Week 1 Individual Assessment
Week 2 Individual Assessment
Summary of article "Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging".
The study uses direct stochastic optical reconstruction microscopy (dSTORM) to investigate STAT1 distribution and localization during stages of the cell cycle. STAT1 belong to the family of 'signal transducers and activators of transcription'. They are transcription factors which become activated via tyrosine phosphorylation and transduce signals from the plasma membrane to the nucleus thus their main function involves activating transcription and regulating gene expression within cells.
The cellular localization of STAT1 is highly related to the transcription process, therefore, within this experiment dSTORM was used to observe localization of STAT1 via the use of small molecular florescent dyes that can be switched on and off during the different stages of the cell cycle. dSTORM allowed the experimenters to observe the varying distribution and localization of STAT1 proteins in the cytoplasm and nucleus of cells during the various stages of the cell cycle.
Method: Hela cells were cultured at fixed times during different phases of the cell. Alexa647 was used as a non-specific IgG antibody for STAT1, the cells were also attached to flourescent green protein dye for imaging. Then, western blot analysis was carried out using primary and secondary antibodies. Cells were then seeded on a microscopy slide, exposed to cytokines and fixed using paraformaldehyde. After this the STAT1 was stained using Alexa647 and a sample was prepared for microscopy imaging by immersing the cell samples in STORM imaging buffer. The Nikon Ti-E was used for STORM imaging and various methods were used to construct the image for cluster analysis of STAT1 in cells during various stages of the cell cycle.
Results: They discovered that STAT1 proteins were found mostly in clusters and with further cytokine stimulation, their size and number of cells in the cluster would increase significantly from G1 to G2 phases of the cell cycle. Furthermore, with epidermal growth factor stimulation, the levels of STAT1 increased gradually as the cell cycle progressed. The increase in size and number of cells from G1 to G2 is believed to occur to allow fast response to signals during signal transduction. In conclusion, the study gained "insight into the mechanism of cell-cycle regulated STAT1 signal transduction."
Week 3 Assessment
Lab 3 Individual Assessment: Functions
<pubmed>24582861</pubmed> This article investigates the function of basement membrane in molecular transport of macro-molecules and nanoparticles and it's potential as a drug delivery barrier using a cell culture of Caco-2 (an intestinal epithelial model). The study concluded that the basement membrane attenuated the diffusion of macro molecules and nanoparticles depending on their size and thus had selective permeability. The proteins (laminins, type IV collagen, fibronectin) attached to the basement membrane had a great impact on delivery of drugs through the basement membrane. These proteins of the BM mainly function in cell migration, proliferation and differentiation. Some of the main materials/techniques they used in the experiment were cell cultures of Caco-2, tight junction immunostaining, immunoflouresence to measure permeability characteristics and scanning electron microscopy.
<pubmed>24078382</pubmed> This is a review article which examines the structure and function of basement membranes, specifically the corneal BM. The basement membrane is a part of the extra cellular matrix which connects the extra cellular matrix to the intracellular matrix. It provides cellular anchoring, scaffolding during embryonic development, participates in migration, differentiation, maintenance of differentiated phenotype of epithelial cells. It also controls cellular function by regulating the amount of growth factor and cytokines as well as control cell polarity, cell adhesion and migration via their effects on cytoskeleton. However, the functions of BM can vary depending on tissues. The BM has shown to be very important in corneal wound healing of the cornea by balancing epithelial to stroma and stroma to epithelial communications by regulating the movement cytokines, growth factors from one cell layer to another.
<pubmed>8645562</pubmed> Basement membrane and it's components have been observed as being required for cell polarisation, cell adhesion and migration during wound healing and regeneration; consequently, many genetic diseases relating to the basement membrane are caused my mutations to components of the BM such as in Alport syndrome. In the experiment they use a dystropic mutant mouse dy (lacking laminin-2, an essential component of BM) to analyze function of the laminin-2 in different tissues and thus also study the general function of the basement membrane. The study stems from the hypothesis that "basement membranes are spatially and temporarily unique" suggesting that it is more variable with less distinction of it's various structural components than previously thought. The study showed that laminin-2 abscence is essential in causing a severe and progressive form of muscular dystrophy. Some of the techniques the study used included immunoflourescence and immunoblotting.
<pubmed>23696606</pubmed> This study used electron microscopy to study the ultrastructure of the epithelial basement membrane in rabbit corneas with and without haze. They used photorefractivekeratectomy (PFK) to induce haze and an immmunohistochemical analysis was done to detect smooth muscle actin (a marker for myofibroblast). Then the study used electron microscopy to analyze the ultrastructure of the BM and stroma. The study found that the BM is a critical modulator of corneal wound healing and that functional/structural limitations caused by abnormal regeneration of the basement membrane can lead to prolonged stromal haze via myofibroblast development. This can result as as the basement membrane functions as a critical modulator of epithelial-stromal interactions mediated by cytokines.
1. Anti-Laminin antibody
2. Rabbit polyclonal to Laminin
3. optimal concentration: 5 ug/ml
4. Rabbit IgG (H+L) Polyclonal Secondary Antibody A-11037
Mouse Cell Line Growth Medium: Dulbecco's modified Eagle's medium with 4.5 g/L glucose and 10 mcg/ml insulin, 90%; fetal bovine serum, 10%
Human Cell Line Growth Medium: Minimum essential medium (Eagle) in Earle's BSS with 0.015 mg/ml 5-bromo-2'-deoxyuridine, 90%; fetal bovine serum, 10%
Group 1 I think your page is set up well. You have a brief description outlining what makes up your topic of choice and when you first click on the project page, it doesn’t look boring because of the picture you have included of the proteoglycan. The images used throughout the page make it more interesting and give an appropriate visual representation of your topic. It is also very helpful that you have added a glossary at the end of your page as I did struggle to understand some of the acronyms. Looking at the contents of your page, it is well set out. You have divided each topic such as history for example into smaller subtypes which makes it more organized and easy to read. It’s also good that in your referencing you have started to differentiate between review articles and clinical trials but this is not yet continued throughout the page. On the other hand, you do still need to add a lot of information. I get the impression that a lot of it is not finished yet. The disease section is also very extensive and I think it would be more effective if it was a little more simplified. It might also be good to consider drawing your own diagrams instead of using one from an article, will make it look more interesting and also be easier to understand. The table you have included of the various different proteoglycans looks a little awkward on the page, it is not formatted properly or the size might be too big.
