User:Z3399239

From CellBiology
   Add your own student page to the site.


   Add your signature for Lab attendance.

--Z3399239 (talk) 15:45, 13 March 2014 (EST)

   Add a sub-heading.

Sub-heading

   Add an external Link.

z3373930 z3420257

   Add an internal Link.

Lab 1

Cell Biology


Sodium Sulfate[1]


Susan R Compton, Carmen J Booth, James D Macy Lack of Effect of Murine Astrovirus Infection on Dextran Sulfate-induced Colitis in NLRP3-deficient Mice. Comp. Med.: 2017, 67(5);400-406 PubMed 28935001

Gang Liu, Qian Jiang, Shuai Chen, Jun Fang, Wenkai Ren, Jie Yin, Kang Yao, Yulong Yin Melatonin alters amino acid metabolism and inflammatory responses in colitis mice. Amino Acids: 2017; PubMed 28929336

Jun Xiao, Xu Wu, Wenbo Yu, Sha Liang, Jiangwei Yu, Yueyuan Gu, Huali Deng, Jiukun Hu, Keke Xiao, Jiakuan Yang Migration and distribution of sodium ions and organic matters during electro-dewatering of waste activated sludge at different dosages of sodium sulfate. Chemosphere: 2017, 189;67-75 PubMed 28926790

Julie Hablot, Laurent Peyrin-Biroulet, Tunay Kokten, Reine El Omar, Patrick Netter, Claire Bastien, Jean-Yves Jouzeau, Harry Sokol, David Moulin Experimental colitis delays and reduces the severity of collagen-induced arthritis in mice. PLoS ONE: 2017, 12(9);e0184624 PubMed 28926599

Troy Perry, Michael Laffin, Richard N Fedorak, Aducio Thiesen, Bryan Dicken, Karen L Madsen Ileocolic resection is associated with increased susceptibility to injury in a murine model of colitis. PLoS ONE: 2017, 12(9);e0184660 PubMed 28922370

References

  1. Kentaro Yuge, Tomoyuki Takahashi, Ngin Cin Khai, Kazuko Goto, Takako Fujiwara, Hisayoshi Fujiwara, Ken-Ichiro Kosai Intramuscular injection of adenoviral hepatocyte growth factor at a distal site ameliorates dextran sodium sulfate-induced colitis in mice. Int. J. Mol. Med.: 2014, 33(5);1064-74 PubMed 24604303

Individual Assessments

Lab 1

Bacillus Cell Membrane

Reference

Felix Dempwolff, Christian Reimold, Michael Reth, Peter L Graumann Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system. PLoS ONE: 2011, 6(11);e27035 PubMed 22069484


Copyright

Copyright: © 2011 Dempwolff 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.

--Z3399239 (talk) 15:07, 20 March 2014 (EST)

Lab 2

Nuclear membrane.png

Image of Nuclear membranes

Reference

Marie Garnier-Lhomme, Richard D Byrne, Tina M C Hobday, Stephen Gschmeissner, Rudiger Woscholski, Dominic L Poccia, Erick J Dufourc, Banafshé Larijani Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides. PLoS ONE: 2009, 4(1);e4255 PubMed 19165341


Copyright

© 2009 Garnier-Lhomme 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.

--Z3399239 (talk) 19:04, 20 March 2014 (EST)

Super-Resolution Microscopy Strategies in Cell Biology Using a Spinning Disk Microscope

Hosny N.A. et al. (2013) present a comparative study of super-resolution microscopy strategies using two methods of super-resolution microscopy; Photoactivation Light-Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), in conjunction with Spinning disk super-resolution imaging (SDSI) or Structured Illumination microscopy (SIM) and differing image analysis algorithms (RainSTORM, QuickPALM, GLRT, SOFI, 3B, Deconvolution-STORM (DeconSTORM), and Faster-STORM). Standard confocal microscopy has a resolution limit of 200nm, which has prevented further research into small molecular structures e.g. nuclear ultrastructure. Super-resolution microscopy has bypassed this resolution limit (described by Abbe's Law) and allowed for observation of structures as small as 30nm in size.

The study suggests that; 1) Multi-spectral SDSI can collect super-resolution images with good signal-to-noise (S/N), resolved in any selected axial plane within a cell. 2) PALM and STORM can both be used separately or in conjunction to produce super-resolution data. 3) SOFI has the best retention of image intensity information and provides the most accurate data reconstruction, in terms of spatially assigning all of the emission data found in the original images. 4) SIM was more appropriate for imaging 3D structures. 5) PALM/STORM SDSI could generate higher resolved data than SIM for single plane imaging dependent on the image processing algorithm used

Reference

Neveen A Hosny, Mingying Song, John T Connelly, Simon Ameer-Beg, Martin M Knight, Ann P Wheeler Super-resolution imaging strategies for cell biologists using a spinning disk microscope. PLoS ONE: 2013, 8(10);e74604 PubMed 24130668


--Z3399239 (talk) 12:13, 27 March 2014 (EST)

--Mark Hill (talk) 14:30, 3 April 2014 (EST) Good.

