User:Z3336051

From CellBiology

Lab Attendance

--Z3336051 15:30, 8 March 2012 (EST)

--Z3336051 14:24, 15 March 2012 (EST)

--Z3336051 14:20, 22 March 2012 (EST)

--Z3336051 15:04, 29 March 2012 (EST)

--Z3336051 15:25, 5 April 2012 (EST)

--Z3336051 14:00, 19 April 2012 (EST) (Sorry I forgot to log attendance. You can find my in-lab work for this lab, done on that day, below.)

--Z3336051 15:57, 26 April 2012 (EST)

--Z3336051 14:17, 3 May 2012 (EST)

--Z3336051 14:13, 10 May 2012 (EST)

--Z3336051 15:08, 17 May 2012 (EST)

--Z3336051 14:58, 24 May 2012 (EST)

--Z3336051 15:08, 31 May 2012 (EST)

Lab 1

External link

http://www.jove.com/basic | JOVE

Internal link

Wiki editing help


Group 5

User:Z3333421

User:Z3223095

User:Z3289738

User:Z3336051

Lab 2

Lipids pause in Golgi.jpg

Homework

1. Identify a reference article that uses the "superresolution" microscopy technique.

Lothar Schermelleh, Peter M Carlton, Sebastian Haase, Lin Shao, Lukman Winoto, Peter Kner, Brian Burke, M Cristina Cardoso, David A Agard, Mats G L Gustafsson, Heinrich Leonhardt, John W Sedat Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science: 2008, 320(5881);1332-6 PubMed 18535242


2. What did the paper show that normal microscopy could not show?

A normal light microscope cannot detect detail beyond the diffraction limit - images with resolution of higher quality than half the wavelength of the emitted light. The paper demonstrated the potential for the 3D-SIM (three-dimensional structured illumination microscopy) method to resolve cellular structural detail just beyond the diffraction threshold of conventional light microscopy. This enabled quantitative analysis of macromolecular structures such as pores in the DAPI channels after staining, which would be indistinguishable otherwise.

Lab 3

Homework

Chemical: Methanol

Physical properties:

  • State: Liquid
  • Colour: colorless
  • Odor: Alcohol-like, weak odor
  • Boiling point: 64.7°C@760mmHg
  • Specific Gravity: 7910g/cm3@20°C
  • Vapor Pressure (mm Hg): 128mmHg @20°C
  • Vapor Density (AIR=1): 1.11
  • Flash Point: 12°C
  • Solubility in Water: miscible

Hazards:

  • Danger! Poison! May be fatal or cause blindness if swallowed. Vapor harmful. Flammable liquid and vapor. Harmful if swallowed, inhaled, or absorbed through the skin. Causes eye, skin, and respiratory tract irritation. May cause central nervous system depression. Cannot be made non-poisonous.

Target Organs: Eyes, nervous system, optic nerve.


Potential Health Effects

  • Eye: May cause painful sensitization to light. Methanol is a mild to moderate eye irritant. Inhalation, ingestion or skin absorption of methanol can cause significant disturbance in vision, including blindness.
  • Skin: Causes moderate skin irritation. May be absorbed through the skin in harmful amounts. Prolonged and or repeated contact may cause defatting of skin and dermatitis. Methanol can be absorbed through the skin, producing systemic effects that include visual disturbances.
  • Ingestion: May be fatal or cause blindness if swallowed. Aspiration hazard. Cannot be made nonpoisonous. May cause gastrointestinal irritation with nausea, vomiting and diarrhea. May cause systematic toxicity with acidosis. May cause central nervous system depression, characterized by excitement, followed by headache, dizziness, drowsiness, and nausea. Advanced stages may cause collapse, unconsciousness, coma, and possible death due to failed respiratory failure. May cause cardiopulmonary system effects.
  • Inhalation: Methanol is toxic and can very readily form extremely high vapor concentrations at room temperature. Inhalation is the most common route of occupational exposure. At first, methanol causes CNS depression with nausea, headache, vomiting, dizziness and incoordination. A time period with no obvious symptoms follows (typically 8-24 hrs). This latent period is followed by metabolic acidosis and severe visual effects which may include reduced reactivity and/or increased sensitivity to light, blurred, double and/or snowy vision, and blindness. Depending on the severity of exposure and the promptness of treatment, survivors may recover completely or may have permanent blindness, vision disturbances and/or nervous system effects.
  • Chronic: Prolonged or repeated skin contact may cause dermatitis. Chronic exposure may cause effects similar to those of acute exposure. Methanol is only very slowly eliminated from the body. Because of this slow elimination, methanol should be regarded as a cumulative poison. Though a single exposure may cause no effect, daily exposures may result in the accumulation of a harmful amount. Methanol has produced fetotoxicity in rats and teratogenicity in mice exposed by inhalation to high concentrations that did not produce significant maternal toxicity.


