User:Z3418837

From CellBiology

Add your own student page to the site.

Add your signature for Lab attendance.

--Z3418837 (talk) 15:45, 13 March 2014 (EST)

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Add a sub-heading.

Add an external Link.

PubMed

Add an internal Link.

Mickey Mouse

This is a wonderful picture of two cells.

Reference

Tomohiro Shimada, Yukiko Yamazaki, Kan Tanaka, Akira Ishihama The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS ONE: 2014, 9(3);e90447 PubMed 24603758


This is about Prokaryotes.[1]

Cristina Y Zamora, Nathaniel S Schocker, Michelle M Chang, Barbara Imperiali Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars. Meth. Enzymol.: 2017, 597;145-186 PubMed 28935101

Aharon Oren On names of genera of prokaryotes that are later homonyms of generic names with standing in the zoological or the botanical nomenclature. Proposal of Neomegalonema gen. nov. and Neomegalonema perideroedes comb. nov. as replacements for the prokaryotic generic name Meganema and the species name Meganema perideroedes. Int. J. Syst. Evol. Microbiol.: 2017; PubMed 28933323

Dekang Liu, Guido Keijzers, Lene Juel Rasmussen DNA mismatch repair and its many roles in eukaryotic cells. Mutat. Res.: 2017, 773;174-187 PubMed 28927527

Joseph W Briggs, Ling Ren, Kristi R Chakrabarti, Yien Che Tsai, Allan M Weissman, Ryan J Hansen, Daniel L Gustafson, Yousuf A Khan, Jonathan D Dinman, Chand Khanna Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus. PLoS ONE: 2017, 12(9);e0185089 PubMed 28926611

Reza Hosseini Ghatar, Tahereh Soltantoyeh, Motahareh Bahadori, Jalal Khoshnoodi, Forough Golsaz Shirazi, Mahmood Jeddi Tehrani, Mohammad Mehdi Amiri, Fazel Shokri Polyclonal Antibody against Different Extracellular Subdomains of HER2 Induces Tumor Growth Inhibition in vitro. Iran J Immunol: 2017, 14(3);200-214 PubMed 28919583


  1. Tomohiro Shimada, Yukiko Yamazaki, Kan Tanaka, Akira Ishihama The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS ONE: 2014, 9(3);e90447 PubMed 24603758


Individual Assessments

Lab 1

Chloroplast present in Eukaryotes and not in Prokaryotes.png

Chloroplast present in Eukaryotes and not in Prokaryotes [1]

  1. Gregory P Henderson, Lu Gan, Grant J Jensen 3-D ultrastructure of O. tauri: electron cryotomography of an entire eukaryotic cell. PLoS ONE: 2007, 2(8);e749 PubMed 17710148 | PLoS One.

Copyright: © 2007 Henderson 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.

--Z3418837 (talk) 21:05, 18 March 2014 (EST)

Lab 2

1. On your own student page upload an image with the reference using the Pubmed formatting shown in the practical class tutorial last week.

Plant Nucleus.png

Plant Nucleus [1]

  1. Ruimin Gao, Peng Liu, Sek-Man Wong Identification of a plant viral RNA genome in the nucleus. PLoS ONE: 2012, 7(11);e48736 PubMed 23155403 | PLoS One.

Copyright © 2012 Gao 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.

--Z3418837 (talk) 21:42, 23 March 2014 (EST)


2. Identify a recent research article (not review) that uses either confocal microscopy or super-resolution microscopy as one of the study's techniques. Explain briefly (1 paragraph) how the microscopy technique specifically contributed to the article's findings.

In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach.

The purpose of the study was to measure the intracellular pH of organelles in situ using the physics behind confocal microscopy. This was achieved by extracting the colonies of Saccharomyces cerevisiae which were associated at various developmental stages. Confocal fluorescence microscopy was then used to monitor the variation in intracellular pH by targeting fusion proteins to subcellular compartments and then quantifying the fluctuations in the emission wavelengths obtained. The use of confocal microscopy imaging enabled researchers to distinguish between the various fluorescent fractions that were present in single cells. It also provided enhanced readings of emissions by the decreased photobleaching and amplified signal-to-noise ratios which detected a wide range of fusion proteins and intracellular pKa levels leading to precise quantifications of organellar pH. Ultimately, the use of confocal microscopy imaging has advanced to such a high level that determining intracellular level of pH is simple and efficient compared to the previous methods of microscopy and spectrofluorometry used. Confocal microscopy also promises much greater result as research proceeds.[1]

  1. Albert Pineda Rodó, Libuše Váchová, Zdena Palková In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach. PLoS ONE: 2012, 7(3);e33229 PubMed 22470445

--Z3418837 (talk) 23:45, 23 March 2014 (EST)

Lab3

1.Select 4 reference papers related to your selected topic sub-section. Read these papers and write a brief description of their findings and relevance to the selected topic sub-section. The reference along with your description should then be pasted on both your group discussion page and your own personal page.