Lab 3

Research on trans-endocytosis; Trans-endocytosis is a process whereby material created in one cell is incorporated into another cell through endocytosis.

Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47–SHPS-1 system

Shinya Kusakari, Hiroshi Ohnishi, Feng-Jie Jin, Yuka Kaneko, Takaaki Murata, Yoji Murata, Hideki Okazawa, Takashi Matozaki Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47-SHPS-1 system. J. Cell. Sci.: 2008, 121(Pt 8);1213-23 PubMed 18349073

This article looks at trans-endocytosis of transmembrane proteins CD47 and SHPS-1. The study suggests that CD47 and SHPS-1 interaction initiates the transfer of CD47 from CD47-expressing cells to neighboring SHPS-1-expressing cells followed by the internalization of the ligand-receptor complex into the SHPS-1-expressing cells. SHPS-1 was found to undergo trans-endocytosis from SHPS-1-expressing cells to neighboring CD47-expressing cells, suggesting that trans-endocytosis of CD47 and SHPS-1 occurs bidirectionally. The study suggests that CD47 trans-endocytosis is implicated in the regulation of the CD47–SHPS-1 system.

This article is relevant to the sub-topic of trans-endocytosis as it describes the mechanism and physiological roles of endocytosis and gives a specific example of trans membrane protein trans-endocytosis.


Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell-Extrinsic Function of CTLA-4

Omar S Qureshi, Yong Zheng, Kyoko Nakamura, Kesley Attridge, Claire Manzotti, Emily M Schmidt, Jennifer Baker, Louisa E Jeffery, Satdip Kaur, Zoe Briggs, Tie Z Hou, Clare E Futter, Graham Anderson, Lucy S K Walker, David M Sansom Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science: 2011, 332(6029);600-3 PubMed 21474713


Qureshi et al. (2011) investigate the cell-extrinsic mechanisms of Cytotoxic T lymphocyte antigen 4 (CTLA-4). CTLA-4 is thought to be an important factor in the prevention of autoimmune disease. Trans-endocytosis of CD80 and CD86 (ligands shared by both CLTA-4 and CD28, a stimulatory receptor) inhibits co-stimulation of CD28. The study suggests trans-endocytosis as a possible cell-extrinsic model of CLTA-4 function whereby co-stimulatory ligands are removed from antigen presenting cells (APCs).

This study is relevant as it outlines a mechanism whereby trans-endocytosis is used to inhibit co-stimulation of another cell. The study also shows that trans-endocytosis can play a regulatory role in autoimmune diseases.


β-Arrestin1 Mediates the Endocytosis and Functions of Macrophage Migration Inhibitory Factor

Lishi Xie, Xiaohang Qiao, Yanfang Wu, Jie Tang β-Arrestin1 mediates the endocytosis and functions of macrophage migration inhibitory factor. PLoS ONE: 2011, 6(1);e16428 PubMed 21283538


Xie et al. (2011) investigate the effects of Macrophage migration inhibitory factor (MIF) and β-Arrestin on MIF endocytosis. The study suggests that MIF utilises β-arrestin1 as a molecular scaffold to maintain integrity and specificity of signalling.

This study relates endocytosis to cytokines and chemical mediators involved regulating inflammatory and immune responses.

Uptake of MIF via CPZ sensitive endocytosis.png

Image of Uptake of MIF via CPZ sensitive endocytosis

Reference

Lishi Xie, Xiaohang Qiao, Yanfang Wu, Jie Tang β-Arrestin1 mediates the endocytosis and functions of macrophage migration inhibitory factor. PLoS ONE: 2011, 6(1);e16428 PubMed 21283538


Copyright

© 2011 Xie 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.


Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor

Joyce Putters, Ana C da Silva Almeida, Peter van Kerkhof, Agnes G S H van Rossum, Ana Gracanin, Ger J Strous Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor. PLoS ONE: 2011, 6(2);e14676 PubMed 21347402


Putters et al. (2011) investigate the ability of Janus kinase 2 (Jak2) to bind to the growth hormone receptor, preventing endocytosis of growth hormone receptor (GHR). The article shows that Jak2 specifically inhibits GHR endocytosis independent of its kinase activity and Growth Hormone-induced and constitutive endocytosis undergo the same mechanism of endocytosis.

This article is relevant because it shows that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, therefore strongly suggesting that the modes of endocytosis are similar, if not identical.

