--Z3331639 16:05, 22 March 2012 (EST) --Z3331639 15:13, 8 March 2012 (EST) --Z3331639 14:58, 15 March 2012 (EST) --Z3331639 14:16, 29 March 2012 (EST) --Z3331639 14:22, 5 April 2012 (EST) --Z3331639 14:18, 19 April 2012 (EST) --Z3331639 14:19, 10 May 2012 (EST) Hello there
JOVE - External
Lecture 2 - Internal
Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
Timothy A Brown, Ariana N Tkachuk, Gleb Shtengel, Benjamin G Kopek, Daniel F Bogenhagen, Harald F Hess, David A Clayton Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol. Cell. Biol.: 2011, 31(24);4994-5010 PubMed 22006021
The article looks at the appearance of mitochondrial DNA and their compartmentalisation of their DNA. The paper articulates that normal microscopy has indeed been useful, however it did not show the specific detail and structural components of the mtDNA. It showed that in normal microscopy, the diffraction of light restricted the imaging resolution of particular objects and hence this limitation has been overcome through the use of both 2 and 3 dimensional approaches involving superresolution microscopes. The fast advancements in this technology as well as their detail in imaging allow for them to be of great importance in the identifying of finely detailed structures such as that observed in the mitochondria of the DNA.
Physical and Chemical Properties of Paraformaldehyde
Solubility (water) INSOLUBLE
Odour PUNGENT ODOUR
Specific gravity NOT AVAILABLE
pH 4.0 to 5.5
%Volatiles NOT AVAILABLE
Vapour pressure NOT AVAILABLE
Flammability FLAMMABLE SOLID Vapour density NOT AVAILABLE
Flash Point 70°C (cc)
Boiling point NOT AVAILABLE
Upper Explosion Limit NOT AVAILABLE
Melting point 163.0°C to 165.0°C
Lower Explosion Limit NOT AVAILABLE
Evaporation rate NOT AVAILABLE
Autoignition temperature 370°C
Bulk density 500.0 to 800.0 kg/L
Decomposition temperature NOT AVAILABLE
Density 0.88 g/cm³
Partition coefficient NOT AVAILABLE
Viscosity NOT AVAILABLE
Health Hazard Summary Low to moderate toxicity - irritant. This product has the potential to cause adverse health effects with over exposure. May release formaldehyde, which is a skin and respiratory sensitiser and classified as a confirmed human carcinogen (IARC Group 1).
Eye Severe irritant. Contact may result in irritation, lacrimation, pain, redness and blurring or dimness of vision. Prolonged contact may result in corneal burns and possible permanent damage.
Inhalation Irritant. Over exposure to dust may result in mucous membrane irritation of the respiratory tract, coughing and breathing difficulties. Upon decomposition, this product may generate formaldehyde, which is a potential sensitising agent and may result in asthma-like symptoms.
Skin Irritant. Contact may result in irritation, redness, rash and dermatitis. Prolonged or repeated contact may result in burns. May be absorbed through skin with harmful effects. May cause sensitisation by skin contact.
Ingestion Low to moderate toxicity. Ingestion may result in gastrointestinal irritation, nausea, vomiting, headache and diarrhoea. However, due to product form ingestion is considered unlikely. Maintain good personal hygiene standards.
Toxicity Data PARAFORMALDEHYDE LC50 (inhalation): > 170 mg/m³/1 hour (rat) TDLo (ingestion): 9 g/kg/90 day-continuous (rat) LD50 (ingestion): 800 mg/kg (rat) LDLo (skin): 10000 mg/kg (rabbit)
References for Extrinsic Apoptosis
Kari Högstrand, Eduar Hejll, Birgitta Sander, Björn Rozell, Lars-Gunnar Larsson, Alf Grandien Inhibition of the intrinsic but not the extrinsic apoptosis pathway accelerates and drives MYC-driven tumorigenesis towards acute myeloid leukemia. PLoS ONE: 2012, 7(2);e31366 PubMed 22393362
Looks at the important role of Myc in tumour developement and the association of that with acute Myeloid Leukemia as well its inducive relationship with extrinsic apoptosis.
