User:Z3420257

From CellBiology

Add your own student page to the site. Add your signature for Lab attendance. --Z3420257 (talk) 15:52, 13 March 2014 (EST) Add a sub-heading.

Max, the talkative fellow

Add an external Link. Max

Add an internal Link. Lab 1

50pc

This is table on the cell membrane.

Figure 2: Cell membrane disruption induces phosphorylation of CREB, not only in wounded cells, but also in neighboring cells.

(A) Western blot analysis of extracts from MDCK and SK-N-MC cells, untreated or forskolin- and IBMX-treated, using anti-phospho-CREB (Ser133) (87G3) rabbit mAb with or without preincubation with phospho-peptide. Experiments were repeated three times. (B) Cells were wounded by scratching in the presence of fixable Alexa 488-dextran in 1.8 mM Ca2+ Ringer's solution, fixed 1 hour later, and then immunostained with anti-phospho-CREB antibody. Cells that were wounded and survived plasma membrane disruption show cytosolic labeling with the marker dye, Alexa 488-dextran. Immunostaining of the nuclear region with an anti-phospho-CREB antibody was observed in wounded cells (arrows). In addition to these cells, nuclear staining with anti-phospho-CREB antibody was also observed in neighboring cells. (C) Schematic drawing of nuclear localization in the image shown in (B). The nuclei of wounded cells were labeled as W, nuclei adjacent to Nucleus W were labeled as 1, nuclei adjacent to Nucleus 1 were labeled as 2, and so on. (D) Quantitative analysis of the immunostaining intensity of cell nuclei. Data were collected from three different images including (B), and are expressed as mean ± s.e.m. Numbers of cells observed are indicated in parentheses. *, P<0.01 versus “no wound”; #, P<0.01 versus W; ¶, P<0.01 versus Nucleus 1.


doi:10.1371/journal.pone.0042885.g002


Reference

Lijian Shao, Yingying Wang, Jianhui Chang, Yi Luo, Aimin Meng, Daohong Zhou Hematopoietic stem cell senescence and cancer therapy-induced long-term bone marrow injury. Transl Cancer Res: 2013, 2(5);397-411 PubMed 24605290


<reference/>


Sonya Levine, Laura Grabel The contribution of human/non-human animal chimeras to stem cell research. Stem Cell Res: 2017, 24;128-134 PubMed 28941410

Sean V Murphy, Aleksander Skardal, Lujie Song, Khiry Sutton, Rebecca Haug, David L Mack, John Jackson, Shay Soker, Anthony Atala Solubilized Amnion Membrane Hyaluronic Acid Hydrogel Accelerates Full-Thickness Wound Healing. Stem Cells Transl Med: 2017; PubMed 28941321

Thorsten R Doeppner, Mathias Bähr, Dirk M Hermann, Bernd Giebel Concise Review: Extracellular Vesicles Overcoming Limitations of Cell Therapies in Ischemic Stroke. Stem Cells Transl Med: 2017; PubMed 28941317


Lab 2

--Z3420257 (talk) 15:06, 20 March 2014 (EST)

120pix

(A) Western blot analysis of extracts from MDCK and SK-N-MC cells, untreated or forskolin- and IBMX-treated, using anti-phospho-CREB (Ser133) (87G3) rabbit mAb with or without preincubation with phospho-peptide. Experiments were repeated three times. (B) Cells were wounded by scratching in the presence of fixable Alexa 488-dextran in 1.8 mM Ca2+ Ringer's solution, fixed 1 hour later, and then immunostained with anti-phospho-CREB antibody. Cells that were wounded and survived plasma membrane disruption show cytosolic labeling with the marker dye, Alexa 488-dextran. Immunostaining of the nuclear region with an anti-phospho-CREB antibody was observed in wounded cells (arrows). In addition to these cells, nuclear staining with anti-phospho-CREB antibody was also observed in neighboring cells. (C) Schematic drawing of nuclear localization in the image shown in (B). The nuclei of wounded cells were labeled as W, nuclei adjacent to Nucleus W were labeled as 1, nuclei adjacent to Nucleus 1 were labeled as 2, and so on. (D) Quantitative analysis of the immunostaining intensity of cell nuclei. Data were collected from three different images including (B), and are expressed as mean ± s.e.m. Numbers of cells observed are indicated in parentheses. *, P<0.01 versus “no wound”; #, P<0.01 versus W; ¶, P<0.01 versus Nucleus 1.

doi:10.1371/journal.pone.0042885.g002


Reference

Tatsuru Togo Cell membrane disruption stimulates NO/PKG signaling and potentiates cell membrane repair in neighboring cells. PLoS ONE: 2012, 7(8);e42885 PubMed 22880128


Copyright

Copyright: © Tatsuru Togo. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Note - This image was originally uploaded as part of a student project and may contain inaccuracies in either description or acknowledgements. Please contact the site coordinator if the uploaded content does not meet the original copyright permission or requirements, for immediate removal.