K Willert, R Nusse Beta-catenin: a key mediator of Wnt signaling. Curr. Opin. Genet. Dev.: 1998, 8(1);95-102 PubMed 9529612

A review of the Wnt/beta-catenin pathway and the protein and receptor interactions involved.


K M Cadigan, R Nusse Wnt signaling: a common theme in animal development. Genes Dev.: 1997, 11(24);3286-305 PubMed 9407023

Outlines the evidence for the interaction of the Frizzled receptor with the Wnt protein.


W James Nelson, Roel Nusse Convergence of Wnt, beta-catenin, and cadherin pathways. Science: 2004, 303(5663);1483-7 PubMed 15001769

Explores the interrelationship between the Wnt, beta-catenin and cadherin pathways by examining the role of receptor activation and repression to control gene expression.


Amy Bejsovec Wnt pathway activation: new relations and locations. Cell: 2005, 120(1);11-4 PubMed 15652476

Newer research made possible by advancements in imaging technology have better elucidated the finer interactions in the Wnt/beta-catenin pathway.

Lab 4

Musashi protein

--Z3336051 15:04, 29 March 2012 (EST)

  • RNA binding protein; stem cell marker [1]
  • first discovered 1996 [2]
  • Molecular size: 1551 bp, i.e. 170.61 kD


Antibodies that recognise the Musashi protein:


  1. Michael G Kharas, Christopher J Lengner, Fatima Al-Shahrour, Lars Bullinger, Brian Ball, Samir Zaidi, Kelly Morgan, Winnie Tam, Mahnaz Paktinat, Rachel Okabe, Maricel Gozo, William Einhorn, Steven W Lane, Claudia Scholl, Stefan Fröhling, Mark Fleming, Benjamin L Ebert, D Gary Gilliland, Rudolf Jaenisch, George Q Daley Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia. Nat. Med.: 2010, 16(8);903-8 PubMed 20616797
  2. S Sakakibara, T Imai, K Hamaguchi, M Okabe, J Aruga, K Nakajima, D Yasutomi, T Nagata, Y Kurihara, S Uesugi, T Miyata, M Ogawa, K Mikoshiba, H Okano Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev. Biol.: 1996, 176(2);230-42 PubMed 8660864


Lab 5

List the key differences in the generation of transgenic and gene knock-out mice.

  • In transgenic
    • Advantage is that it is quick and easy – takes a few weeks
    • Disadvantage is that the nonspecific targeting of gene insertion may lead to undesired expression in the body
    • More than one copy of the same gene could become inserted in the genome
      • Could lead to easier identification
      • Could lead to toxicity and death of the animal
    • Tissue specific promoters can circumvent the issue of mutation specificity
    • Mainly used to look at dominant disorders
  • In gene knock-out
    • Advantage is its specificity in inserting a gene, in the same proportions found in the human genome
    • Disadvantage is its time and cost – takes a few years to develop a KO mouse
    • Could lead to the perpetually turned on or off state of an enzyme
    • KO is a modification which the activity of the gene is eliminated
    • KI is a modification in which a specific mutation or rearrangement is introduced and the gene remains functional
    • Mainly used to look at recessive disorders


