Sunday, January 26, 2020
Effects of Wnt-3a and Wnt-5a on Proliferation of HEK293 Cell
Effects of Wnt-3a and Wnt-5a on Proliferation of HEK293 Cell Letong YUAN 1. Outline how you decided how many cells, how many wells and how much Wnt to use. Please do not repeat anything in the protocols your answer should just explain concisely and clearly how and why you designed the experiment in the way that you did (250 words max). We were provided with three cell lines but we decided to investigate the effect of both Wnt on the proliferation of one cell line, HEK293 cells, limiting the number of variables in the experiment. Unlike MDA-MB-231 and MCF7 which are breast cancer cell lines, HEK293 is derived from human embryonic kidney cells in tissue culture and are non-pathological. As the average doubling time of HEK293 is 24hours, we decided to seed HEK293 suspension cells at 1X105 cells/ml (suggested by Thermofisher ©) in each well containing 500 µl medium. We used 16 wells in total on two 24-well-culture plates, i.e. 8 wells on each plate as illustrated in figure 1. We used two identical plates as cells are not viable after PFA fixation. After 24 hours, as the cultures are in log phase, an escalating amount of Wnt, namely, 0, 5, 20 and 50ng, was added for the investigation of HEK293 cell proliferation in a concentration-dependent manner. The amount of Wnt added was suggested by Reischmann et al, 2015 and limited by the amount of Wnt provided. 100 and 200ng could also be tested if enough Wnt were provided. Figure 1 Schematic diagram of amount of Wnt added in each well containing HEK293 cells. Two of these wells are used as cells on one plate was fixed after every 24 hours. 2. Describe your cultures non-quantitatively (i.e. qualitatively) (200 words max). At 0h, cells tend to be spherical or have elongated shapes and plump with a high density, then they were passaged. After 24h, cells attached to well surfaces as they settle down and then started to differentiate. Some cells have developed one or more antenna-like projections for better attachment and nutrition, while spherical ones are non-adherent. 2-9 cell clusters could be observed, whereas few dead cells were present. At 72h, cell clusters with more than 20 cells were normally seen. Being polar, HEK293 are fibroblastic cells. 3. Analyse one characteristic of your cultures quantitatively including a well-presented and informative graph and a photograph that illustrates the graphed result (think about what makes an informative graph). Have a look at some primary cell culture papers to see how data can be presented clearly and thoroughly. What should you always have on the graph of an experiment looking at the effects of a cell signal on cultured cells? The number of cells show an increasing trend with an increase amount of Wnt-5a added. The addition of 50ng results in almost twice as many cells as in 0ng. Wnt-5a triggers noncanonical signalling pathway. Anomalies in data are possibly because of a non-representative image taken. Its effect can be minimised by having replicates in this experiment. Another contributing factor is the inaccurate particle analysis by imageJ, whose effect can be minimised by taking the mean of numerous results. 4.Briefly suggest a possible mechanism behind your result or, if the ligand has no effect, suggest why this happened. Please note: no effect is not a failure there could well be solid ICB reasons why there was no effect recorded and you can still produce an excellent write-up (200 words max).   Wnt activates signalling cascade through canonical/ ÃŽ ²-catenin Wnt signalling pathway (e.g. Wnt-3a) or noncanonical/ ÃŽ ²-catenin independent Wnt signalling pathway (e.g. Wnt-5a). In canonical pathway, Wnt binds to frizzled 7TM receptors. Then LRP 5 or LPR6 activates dishevelled (Dvl), triggering the inhibition of GSK3. ÃŽ ²-catenin accumulates in the cytoplasm because of the disassembly of the destruction complex containing APC, Axin and GSK3. In noncanonical pathway, ÃŽ ²-catenin is not involved. Daam1 activates small G-protein Rho, which activates ROCK. Figure 3 In the absence of Wnt, ÃŽ ²-catenin interacts with APC, axin, GSK3, and CK1 forming a degradation complex. The phosphorylation of ÃŽ ²-catenin triggers its degradation. Croucho kept Wnt-responsive genes inactive by binding to LEF1/TCF. The presence of Wnt clamps LRP and Frizzled protein together. The phosphorylation of cytosolic tail of LRP by members of degradation complex causes the disassembly of the destruction complex, which prevents the phosphorylation of ÃŽ ²-catenin and allow its accumulation. Excess ÃŽ ²-catenin can be translocated into the nucleus without degradation and it replaces Groucho and activates the transcription of target genes mediated by LEF1/TCF. These genes are essential to processes like cell attachment, cell proliferation and differentiation. This explains the addition of Wnt promotes cell proliferation and differentiation. 5. Suggest a possible future experiment to investigate if/how this ligand has an effect on the cells you tested (200 words maximum). Be bold and creative here and try to push yourself beyond, try different concentrations. To see if and how the ligand influences the cells, one way is to assay Wnt-responsive gene product, e.g. ÃŽ ²-catenin, by Western blotting. After Bradford assay determining protein concentration in cell lysate, run a SDS-PAGE gel and transfer the gel onto a nitrocellulose membrane. Analyse the membrane with primary antibody against ÃŽ ²-catenin, then incubate with corresponding secondary antibody. It is possible to add an inhibitory component of the Wnt signalling pathway, for example Apc protein (Albert et al, 2015) that binds to ÃŽ ²-catenin and induces its degradation. Wnt-responsive genes should be kept silence by Croucho bound to transcription regulator LEF1/TCF.