Lab 2 Individual Assessment Part 1

--Z3420257 (talk) 22:42, 26 March 2014 (EST)

Nuclear Lamin

Nuclear lamin.png


A) Correlated image of Lamin B1-mEos2 signal with electron micrograph. White arrows point to Au beads used for alignment. (B) Higher magnification view of an area from A showing a possible nuclear pore (red arrowhead) with corresponding gap in Lamin B1-mEos2 signal. (C) SEM only image of panel B to show cellular ultrastructure. M, mitochondrion. G, Golgi apparatus. doi:10.1371/journal.pone.0077209.g003

Reference

Benjamin G Kopek, Gleb Shtengel, Jonathan B Grimm, David A Clayton, Harald F Hess Correlative photoactivated localization and scanning electron microscopy. PLoS ONE: 2013, 8(10);e77209 PubMed 24204771

| PLoS One.

Copyright

© 2013 Kopek et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Note - This image was originally uploaded as part of a student project and may contain inaccuracies in either description or acknowledgements. Please contact the site coordinator if the uploaded content does not meet the original copyright permission or requirements, for immediate removal.

Lab 2 Individual Assessment Part 2

In the study conducted by Braga et.al, the authors aim to prove how Reflectance Confocal Microscopy (RCM) can assist in the diagnosis of skin melanoma through a non-invasive method. Although the study used RCM alongside the analysis of basic transverse section, the RCM technique enabled them to make a pre-surgical diagnosis. RCM is able to give a 3D image of the skin cells, allowing the analysis of the different histological features, enabling pathologists and dermatologists to predetermine whether a lesion is malignant or benign. This concept, according to the authors, improves diagnostic accuracy.

Juliana Casagrande Tavoloni Braga, Mariana Petaccia Macedo, Clovis Pinto, João Duprat, Maria Dirlei Begnami, Giovanni Pellacani, Gisele Gargantini Rezze Learning reflectance confocal microscopy of melanocytic skin lesions through histopathologic transversal sections. PLoS ONE: 2013, 8(12);e81205 PubMed 24339910

| PLoS One.

--Z3420257 (talk) 22:20, 1 April 2014 (EST)

Lab 3 Invididual Assessment

Insulin-DependentEndo.png

Localization of insulin receptor and caveolin-1 in endosomes by immuno-gold electron microscopy. Isolated adipocytes were incubated with insulin at 100 nM for 10 min. Cells were then homogenized and the endosomal fraction isolated. Endosome vesicles were attached to grids, immunogold-labeled against caveolin-1 (6 nm gold particles) and the insulin receptor (15 nm gold particles), lyophilized and sputtered with a 2-nm tungsten film before examination by transmission electron microscopy. C and D are blow-ups from B; arrowheads indicate patches of caveolin-1 labeling; arrows indicate insulin receptor labeling. One experiment of three with similar results is illustrated. doi:10.1371/journal.pone.0005985.g004

Reference

Siri Fagerholm, Unn Ortegren, Margareta Karlsson, Iida Ruishalme, Peter Strålfors Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes. PLoS ONE: 2009, 4(6);e5985 PubMed 19543529

| PLOS One

Copyright

© 2009 Fagerholm et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Note - This image was originally uploaded as part of a student project and may contain inaccuracies in either description or acknowledgements. Please contact the site coordinator if the uploaded content does not meet the original copyright permission or requirements, for immediate removal.

--Z3420257 (talk) 13:51, 3 April 2014 (EST)

Journal Summaries

Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes

Mohammad F Saeed, Andrey A Kolokoltsov, Thomas Albrecht, Robert A Davey Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog.: 2010, 6(9);e1001110 PubMed 20862315

| PLoS One

Zaire ebolavirus (ZEBOV) is studied in this publication as it posts a threat to the general public. The study mentions different methods sited previously on how the virus infects the host cell. In this particular study, Saeed et. al has establish micropinocytosis as the pathway involved. The paper goes through the factors present in the pathway such as the requirement of free membrane cholesterol (lipid rafts), and the role actin filaments play in macropinosome formation, which involves a ruffle filament folding back on itself, creating a large cavity referred to as a macropinosome.

Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Fabienne Haspot, Amélie Lavault, Christian Sinzger, Kerstin Laib Sampaio, York-Dieter Stierhof, Paul Pilet, Céline Bressolette-Bodin, Franck Halary Human cytomegalovirus entry into dendritic cells occurs via a macropinocytosis-like pathway in a pH-independent and cholesterol-dependent manner. PLoS ONE: 2012, 7(4);e34795 PubMed 22496863

| PLoS One

Supporting the findings of the study conducted by Saeed et.al, this study also demonstrates the ability of cells to engulf materials via macropinocytosis. The involvement of actin filaments was demonstrated by incorporating cytoskeleton pharmacological inhibitors, resulting in the absence of macropinocytosis. Macropinocytosis is generally observed in larger viruses rather than small ones, which have been found to enter the cell via a different form of endocytosis. As demonstrated by both articles, macropinocytosis is a good area of study as it is involved in many disease pathways, especially those of a viral nature.

The Role of Flotillins in Regulating Aβ Production, Investigated Using Flotillin 1-/-, Flotillin 2-/- Double Knockout Mice

Vassilis Bitsikas, Kirsi Riento, Jonathan D Howe, Nicholas P Barry, Benjamin J Nichols The role of flotillins in regulating aβ production, investigated using flotillin 1-/-, flotillin 2-/- double knockout mice. PLoS ONE: 2014, 9(1);e85217 PubMed 24465508

| PLoS One

Flotillins are involved in many cell functions such as cell adhesion, signalling, cell membrane interactions and endocytosis. They generally function as adhesion proteins allowing for many cell interactions to occur. Its absence is seen to have an overall negative effect on endocytosis. While this study is not solely concentrated on flotillin’s involvement in endocytosis, it’s relevance lies in the demonstration of its role in cell-cell adhesion.

Although extensive research was conducted on this topic, there is a minimal number of sources covering the role of Flotillin in endocytosis. Any study found to have done so has seen to have lacked in depth analysis of the role it plays in endocytosis. Thus, it can be said that this topic is a good research option for future interests on endocytosis.

Roles of AP-2 in Clathrin-Mediated Endocytosis

Emmanuel Boucrot, Saveez Saffarian, Rongying Zhang, Tomas Kirchhausen Roles of AP-2 in clathrin-mediated endocytosis. PLoS ONE: 2010, 5(5);e10597 PubMed 20485680

| PLoS One

Many studies on endocytosis demonstrate the role of clathrin and the resulting clathrin coated vesicles. This particular study demonstrates the role of the heterotetrameric chalthrin adaptor complex, which is needed for the occurrence of clathrin-related endocytosis. This study focuses on how LDL’s enter the cell via this specific pathway. The absence of AP-2 is suggested to hinder the process of endocytosis in this instance. It is, in conclusion, a very important component of the clathrin-based endocytic pathway. Although not specific to macropinocytosis, this study is able to demonstrate that there is present a number of endocytic pathways involving different proteins.


--Mark Hill (talk) 14:20, 1 May 2014 (EST) Your references relate to the project topic, though how are you going to use them in the final project? If you remove the space in the pubmed reference format and make a few other changes, it will appear correctly as shown in the example below.

Emmanuel Boucrot, Saveez Saffarian, Rongying Zhang, Tomas Kirchhausen Roles of AP-2 in clathrin-mediated endocytosis. PLoS ONE: 2010, 5(5);e10597 PubMed 20485680

| PLoS One

Lab 4 Individual Assessment

--Z3420257 (talk) 15:12, 3 April 2014 (EST)

Purified anti-human CD144 (VE-Cadherin) Antibody

Adhesion junction: Tight junctions

Species raised in: Mouse

Species reacts against: Human

Types of application uses: Clone BV9 has been shown to block VE-cadherin, causing a redistribution of VE-cadherin away from intracellular junctions.6 Additional reported applications (for the relevant formats) include: Western Blotting1,2, immunofluorescence microscopy1,3, immunoprecipitation1,4, blocking angiogenesis in vitro4,5, inhibiting VE-cadherin reorganization4, and inducing endothelial cell apoptosis4. The LEAF™ purified antibody (Endotoxin <0.1 EU/μg, Azide-Free, 0.2 μm filtered) is recommended for functional assays


1. Almagro S, et al. 2010. Mol. Cell Biol. 30:1703. (WB, IF, IP) 2. Zhang F, et al. 2004. J. Biol. Chem. 279:11760. (WB) 3. Iurlaro M, et al. 2004. Am. J. Pathol. 165:181. (IF) 4. Corada M, et al. 2001. Blood 97:1679. (IP, Block) 5. Kooistra M, et al. 2005. FEBS 579:4966. (Block) 6. Corada M, et al. 2001. Blood 97:1679. (Block)


