- 1 Attendance
- 2 Lab Assignments
- 2.1 Lab Assignment 1
- 2.2 Lab Assignment 2
- 2.3 Lab Assignment 3
- 2.4 Lab Assignment 4
- 2.5 Lab Assignment 5
- 2.6 Lab task 6
- 2.7 Peer Reviews
- 2.8 Micro array assessment
- 3 Reference
Lab 1 Work
Lab 3 Work
Lab Assignment 1
Lab Assignment 2
A new approach for the study of the chemical composition of bordered pit membranes: 4Pi and confocal laser scanning microscopy.
Intro/Aim Study aimed to investigate the interesting and complex microstructures of lignified cell wall present on coniferous bordered pits. The bordered pits assist in maintaining xylary water columns. The seperate Margo and torus regions reducing air intrusion by pit aspiration. Borders control liquid flow via shrinkage and swelling of pectin. The new approach in this study uses new antibodies to label pectin and cellulose which while be observed in 3 dimensions using CLSM and 4Pi microscopy. Compared to previous studies the cells can be observed in a nondestructive manner and more flexibility to modify the experiment without altering the cell.
Methods The conifer used was Pinus strobus as 5x10x20mm blocks. Pectin in sample is fluorecent tagged with the IgG antidoby Jim 5.Crystaline cellulose in sample is fluorescent tagged by Tis-tagged CBM3a.Leica TCS 4Pi and Confocal laser scanning microscopy generated 3D images of pit membranes
Results 3D imaging displayed that unaspirated pits had margo strands tagged from outer margin continuing down into the torus. The torus had a small amount of of margo. Pectin creates a ring encircling and inside the torus as a uniform G-ring.
Significance of study In unaspirated pits the torus pectin and cellulose surround a central matrix(void). In aspirated pits the microscrope the only region which remains well labelled is within the pit aperture and a pectin L-ring at the edge of torus. Concluded CLSM and 4Pi microscopy in conjunction with the fluorescent antigens to analyze composition of pit membrane and the 4Pi microscope would have further application in cell wall analysis in which destruction of the cell is not prefered
Lab Assignment 3
The concave face of decorin mediates reversible dimerization and collagen binding. 
Decorin is a family of proteoglycans, called small leucine-rich proteoglycans(SLRPs)which is a reptiion of leucine rich repeats which are bordered by cysteine-rich cap regoins. It is important in regulating collagen fibrillogenesis, growth factors and receptor tyrosine kinase. They bind within gap region of the D-period.
Experimental: decorin DNA residues of mouse is amplified by PCF form cDNA clone. Proteins were produce in human embryonic kidney in a Dulbecco's modified Eagle Medium at 37C and later frozen. The samples at a concentration of 3mg/ml was thawed and Analysied with Size exclusion Chromatography with Laser Light scattering. Furhter analysed by Differential Scanning Calorimetry, Differential Scanning Fluorimetry, Collagen Fibrillogenesis Assay and Solid-phase binding assay.
Significant results: Mutational disruption of decorin Dimer Interface; The decorin core protein had 4 designed mutations(Y51A/R52A/Q54A0), R151E, Q61N and Y130N that could potentially interrupt dimer interfacing. With the mutations strong yields were still present, Q61N had a higher molecular weight since site 61 is modified by glycans, electrophoretic mobility of Y130N mutation refelcted that of wild-type, (Y51A/R52A/Q54A0)mutation results in two mutant engineered glycosylation sites.
Oligermic States Wild vs Mutant; unmodified molecular mass 64.4kDa close to value of calculated dimer mass(72.4kDa). Modified protein gave a molecular mass of 84.6kDa
Stability of Wild vs Mutant; Wild type unfolded at 50-52C, Mutant type unfolded at 49-51C. Difference in stability not significant
Collagen binding Wild vs Mutant; To determine if decorin binds to collagen as a monomer or a dimer. Of the four dimer interface mutations only (Y51A,R52A/Q53) delayed fibrillogenesis like wild type. Q61N, Y130N, R151E all were inactive. Additionally Wild and (Y51A,R52A/Q53) mutant showed stronger collagen binding then the other three mutants.
