Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths).
Protocol: 1. Microwave 2% Agarose Gel for 4 min 2. Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins 3. Pour 2% Agarose solution into gel tray with the well comb in place 4. Let sit at room temperature for 20-30 mins, until it has completely solidified https://www.addgene.org/protocols/gel-electrophoresis/
2017/7/30 Tobacco smoke contains many dangerous chemicals Chemicals found in cigarettes Scientists have identified over 5,300 different chemicals in tobacco smoke. The International Agency for Research into Cancer (IARC), the gold standard for establishing the causes of cancer, state that there are more than 70 chemicals in tobacco smoke that have been found to cause cancer in studies involving people or in the laboratory. And many of the other thousands of chemicals are toxic and harmful to your health, including carbon monoxide, hydrogen cyanide and ammonia.
Nicotine is a highly addictive drug Most smokers do not smoke out of choice, but because they are addicted to nicotine]. This was highlighted in a report by The Royal College of Physicians into the effects of nicotine. They also compared nicotine to other supposedly ‘harder’ drugs such as heroin and cocaine. They looked at many things including how these drugs cause addiction, how difficult it is to stop using them and how many deaths they caused. The report concluded that nicotine is a highly addictive substance, particularly when people are exposed to it through using tobacco, and that tobacco dependence is at least as serious as addiction to ‘harder’ drugs. Smoker may be more stressed than non-smokers People associate smoking with feeling less stressed and anxious, but the evidence suggests this is only because it temporarily relieves the unpleasant symptoms of nicotine withdrawal. If anything, current smokers seem to feel more stressed and anxious than ex-smokers or people who have never smoked. Smokers can also make mental associations with abstract things like the smell of cigarettes, objects related to smoking like ashtrays and lighters, and situations in which they usually smoke. These can all act to reinforce the addiction to smoking. http://www.eduhealthfood.com/article-no-smoking.html
2017/8/3 Effects of Nicotine on Exosome Biogenesis February 2, 2017 Exosomes are extracellular vesicles that act as biomarkers of certain diseased states. Specific characteristics of these extracellular vesicles have been found to be elevated in particular diseased states including malaria, various cancers, and autoimmune diseases. Our objective is to measure the effects of nicotine on exosome biogenesis in liver cells. We hypothesize that nicotine administration will affect exosome biogenesis. Our findings will provide additional information related to how nicotine usage affects cellular mechanisms in the human body. The liver cell lines, were either treated with varying concentrations of nicotine or untreated (control). Exosomes were purified from the media via high speed ultracentrifugation or commercial kits. Exosome protein quantity was determined by standard quantitation method. Enzyme-Linked Immunosorbent Assay (ELISA) and Western blot analysis was used to detect specific antibodies in the exosomes. Lastly, NanoSight technology was used to analyze the specific characteristics of the exosomes including size and concentration. Our findings indicate that the size and concentration of exosomes produced were directly related to nicotine dosages. Overall, these results strengthen the stigma that nicotine negatively affects the human body and that exosomes act as a strong precursor to particular diseased states. Future research involves analyzing the effects of nicotine on exosome biogenesis in various cell lines including lung and skin. http://new.emerging-researchers.org/projects/12769/
Reports from cigarette smoke extract:
Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants a Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands b Department of Respiratory Medicine, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands c Department of Pharmacology and Toxicology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands d Department of Medical Microbiology & Infection Control, VU University Medical Center, Van der Boechorststraat 7, 1081BT Amsterdam, The Netherlands https://doi.org/10.1016/j.freeradbiomed.2017.03.026 Highlights •Cigarette smoke extract (CSE) enhances exosome release by airway epithelial cells. •This is mediated by thiol-reactive CSE components such as acrolein. •CSE depletes cell surface thiols, which is sufficient to trigger exosome release. •CSE-induced exosome release can be prevented by N-acetyl-l-cysteine and glutathione. Graphical abstracthttp://www.sciencedirect.com/science/article/pii/S0891584917301752 Does smoking, age or gender affect the protein phenotype of extracellular vesicles in plasma? Abstract Extracellular vesicles (EVs) are involved in several diseases, which have formed the basis for the potential use of EV analyses in a clinical setting. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. With this extensive study of 161 healthy individuals it was elucidated that certain markers of plasma EVs are influenced by demographic variations such as gender, age and smoking status. When the purpose is to use EVs as a diagnostic tool, it should be emphasized how important it is to choose the correct demographic group when comparing marker levels of plasma EVs. http://www.sciencedirect.com/science/article/pii/S1473050216300805
2017/8/6
Testing various volume of water 1000µl 200µl 20µl 10µl 2µl 0.62µl left on 1000µl tip. Ran centrifuge so that all water could flow down to the bottom of the test tube. Calculated error rate: 0.62/1000 * 100 (Percentage) = 0.062% 0.062% is a bad error rate Strive for perfection Reason for bad error rate Tip of the pipette didn't fully touch the lowest point of water While collecting water from the test tube, not all of the water fully went into the tip, some remained on the surface of the tube. Ways to improve Precision/Accuracy Eye-level, see solution clearly Do not rush procedures Firmly (but not fully) press down on the pipette When putting solution into another tube, make sure to extract solution on the bottom of the surface/(touching the) wall for greater accuracy and less error rate
Goal for next trial Get error rate at least below 0.03% At least 50% improvement from this test 2017/8/30 Exosomal Micro RNA purification and quantification
1. Cell culture media from Ten 100 mm culture plates (5.5 x 107 cells) with CSE and chemicals were used to purify exosomal miRs with mirVana™ miRNA Isolation Kit (Invitrogen). 2. The purified miRs were synthesized to cDNA using miScript II RT Kit (Qiagen). 3. Quantification of miRs were peformed using miScript SYBR Green PCR Kit (Qiagen) 4. qPCR was performed with a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) with normalization to an external spike-in synthetic oligonucleotide of C. elegans miR-39 (Qiagen)
2017/8/31
Methods of how to extract cigarette smoke?
This is how my team collected the extract of chemicals from cigarette smoke.
2017/9/01
CSE were treated on A549 lung cancer cells. More than 10% of CSE killed the cells. Therefore, we used 5% of CSE concentrated PBS for further experiments.
2017/9/03 Isolation of exosome using ultracentrifugation:
2017/09/04 We found that exosomal miR23-a was increased in CSE treated A549 cells
2017/9/5 Ideas for water project Making clean water Using charcoal to make clean water, 소독. Or any other material that could be used to clean water. Different methods.
2017/9/7 Went over ideas such as determining the turbidity of water just by taking a simple photo. Researched about past examples, there's one notable source. Today we experimented with charcoal powder. We added 1 gram of powder into a 250ml water filled beaker.
Update Add more pictures here... I should honestly start posting these updates via CAS journal to make sure that I am actually doing this project with the correct dates.
2017/10/30 The Current Definition of Exosomes Definition from PMC: Exosomes are best defined as extracellular vesicles that are released from cells upon fusion of an intermediate endocytic compartment, the multivesicular body (MVB), with the plasma membrane.
Why are people interested in exosomes? 1. Exosomes are (thought to be) related to the communication and transportation of macromolecules between cells. 2. Exosomes contribute to the spread of some macromolecules as well as diseases. 3. Exosomes might be useful vectors for drugs.