Article 1 - Charge as a Selection Criterion for Translocation through the Nuclear Pore Complex.

Nuclear pore complexes (NPCs) are porous structures that selectively control the components that pass through the nucleus to the cytoplasm and vice versa. A common understanding on the selective aspect of NPCs comes from the various diameter sizes that NPCs can occupy, however scientists have been focusing on another governing principle of translocation that they believe is related to charge. It is known that proteins that undergo rapid translocation are highly negatively charged, whereas proteins that are blocked from this translocation process are positively charged. Analysis has shown that, proteins that occupy the inside of the pore channel are net positively charged and transport receptors are negatively charged. This indicates that translocation rates within NPS are dependent on the electrostatic interactions between transport receptors and NPC due to an immense gain in energy. Overall, the investigation suggests that the negative charge plays a significant role in determining which components pass through the NPC.[1]

Article 2 - Role of Molecular Charge in Nucleocytoplasmic Transport.

Similarly to article one, this study focuses on the role of charge in the selectivity of Nucleocytoplasmic transport. It highlights the significance of phenylalanine-glycine (FG) nucleoporins (Nups) in creating a selective barrier with net positive charges in the nuclear pore complex. It is known that the positive charge accounts for the passive diffusion of small molecules and transport-receptor facilitated diffusion of cargo molecules. However, it was recently hypothesized that a negative surface charge plays a significant role in determining which components pass through the NPC. This study aims to unpack how these charge interactions may impact transport kinetics and spatial transport routes for both passive diffusion and facilitated translocation. By using high-speed fluorescence microscopy, scientists were able to determine that the electrostatic interactions between the negatively charged surface receptors and positively charged FG-Nups although increased the likelihood of NPC binding, did not reveal the nuclear transport mode or spatial transport routes. Instead, Nucleocytoplasmic transport was found to be dependent on molecular size, signal and surface charge and not one or the other.[2]

Article 3 - Higher Nucleoporin-Importin β Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import.

For many years scientists have debated on whether the presence of an affinity gradient in NPCs for the import receptor Importin β increased or decreased Nucleocytoplasmic Import. However the answer to this postulation remained obscure. This study aimed to understand how this affinity gradient may have enhanced Nucleocytoplasmic Import by using agent based modelling (ABM) that looked at the association between rate constants and molecular binding. By employing different values of the affinity gradient, they have found that the rate of transport had increased by 10% compared to the pores lacking an affinity gradient. They also found that this effect was maximised at 200 µM for Importin β. Overall, this study highlighted the significance of Importin β Affinity in Increasing Nucleocytoplasmic Import rate.[3]

Article 4 - Assembly of Nsp1 Nucleoporins Provides Insight into Nuclear Pore Complex Gating.

Our current understanding suggests that the central transport channel which is a part of the NPC is formed by repeating subunits of FG-nups and serves as the selective barrier for incoming material. By analysing the spatial arrangements of nups, scientists can understand how NPC serves as a filter for macromolecules and allows for diffusion of small molecules below 40kDA. In this study, scientists used molecular dynamics to model the possible variations of Nsp1 (Nsp1-FG). They have discovered brush-like structures that consist of bundles that have been cross-linked to various nups. They have also found that transport factors are tightly associated with multiple FGS in cross-linking zones and also dissociate the bundles to widen the pores and allow molecules to enter. Overall, the model of the nuclear pore complex gating shows that the periphery of the NPC central channel consists of a few brushes with many cross-linked bundles due to the tethering of nups. However, the central regions shows a sieve-like structure of bundles with repeated cross-links as tethering of nups is less prominent.[4]