Conclusion: Concave face mutation stopped decorin collagen binding regardless of wether the protein could still create a dimer. There for the concave face is critical in collagen binding and must dissociate form the dimer of bind with collagen
Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta.  Study looked at the interaction of three short leucine rich proteoglycans interaction with TGF-beta. The proteoglycans were expressed from human DNA and fused with E.coli maltose binding protein. the maltose binding protein displays very limited binding to other growth factors. Intact bovine decorin, biglycan and fibromodulin was able to compete well with fusion proteins for TGF-beta binding sites. the Kd values of the bonding distant her 50-200nM for low affinity bonding sites and 1-20nM for high affinity bonding sites. Fibromodulin and the fusion protein was a more successful at TGF-beta binding then the decorin anf biglycan. In the proteoglycans typical only one in ten of the core proteins displayed a high affinity binding sight and one low affinity binding sight per protein. Removal of chondroitin/dermatan sulphate chains in the decorin and biglycan resulted in a marked increase in binding activity. Displays that binding activity with TGF-beta could be hindered by glycosaminoglycan chains.
Affinity labelling showed in Mv 1 cells TGF-beta binds to betagylcan and type-II receptors, binding to endoglin in endothelial cells was reduced and Type-II binding was unaffected. Showing that different decorin type proteoglycans have slightly altered binding properties to TGF-beta and other potential binding sites. Additionally can regulate TGF-beta activity by sequestering TGF-beta out to the extra cellular matrix
Biglycan is an extracellular MuSK binding protein important for synapse stability  MuSK is a protein crucial is nerve-muscle synapse formation and maintenance. It pre-patterns the endplate zone and is a signal receptor. Though its processes are poorly understood the protein biglycan plays a role in maintaining stability. In the developmental stages there is no significant deviation from biglycan expressing fetus and biglycan null fetus. 5 weeks after birth nerve muscle synapse in biglycan null mice display abnormal perijunctional folding, segmentation and misalignment. THis indicates that presynaptic and postsynaptic regions were vacated. Expression of MuSK phosphorylation proportional to presence of biglycan. Null biglycan results in ACrH clustering to degrade once cluster has reached a certain size.
Biglycan is an extracellular ligand for MuSK and crucial for prolonged synapse stability
Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding. 
Decorin structure is main small-leucine rich proteoglycan present in the vertebral extracellular matrix. THe Glycosaminoglycuronan chain is key in giving inter-fibrillar organization. Decoron the core binding protein in decorin binding with the d and e bands of type I collagen fibrils.
Previous models suggested that highly curved decoron binds with a single collagen molecule creating strong non-specific binding. It is now apparent the the way collagen aggrates it prevents the one on one binding.
THe respective crystal structures of the decoron-fibril complex shape is complementary. One molecule of decoron interacts with 4-6 collagen molecule not one like previous hypothesis. Relying on many hydrogen bonds and elctrostatic interactions
Lab Assignment 4
Lab Assignment 5
Identify an antibody that can been used in your group's extracellular matrix project. DCN Antibody (Anti-DCN / Decorin Antibody (C-Terminus) LS-C46802)
Identify the species deriving the antibody Rabbit
Identify the working concentration for the antibody: 0.5 mg/ml, PBS, 0.01% sodium azide, 50% glycerol.
Identify a secondary antibody that could be used with this antibody. Anti-Biglycan antibody (ab54855)
Research paper <pubmed>21683670</pubmed>
Lab task 6
General Cell Collection: AT-2.1 (Rat prostatic cancer line); https://www.phe-culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=94101450&collection=ecacc_gc
Media; RPMI 1640 + 2mM Glutamine + 250nM Dexamethasone (DXMT) + 10% Foetal Bovine Serum (FBS).