Maike A Laussmann, Egle Passante, Christian T Hellwig, Bartlomiej Tomiczek, Lorna Flanagan, Jochen H M Prehn, Heinrich J Huber, Markus Rehm Proteasome inhibition can impair caspase-8 activation upon submaximal stimulation of apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) signaling. J. Biol. Chem.: 2012, 287(18);14402-11 PubMed 22408249
Looks at factor-related apoptosis and how that can induce extrinsic apoptosis. The relationship between caspase-9 activation and extrinsic apoptosis is also explored and transcription-dependent signalling is looked at for a relationship with tumour formation.
Georg Häcker, Anette Bauer, Andreas Villunger Apoptosis in activated T cells: what are the triggers, and what the signal transducers? Cell Cycle: 2006, 5(21);2421-4 PubMed 17102629
Overview of the effect of apoptosis on T-cells and their reduction due to signalling and activation.
Stuart A Rushworth, Lyubov Zaitseva, Susana Langa, Kristian M Bowles, David J MacEwan FLIP regulation of HO-1 and TNF signalling in human acute myeloid leukemia provides a unique secondary anti-apoptotic mechanism. Oncotarget: 2010, 1(5);359-66 PubMed 21307400
The paper expolores the effects of signalling on cancerous Acute myeloid leukemia (AML)cells as well as the counteractive methods by which protection against extrinsic apoptosis can occur in order to prevent cell death and tumour formations.
Musashi expresses in B-cell coardination of insulin through a response to apoptosis and proliferation in response to endoplasmic reticulum stresses in diabetes. Musashi genes regulate cell fate via Notch signaling, which regulates B-cell survival.
M Szabat, T B Kalynyak, G E Lim, K Y Chu, Y H Yang, A Asadi, B K Gage, Z Ao, G L Warnock, J M Piret, T J Kieffer, J D Johnson Musashi expression in β-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes. Cell Death Dis: 2011, 2;e232 PubMed 22113197
Anti-Mushashi: Primary Antibody:
Store at -20°C. Stable for 12 months at -20°C
PBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%
Tissue culture supernatant
Stem Cells >> Neural Stem Cells >> Glial Restricted Lineage >> Astrocyte
Label or Dye:
Alexa Fluor® 488
Transgenic- random chromosomal integration of foreign DNA. Homologous Recombination-knock out (KO) or knock in(KI)
-conditional KO/KI; tissue specific and inducible
TYPES OF TRANSGENICS: 1) Target protein may be over expressed: Excessive amounts of normal protein expressed in tissues which normally express it. E.g GH transgenic. 2) Target protein may be ectopically expressed: protein expressed in tissues which don not normally express it. 3) Mutated protein is expressed to produce consitutively active (gain-of-function) or dominant negative (loss of function) form of a protein or to mimic a mutated protein observed in a human genetic disease.
CONTEXT-SPECIFIC EXPRESSIION OF THE TRANSGENE
Design transgeinc construct that contain transcriptional regulatory elements that direct expression to a specific cell type or developemental stage.
1) do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?
Fan: No alteration in cell numbers between control and TM4 group.
Broken Fan: TM4 group seems to be reduced by 20%, indicating that TM4 possibly will inhibit this cell phenotype.
Stumped: There is a small increase in TM4 group about 4% which may not include the role that TM4 played in this particular phenotype.
Pronged and stringed: TM4 groups expressed an increase about 10 and 8 percent respectively. This may indicate that TM4 is actively involved in the events of neurites growth for these types of phenotypes.
Pygnotic: No cells found for the TM4 group. This may possibly suggest that TM4 entirely inhibits this phenotype.
It seems that the morphology of the control cells revealed the opposite characteristics in comparison to the Tm4 induced cells. Hence it can be concluded that Tm4 cells are involved in cell motility, and thus exposing these defined characteristics.