  1. Lucy J Colwell, Michael P Brenner, Katharina Ribbeck Charge as a selection criterion for translocation through the nuclear pore complex. PLoS Comput. Biol.: 2010, 6(4);e1000747 PubMed 20421988
  2. Alexander Goryaynov, Weidong Yang Role of molecular charge in nucleocytoplasmic transport. PLoS ONE: 2014, 9(2);e88792 PubMed 24558427
  3. Mohammad Azimi, Mohammad R K Mofrad Higher nucleoporin-Importinβ affinity at the nuclear basket increases nucleocytoplasmic import. PLoS ONE: 2013, 8(11);e81741 PubMed 24282617
  4. Ramya Gamini, Wei Han, John E Stone, Klaus Schulten Assembly of Nsp1 nucleoporins provides insight into nuclear pore complex gating. PLoS Comput. Biol.: 2014, 10(3);e1003488 PubMed 24626154

2.Select an image related to your selected topic sub-section (this can be from one of the 4 above or from elsewhere). The image should be uploaded (with all the required information: description, reference, copyright and student template) and pasted onto the project page sub-section and onto your own personal page.

Charge as a selection criterion for nuclear transport.png

Charge as a selection criterion for nuclear transport [1]

  1. Lucy J Colwell, Michael P Brenner, Katharina Ribbeck Charge as a selection criterion for translocation through the nuclear pore complex. PLoS Comput. Biol.: 2010, 6(4);e1000747 PubMed 20421988 |PLoS One.

© 2010 Colwell 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.

--Z3418837 (talk) 05:36, 3 April 2014 (EST)


Lab 4

1. Identify an antibody that can been used in your group's transport project.

Anti-Nuclear Pore Complex Proteins [Mab414] antibody - ChIP Grade (ab24609)

2. Identify the species deriving the antibody.

Mouse and it is monoclonal

3.Species reactivity

Reacts with: Mouse, Rat, Cat, Human, Saccharomyces cerevisiae, Xenopus laevis, Caenorhabditis elegans, Zebrafish, Vertebrates, Yeast.

4. IsoType

IgG1

5. Epitope and use.

ab24609 recognizes the conserved domain FXFG repeats in nucleaporins such as the p62, p152, p90 and this NPC family is used to study the morphology and composition of the nucleus and nuclear envelope.

6. Identify the working concentration for the antibody.

100 µl at 1 mg/ml

7. Applications:

WB: 1:5,000, IF: 1:5,000, IHC: 1:500, IP: 1:5,000, IEM: 1:5,000, ChIP: use at an assay dependent dilution.

8. Identify a paper that has used this antibody.

Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins.[1]

  1. J P Aris, G Blobel Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins. J. Cell Biol.: 1989, 108(6);2059-67 PubMed 2661560


--Z3418837 (talk) 23:24, 9 April 2014 (EST)

Lab 5

Knockout exercise

Lab 6

Z3418837 tm4 correct.jpg

Lab 7

Confocal Worksheet

Lab 8

Group 1:

- The introduction seems well informed and covers many aspects about phagocytosis which can be well understood by students of a non-science background. However, it should include the link to phagocytosis to transport. Also, I’m not sure if the links under introduction are temporarily there so you can reference them properly later, however, I suggest you reference them as soon as possible. If the links are just placed there for general purposes for others to click on then I think you should make a separate subheading for ‘links’.

- As for the structure of Plasma Membrane, there seems to be enough detail to describe the structure. However, I believe there needs to be images of the plasma membrane showing the components of the lipid bilayer. The text should also be properly referenced.

- Similarly for receptors, the detail presented is adequate and understandable but there needs to be images showing the different receptors and the text should be referenced properly.

- Mechanism of Phagocytosis: this section is good as it describes how everything you’ve mentioned about phagocytosis relates to transport including the structures of plasma membrane and receptors. There are also images of the Zipper model which is useful in allowing others to understand the concept more easily. However, I suggest you reference the text correctly and include a glossary for words like pseudopods, macrophages, etc… in case non-science background students don’t understand. Also, the definition of phagocytosis shouldn’t be placed in that section but should be at the introduction instead.

- As for diseases and current research, there needs to be more information and images. There should also be more references.