Saturday, January 18, 2020
Poliomyelitis Paper Essay
Poliomyelitis is a viral disease that mainly affects the young children, it is highly infectious that caused by three types of poliovirus which targets the nervous system of the person, it may result to be abortive poliomyelitis, non- paralytic and paralytic poliomyelitis. There are many initial symptoms of polio fever (up to 103 °F or 39.5 °C), less of appetite, vomiting, constipation and pain in the limbs. We can diagnose this by having complete physical examination; having a complete test of throat, urine, lumbar puncture or spinal tap. Medical history of a child who does not have his/her polio vaccine or not complete polio vaccines series is also one of the caused of this infection. The treatment for this is determined by your physician base on your age, tolerant for specific medications, procedures or therapies. Although we have come to prevent this disease not to infect young ones by having a complete vaccines which have two versions Inactivated Polio Vaccine (IPV) and Oral Polio Vaccine (OPV) and the best way for this is to have good hygiene and clean surroundings but for those who had already been infected will have no cure, the supportive means of treatment is the only way to help them to ease some pains like analgesics, a bed rest to reduce fever, a good balance diet, hot compress for the pain of your muscles, if the patient is paralytic poliomyelitis it requires a hospitalization. In early days infectious disease like this maybe a threat to our society but its a good thing to know that now we have the treatments and the best of this is that to follow the first step of cure which is prevention. References World Health Organization (2007). Poliomyelitis. Retrieved December 10, 2007 from            http://www.who.int/topics/poliomyelitis/en/.  University Health Care (2003). Poliomyelitis. Retrieved December 10, 2007 from            http://healthcare.utah.edu/healthinfo/adult/infectious/polio.htm
Friday, January 10, 2020
Factors influencing News editors’ decisions Essay
Factors influencing News editors’ decisions Introduction            News form an important part in the society because it helps in providing people with information as well as keeping people updated on events and happenings that are taking place across the world. The factors which I think news editors consider in making a decision include: Age of the targeted audiences. The editors have noted that the age group of those who read the Newspapers and those who watch TVs is different in that the older generation prefers written content unlike the younger generation who prefer visuals and audio which is available through television (Halan, 2009).            Secondly, the difference in taste and preference of the audiences also affects the choice of news presentation by the editors. There are people who prefer a combination of the visual and audio (Islam, 2008). Therefore, the use of TV to present some news makes a greater impact than when the newspapers are used. Furthermore, news editors aim at being providers of exclusive news for their audiences. Therefore, to maintain exclusiveness with their news as compared to their competitors, news editors would choice an alternative that would make them exclusive to the audience(Halan, 2009). This will enable them to achieve higher sales in terms of newspapers or high viewing rate if they use the TVs.            Finally, the other factor that I think may be an influence on the choice of news presentation, whether through a newspaper or television is whether the news is meant for international or the national audience(Islam, 2008). To reach a wider audience internationally or nationally, news editors would prefer a means that is easily accessible to majority of the targeted audience.            Therefore, in conclusion, it is my view that age, exclusivity, taste and preference of the targeted audience, and national or international interest that the news is aimed at form the factors that influence the decision of the editors. The end result is for them to achieve more sales, higher viewing rates as well as enhanced reputation. References Halan, Y. C. (2009). Communication skills: Effective editing; a practical handbook to develop good editing skills. New Delhi: Sterling Publishers. Islam, R. (2008). Information and public choice: From media markets to policy making. Washington, D.C: World Bank. Source document
Thursday, January 2, 2020
Essay about The Fight to Woment to Obtain Their Rights and...
Growing up as a female has never been easy. Men assumed women only had two jobs, keeping the house clean and bearing children. Luckily, many women in history have paved the way making it easier for all women in the 21st century to work outside the house, vote, earn more money, hold positions of power and achieve all their dreams. Susan B. Anthony, Elizabeth Cady Stanton, Madeleine Albright, and Lucy Stone, just to name a few, each had a hand in helping little girls dream big dreams and realize that their dreams were achievable. Through their hard work, determination and suffering, women are more equal than ever. Historians typically like to breakdown the history of the United States into different â€Å"eras†. One era that was important to†¦show more content†¦Anthony and Stanton, as well as other leaders, worked endlessly lobbying congress and circulating petitions around the country to pass a consititutional amendment to give women the right to vote. (Reforming) The National American Women Suffrage Association (NAWSA) and the National Women’s Party (NWP) helped spread the work through campaigning, lobbying the President and even picketing the White House. (Reforming) In 1920, the 19th Amendment was passed giving women the right to vote as a US citizen. (Reforming) There were many courageous women in American history that fought for the rights of all women. One of the most notable leaders was Elizabeth Cady Stanton. Stanton was born in New York on November 12, 1815. (Ward) Her family was wealthy, enabling her to go to some of the best school and colleges to earn her e ducation. Like many other leaders, Stanton was involved in many causes. While she was working on the abolition movement, she met her husband, Henry B. Stanton, who also shared her passion for ending slavery. (Nash) In 1848, Stanton met several other women who were working within the Women’s Right’s movement. Together with these women, Lucretia Mott, Martha Wright, and Mary Ann McClintock, they formed the first Women’s Right Convention later called the Seneca Falls Convention. (Ward) Before the meeting, the women met and drafted a document called the Declaration of
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