Describe how genetically modified mice have been used to study the biological function of tropomysin

  • The type of cell used
    • Adipocytes were the cell type studied to observe tropomysin expression
  • The measurements used to determine whether any phenotypic changes were observed
    • A number of tests were used to monitor phenotypic changes:
      • Adipocyte proliferation rate – organ weight
      • Glucose tolerance test – glucose uptake levels
      • Western blot – gene expression
  • A model to explain how actin and changes in the levels of tropomysin can lead to the observed phenotypic changes
    • Overexpression of tropomyosin leads to an increase in filamentous actin filaments, which increases GLUT4 transporter translocation to the membrane, thus increasing glucose uptake.

Lab 6

Char.JPG

Observations with regard to cell morphology

  • cell population density was far greater in A than in B with most columns having greater TM4 counts.


Description of phenotypes in sample images (differentiated B35 cells)

  • In Genotype A
    • cytoskeleton pink
    • more branching and processes
    • nuclei pink
  • In Genotype B
    • cytoskeleton red
    • less branching and processes
    • blue nuclei


Lab 7

Homework

Key players in Wnt/β-catenin Signalling

  • Frizzled receptor
    • seven transmembrane receptor-like protein with an extracellular amino terminal rich in cysteine residues PMID 9407023
  • Wnt proteins
    • secreted lipid-modified signaling proteins that influence multiple processes in animal development
    • Evolutionarily conserved across species PMID 9407023
    • Wnt interacts with Frizzled proteins and LRPs to initiate signal transduction PMID 9407023
    • 19 proteins have thus far been identified in this protein family PMID 9407023
  • beta-catenin
    • transcription cofactor with TCF and LEF PMID 17693601
    • may play an important role in cell-cell adhesion by coordinating rearrangement of the actin cytoskeleton[1] through binding dynein at the "plus end" of actin filaments, close to the cytoskeleton[2]
    • beta-catenin undergoes ubiquitination by phosphorylation through the serine/threonine kinases casein kinase I (CKI) and glycogen synthase-3-beta (GSK-3-beta) and degradation by the 26S proteosome PMID 9312064
    • excess E-cadherin inhibits translocation of beta-catenin into the nucleus[3][4]
  • Cadherin protein
    • Prevents beta-catenin transmigrating into the cell, by binding it at the cell membrane PMID 15001769
  • Frizzled proteins


Future research

  • Taken from Wnt signaling: a common theme in animal development [5]
    • How do the Fz receptors work?
    • What is Dsh doing to transduce the signal?
    • What is the relationship between APC and Wnt signaling?
    • How does Arm/β-catenin get into the nucleus?

Lab 8

Homework

Mammalian cell line

HL-60 - promyelocytic leukemia


Tissue of origin

human peripheral blood


Original paper

S J Collins, R C Gallo, R E Gallagher Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature: 1977, 270(5635);347-9 PubMed 271272


Lab 9

Homework

Peer review

Group 1

I can tell from the wording that the history was not just regurgitated, but that the writer took the time to synthesise and summarise the content in the tables. Also regarding "normal function", the section is clear and well set out and easy to read. I found the use of tables in "clinical uses" to be effective in presenting the current clinical uses of testosterone.

However, there are also some areas that need improvement or modification. Aside from the spelling, punctuation and grammar errors (which should be fixed, by the way), where's the citation for figure 1? Also, the second citation in the reference list didn’t properly show up. And I also noticed you have two wikipedia images - we're allowed a maximum of one, so take at least of them off if you don't want to lose marks. You might also like to add a citation to the testosterone structure image. There are numerous other places with incorrect referencing as a result of incorrect coding, or sometimes no attempt at finding the reference at all.

