- 1 Lab Attendance
- 2 Labs assessments
- 3 Lab Notes: week 1
- 4 Individual Assessments
- 5 Lab 1 Individual Assessment
- 6 Lab 2 - Image Referencing
- 7 Lab 2 Individual Assessment
- 8 Lab 3
- 9 Lab 3 Individual Assessment
- 10 Lab 5 Individual Assessment
- 11 Lab 6 Assessment
- 12 Lab 9 Assessment
- 13 Group Peer Review
--Mark Hill Thank you.
Lab Notes: week 1
Lab 1 Individual Assessment
Fig.1 DDX3 molecules
a) DDX3 molecules from top 6 docked structures overlapped together.
Copyright: © 2015 Mahboobi 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.
--Mark Hill Very good. You did not need to add all the information here on your page, it is only required in the image summary box. On the page where the image appears you simply correctly reference the image as described in class.
Lab 2 - Image Referencing
DDX3 molecules from top 6 docked structures overlapped together.
Lab 2 Individual Assessment
Brain activity occurs via chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are arranged to realize the synaptic transmission. The calcium channels are fundamental to the synapse and occupy a central position in AZ. Predictions of channel properties, numbers and positions on the nanometer scale has been provided through combination of quantitative functional studies with modeling approaches. However, without the elucidation of nanoscopic organization, the knowledge about molecular synaptic remains incomplete.
The Fluorescence microscopy is the method of choice for visualizing biomolecules in fixed and living cells. However, it fails to show organizational patterns at the molecular scale due to its limited spatial resolution.
The electron microscopy (EM) can reveal an organized arrangement of synaptic vesicles relative to Ca2+ channels at release sites using the immunogold labelling. However, specific labeling with antibody-coupled gold particles is inefficient and a relation between optimal tissue preservation and structural resolution must be made.
Thus, recently super-resolution microscopy (SRM) techniques have helped to elucidate the molecular structures involved in synaptic transmission. The development of super-resolution microscopy (SRM) techniques allows to bypass the diffraction barrier of ~300 nm in lateral dimensions and to bridge the gap between conventional light microscopy and EM. These emerging technologies offer promising new options for studying nanoscopic structures.
Therefore, the ideal microscopic technique should assemble efficient specific labelling possibilities of optical microscopy with the high spatial resolution of EM. The techniques that have these features are collectively named SRM. These include structured illumination microscopy (SIM), stimulated emission depletion (STED) and single-molecule based localization microscopy methods, such as photo-activated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). Then, SRM can provide better imaging and, consequently, improve our mechanistic understanding of neurotransmission.
--Mark Hill The reference is a good example of recent super resolution microscopy. You should not directly copy the text from the paper onto your own page here. If you do use the text (directly) it needs to be in quotation and referenced.
Lab 3 Reference Searching Search term: Laminin
Lab 3 Individual Assessment
Article 1: “Origin and Evolution of Laminin Gene Family Diversity”: Laminins are a family of multidomain glycoproteins that are important contributors to the structure of metazoan extracellular matrices. To investigate the origin and evolution of the laminin family, the researchers characterized the full complement of laminin-related genes in the genome of the sponge, Amphimedon queenslandica. Five Amphimedon laminin-related genes possess the conserved molecular features, and most of the domains found in bilaterian laminins, but all display domain architectures distinct from those of the canonical laminin chain types known from model bilaterians. This finding prompted to perform a comparative genomic analysis of laminins and related genes from a choanoflagellate and diverse metazoans and to conduct phylogenetic analyses using the conserved Laminin N-terminal domain in order to explore the relationships between genes with distinct architectures. Together, results suggest that gene duplication and loss and domain shuffling and loss all played a role in the evolution of the laminin family and contributed to the generation of lineage-specific diversity in the laminin gene complements of extant metazoans.
Article 2: “Laminins in basement membrane assembly”: The heterotrimeric laminins are a defining component of all basement membranes (BM) and self-assemble into a cell-associated network. The three short arms of the cross-shaped laminin molecule form the network nodes, with a strict requirement for one α, one β and one γ arm. The globular domain at the end of the long arm binds to cellular receptors, including integrins, α-dystroglycan, heparan sulfates and sulfated glycolipids. Collateral anchorage of the laminin network is provided by the proteoglycans perlecan and agrin. A second network is then formed by type IV collagen, which interacts with the laminin network through the heparan sulfate chains of perlecan and agrin and additional linkage by nidogen. The difficulty of imaging BMs in situ, without harmful extraction from tissues, has prevented a clearer understanding of BM architecture and our current model is largely based on a multitude of indirect clues rather than on direct observation. An intriguing aspect is that the thickness of a typical BM is of the same order as the dimensions of a single laminin molecule, which makes it unlikely that laminins are standing erect on the cell surface. Another question is whether the researchers actually know all the molecular interactions that are important for BM assembly and maturation. Some of current knowledge is based on early experiments with relatively crude protein preparations, and a search for additional interactions using modern recombinant and proteomic techniques might prove fruitful.