B: If so, how could Tm4 over-expression lead to this difference?
Tropomyosin (Tm) is one of the two major components of the actin filaments with the other component being troponin. Tm contains two polypeptide chains and is a long thin molecule. They are bound head to tail, forming a polymer that run over the actin subunits alongside the outer edges of the groove between the twisted actin strands. It binds to actin and acts as a molecular barrier. In relaxed muscle, Tm blocks myosin-binding sites of actin and prevents the crossbridge cycle from occurring. After the release of calcium ions, their binding causes a conformational change to troponin, shifting Tm's position on the actin filament and exposing the myosin binding sites. As a consequence Tm4 cells result in altering the cells motility, Thus exposing the observed characteristics.
C: What are the differences in phenotype (morphology) between cAMP over-expressing cells and control cells?
Genotype A (cAMP induced)
1) More processes are seen
2) Shorter and wider processes
3) Less dense and flourescent nucleus
4) Lamella appears yellowish on the edges
1) Less processes are seen
2) Longer and thinner as well as more compacted processes
3) Denser nucleus with a shinier appearance
4) Lamella appears reddish on the edges
D: If so, how could cAMP over-expression lead to this difference?
cAMP is mainly used for intracellular signal transduction, such as transferring into cells the effects of hormones like glucagon and adrenaline, which cannot pass through the cell membrane. It is derived from adenosine triphosphate and used for intracellular signal transduction in many different organisms, cAMP is also important in the binding and regulation of the function of specific ion channels. The main effect of cAMP in eukaryotic cells is its activation of protein kinase. It binds to specific locations on the regulatory units of the protein kinase, causing dissociation between the regulatory and catalytic subunits amd hence activating the catalytic units and allowing them to phosphorylate substrate proteins. These differing characteristics of Genotype A are hence articulated, as a result of these stimulating effects of cAMP on the cell.
LAB 7 TASK
Discussed the Introduction with group as well as identified research information and papers. I also finished editing and researching the History that was allocated to me. Now I'm researching the first part of TNF.
1842: Carr Vogt establishes that that in the process of vertebrate development the occurrence of cell death occurs.
1951: Glucksmann writes a prominent review on apoptosis and cell death in vertebrate developement lighting interest in the field of apoptosis once again.
1965: John Kerr writes his first paper on cell death
1972: John Kerr describes the process of cell death during vertebral development as ‘apoptosis’. He initially recognises the death of hepatocytes as shrinkage necrosis, however he further lights the difference between apoptosis and shrinkage necrosis was that there was no inflammation in apoptosis and hence he concluded that the two processes were different. He affirmed his hypothesis through observing the morphological structural difference between apoptosis and necrosis.
1974: The observation of DNA ladders and the linkage of cell death and DNA degradation was recognised.
1976: Programmed Cell Death was first seen in the nematode Caenorhabditis elegans by J.E. Sulston where it was described to be ‘a defined pattern of cell deaths’.
1980: Wyllie continues the relationship observed between cell death and DNA degredation and states that the process had a relationship with apoptosis and intracellular signalling.
1982-1988: Horvitz describes the process of cell death as a natural developemental process of the nematode Caenorhabiditis elegans. It was identified that the worm had over 1000 somatic cells before reaching its mature state, by which over 100 cells died through apoptosis. This difference in somatic number caused discovery of the genetic pathway recognising this programmed cell death and the generation of first cell death mutants; ced-1, ced-2 later occurred. Ced-3 and Ced-4 were later described in 1986 sparking immense discoveries and generating a great deal of interest in the apoptosis research. In the late 1980’s Vaux identified a component of the apoptosis mechanism naming it bcl-2 recognising it as an important cell death inhibitor. Cloning of bcl-2 also occurs.