I'm not sure you need a table under "biosynthesis".. Sometimes less is more. Also and instead, I would prefer to see subheadings for the four steps you mentioned. That would be helpful.

What would also be helpful would be a diagram for the "regulation" section from the peer reviewed literature supporting what you're writing.

Also, take "proteins" and "structure" out of "signalling pathway". They're not elements of the pathway! Put them under a different section, or make separate sections for them.

Finally, I think a few external links wouldn't do you harm, to such web pages as maybe major facilities/journals/institutes for the research of testosterone, or complementary info or diagram web pages.

Hope that helps.

Group 2

I am quite impressed by the layout of the "research" section, as well as its referencing and the use of relevant images to support your points. The incorporation of the table to summarise the study's findings was also effective. And I like the nice touch of the "related studies" links. But one question: besides the anti-VEGF research, why did you only talk about research relating to AMD? Why not the other disorders as well, since you discussed one of them?

The "abnormal function" section is also well laid out. However, at the same time, there are a few blatantly concerning issues. The least of issues is that the first box in the "description" column is not referenced. The more major issue is that there are three wikipedia images!!! Mark clearly specified we are only allowed a maximum of one. Please remove at least two of them, if you don't want to be marked down.

There were a few sections where I didn't feel that the headings matched their content. Firstly, the "introduction" sounds more like an essay opening than a wiki page opening. Go take a look at some cell signalling pages on wikipedia for an idea of structuring intro for a wiki page. Secondly, the content of the "function" section isn't talking about the function of VEGF. It would more appropriately be named "production of VEGF". As for the function of VEGF, you mentioned vasculogenesis, angiogenesis and lymphangiogenesis, which 'are' relevant - you should have expanded on these instead. Third, the "signalling pathway" section has a number of flaws. The table is good, but it is a summary of information presented. There is so far no content posted that the table is summarising!Also, the table lacks references. I also noted that the last sentence of VEGFR-1 is incomplete. The rest of the receptor pathways, as well as the image in the "signalling pathway" section, all need referencing.

Going through your images, I noticed that your first image also requires a reference. And the copright notice for "Chart-VEGFR2.gif" is broken. Anyway, the relevant permissions section should be copy-pasted into the description box for the uploaded image, not linked to.

I hope the feedback has been constructive, albeit leaning on the negative side, and I hope my comments help you to improve your wiki page.

Group 3

NO REFERENCES?!?!?!?!?!

It is understandable if one member of the group has technical difficulties in inserting citations, but for there to be no coding for any references to appear at the end of the wiki by any member of the group? It's a bit hard to accept. Typing up content without referencing is like borrowing a stranger's car without asking and without expressing thanks. You could wait till you return the car to say thanks, but it's just not the same.

I do, however, like the simplified concise explanation of the concept of apoptosis in the intro. For a reader new to the concept it is helpful to express it in layman's terms. But due to the lack of referencing I don’t know if that was an original concept, or one taken from another source.

Another thing I liked was the good depth of research conducted on the current research on apoptosis. A suggestion could be looking for research that people look to do in the future.

Something not so praiseworthy is the last entry under history, which is unbelievably condensed…….. you might want to expand a bit on that. And then find the individual references for each paper that demonstrated the landmarks in apoptosis history.

Furthermore, half the content under "function" wasn't about function, and the half that was was all about caspases……. I was a bit lost as to why only caspases were mentioned. Reading it made me feel like I was reading a wiki page solely focusing on capase signalling.

As for "signalling pathway", an image would've been nice. Besides the fact that this wiki page has a blatant absence of images, even one image depicting a section of the signalling pathway would have been helpful. At the moment I am clueless as to how they relate nor what they do. And also, you have 1 week to (theoretically) write about the other 15 proteins that supposedly only comprise "some of the proteins involved". Regarding that point, you will need to come up with a qualification for why you decided to showcase these 15 and not the rest.