Lab 4

--Z3399239 (talk) 15:11, 3 April 2014 (EST)

   Identify an antibody against an adhesion junction protein that is commercially available.

Anti-E Cadherin [HECD-1] antibody

   Add a link to the original data sheet page and identify the type of adhesion junction.

[1] - Tight junctions (present in all cells but most prevalent in epithelial cells and also forms the blood brain barrier)

   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 antibody. 

Type of Antibody: Monoclonal

Species raised in: Raised in humans

Species reacts against: Does not react with Mouse or Rat

Types of Application: Flow Cytometry, Immunocytochemistry, Immunoprecipitation, Immunohistochemistry, Immunohistochemistry (frozen sections), Immunocytochemistry/Immunoflourescence, Western Blot

References: N R West, J I Murray, P H Watson Oncostatin-M promotes phenotypic changes associated with mesenchymal and stem cell-like differentiation in breast cancer. Oncogene: 2014, 33(12);1485-94 PubMed 23584474

I Garcia-Murillas, R Sharpe, A Pearson, J Campbell, R Natrajan, A Ashworth, N C Turner An siRNA screen identifies the GNAS locus as a driver in 20q amplified breast cancer. Oncogene: 2014, 33(19);2478-86 PubMed 23752180

Tove Lekva, Jens Petter Berg, Ansgar Heck, Stine Lyngvi Fougner, Ole Kristoffer Olstad, Geir Ringstad, Jens Bollerslev, Thor Ueland Attenuated RORC expression in the presence of EMT progression in somatotroph adenomas following treatment with somatostatin analogs is associated with poor clinical recovery. PLoS ONE: 2013, 8(6);e66927 PubMed 23825587


--Z3399239 (talk) 11:31, 10 April 2014 (EST)

[2] - Junctional Adhesion Molecule 1(present in tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability)

Type of Antibody: Monoclonal

Species Raised in: Rabbit

Species reacts against: Reacts with Rabbit, Mouse, Human

Types of Application: Western Blot, Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)

References: Mirko Manetti, Serena Guiducci, Eloisa Romano, Irene Rosa, Claudia Ceccarelli, Tommaso Mello, Anna Franca Milia, Maria Letizia Conforti, Lidia Ibba-Manneschi, Marco Matucci-Cerinic Differential expression of junctional adhesion molecules in different stages of systemic sclerosis. Arthritis Rheum.: 2013, 65(1);247-57 PubMed 23001478


J Sundqvist, K L Andersson, G Scarselli, K Gemzell-Danielsson, P G L Lalitkumar Expression of adhesion, attachment and invasion markers in eutopic and ectopic endometrium: a link to the aetiology of endometriosis. Hum. Reprod.: 2012, 27(9);2737-46 PubMed 22767451


Types of Application

Lab5

--Z3399239 (talk) 15:09, 10 April 2014 (EST)

Lab 6

--Z3399239 (talk) 15:29, 17 April 2014 (EST)

B35 graph.png

Lab 7

--Z3399239 (talk) 16:55, 1 May 2014 (EST)

Lab 8

--Z3399239 (talk) 12:42, 15 May 2014 (EST)

Group 2 Peer-Review

Introduction: Good introduction and outline of mitochondria that shows development of knowledge beyond lecture material. Significant research is evident and well referenced/cited. Good use of imaging though possibly more descriptive diagrams could be used? Consider changing the first sentence from "The mitochondria are..." to just "Mitochondria are...". Make sure the image of the mitochondria has the relevant copyright clearance.

Protein Transport Into Mitochondria: Well referenced, relevant material. The diagram was not very well explained in my opinion and could use further development in describing what it is illustrating. The information presented looks good. I was confused by the statement "Such subunits are TIM17, TIM23 and TIM44 which are found in a ratio of roughly 2:2:2" would the ratio be the same as 1:1:1 or are you making a point that they function as dimers?

Pyruvate Into the Mitochondria: Good referencing. Looks a little under-developed but that may be due to lack of current understanding. Diagrams would help greatly here.

What can go wrong with Transport into Mitochondria: Good referencing up until the last two paragraphs. In line 4 should it be "It is made in the cytosol and imported into the mitochondria via the (TOM) complex.". Consider revising "When disrupted and the mitochondrial is damaged and it loses its membrane potential," as the sentence structure is confusing. This section could also be further developed and the picture associated explained and referenced.

Current or Future Research: Needs further development and information as well as diagrams. Some referencing present.