1991: Yonish identifies p53 as a prominent inducer of apoptosis
1994: Birnbaum identifies an apoptosis inhibiting gene , cloning of Ced-9 occurs, the identification of the ‘reaper’ and Inhibitor Apoptotic Proteins (IAPs) then follows along with their description of functionality.
1995-2000s: Greater identification of genes and proteins were made; inducers and inhibitors of apoptosis, regulatory mechanisms and functions were also understood to a greater degree. Receptor and signalling pathways, along with important apoptotic factors such as that of IAPs, BAX (pro-apoptotic intracellular enzyme) TNF (tumour necrosis factor) and caspases (proteolytic intracellular enzymes) were also discovered. Through further research and greater understanding of apoptosis, we are able to futher our knowledge on the mechanisms by which diseases such as Cancers and AIDS work and the implications that apoptosis has on these diseases.
D L Vaux Apoptosis timeline. Cell Death Differ.: 2002, 9(4);349-54 PMID:11965486
1) Colon Carcinoma: T84 (CCL-248)
-Disease: colorectal carcinoma
-Derived from metastatic site: lung
3) K Dharmsathaphorn, J A McRoberts, K G Mandel, L D Tisdale, H Masui A human colonic tumor cell line that maintains vectorial electrolyte transport. Am. J. Physiol.: 1984, 246(2 Pt 1);G204-8 PubMed 6141741
Group 1 Feedback:
The main aspects of the marking criteria has been met on a relatively good level. The project page seems to demonstrated interesting propositions with testosterone and a good understanding of the topic seems to be shown. The colouring in the history page allows for easy understanding as well as the images seem to show interesting graphs. There is however, no hand drawn image and grammatical errors in the introduction should be addressed.
Group 2 Feedback:
Maybe a bit more info would be good, main point and concept was put through
Good use of table, well referenced and the colour choice was good, easy to read
Normal & Abnormal Function
I thought normal function should come just before abnormal function so that a comparison could be drawn and the signalling pathway could be better placed just after the introduction. Regardless, it was thoroughly presented with tables making it easy to read and understand with relevant images to each of the sub topics.
I thought that maybe the information in this section was a brief, could use a bit more information since it is a major part of the assessment. Also, it would be nice if there were some info under VEGF, so it won't seem like theres nothing done on it.
The information was well researched, but the layout wasn't as clear. Maybe the pictures should all go on one side of the page, to present a clearer page.
This assessment seems to be going in the right direction. With slightly more 'touching up' to do, it will be ready for submission.
Group 4 Feedback:
The information given seems to be good, however it lacks a bit more information and elaboration, specifically in the introduction. The information provided on the Normal functioning seems to be well elaborated and researched. However, there needs to be a show of abnormal functioning on the pathological level to allow for a compare and contrast of the project. The use of videos seems to be a great idea and is well integrated within the assessment. Self made drawing on the page is also needed in the near future and further research and referencing is recommended to allow for greater expansion on the information at hand. Overall, this assessment seems to be going in the right direction.
Group 5 Feedback:
-Introduction: I thought that the introduction was well written. It was put thoroughly and insightfully -History: I thought the history was good. It was well researched and easy to follow. -Function: I thought that this section was short and simple to read. The pictures were good and made things easy to follow. The video really is a good idea and gave me something to think about for my project. However it does seem slightly messy, so a slight organisation of the content would make it easy to follow. Overall content was good. -Abnormal function: I thought that the table really demonstrated the research they had done. However, greater referencing of this section is needed. -Further research: I didn’t think that it was simple to follow. It needs to be concise and follow a certain progression. Slightly all over the place and hence some focus is needed on organising the content in a logical manner Overall I thought the assessment was insightful, and showed a great deal of success in its contents and the way the assessment was presented with images, videos and tables allowing for interactivity. There needs to be more referencing is some sections as well as organisational work of the content to make it easy to follow.