Article 3: "The laminin family": Laminins are large molecular weight glycoproteins constituted by the assembly of three disulfide-linked polypeptides, the α, β and γ chains. The human genome encodes 11 genetically distinct laminin chains. Structurally, laminin chains differ by the number, size and organization of a few constitutive domains, endowing the various members of the laminin family with common and unique important functions. In particular, laminins are indispensable building blocks for cellular networks physically bridging the intracellular and extracellular compartments and relaying signals critical for cellular behavior, and for extracellular polymers determining the architecture and the physiology of basement membranes. Therefore, the cell adhesion-promoting activity of laminin isoforms is now well characterized, also at the structural level. However, the specificity, if any, of the signaling pathways activated by the different laminin-binding integrins is not known.
Articule 4: "Laminin α4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain.”: A better understanding of the mechanisms regulating adipose tissue expansion could lead to therapeutics that eliminate or reduce obesity-associated morbidity and mortality. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. However, there is little understanding of its function in adipose tissue. These article describes the role of laminin α4, a specialized ECM protein surrounding adipocytes, on weight gain and adipose tissue function. Adipose tissue accumulation, lipogenesis, and structure were examined in mice with a null mutation of the laminin α4 gene (Lama4-/-) and compared to wild-type (Lama4+/+) control animals. Lama4-/- mice exhibited reduced weight gain in response to both age and high fat diet. The results suggest that laminin α4 influences adipose tissue structure and function in a depot-specific manner. Alterations in laminin composition offers insight into the roll the ECM potentially plays in modulating cellular behavior in adipose tissue expansion.
Figure 1. Domain structure and self-assembly of laminin-111.
(A) Schematic drawing of the laminin-111 heterotrimer. The three short arms of the cross-shaped molecule have a common domain structure and consist of laminin N-terminal (LN) domains, laminin-type epidermal growth factor-like (LE) domains, and L4 domains, as indicated for the α1 chain. The α1 chain uniquely contains five laminin G-like (LG) domains. LG1-3 likely interact with the C-terminal residues of the γ1 chain. (B) The three-arm interaction model of laminin self-assembly. The ternary nodes in the network are formed by the N-terminal regions of one α, one β and one γ chain. The long arm of the laminin heterotrimer is not involved in network formation. 
Copyright © 2012 Landes Bioscience This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
--Mark Hill 4 relevant references and a related image. You do not need to include the copyright information here, just in the image summary box.
Lab 5 Individual Assessment
B35 in TM4 overexpressed and wild type cells
--Mark Hill I will give you the mark for this practical when Dr Fath has completed his assessment.
Lab 6 Assessment
1.Identify an antibody that can been used in your group's extracellular matrix project.
Anti-Laminin antibody (ab11575). Function: binding to cells via a high affinity receptor, laminin is thought to mediate the attachment, migration and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components.
2.Identify the species deriving the antibody.
Rabbit polyclonal to Laminin
3.Identify the working concentration for the antibody.
Concentration batch dependent within range: 250 µl at 0.54 - 0.72 mg/ml. The referenced paper does not quote the concentration that was used.
4.Identify a secondary antibody that could be used with this antibody.
Some examples that could be used with this antibody:
Biotin-conjugated goat anti-rabbit IgG polyclonal (1/1000)
TRITC-conjugated donkey anti-rabbit IgG (H+L) polyclonal (1/200)
FITC conjugated goat anti-rabbit IgG used at a 1/100 dilution.
5.Identify a paper that has used this antibody.
Matthaei M et al. Endothelial cell microRNA expression in human late-onset Fuchs' dystrophy. Invest Ophthalmol Vis Sci 55:216-25 (2014). Human. Read more (PubMed: 24334445) <pubmed>24334445</pubmed>
--Mark Hill Very good. Note the different primary antibody concentration used for different techniques. Have you thought why that might be?
Lab 9 Assessment
Human Cell Line
Complete Growth Medium Vascular Cell Basal Medium: http://www.atcc.org/products/all/PCS-100-030.aspx#generalinformation
Medium: Vascular Cell Basal Medium (Vascular Cell Basal Medium contains essential and non-essential amino acids, vitamins, other organic compounds, trace minerals and inorganic salts)
Mouse Cell Line http://www.atcc.org/Products/All/SCRC-1045.aspx
Description The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria. Penicillin was originally purified from the fungi Penicillium and acts by interfering directly with the turnover of the bacterial cell wall and indirectly by triggering the release of enzymes that further alter the cell wall. Streptomycin was originally purified from Streptomyces griseus. It acts by binding to the 30S subunit of the bacterial ribosome, leading to inhibition of protein synthesis and death in susceptible bacteria. This solution contains 10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin.
--Mark Hill All correct here.
Group Peer Review
The page gives important information about proteoglycans. It focuses only on the Small Leucine-Rich Proteoglycans which is understandable because proteoglycans is an extensive subject. Therefore, other families should be at least mentioned, to give a notion of the vastness of proteoglycan families.
The history was very well presented, giving a good idea of the evolution of research, but I consider that the “future researches” need improvements. About Synthesis, it is greatly simplified; I think you could explain in a little more details, but I consider that the webpage is not finished yet. Regarding to structure, I do not understand why you divided decorin/byglycan and fibromodulin/lumican in different sections. Furthermore, it lacks some information about lumican, but I believe that the page is in progress and you will add it.
