Transcript: Difference-gel electrophoresis is.... Confirmation of protein regulation by Western blotting For this study, we used a 2D-DIGE approach to identify proteins associated in natural resistance mechanisms to sulfadiazine (Supplemental data 2). Protein abundance was compared between sulfadiazine sensitive and resistant strains from T. gondii with same genotype. Introduction Identification of differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii using difference-gel electrophoresis (DIGE) Discussion and Conclusion. Seven strains of T. gondii tachyzoites were used in this study: RH and ENT (Type I, sensitive strain), TgA 103001 (Type I, resistant strain), ME-49 and PRU (Type II, sensitive strain), TgH 32006 (Type II, resistant strain) and TgH 32045 (defined as Type II variant, resistant strain). Identification of differentially expressed proteins. Sydney Smith In conclusion, we have identified 31 proteins which are differentially modulated between sulfadiazine resistant and sensitive strains of T. gondii according to their genotype. These proteins were predicted to be involved in several different mechanisms such as carbohydrate metabolism, host cell interaction and protein translation. Although none of them allow us to identify directly resistance mechanisms to sulfadiazine at this stage, several of these proteins represent encouraging potential targets to be followed-up. We confirmed by Western blotting proteins regulation of several proteins identified in DIGE: ENO2, IMC1, ROP2, MIC2 and GRA7 in the different strains studied. We also analyzed these proteins regulation in two others sensitive strains of genotype I (ENT) and genotype II (PRU) Ajioka, J.W., Soldati, D., 2007. Toxoplasma Molecular and Cellular Biology. Horizon Biosciences, Norfolk. Ajzenberg, D., Bañuls, A.L., Su, C., Dumètre, A., Demar, M., Carme, B., Dardé, M.L., 2004. Genetic diversity, clonality and sexuality in Toxoplasma gondii. Int. J. Parasitol. 34 (10), 1185–1196. Ajzenberg, D., Collinet, F., Mercier, A., Vignoles, P., Dardé, M.L., 2010. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J. Clin. Microbiol. 48 (12), 4641–4645. Alban, A., David, S.O., Bjorkesten, L., Andersson, C., Sloge, E., Lewis, S., Currie, I., 2003. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44. Andrade, H.M., Murta, S.M., Chapeaurouge, A., Perales, J., Nirdé, P., Romanha, A.J., 2008. Proteomic analysis of Trypanosoma cruzi resistance to Benznidazole. J. Proteome Res. 7 (6), 2357–2367. Baatz, H., Mirshahi, A., Puchta, J., Gumbel, H., Hattenbach, L.O., 2006. Reactivation of Toxoplasma retinochoroiditis under atovaquone therapy in an immunocompetent patient. Ocul. Immunol. Inflamm. 14, 185–187. Bohne, W., Gross, U., Ferguson, D.J., Heesemann, J., 1995. Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants. Mol. Microbiol. 16 (6), 1221–1230. Methods and Materials Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that infects more than one-third of the world’s human population. The population structure of T. gondii consists of three main clonal lineages (Types I, II and III) correlated with virulence expression in mice. Recently, a study reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Results. What is Toxoplasma gondii? a. protozoan parasite b. bed bug c. a protein a. 2G DIGE b. DIGE c. analyzed gel In total, 31 proteins, including four hypothetical proteins, were identified from the three experiments, 44% were overexpressed in resistant strains and 56% were over-expressed in sensitive strains. Interestingly, GRA7 was identified in two gel spots and showed contradictory expression changes in these gel spots: one appearing more abundant in TgH 32045 and the other one in ME-49 There was a variety of methods used; cell structure, Preparation of tachyzoites, DIGE, Sypro Ruby staining, Image analysis and statistics, In-gel tryptic digestion and Western blot analysis Quiz Time!!!! References. How many different strains of T. gondii tachyzoites were used in this study? a. 31 b. 7 c. 1,000,000 There is increasing evidence for the emergence of strains of T. gondii that are resistant to treatment with sulfonamide and/or pyrimethamine-based compounds. In collaboration with Meneceur et al. (2008), we have recently isolated three strains of T. gondii resistant to sulfadiazine. As there is no apparent correlation with strain