Group 2 The page is looking very well so far. However, I think there is still quite a lot of information that needs to be added for example in the time line section and future research. I also think that maybe you should consider joining the ‘discovery’ subtopic with the ‘timeline’. It might be more coherent that way. Some of the referencing is lacking for example in the ‘structure’ section but that can be added in later. It is good that you drew a picture of how integrin react with other proteins. The diagram is well done and easy to understand. The introduction also does provide a brief overview of what integrins are which is helpful when you start browsing the page. The addition of the video is also very helpful in understanding integrin’s interaction with the ECM. I would suggest that you add a couple of pictures to the ‘role in diseases’ possibly illustrating the clinical manifestations of the disease. It might make it more interesting. In general, I think there is still some parts to edit over and fix up but overall I think is a very good start.
Group 3 The page is looking good. The introduction is very well written and appropriately summarizes the whole page. However, I noticed that you don’t have a history/timeline/current/future research section. It might be good to include those sections briefly at the beginning as it would make the page more interesting and provide context for your reader. In the structure and components section, it might be beneficial to include a diagram of the molecular structure of elastic fibers or possibly draw something so the reader can relate what you’re describing in the text to a visual diagram. I thought the diagrams you used in displaying elastic fiber assembly were very effective but that section might be better if the process was more simplified in text form as well. It would be great to have visual representations of the diseases such as Ebola in the clinical significance section as it would make it more interesting. It might also be better to simplify some of that information as there seems to be a lot of text. The functions section is good but it might be more effective if you added a subtopics into that section dividing it into different parts e.g. heart, skin. Overall, I think the page is coming together well.
Group 4 The page seems to have a lot of sections and looks like it has been divided very effectively into the different sections. My main suggestion for this page would be to include more illustrations demonstrating what the text is describing, this could be used very effectively in the structure section. The use of dot points really helps because sometimes it seems like there is too much text (the beginning of structure). It would be great if you had text caption for the picture in the structure section describing the image otherwise the reader might be confused why it is being used. The functions section is also very comprehensive. You talk about a lot of aspects including wound healing, role in tumors, atherosclerosis, blood vessels e.t.c and it is quite concise and well written so easy to understand as a reader. However, again it would be great if you had more illustrations. Having illustrations for abnormalities/defects would also make your page very interesting, maybe showing some of their clinical manifestations? Also, I think the section on current research would be more effective if it was placed at the beginning of the page, possibly before or after the introduction. Overall, the project is coming along very well and it super organized. I would say you need to add more illustrations throughout the page but other than that it’s really good!
Group 5 The introduction was very good, concise and succinct and it mentioned all of the important information. The way you guys have done referencing on this project is a little different to what I have seen in the other pages but I think that it is working well for you guys, but sometimes it does look a little messy? So maybe that is something you should consider. The history section might be more effective if it was done in dot point form and kept very brief. The information on the structure of laminin is quite extensive! It is a lot to get through as a reader. Maybe try to simplify some parts that are not quite as important? However, the diagram works very well in explaining the text and illustrating the structure of laminin. The functions section also looks very comprehensive and detailed and I think if you do in paragraphs it will too overwhelming and will look boring. Since you are discussing the different laminin functions, maybe tabulating what you find would be a more efficient way to discuss the function. It would be easier to read and understand for the reader. Illustrations are also lacking in this section, so definitely try and draw something or add an image if possible. The abnormalities section is good, it might again be more effective if you used some illustrations to highlight the clinical manifestations of some of these diseases. The current research section is also very very extensive, but I’m not sure it needs to be so detailed? Also the antibodies section at the end, I’m not sure if that’s supposed to be there or not. If not, I’m sure you guys will remove it. Overall, I think the project is looking very good. It just needs more illustration and the information needs to be more concise and presented in an easy to understand manner (dot points, tabulated).
Group 6 I think the project has been divided well into the different sections. However, information is missing from the beginning half of the project (introduction and historical findings) so that needs to be added. I would recommend using dot points for the historical findings. The structure is a bit patchy, it seems as though not all of the information is in the section yet e.g. the proteins, types of fibers. From what is written, it needs to be edited and more concise. The image in the section is good for illustrating the structure but I think more images should be used. Maybe also consider moving the picture you have from the functions section on the structure to the structure section? Also reading the functions section, there seems to be a lot on the actual structure of the cartilage rather than its function so maybe shift some of it to the structure section and emphasize the functions of the cartilage in that section. The recent findings should be created as a separate section or joined with the current understanding/areas of research? The abnormalities needs to be more simplified. Some of that is hard to understand having not read the actual article, also more information needs to be added to the other abnormalities. Make sure to include some pictures of the abnormalities to make it more interesting. The current areas of research seems to be more organized but I’m not sure so much detail is needed? Maybe simplify it a little more and focus on the other sections. The glossary also needs to be expanded a little because there are a lot of terms in the project that aren’t explained. Overall, the page needs more work but it’s heading in the right direction.
Week 13 lab assessment
Next generate genome sequencing in relation to basement membrane:
Lab 2 - away