The section of the role of SLRPs in disease was best described so far. I liked the resources and images.
Finally, I liked the pictures and diagrams helping to understand better about it. Also, I like the references used. Overall, I consider a good page.
First, key points relating to the Integrins are clearly described. The contents are well-organised because they are divided in relevant sections. The introduction explained what is Integrins is very clearly and briefly and I consider this good for the reader (the only problem is the website link in the final should be placed in reference section).
The History section is not completely done, but I know the page is still in progress. The graphic showing the interest in researching Integrins was a very good idea and gives to the reader an interesting impression about Integrins history and perspectives.
The Structure section is very good and clearly, but I think the introduction in this section was a bit repetitive and very similar with the function. In the Function section, it is not clearly what is the “two functions” cited in the beginning of explanation, but even it is possible to understand the functions. Furthermore, the “interactions with the Extracellular matrix” is a amazing point that you explained.
Finally, the role of integrins in disease is clearly but it would be good if you put some images. The glossary was a very good idea, but I think it is still in progress.
Overall, considering that you are still working on the page, it is a good job so far. I think when you finish the page, including the history section, adding images and completing some sections, it will be very useful for learning about Integrins.
The page is very good. The introduction is clearly and briefly and it gives a good idea about Elastic Fibres. The structure and components section is clearly, but I think a image would help to understand. Finally, I noticed that you did not put something about history and future researches that would be good in the beginning of the page.
The “Assembly of Elastic fibre” is good too, and I really like the draw about the process of elastic fibre assembly. The other image (Role of MFAP-4 in Elastic fiber assembly) was well-chosen too. Finally, I like the step-by-step that you made in the final of section, because it summarizes the process being very informative and the reader can see the images at the same time. However, the images should be referenced correctly.
The functions related with organs, like heart, lungs and skin was a great idea. Also, the clinical significance you chose relevant diseases to relate but I think you could explain more deeply how the elastic fibres are related with this diseases. Also, I think some images in this section would be good to break up the text and the reader would feel stimulated to read at all.
Overall, the page is very clearly and informative so far. I think there are few little problems such as images referencing but I am sure that the page is still in progress and you will fix it. Also, it would be more informative if you include more images.
First, the page is well-organised, even though there are a lot of sub-sections but I think it is not a problem because the sub-sections are brief. The introduction is very clearly and helpful for those who do not have any knowledge about the fibronectin. The history section is brief but I consider satisfactory informative.
The structure section is good. However there are few problems, for example I think the first two sentences the term “fibronectin” is repetitive. Furthermore, it would be more informative if you put a fibronectin structure that is more clear and simple. The assembly section is clear, but I think it would be better with a image.
The function section is very informative. In the subsection “wound healing” there are some sentences that are a little similar in the paragraph. The subsections was great divided in relevant issues. However I think that if you put more images would be better. The Abnormalities/defects section is also very clear and informative. However, I think that it should be more images too, because it would illustrate better the diseases and break up this extensive text. Overall, I really like the page and it is very informative so far.
The page is well-organised but I think you should finish some sections. The Introduction is not still finish and I did not understanding why the type II collagen was divided in a section (the first section). The Structure section is clearly but it is incomplete like sub-sections “protein” and “types of fibres”.
The general function is very brief when comparing with articular cartilage. I think you could describe more functions and then focus on the cartilage. The image of cartilage is very informative. The Abnormalities section is very interesting (the issues chosen are very relevant) but it is still incomplete. For example, I do not understand clearly about Kniest Dysplasia and Rheumatoid Arthritis. I think you can explore more and use images, for example. Furthermore, the studies cited in abnormalities were confused. The Current Understanding and Areas of Research section is also informative and gives a good idea about the perspectives related to type II Collagen.
Overall, considering that the page is still in progress, there are few things to finish and do not forget to look over the references. I think it will be a great page.
The key points were clearly described. The sections were divided in relevant issues and I like the organization. The introduction is very informative and easy to understand. The “history” section is brief but I consider that it is still in progress. The “current research” you should summarize something and not only list some articles. The “Formation, Plasticity and Regeneration” and “Functional Layers” sections are also clearly and informative. The images were well chosen.
In the “Structure Components” you use an abbreviation (GAG) without explain it before. It can cause confusion for those people do not have any knowledge in biological sciences. Despite of this, the section is very well structured but it is not complete. For example, the perlecan section is not finished.
The “Function” section is very informative. The relations between organs and Basement Membrane were very well explained. However, in the beginning, the sub-section “Heterogeneity of Basement Membrane” seems random or confused. The “Abnormalities” section is the best section so far. It is very clear and informative, with great hand drawn image in the expand section. Overall, considering the page is still in progress, it is a very informative page. There are few issues to complete or finish, but I am sure that you will fix up them.
--Mark Hill Very good critical feedback, with good balance shown in your writing.
--Mark Hill You have not included the lab 12 assessment? It was to find a reference related to your project based on the techniques described in the Centre visit.