Transcript: BioTech Presentation -CRISPR- Michelle Seymore, Sofia Bufano, Genavieve Goss CRISPR Introduction Overview of CRISPR CRISPR is a natural process that frequently occurs in nature, especially in Bacteria. Today thanks to research, CRISPR is a way to cut and rewrite the code of life. In 2012 scientists figured out a way to use CRISPR not just to fight viral DNA but they discovered a way to apply this knowledge to any kind of DNA. Who discovered CRISPR: CRISPR was discovered by two women, Jennifer Doudna and Emmanuelle Charpentier and they received a Nobel Prize in Chemistry in 2020. In 2011 their research started when Dr. Charpentier was studying TracrRNA and traced it back to a bacteria’s immune system, the CRISPR/Cas that targets viruses DNA. Dr.Charpentier and Dr. Doudna worked together and they were able to recreate the same procedure on a test tube. What does that mean? CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), can be defined by “a segment of DNA containing short repetitions of base sequences, involved in the defense mechanisms of prokaryotic organisms to viruses.” This DNA sequence is used to edit the base pairs of proteins in humans. Cas9 is a protein used in CRISPR to influence a change in the DNA sequence by cutting the DNA. TracrRNA, or Trans-activating CRISPR RNA, is a type of RNA that aids in the CRISPR mutation process. In this process, TracrRNA base pairs with crRNA (CRISPR RNA) and forms a functional guide RNA (gRNA). Cas9 protein uses this RNA as a handle, while the crRNA sequence directs the complex to a matching viral sequence. Important Terms CRISPR Process Visual Explanation Genetic Process of CRISPR Prokaryotic Vs. Eukaryotic Prokaryotic Vs. Eukaryotic Although CRISPR can occur naturally, enginers are learning how to tailor Eurkaryotic genes. The CRISPR process occurs differently in Prokaryotic and Eukaryotic cells. Eukaryotic Eurkaryotic Process : 2 The CRISPR RNA molecule binds to an endonuclease called Cas9. Cas9 cuts both strands of the DNA within the target sequence. 3 TracrRNA creates a guide for crRNA to follow. crRNA follows the TracrRNA guide and base pairs to the DNA strand. 4 1 Prokaryotic 2 Prokaryotes Segments of DNA (CRISPR) get cut by Cas 3 The viral DNA is inserted in the sequence CRISPR RNA is made CRISPR RNA targets virus that comes in 4 1 CRISPR in Bacteria Diagram CRISPR's Contributions to Science and Nature CRISPR in Society CRISPR is a known advance in the process of genome editing, the change of a DNA sequence. CRISPR specifically uses the CRISPR enzyme (Cas-9 protein and guide RNA) to cut a part of a DNA sequence and a different sequence can be added in place of it. The advance of gene editing with CRISPR has many effects on nature and society. Changing a DNA sequence can cure genetic diseases, and has the possibility to cure the following: Cancers Blood disorder Blindness Aids Cystic fibrosis Muscular dystrophy Huntington’s disease How It Affects Science, Society, and Nature How It Affects Science, Society, and Nature CRIPSR contributes to science by advancing genome editing, which can then advance the health of people who have their DNA edited. CRISPR can affect nature entirely by causing offspring of animals with certain expressed genes to not carry their original genes anymore. Many controversial topics have been touched because of CRISPR because of moral dilemmas on changing natural occurrence of genes. Example of curing a blood disorder using genome editing Victoria Gray Victoria Gray, from Forest, Mississippi, was affected by sickle cell disease; a disease that causes bone marrow to produce a defective protein that makes blood cells sickle-shaped. In 2019, she received treatment using CRISPR at the Sarah Cannon Research Institute in Nashville, Tennessee. Using CRISPR, scientists create fetal hemoglobin with the patient's bone marrow. They then undergo chemotherapy as a part of a bone marrow transplant, and that wipes out cells with the existing defect. They receive billions of their own new cells. Victoria Gray Carlene Knight Carlene Knight is a CRISPR patient that was born with a rare genetic eye disease. She was one out of seven people with a rare genetic eye disease to volunteer to use CRISPR to edit their DNA. When using CRISPR for these patients, it was injected directly into the cells of the patient's bodies. For Carlene, the genes in her retina were edited. Knight has said that CRISPR has given her the ability to see much brighter and more vivid colors and images. Before CRISPR, she couldn't move around her workplace, even with a cane. Carlene Knight CRISPR Research CRISPR research is currently being used to cure illnesses such as common blood disorders and cancer. This research is proving effective in this respect. CRISPR Research CRISPR in Blood disorders CRISPR is a relatively new technology that is constantly being tested to see how this method can be used to cure diseases. In 2019 this method was tested with sickle cells and
Transcript: Jeffreys Big Discovery Jeffreys put together all of the research he had and figured out a way to put the information together The information included a group of messy genetic material that didnt give a clear conclusion https://web.mit.edu/iment/iou/jeffreys.html http://genome.welcome.ac.uk/doc.wtd020877.html http://www.thebrightesthub.com Jeffreys observations led to the last step of his research projects This project was created to look deeper and closely into the different inherited types of genes After coming up with a complicated plan on how to do his research, Jeffreys noticed a new type of DNA formation Jeffreys Shocking Dicovery Jeffreys Research In England Jeffreys learned how to detect single copies if human genes Makes the first observations of introns (non-coding sections of DNA that split up genes) Citations