Group 4

The "normal function" section is especially well done. I am impressed by the clear layout, referencing, and relevance in the picture. Together it facilitates in drawing me in and engaging my interest. I'd like to point out, however, you're missing the reference for the figure.

Frankly, the rest of the page gives the impression of, at best, half-heartedness. I won't comment on "abnormal function", which currently does not exist. For example, the introduction sounds like an essay intro and not a wiki. This may have been an unintentional error arising from the unfamiliarity of how wiki intros are set out, and if that's the case, I suggest looking up some examples of introductions of cell signalling pathways on Wikipedia. Another example is "history". It seems awfully short to me… surely more has taken place in the way of scientific advances for notch signalling from 1917 till 2012? Like what about the gap between 1930-1978 and also 1980-1995? Additionally, I think the image linked under "proteins and receptors" would be more appropriate under the "pathways" section. Instead, images depicting the gene sequences or even the 3D crystal structures should be included under "proteins and receptors".

Just another suggestion is beginning your wiki page with the name of the signalling pathway you are talking about. That way it's clear from the beginning what you are writing about.

Group 6

I really enjoyed reading the "signalling pathway" section. It was clearly set out, with good use of subheadings. The way the text closely related to the image also helps new readers to follow along easily. I also thought the "abnormal function" section was well set out. A few images would make it more helpful for readers.

Some improvements that I think can be made include adding a page heading with the signalling pathway, before "introduction". I was reading "Introduction" and I wasn't sure what exactly your page was focusing on. I'm guessing it's insulin signalling, in which case I don't think the content on abnormal and normal function belongs in the intro; they should be brought up in separate sections later on (which you do have). Having said that, I think something like "structure" does belong here, if you're not sure of what additional content to add. Another point to note is that you should add a caption to the image and reference it; the same comment goes for all your other images.

The "history" section needs expanding - how does the discovery of things like GSK3 and PI3K relate to the advancement of our understading of insulin signalling? It's not detailed enough to be relevant.

The "insulin receptor" section is awfully empty. Furthermore, the way the content is written didn't really engage me - it sounds very monotonous. Additional things I would've expected to learn about in this section would've been along the lines of what gene(s) code for it, and since your image depicts it, what the role of the juxtamembrane motif, ATP binding site, regulatory loop, and C terminus do.

There was a prominent lack of referencing in "normal function". Did you refer to one source only for all that information, or did you just not bother to acknowledge the rightful authors?

Finally, I noticed you have three images on your page (there should be four members in your group), and two of them don’t provide citations and two of them again don't provide copyright information. Did you get them straight from wikipedia? Note that we're only allowed a maximum of one wikipedia image.

I hope these comments are helpful in pointing you toward what can be improved.

Group 7

I'm impressed at the number of student drawn images. They are very helpful in helping me to visually picture the explanations given. However, you still need to remove at least one wikipedia image. We are only allowed one on the page before being marked down. Also, the non-student drawn images still need proper captioning and citation.

The "receptor structure" section was very informative, and I'm looking forward to when the beta-1-adrenoceptor structure section is complete. As for the "pathway" and "normal function", I think they should be divided up into separate sections for clarity. It would also be nice to add a section at the bottom outlining the research being conducted in this field nowadays. But overall, good effort.

Group 8

You are one of the few groups to have a student drawn image for pathway, and the image is very relevant to the content on the pathway. The sections "abnormal function" and "proteins" are structured and presented well, although some pictures of the effects of LAD would be helpful for "abnormal function", and the integrin picture in "proteins" needs a caption and proper citation.

Besides the fact that there is nothing under "history", I think that section should go before "pathway". As for "introduction", "pathway" and "normal function", where are the citations??? And for the person doing "normal function", are you sure catalysation is a real word? If it is, shouldn't it be in the glossary?

Group 9

Firstly, good number of references posted, especially for the little amount of content on the page already, although I noticed a signficant number of repeated references - check the tutorial for how to combine them. Also, you might want to remove your z ID tags from this page. They don’t belong on wikis.