Overall: Some good content and referencing. Knowledge and research shows understanding that goes beyond lecture material. Relatively accessible to peers though more diagrams with descriptions would be helpful. The Glossary was a nice addition and could be further expanded. There are a number of repeats in the reference list, namely "Sébastien Herzig, Etienne Raemy, Sylvie Montessuit, Jean-Luc Veuthey, Nicola Zamboni, Benedikt Westermann, Edmund R S Kunji, Jean-Claude Martinou Identification and functional expression of the mitochondrial pyruvate carrier.". Headings and sub-headings are appropriate and easy to follow.


Group 3 Peer-Review

Nuclear Envelope: Referencing not complete yet and a very general overview of the topic. Lacks diagrams. Looks like its still a work in progress at this point. Some of the sentence structure does not flow well e.g. "The outer membrane faces the cytoplasm and is continuous and closely associated with the rough endoplasmic reticulum and has ribosomes attached to it.".

Nucleoporins: Good introduction to nucleoporins. FG-Nups section is well referenced and provides a good insight into the topic. Good use of tables.

Nuclear Pore Complex (NPC): Good referencing. Well structured and accessible to peers. Could use some diagrams.

RanGTP and RanGDP: Well referenced. Good information. Diagram is useful, referenced, and accessible.

Nuclear Transport Receptors: Well referenced. Good information. Consider revising the wording in "As these nucleocytoplasmic transport receptors can go both ways of nuclear envelope," in line 2. "It is understood that distinct associations are formed with different substrates[19] When it binds to a molecule aiming to take it from nucleus to cytoplasm the transport mediator is called exportins, whereas when it goes the opposite way they are called importins." is missing a full-stop between substrates and When.

Importins: Some referencing. Still a work in progress.

Exportins: Still a work in progress. Video lacks audio and is therefore somewhat hard to follow. Make sure the video is referenced correctly.

IMP and Ran-independent nuclear import pathways: Good overview and outline of β-catenin. Still a work in progress for other sub-heaings.

Overall: Certain sections are well put together e.g. RanGTP and RanGDP, but many parts are still missing. There are some useful diagrams but more could be used to help illustrate concepts. Referencing varies throughout the project but in general is good. At this point the project shows that some key concepts are understood but there are some that still need to be researched and added to. Relatively accessible information that goes beyond lecture material. Adding a Glossary could be useful?


Group 4 Peer-Review

Introduction: No references or images. Looks as if still a work in progress.

Fast and Slow Transport: Bullet points of basic information. Good references. Accessible information. Also a work in progress.

Motor Proteins: Good use of table to show differences between Kinesins and Dyneins (misspelled in table). Good references. Good description of role of different kinesins. Good outline of kinesins and dyneins. Informative and accessible diagram. Promising section of the project. Good work!

Overall: Some development of concepts beyond lecture material. The Motor proteins sections looks good though could use a few more illustrations. Generally still needs more information/diagrams and content. Reference list for certain parts are good. Relatively accessible information in the Fast and Slow Transport sub-heading. Use for the images next to reference list?

Lab 9

--Z3399239 (talk) 17:08, 15 May 2014 (EST)

Hypothesis

The normal mechanism through which cell death in the grown tissue culture 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

Hoecht staining of apoptotic nuclei (with Hoescht 33342 as a blue stain) to determine the condensation and fragmentation of the nuclei. Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios.

FLICA (flourochrome inhibitor of caspase) is a simple yet accurate method to measure apoptosis via caspase activity in whole cells. It applies the green fluorescent inhibitor probe FAM-VAD-FMK to label active caspase enzymes in the cell samples. FLICA probes are comprised of an inhibitor peptide sequence that binds to active caspase enzymes, a fluoromethyl ketone (FMK) moiety that facilitates an irreversible caspase binding event, and a fluorescent tag (either carboxyfluorescein or sulforhodamine B) reporter. The green poly caspase inhibitor FAM-VAD-FMK utilizes the fluorophore carboxyfluorescein and the multi-enzyme recognition sequence valine-alanine-aspartic acid (VAD). Upon encountering active caspases, the FLICA probe interacts with the enzymatic reactive center of the enzyme via the peptide recognition sequence, forming a covalent thioether adduct with the enzyme through the FMK moiety. After a simple wash step, the remaining green fluorescent signal is a direct measure of caspase activity at the time the probe was added.

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, . FLICA-FAM

The E-Gel® Low Range Quantitative DNA Ladder with ethidium bromide staining. 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

Outcomes

If apoptotic blebs and DNA fragmentation are detected by flow cytometry, the results suggest that the cells died by apoptosis. The Hoechst stain will present the apoptotic nuclei with a blue stained DNA. The FLICA will present a green stain of the caspase that are involved with apoptosis i.e. Caspases 3, 8, 9. The E-gel will present fragmented DNA, from the apoptic cell, will present a DNA ladder in comparison to the unfragmented live cell.