Sir Alec Jeffreys Jeffreys Conclusion Research In Leicester Famous British geneticist Went to Leicester,England to work on his own research in 1997 DNA Fingerprinting Jeffreys' research lead to another discovery. He found out that RFLP's (Restriction Fragment Length Polymorphism), which are segments of DNA that make changes he generic group, were proof of variation in inherited DNA levels After discovering what the RFLP's do, he then pointed out that some DNA segements couldnt inform you about the different types of inherited genes Sir Alec Jeffreys began to see patterns in the genetic formation, he noticed that the DNA this group contained, was the information of a single individual Jeffreys made a conclusion on September 1984, that the projects had led to the discovery of the very first DNA fingerprints ever created
Transcript: Biotechnology Design Team 2007-2 5 3 1 What is Cancer? Cancer: uncontrollably dividing cells that form a tumor This is the most common way to remove tumors where surgeons manually remove the tumor along with some nearby tissue. The Issue: Re-operation 2 Cancer Surgery Surrounding Tissue Surrounding Tissue The surgeon cuts out some surrounding tissue to make sure all possible “hidden” cancer cells have been removed It's hard for surgeons to tell how much surrounding tissue is cancerous during surgery. Post-operative biopsy No left over cancer cells: no re-operation Left over cancer cells: needs re-operation Impacts of the Issue Although re-operation is very common, it is a problematic procedure with far-reaching impacts. Explanation of Impacts Considerations of Re-operation Risk of complications after surgery Psychological and emotional effects Financially Overburdening Increased mortality rate Delayed recovery and treatment Impacts of Re-operation Excessive bleeding, blood clots, or infections in other parts of body $31,000 Average American's salary $150,000 Average cost of cancer treatment May increase anxiety and inflict emotional trauma on patient and family This data was found in a study conducted by the BMJ Journal of Ethics. Mortality rates increase by 27% - 28% Increases the patient's recovery time and delays treatment Risks of no re-operation Risks of Not Re-operating Percent of accurate biopsies 57% 43% "False Negative" - biopsy shows no cancer, but there actually is Percent of Inaccurate biopsies - this means cancer is left undetected and advances to even more dangerous stages. 4 The Solution: iKnife Step 1 1 Electricity heats up the tip of the iKnife 2 Step 2 The hot blade causes cells in the tissue to explode, releasing particles in smoke 3 Step 3 Particles are sucked into a tube, heated, ionized 4 Step 4 The particles in the tube are then fed into a mass spectrometer (which is like an extremely accurate weighing scale) 5 Step 5 A detector on the mass spectrometer outputs this data, which lets surgeons distinguish between cancerous and non-cancerous tissues during the surgery. Overall accuracy of iKnife is Success of iKnife 97.8 % Real Life Scenario Real-Life Scenarios Ben Normal surgery More hidden cancerous tissue left Needed re-operation. Jake False negative Later found that cancer had spread More complicated re-operation was needed Clara Surgery with iKnife All cancerous tissue was removed No re-operation was required
Transcript: Who owns the Patent on Your Genetic Code? Yes No Only If... type something Employers? The Military? They could us it discover cures or treatments for fatal or epidemic diseases that can't be treated today. They could use your code to do testing that you are not comfortable with or you think is wrong, like cloning. If it helps you land your dream job. If it helps them save your life during combat. They discovered that your genes can help cure or threat a disease. Medical Authorities? That kind of information shouldn't matter to a potential partner for life. They could use it to find diseases early and either cure them or help you find a treatment option. If there is a great breakthrough on the therapy or cure of something, and they needed you code to help other people. They could see if you are a carrier of some disease that your child could get and be prepare for it That kind of information might be too private for them to know, and they don't really need it. They could find ways to make you immune to diseases that you could be exposed while deployed over seas Prospectec Spouses? If that helps them decide if you are a good choice of partner. That way you can pay for future treatments if you have a disease that takes years to show and that is uncurable You have the risk of someone breaking into the system and stealing your information and use it for who knows what Scientists? They could use you as a tests subject without you knowing, because you are their property To ensure that you are the best suitable employee for the job they require. If you wanted to be ensure for a disease that takes a long time to show and to take any effect on you. Insurance Companies? They could be doing experiment that are controversial and could use your code in them.