General Cell Collection: Ma-Mel-22 (Human melanoma, metastasis); https://www.phe-culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=13012452&collection=ecacc_gc
Media; RPMI 1640 + 2mM Glutamine + 10% Foetal Bovine Serum (FBS).
RIN-m5F (ATCC® CRL-11605™) 9 (Rat pancreas/islet of Langerhans); http://www.atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Animal/Rat/CRL-11605.aspx
Media; RPMI-1640 Medium, Catalog No. 30-2001 ( RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate)
CCD-112CoN (ATCC® CRL-1541™) (Human colon); http://www.atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Human/Alphanumeric/CRL-1541.aspx
Media; Eagle's Minimum Essential Medium, Catalog No. 30-2003 (Earle's Balanced Salt Solution, non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate.)
Penicillin: active against Gram-positive bacteria, inhibits the last cell wall synthesis, destroys peptidoglycan strands by interfering transpeptidase
Streptomycin-sulfate: active against Gram-negative bacteria and Mycobacteria, stops the start of protein biosynthesis
Peer Review 2
The content and structure of this page is well presented with the exception of history and future research. The introduction is well written and gives a general explanation of integrin. I believe the image accompanying the introduction is too complex with too many moderately complex structures identified, if an image is be used in the introduction it shown be more simplistic. The actual image is draw especially well and is quiet interesting but I think its placement is incorrect and scanning instead of taking a picture would make it more attractive. It could be moved to function section, though that may result in an over saturation of visual aids in the functions section. The history is brief because of its recent discovery there isn’t much that would be described as historical research but it would be preferable to add more content. Structure and function content was good except for limited reference to the method of synthesis and regulation since obviously these molecules require certain circumstances to form. The functional structure subheading I believe should be integrated into a small introduction paragraph for the function heading. As you have stated you need to reword the statement that defects in integrin doesn’t directly cause disease to allow a better transition into how it behaves in different diseases. So far there are only 2 subheading under recent research, it would be best to add an additional 1 lines of research, you don’t require as much as other groups since the heading covered so far are quite broad. To eliminate the dullness walls of text in the recent research section add images or cut down appropriate content to make it more appealing.
The referencing is consistent throughout the whole project with all statements properly supported. In terms of formatting the clutter of images and video around the function heading should be addressed, the copied table creating instructions under the glossary should be removed and some of the headings need to be cleaned up.
Overall your project is good with seemingly perfect referencing but a small short flaw in written content and images in certain areas.
Peer Review 3
This page is close to finished with all of heading filled out with decent amounts of content. The introduction doesn’t properly represent the rest of the article, redoing it would be preferable. All sections have enough content to grasp the concepts being presented. The main thing I would like to see subheadings on brief overview on historical research on elastic fibres and some moderately in-depth examples of recent research.
Structure and components is well organised addressing structural aspects and some relations to process of discovery. This section displays bolding of key molecule/structures/substances which obviously highlights their importance at the sacrifice of the paragraphs attractiveness and creates some confusion. Elimination of bolded text and would be recommended. Assembly content is strong with good referencing at the start which lacks in the later paragraph and the Assembly list. The assembly list should be more distinguished from the paragraph above it a table with alternating colours would make a visually appealing presentation. Structure serves as function section had a lot of words without any reference; even though the content is general the lack of referencing is unappealing. Function section is structured as if it is reviewing individual articles instead of taking information from multiple sources of information to create an overview on a specific field. This results in the this section to have an unnatural flow from overly specific topic to overly specific topic. The key thing I would change here is the long subheadings and first sentences. A nice variety of clinically related conditions are presented with nice amount of content though it seems information was sourced from.
The referencing for the majority page was good except for clinical section in which each condition only had a single reference more references are required to increase credibility. The image used is placed near relevant content though those in the functions section are not formatted properly/referenced. An addition of two images to the structure section and one for each of the clinical conditions would break up the boring walls of text.
