6 months post treatment, colony formation, MTT assay, xenograft and cell number evaluation was done to assess improvement or no improvement in breast cancer tissue and cells i. Explain colony formation ii. Explain MTT assay iii. Above tests will determine cell numbers of breast tissue seen D. Every 3 months/90 days, CT scans were done to check for tumor growth i. Explain CT scan E. Monthly journal log that documented food intake, exercise/fitness routines and mood. This journal was given to case manager for review and discussion at monthly check up. F. Monthly support and rehab meeting to offer program support to breast cancer survivors G. Every four months breast cancer patients had a mammogram done to detect the presence or absence of cancer cell growth V. H. In the case that participants passed away, their death certificates were requested I. After five-year study, post evaluation tests were done to include colony formation assay and MTT assay on 24 and 48 hours to determine cell number differentiation Data Analysis A. Calculation of Participant Survival Rates i. 2/10 participants dropped out who were part of the placebo group due to non-compliance ii. 23 total survivors remained iii. 3/18 participants died. 2 died who was diagnosed with stage 4 cancer and 1 died who was diagnosed with stage 3 cancer B. Calculation of Participant Retention Rates i. 23/28 participants remained in the 5-year clinical study 3 C. Tumor Grading Description i. Use of Nottingham grading system - this grading system is a reference tool to describe tumor biology ii. Low-grade tumors (DESCRIBE) iii. High-grade tumors (DESCRIBE) D. Tumor Size Evaluation i. Comparison of 4T1 Wnt/B-Catenin (estrogen receptor positive), MDAMB 231 Wnt/B-Catenin (estrogen receptor positive), and MCF7 activation of capsase-8 (estrogen receptor positive signaling pathway) cancer cell lines to monitor the proliferation, cell differentiation or apoptosis of breast cancer cells ii. CT scan (CAT scan, also known as computerized tomography scan) to check growth of tumor a. x-ray technique to provide health care providers 2-dimensional cross sections of internal body parts iii. In Situ TUNEL Assay to check if fucoidan induced apoptosis in tumor tissue a. TUNEL assay tests DNA fragmentation by light microscopy REFERENCES About Breast Cancer. (n.d.). Retrieved May 5, 2015, from http://www.nationalbreastcancer.org/about-breast-cancer 4 Funahashi, H., Imai, T., Mase, T., Sekiya, M., Yokoi, K., Hayashi, H., Shibata, A., Hayashi, T., Nishikawa, M., Suda, N., Hibi, Y., Mizuno, Y., Tsukamura, K., Hayakawa, A. and Tanuma, S. (2001), Seaweed Prevents Breast Cancer? Japanese Journal of Cancer Research, 92: 483–487. doi: 10.1111/j.1349-7006.2001.tb01119.x Hsu, H., Lin, T., Hwang, P., Tseng, L. Chen, R., Tsao, S., & Hsu, J. (2013). Fucoidan induces changes in the epithelial to mesenchymal transition and decreases metastasis by enhancing ubiquitin-dependent TGFB receptor degradation in breast cancer. Carcinogenesis, 34(4), 874-884. Sekiya, M., Funahashi, H., Tsukamura, K., Imai, T., Hayakawa, A., Kiuchi, T., & Nakao, A. (2005). Intracellular signaling in the induction of apoptosis in a human breast cancer cell line by water extract of Mekau. International Journal Of Clinical Oncology, 10(2), 122-126. doi:10.1007/s10147-004-0469-2 Smyth, P.A. (2003). Role of iodine in antioxidant defense in thyroid and breast disease. Biofactors, 19(3/4), 121-130. Teas, J., Vena, S., Cone, D., & Irhimeh, M. (2013). The consumption of seaweed as a protective factor in the etiology of breast cancer: Proof of principle. Journal of Applied Phycology, 25(1),771-779. doi: 10.1007/s10811-012-9931-0 Vogel, V.G. (2015). Ongoing data from the breast cancer prevention trials: opportunity for breast cancer risk reduction. BMC Medicine, 13(1), 1-4. doi:10.1186/s12916-015-0300-0 Yang, Y., Nam, S., Kong, G., & Kim, M. (2010). A case–control study on seaweed consumption and the risk of breast cancer. British Journal of Nutrition, 103(9), 13451353. doi:10.1017/s0007114509993242 5 Capstone Methods Outline • Method A. Hook: In our stem cell transplant for SCD, stem cells are taken from the patient in utero, specialized to repair the defective gene, and implanted back into the fetus’s bone marrow. • #1 Subtopic: Overview of autologous stem cell transplant 1. Population criteria 2. Risks and benefits of autologous stem cell transplant 3. Surgical methods and tools of normal stem cell recovery and transplant • #2 Subtopic: Overview of pinpoint gene therapy 1. Zinc finger nucleases method 2. The study seeks to amend the change in the beta-globin chain so bone marrow stem cells produce ordinary, circular shaped blood cells 3. Utilized extraordinarily engineered enzymes • #3 Subtopic: Overview of fetal surgery 1. Risks associated with surgery 2. Minimally invasive fetal surgery that will be used in experiment 3. An ultrasound to see the baby movement 4. To inject the baby with zinc fingers to prevent sickle cell 5. Safety and complications. 6. Sign consent for procedure