Group 2:

Introduction:

- Instead of using the phrase, “this leads to the idea” in the first sentence, you should use “this shows that…” because it makes it sound like it’s more of a postulation and not 100% solid fact. - It’s great that you have differentiated between the structure of the inner and outer mitochondrial membrane. This gives a better insight to the different roles they play in transport and the various mechanisms. - However, I think there is too much emphasis on the structure and role of the mitochondria in the introduction when it should also include information on the transport from the cytoplasm to the mitochondria. - The use of the mitochondria image is good in letting the viewers observe the major features of the mitochondria.

Proteins used in transport:

- This section is really well written as it talks about the various proteins used in transport. - The use of the image highlighting the various proteins is great for understanding the mechanisms involved.

Pyruvate transport into the Mitochondria:

- This section seems is good as it incorporates the proteins and signalling molecules needed for the transport of pyruvate. However, there needs to be more paragraphs on the transport of other important molecules to show why mitochondrial to cytoplasmic transport is important. - There should also be images to show the transportation of various molecules.

Diseases and current research:

- This section is very well written and is easy to understand. The use of the image relates to the text and is easy to navigate. - The section on current research in incomplete, however judging on the content written so far, it is really good. - It would be a good idea to use images for the current research section

The use of the glossary list really helps viewers understand and locate words they otherwise wouldn’t have known. The references are also correctly listed with each section having 3-4 references for each paragraph which is good. However, there is definitely more that needs to be written on the pages such as the transport of molecules other than pyruvate to make the page look more complete. Also, maybe a subheading for history should be placed to make the page look even more detailed and interesting. The history could include dates to which the structure of the mitochondria was found, the various imaging techniques that enabled us to view the mitochondria in a molecular level and also the discovery of important proteins included in the transport from the cytoplasm to the mitochondria. All in all, I think group has so far done a great job but needs to work on the criticism listed above.


Group 4:

- Most of the sections seem to be lacking information. The use of dot-points in the ‘fast and slow transport’ should be changed to paragraph form with more flow that could improve readability.

- There should definitely be more images along with the text which can explain certain processes.

- The kinesin section seems well constructed which is easy to read and more professional.

- I am impressed with the table form which is seen under motor proteins as it makes it easier to differentiate between the two motor proteins.

- There seems to be errors in the referencing which I suggest you guys should fix as soon as possible.

- There should also be a glossary list.

Lab 9

1. Write a hypothesis that you are going to test.

Large quantities of cytochrome C will be detected in the cytoplasm of the cell which can only be released via the intrinsic pathway during apoptosis.

2. Write aims of your experiment.

To measure the amount of cytochrome C within the cytoplasm.

3. Identify key techniques and procedures used in your investigation (Spell these out in some detail).

Centrifugation – To separate the supernatant from the tissue culture.

Western blotting – analytical technique used in this experiment to detect cytochrome C.

4. Identify suppliers that have resources that you will need for your study (create links to the supplier resource pages, kits, antibodies etc).

http://www.abcam.com/cytopainter-mitochondrial-staining-kit-green-fluorescence-ab112143.html

http://www.cellsignal.com/product/productDetail.jsp?productId=4272

http://www.lifetechnologies.com/order/catalog/product/KHO1051

http://www.mblintl.com/products/datasheets/JM-K257.pdf

5. Now prepare a flow diagram of how the experiment will be carried out and analysed.

Tissue Add 1ml of mitochondria Extraction Buffer and 1 ml of Cytosol Extraction Buffer individually Centrifuge Wash cells with PBS Incubate on ice Homogenize cells in an ice-cold dounce tissue grinder Collect supernatant as Cytosolic Fraction and Mitochondrial Fraction Load 10 μg of each cytosolic and mitochondrial fraction on a 12% SDS-PAGE Western blotting and probe with cytochrome c antibody.

6. What will different experimental results (outcomes) mean.

After using the Assay kit we will see a large amount of Cytochrome C within the Cytoplasm, and by analysing the Mitchondria, we will see pores formed on the outer membrane. Thus, proving that Apoptosis occurred due to the Intrinsic Pathway.

--Z3418837 (talk) 17:10, 15 May 2014 (EST)

Lab 11

How many Gbases (Gb) would you require to sequence the human genome to 40x coverage?

120Gb

Describe an advantage of using next-generation sequencing technology over microarrays to study gene expression?

Next-generation sequencing is advantageous over microarrays when it comes to discovering novel transcripts. This is because microarrays can only detect transcripts that correspond to existing genomic sequencing information, whereas next-generation sequencing can detect both known undiscovered transcripts.