Peer Review 4
Organisation of Content is strong with simple easy to understand subheadings. The Introduction is very in-depth addressing many of the technical aspects of fibronectin, potentially to excess. Some of the over the top information include the size of the monomer, concentration in blood and multiple unexplained acronyms; ED-A, ED-B, RGDS. I would recommend the removal of such in-depth description and instead mention that is has roles is wound healing, blood vessels, inhibition of cell adhesion and embryogenesis along with mention of the most significant abnormalities. The history is good but could be slightly improved by increasing articles to include those up to 1990 and put it into a table to make it more attractive.
Structure section is well made with sufficient content, the dot points are a good way to reduce amount of words and put down a lot of technical information easily. You should pair the structural picture to the right of these dot points to avoid a large void of white on the right of the screen. Additionally other you could find alternative more simplified images and have the present one have the capability to expand since its complicated nature can cause excessive confusion. Assembly, Function had sufficient detail well written information with good referencing, though significant lack of pictures. AN addition of 4-6 pictures in the latter half of your page would make it more visually appealing. Some of the abnormalities only focus of the molecular process without reference of the clinical symptoms that they cause (Ehlers-Danlos Syndrome). At the bottom of the page there is a current research heading, this should be expanded on show the page is up to date with present advancement in the area of fibronectin. Additionally you will require to add an image you created yourself to meet one of the assignment requirements.
To summarise page is well constructed, though multiple images need to be added and introduction should be toned down.
Peer Review 5
The paragraph section of the introduction read well and presented the required general information with nice amount of citation something usually lacking in an introduction. The dot points seem bland and out of place it would be advised to make them flow on from the paragraph. The formatting of the references has obviously been done list at the bottom individual students sections which is understandable, just make sure in the final product the referencing is compiled at the bottom of the page to allow better presentation of content. The only places which might need more references to back up the content is the second half of the history section and the abnormalities/future research section, it is understandable to have only a few references for each abnormality/future research because of limited information available but it should be more than just a single reference. The use of images are sparse compared with written content try to find relevant images for the following sections; history could have a graph showing papers published per year on laminins, for function a diagram of one of the more important laminins interacting with the basement membrane would be a nice inclusion also more images in the future research would be preferable. The images presented in the structural and abnormality sections are sufficient in number and relate well to discuss content.
The amount of content is quite vast you have obviously done extensive research; the only place in which it is lacking is for function of separate laminins which are standing as lone headings without content. I would advise that you only address the most significant laminins and remove the ones which less research or integrate many of similar function into a single paragraph. Some of the information presented at the end of structure should be removed it focusses to heavily on the function of the laminins resulting in overlap with the functions section. You can include some reference to function but not to the extent presented in the final two paragraphs of structure. The contents displayed in Future research are too extensive making the walls of text uninteresting, to solve either cut down information or utilise more visuals to make it more entertaining. In the later sections of the project you often refer to previous studies, examples of these previous studies would show you know more of the background to the future studies/abnormalities.
Overall your page is quite well put together just changes to formatting and making the page more entertaining is required.
Peer Review 6
The project topics have been divided well though the introduction and history have no content present. Each heading has its one mini intro before the more complex content is introduced, even though it results is the repetition of ideas I quite enjoyed it making the transition for heading to heading quite easy. The plan of leaving the introduction blank until all other parts of the project are complete is a viable one, though the history is required to give context to the methods used to get to our current understanding and the basis for further research. The content isn’t fully developed especially sections of antibodies and Models. If these are to be included in the final page I recommend making the antibodies either expand/minimise to save space and have any models content be put in current research. Additionally abnormalities require more content on Dysplasia and arthritis. The subheadings of Skeletal bone development and fracture healing require more information an extra 2-3 sentences each would suffice The structure, function and current research content seems to be mostly there with some further small additions needed. The references are formatted correctly except the Type II Collagenopathies section in which there is evident referencing not formatted using the proper script. There seems to be a slight lack of reference in biosynthesis subheading, references is key in providing support for the concepts you are presenting. You will require more than 2 images, preferably at two for each section. Some recommendations would be fracture healing, skeletal bone, number of articles published per year graph, pathways during synthesis but most importantly is clinical pictures of abnormalities. I really like the DIY articular cartilage picture the drawing is very clean. Additionally the collagen triple helix doesn’t seem to have any referencing which could get hairy since the copyright information doesn’t seem to be present in its description. There seems to be multiple grammatical errors which would need to be addressed before submission, this is particularly prevalent in the structure section. Though you seem to not have all you content up, all of the flaws in your project are straight forward to address, Best of luck
Peer Review 7
As a general remark the project looks close to completion with small additions required in content pictures and some formatting changes. Your main strength is in the writing style it flows well with minimal mistakes and technical terms are used but not to excess.