Include the following information: type of antibody (polyclonal, monoclonal), species raised in, species reacts against, types of application uses, and if available any reference using that antibod

| Biolegend


Anti-Early Endosomal Antigen 1 (C-terminal) antibody produced in rabbit

--Z3420257 (talk) 05:52, 10 April 2014 (EST)

Adhesion junction: Tight junctions

Type of antibody: Polyclonal

Species raised in: Rabbit

Species reacts against: Rat, Mouse, Human

Types of application uses:

immunoblotting (~160 kDa) and immunofluorescence. Detection of the EEA1 band by immunoblotting is specifically inhibited by the immunizing peptide

References 1. Stenmark, H., et al., J. Biol. Chem., 271, 24048-24054 (1996). 2. Gaullier, J.M., et al., J. Biol. Chem., 275, 24595-24600 (2000). 3. Callaghan, J., et al., Biochem. J., 338, 539-543 (1999). 4. Lee, S.A., et al., Proc. Natl. Acad. Sci. USA, 102, 13052-13057 (2005). 5. Simonsen, A., et al., Nature, 394, 494-498 (1998). 6. Gillooly, D.J., et al., Biochem. J., 355, 249-258 (2001).


Include the following information: type of antibody (polyclonal, monoclonal), species raised in, species reacts against, types of application uses, and if available any reference using that antibod

| Sigma Aldrich Data Sheet


--Mark Hill (talk) 14:21, 1 May 2014 (EST) All OK, perhaps next time include a link to the reference rather than just cite those as listed on the data sheet.

Lab 5 Individual Assessment

Done in class (please note that I forgot to log in, but proof of attendance is in the submission of the in-class assessment sheet).

Lab 6 Individual Assessment

--Z3420257 (talk) 16:19, 17 April 2014 (EST)


Graph.jpg

Lab 7

--Z3420257 (talk) 16:56, 1 May 2014 (EST) Discussion on current status of project.

Lab 8

Visit to confocal microscopy lab

Lab 9

Peer reviews/individual assessment

Group 2

The key points of the topic were clearly described. The introduction made good use of the funnel technique by introducing the role of the mitochondria and siting its special properties, ones that other organelles do not possess. It gave a brief understanding of how the process of transport works and a photo was also included to show the basic structure of the mitochondria.

The headings and sub-headings were wisely chosen. The way that the wiki was outlined made if much easier to understand. It was good to use the different macromolecules to look at things, although I can see that information is needed in the sections for fat and RNA transport.

The first section, which pertained to the transport of proteins into to mitochondria, was made easy to understand. You included all the definition in the paragraphs, as well as a colourful diagram relating to the topic. I think the diagram, being coloured, makes it more pleasant to the eye and attracts more attention/interest. I think you could add videos of how these TOM and TIM work to ease understanding.

The section on pyruvate transport was brief and precise. If you have more research papers that discuss it’s specific mechanism, maybe you could also add this. A photo/video could also be added to this section or maybe a photo of the 2 subunits responsible for its transport.

The section on disease was very interesting as it was on Parkinson’s, which is a disease familiar to all. It was made easy to understand. The only downside is that it could be more related to the topics and mechanisms discussed in the previous sections of the page. PINK was never mentioned in the subsequent sections. Maybe you could add a semi introduction of this in the section it is relevant to. The section on future research was also easy to understand. You should also add this to your glossary.

Overall, I think the topic was well outlined. It is easy to understand, which is important, as they will be read more so by your peers than academes. More visuals could be incorporated to make the page more interactive and interesting, as mentioned before. The references were well sited and each piece of information was linked to the specific paper it came from. All you need to do is add more information on to topics you have yet to research.

Group 3

Just by looking at the outline alone (the top section indicated by the grey box), I think that the flow of topics could be outlined more clearly. There are heading titles which could be improved and the use of layman terms could be rendered beneficial and the more specific terms be kept as subheadings. This will benefit the reader who is looking into just researching a particular topic, rather than reading the whole wiki. I think the beginning was better outlined than that of the latter half of the wiki. The part that breaks down the introduction, history, nuclear envelope etc. seems to have a better flow in general compared to the specifics (RanGTP etc.). I think a glossary section should be added.