2017/11/30 Writing introduction section:
Introduction Smoking cigarette causes 480,000 deaths every year in United States (1 out of every 5 deaths) and causes more than 4 in 5 cases of lung cancer. Non-small cell lung cancer (NSCLC) consists about 85 percentage of lung cancer. It is known that quitting smoking increased 5-year survival rates after the diagnosis of early- and late stage NSCLC to 37% and 34% respectively [1]. Exosomes are cell derived extracellular vesicles that are present in all body fluids. The diameters of exosomes are identified to be ~30-100nm. Exosomes can be both released from the cells and fused with the plasma membrane. Various molecules such as proteins, RNAs, and lipids are packaged in exosomes and can be secreted into diverse body fluids [2]. Therefore, exosomes have great potential as diagnostic tools and biomarkers for various diseases. MicroRNAs (miRs) are small (19–25 nt), single-stranded, non-coding RNAs that bind to partially complementary sequences to their target messenger RNA (mRNA). miRNAs degrades mRNA and inhibit the translation of target mRNAs [3]. miRNAs are often found in exosomes, which can be taken up by distant cells. Exosomal miRNAs are known to play important role in various disease progression including cancer [4]. Cigarette smoke has been shown to change the expression of genes in airway epithelial cells from smokers [5]. Lung cancer cells exposed to cigarette smoke altered the expression of genes associated with oxidative stress and inflammation [6]. Recent studies indicated that cigarette smoke extract (CSE) induced exosome release in airway epithelial cells. However, the functional role of miRs in exosomes released from CSE exposure has never been tested. In this study we investigated the role of exosomal miR-23a on A549, a model for NSCLC.
2017/12/30 Essay writing for global environment essay competition:
Climate change is a hot potato lately. People living in the 21st century have to face a new crisis on environment and human health. Even though the climate can change in many ways, human activities are the main factor-causing climate to change rapidly because the world depends on fossil fuels for 80% of its energy needs. Therefore, it is very hard to switch from fossil fuels to any other forms of energy. If climate change continues unabated without any preventions, climate could affect plants, animal, and all the other living by changing the condition of the environment such as soil, lakes, and ocean. Therefore, scientists believe that climate change is a direct threat to human health. Climate change can affect human health through spreading infectious disease and initiating dietary deficiency. Climate change affects human infectious disease via pathogen and disease transmission. Climate changes refer to a wide range of climate variables including temperature, wind, precipitation, and sunshine. These changes affect survival and distribution of disease pathogens. Pathogen is an infectious disease agent such as virus, bacterium, parasite germ, and fungi. Climate change affect disease through affecting the life cycle of pathogens in many ways. Firstly, rising temperature may increase the reproduction of pathogens. For example, the time of the reproduction for malaria parasite (P.falciparum) reduces from 26 days at 20oC to 13 days at 25oC (Harvell, 2002). Secondly, temperature change may provide a favorable environment for microorganism reproduction cycles for bacteria and algae. For example, Vibrio spp. bacteria showed an increased growth rate during hot summers in 2006 (Frank, 2006). Therefore, climate change cause death or injury of people by infectious disease via survival and distribution of disease pathogens. Climate change affects world’s food supply. Scientists believe that climate change is poised to affect the world’s food supply in three ways. Firstly, climate change will diminish the quantity of food grown. The rising carbon dioxide level may decrease in food yield around the world. For example, combination of increased levels of carbon dioxide in the atmosphere and high temperatures showed significantly lower yields for corn and wheat (Robin, 2017). Currently, areas that experience increasing temperatures will also likely see an increase in crop pests, which were responsible for 25 to 40 percent of all crop loss. Lastly, climate change will decrease food quality. For example, high levels of atmospheric carbon dioxide decreased proteins, zinc, and iron of the crops (Myers, 2014). The decrease in iron and zinc could deteriorate the health problem of nutrient deficiencies because it is already a huge health problem today. Therefore, in the future more than 200 million people worldwide could develop iron and zinc deficiency due to the climate change. Climate change has many consequences and influences to the Earth and to humans. Climate change affects spreading infectious diseases diminishing world’s food supply. Climate change leads to malaria and food-borne disease through distribution and reproduction of pathogens. Malaria can cause impaired consciousness, coma, and even death through blood sequestration in the blood vessels of the brain. Recent reports indicate that future climate might become more suitable for malaria transmission in the tropical highland regions (Cyril, 2014). Precautions are needed for people who lived in the tropical highland regions. Moderate warming and more carbon dioxide in the atmosphere may reduce food yield and quality. There are many ways to prevent climate change. Save the energy by unplugging computers, TVs and other electronics when not in use. Trim the waste by recycling paper, plastic, metal, and glass. The most important way is to follow the latest news about climate change.