The introduction is well worded and the accompanying image displays multiple simplified aspects of the basement membrane. The history section should be expanded on since it displays the methods used to reach our current understanding and the basis for future research. I like the use of a table with the dash of colour; it is a great way to present many small events and would recommend adding many more important studies/events. Formation section has great information divided as dot points according to source used, these should be integrated into 2-3 paragraphs since which some much text the dot points look out of place. Functional layers I think should be a sub heading under Structural components but if there is a specific reason why it’s not doesn’t worry. The Structural component is the only section lacking in terms of content, further description of laminins, perlecan and collagen IV required. You use GAG in structure components without explain the abbreviation. The Function and abnormalities section both amazing very little problems in the amount and delivery of written content. The expandable box for the rarer abnormalities and the glomerulus is a great idea to include more optional information without making the page looking over complicated. For current research seems to be numerous studies referenced under the heading which hopefully will be expanded on after the peer review, this section is required to make the page seem more up to date and future focused.
The images used are integrated well and formatted to not be oversized I would advise additions of chemical diagrams of the structural components and additional image for basement membrane function to break up the sheer volume of text and an additional 1 or 2 images for the abnormalities section. Referencing is almost perfect except for the article dump under the current research heading and the introduction of abnormalities , even though the information is very general its looks unusually to have so much text without a reference.
Great project, only minor tweaks needed before Completion
Micro array assessment
Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies
Glycosaminoglycans are a key structural component of Small leucine rich proteoglycans
- How many Gbases (Gb) would you require to sequence the human genome to 40x coverage?
The standard single genome coverage is 40x which requires 300 to 350 Giga bases though can be greater
- Describe an advantage of using next-generation sequencing technology over microarrays to study gene expression?
Next-generation sequencing technology allow inexpensive, genome wide sequencing which can be used for a multitude of purposes.
Examples of next generation sequencing technology; 454 pyrosequencing (roche), Solexa/Illumina Genome Analyzer, SOLiD (Applied Biosysytems)
Problems with microassays;
- Needs prior knowledge of genomic feature, which can jeopardizes effectiveness in when background knowledge is incomplete or outdated
- Reliant on cross-hybridization between similar sequences. So it cannot effectively sequence repetitive fraction of genomes and limited in analysis of alike genes, additionally adds bias
- Format and methodology can limit reproducibility
- Reliant on PCR amplification to produce DNA micrograms, can result in errors
- High signal to noise ratio, limits range of of high confidence in results
Methods NGS can solve the problems;
- Knowledge of genomic feature assist but not necessary allowing the sequencing of genomes previously partially known or unknown
- NGS uses single nucleotide resolutions, not cross hybridization. Allowing it to monitor expression at the single-nucleotide resolution, vasting improving ability to analyze repetitive regions of genome.
- All next generation sequencing technologies have same data output, increasing reproducibility of expierment
- Unlimited quantitative range of signalling
- Only requires nanograms of DNA eliminating bias from PCR amplification
Lab Assignment 5
|Modulating substance||Effect on transcription|
|Interleukin-1beta||Down-regulates general proteoglycan|
|Tumor necrosis factor alpha||Down-regulates general proteoglycan|
|Transforming Growth Factor beta||Up-regulates biglycan, decorin, and fibromodulin , |
|Dexamethasone||Up-regulates decorin and down-regulates biglycan |