I strive to stick to constructive criticism, so the comments in my review can help identify specific areas to improve. Inevitably, for a page with much work to be done, there will be proportionately less positive criticism. You can understand that it's hard to do so when half the page is missing. I recognise that we still get a week to complete the page, but looking at the state of your progress after 10 weeks of semester, there is an abnormally large amount of work left to be done so I don't think the level of completion is satisfactory.

I won't make comments on the "receptor" and "proteins" sections, because there is no content; nor on the "abnormal function" section, which has little beyond a quote.

Please reorganise the order of your headings. I feel a lack of coherency when the history comes after the pathway. Also, check your spelling and grammar. I found multiple errors in phrasing and coherency under "introduction" and "pathway" alone.

For "introduction", the third sentence should have a reference, since you referenced all the other content you introduced. And I wonder if its direction should be tailored to better reflect the content to be expounded in the rest of the page.

Regarding the pathway section, it sounds more like "introduction to the pathways". You mentioned three pathways, but haven't said anything about them. Furthermore, you haven't provided any references for the literature supporting your supposed three pathways that have been discovered. The image doesn't help in this respect, because it doesn't show three pathways either. How does it relate to the text in that section?

Finally, you mentioned the glossary comes from citable sources (spelling corrected for "cite-able"). Where are these citations on your glossary entries?

I hope these comments help you work on the parts of the wiki that have seen good progress so far.


--Mark Hill 13:04, 17 May 2012 (EST) This is indeed a comprehensive critical assessment of each project showing extensive effort, well done. You might consider structuring/formatting your assessment so that the group can easily review your comments.

Lab 10

submitted worksheet for assessment item

Lab 11

no assessment item

Lab 12

homework

  1. sequencing-by-ligation

Charles Coutton, Raoudha Zouari, Farid Abada, Mariem Ben Khelifa, Ghaya Merdassi, Chema Triki, Denise Escalier, Laetitia Hesters, Valérie Mitchell, Rachel Levy, Nathalie Sermondade, Florence Boitrelle, François Vialard, Véronique Satre, Sylviane Hennebicq, Pierre-Simon Jouk, Christophe Arnoult, Joël Lunardi, Pierre F Ray MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia. Hum. Reprod.: 2012, 27(8);2549-58 PubMed 22627659

  1. This publication details the utilisation of the sequencing-by-ligation DNA sequencing technique to test the impact of mutations and deletions on the DPY19L2 gene on the prevalence of globozoospermia. This form of DNA sequencing was quite central to the experimental outcome, in elucidating the introns and exons in this gene, as well as to identify the specific deletions, or lack thereof, in the various individuals who participated in the trial. Results of the type of sequencing-by-ligation adapted by the researchers enabled the researchers to arrive at the conclusion that molecular alteration of DPY19L2 was commonly observed in globozoospermia.

Test section

heading

sub-heading

sub-sub-heading

sub-sub-sub-sub-heading
This is the lowest possible header.
  1. Colin Jamora, Elaine Fuchs Intercellular adhesion, signalling and the cytoskeleton. Nat. Cell Biol.: 2002, 4(4);E101-8 PubMed 11944044
  2. L A Ligon, S Karki, M Tokito, E L Holzbaur Dynein binds to beta-catenin and may tether microtubules at adherens junctions. Nat. Cell Biol.: 2001, 3(10);913-7 PubMed 11584273
  3. F Fagotto, N Funayama, U Gluck, B M Gumbiner Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus. J. Cell Biol.: 1996, 132(6);1105-14 PubMed 8601588
  4. J Heasman, A Crawford, K Goldstone, P Garner-Hamrick, B Gumbiner, P McCrea, C Kintner, C Y Noro, C Wylie Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell: 1994, 79(5);791-803 PubMed 7528101
  5. K M Cadigan, R Nusse Wnt signaling: a common theme in animal development. Genes Dev.: 1997, 11(24);3286-305 PubMed 9407023