The introduction and the history have yet to be uploaded. I think it is good that this is part of your wiki as the key points of the wiki could be included in the introduction. You can briefly introduce the topics you are going to be covering in this section. I think you can also cover the flow of the wiki in this section by briefly introducing the different subheadings you are to include. This way, the reader will be able to get a grasp of what you are trying to achieve and someone who is just merely doing research can quickly jump on to the subheading that is of interest to them. I think you might want to include a brief summary of the nucleus’ structure, as well as a photo. A photo including the membrane would be good so that you might be able to describe the transport a bit better. I see that you have also included a section on the nuclear envelope; this is very good as this is the main focus when studying transport. I can see that you have linked a photo, but I think being able to embed this onto the relevant section might be a better visual. Think about it this way, anything considered easy to understand will add to the reader’s interest towards you Wiki page. The history can just be brief, but will still be very interesting. A table might be a better way to write it, rather than doing it in a paragraph form.

I think the section on NPC’s was well introduced and a brief description of what they are was in the beginning. Having the different models laid out as they are is good. Having things divided into subtopics makes them easier to understand and organise in ones’ head. I think if you can find different photos of the different models would help the reader understand everything better. I think add a definition as well for FG-Nups, even if it is found in the glossary. Although it is describes in a different section, it should briefly described in the section where it is first mentioned. This saves the reader from having to revisit the previous sections and having to reread for better understanding. Maybe it might be a good idea to have the nucleoporins sections before the NPC section.

I think a bit of an introduction could be added to the section on RanGTPs. You can also add if Ran actually stands for anything. Everyone might be familiar with GTP, but the significance of Ran would be helpful.

More information is needed for the latter sections.

As a whole, I think the first section of the Wiki should serve as a good example of quality which should be followed all throughout. I think that this was done really well, and it is worth it to reiterate that it was well outlined. More visuals could be included. The glossary can also be added in the end, as you topic seems to have many acronyms. I think you will definitely ace it if you deliver the quality of work you did on the first section. You may also add more disease examples as this just keeps the interest of the reader, especially if it is one that they are familiar with. The topic is also well written and is an easy read for your peers, your target audience.

Group 4

Just based on the outline at the very beginning of the Wiki, I think you have chosen good topic headings and subtopics. I think as whole, this will help the page flow well. In the introduction, I think you should include a brief description of the topic, as well as a breakdown of the subtopics to aid in the understanding and the flow of the page. You should also add a photo of a neuron. Any visuals you might be able to add should be included. I think it is good that you added the section describing the difference between the two modes of transport – as well as other comparative sections in your page. You can keep this section short and brief, as details may be covered in the succeeding topics.

The good thing about the way you have outlined everything is that it is clear and precise. You also made use of a table in the section comparing kinesin and dynein. You may possibly add more sections to it to compare, as well as include a photo.

As much of the research has yet to be uploaded, I can only give a general feedback which is as follows: 1. Add more content 2. Make sure you include your references 3. Do not forget to add photos/any visual tools; maybe a video would be a good addition 4. Add a glossary to the page

I think you will do well if you continue to write the Wiki as you have doing, with the use of bullet points. This always makes things easier to understand. Your Wiki has so far been written in a fashion easily understood by your peers – especially those who have no background knowledge of the topic. You basically need to just fill in the sections which have not been filled as yet.

In-class Lab

--Z3420257 (talk) 16:08, 15 May 2014 (EST)

Hypothesis

The normal mechanism through which cell death occurs is apoptosis

Aims

We will measure apoptotic rate by subjecting the tissue culture to Flow cytometry.

Key Techniques & Procedures

Flow cytometry is used to detect the fragmented DNA Conofocal microscopy to assesses the morphological features of apoptosis such as . Southern blot to detect the DNA fragmentation. The The fragmented DNA will show a DNA ladder in comparision Hoecht staining of apoptotic nuclei (with Hoescht 33342 as a blue stain) to determine the condensation and fragmentation of the nuclei FlLICA (flourochrome inhibitor of caspase)

Suppliers

Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain is a high-quality solution of Hoechst dye for fixed- and live-cell fluorescent staining of DNA and nuclei in cellular imaging techniques

Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain

FLICA™, Fluorescent-Labeled Inhibitor of Caspases, is a simple yet accurate method to measure apoptosis via caspase activity in whole cells. FLICA-FAM

The E-Gel® Low Range Quantitative DNA Ladder The E-Gel® Low Range Quantitative DNA Ladder

Flow Diagram

Cell sample (non-adherent cells) → add necessary fluorescent dye (FLICA™)→ perform flow cytometry → with blebs present, apoptosis is proven to have occurred

===Outcome=== If apoptotic blebs and DNA fragmentation are detected by flow cytometry, the results suggest that the cells have died by apoptosis.