Transcript: C Q&A Frontier Issues & UNFPA T - Benefits of Frontier Tech Overview - Biotech one example - Why should we care about frontier tech? Frontier Technology - CRISPR-Cas9 - In Vitro Fertilization - The combinative value The Technology: The Technology 1 - Revolutionized genetic therapy. - Gene manipulation i.e. "Designer Babies". - Curing previously incurable diseases. For us that means HIV. - Reduce total number of maternal deaths are attributed to HIV. CRISPR-Cas9 2 - Tech isn't cutting edge, its uses are. - Preimplantation Genetic Screening/Diagnosing (PGS/PGD). - Goodbye unwanted inherited traits! No more sickle cell, Huntington's, double X chromosones, hemophilia, etc...wait a minute. - 117 million women missing from Eastern Europe and Asia, with certain areas seeing up to 25% more male births than female. Any country with less regulation of sex selection pose serious threat for female population...or maybe not? In Vitro Fertilization 3 - Current CRISPR-Cas9 treatments cost ~$375,000. - Possible to apply CRISPR treatments via in vitro to reduce price. $20,000-40,000 The Synergy - Germline modification = exponential proliferation of resistant gene. - A couple generations later inoculated population. Business Model How This Fits Additional Benefits/Concerns Global Concerns - Global scale implications of this technology. Unregulated, ungoverned, ethically ambiguous. To name a few... - Pricing = unviable for low-income countries/persons. (SDG 10.3) - 10/90 gap. - Safety is a major concern. The Bad The Bad - Healthcare costs overall reduced. - Bring the research to them/promote economic growth. The Good The Good - Ethically, this tech lacks globally-accepted framework of control/regulation. - Conflicting views on restrictions. Varying restrictions government to government/institution to institution. - CEB has input: need for universal agenda. - At this stage moral complexity of tech is low. Difficult choices are down the road. Ethical Implications Ethical Implications Two major concerns: 1) "Too much potential harm. We should be denouncing this technology instead!" 2) "This is not what we do at UNFPA. We should be focusing on improving our approach to our mandate, not on things that have nothing to do with us." Causes For Concern Causes for Concern Questions? Questions?
Transcript: Nobody knows babies like we do! Quality products . Good Customer service. Every Kid really loves this store.. BABYLOU ABOUT US About Us BabyLou was established in 2004. It has been more than a decade since we started, where we have ensured to take care of every need and want of every child and infant under one roof, true to the caption “NO BODY KNOWS BABIES LIKE WE DO”. Our benchmark is to provide 100% customer service and satisfaction and continue to deliver the same with a wide range of toys, garments and Baby Products. Play and Create We Are Best 01 02 03 Block games Building Blocks help Kids to use their brain. PLAY TO LEARN in Crusing Adventures Our Discoveries Enjoy a sunny vacation aboard a luxury yacht with the LEGO® Creator 3in1 31083 Cruising Adventures set. This ship has all the comforts you need, including a well-equipped cabin and a toilet. Sail away to a sunny bay and take the cool water scooter to the beach. Build a sandcastle, enjoy a picnic, go surfing or check out the cute sea creatures before you head back to the yacht for a spot of fishing. Escape into the mountains Disney Little Princes in Also available for your Babies..... Also... Out of The World… Our reponsibility BABYLOU…. Our Responsibility All children have the right to fun, creative and engaging play experiences. Play is essential because when children play, they learn. As a provider of play experiences, we must ensure that our behaviour and actions are responsible towards all children and towards our stakeholders, society and the environment. We are committed to continue earning the trust our stakeholders place in us, and we are always inspired by children to be the best we can be. Innovate for children We aim to inspire children through our unique playful learning experiences and to play an active role in making a global difference on product safety while being dedicated promoters of responsibility towards children.
Transcript: -all similar procedures -bladder and trachea -simple organs only so far Cell Therapy Surgery, Surgical Implants, and Skin Grafts Tissue Engineering Regenerative Medicine What is going on in the science of growing organs? Regenerative Medicine Project: Pancreas Cardiovascular Bone development Intestinal.... -Regenerative Medicine -Began in Ancient Civilization -Founding of four signs of inflammation, chick embryos, chemical reactions with in cell, and official intro to DNA forms -Homer & Hesiod: Legend of Titan God Prometheus -16th Century: Cosmas and Damian - Scientists want to send amputee home and relieve diabetics Trachea The future? 116,118 Bladder -stem cells taken from bone marrow -grown in a culture -inserted into "scaffold" -transplanted into patient -improving statistics Growing Organs Finally History Controversial subject Current research Success stories -trachea -bladder 18 Procedures -cells are taken and grown in a culture -then added to a "scaffold" -organ continues growth and is later implanted
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