Group 6 Feedback:
The Structure and content of the project seems to be of a good standard. All images used seem to coincide with the content given and the correlation between each section allows for greater understandability and cohesiveness. Despite the assignment meeting much of the criteria, there is room for improvement. Starting with the title of the project page to help understand what the project is all about. The introduction in my opinion lacks as it doesn't sum up the entire project and seems to disintegrate into subheadings which i found confusing. Also the image of 'structure of insulin' does not have its reference and copyright information and hence this needs to be looked upon. Overall, the project seems to meet much of the criteria however as I mentioned, improvement could be made.
Group 7 feedback:
-Introduction: The introduction had sufficient detail and presentation. The images were good and specifically the hand drawn image that complemented the introduction. I would stress a little more on the referencing.
-History: This section was done at a sufficient level and referenced. -Receptors: This section was well done however it still remained unfinished. It had many subheadings which didn't seem to bother me as the content was split into a variety of categories allowing for a logical sequence of events to be understood. The table seems to add some of that emphasis however it lacked detail.
-Pathway: This section was done well, and understood clearly, however I would place more emphasis on the referencing. Specifically for this section. -Normal functions: I thought that this section was also well done, it was clear and concise, and flowed really well. -Abnormal function: This section needs a little bit of formatting, however a great deal of research seems to be put on this part which is clearly shown by the referencing and the flow of the content. - Overall, I would be happy with this project as it went through material in sufficient detail, allowing for the reader to understand what the project is all about. I think with more research and diagrams, as well as referencing, the project will be ready for submission.
Group 8 feedback:
The introduction seems to start off well, however it is still short and there is a lack of hand drawn images. There doesn't seem to be any history present and Current research has not been started. The pathway section was very easy to understand and flow quiet well. There are also diagrams that support the content given. The referencing section needs more work, specifically in the first half of the project. Despite this the flow and content of the assessment seems to be well done. With more focus and improvement on these sections and use of tables and imaging, the assessment will be completed.
Group 9 Feedback:
- I think this part of the assessment should have an image to coincide with the overarching content of the project
- More information is needed on this part of the assessment, Image is rather good, however there should be some sort of logical sequence that could be followed. Perhaps a flow chart.
Receptors and Proteins
- More information is needed for this part, however the start seems to be ok
- This part has been done well, referencing as well as content seemed to be of great detail. I would recommend organising it a little bit so that the info of the table correlates well. So perhaps formatting, otherwise good.
- More info is needed, also this information can be tabulated.
- This section has been done well, however I think that to improve, maybe tabulate and provide greater referencing. As well as go into sufficient detail. Otherwise it is going in the right direction.
- Image seems to stand out to me, however some more information is needed, get into depth and more detail. The pathological aspects of it, otherwise there seems to be a good start to it.
Referencing and glossary seemed to have been done well.
Overall I think this project is heading in the right direction.
--Mark Hill 12:52, 17 May 2012 (EST) You have not completed the peer assessment process yet. If you have made comments on each project page they need also to be pasted here today for me to include in your individual assessment.
1. Identify a current technique used in gene sequencing.
Next generation sequencing
2. Identify a recent cell biology research paper that has used microarray technology.
Tomozumi Imamichi, Jun Yang, Da Wei Huang, Brad Sherman, Richard A Lempicki Interleukin-27 induces interferon-inducible genes: analysis of gene expression profiles using Affymetrix microarray and DAVID. Methods Mol. Biol.: 2012, 820;25-53 PubMed 22131024
3. What aspect of the research findings were contributed by the microarray technique.
Through performing Affymetrix DNA microarray and gene functional analysis using Database for Anootation, Visualisation and Integrated Discovery (DAVID) which is a web-basd bio informatics application that identifies biology associated with large genes which consequently come from genomic experiments such as microarrays. Through the use of this technology, researchers were able to show that IL-27 regulates the gene expression between T cells and macrophages in a different kind of manner. And as this research expands, greater knowledge of the genome will be acquired, allowing for further understanding to human defense mechanisms and counteracting diseases such as Cancer, which is articulated sufficiently in this paper.