Difference between revisions of "User:Z3459996"
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* Various inorganic salts.
* Various inorganic salts.
* L-amino acids.
Revision as of 17:23, 14 May 2015
User page for Z3459996, an otherwise anonymous and overbearingly enthusiastic ANAT3231 student. Check out our Project Page on Small Leucine-Rich Proteoglycans!
- Lab 1
- --Z3459996 (talk) 16:34, 12 March 2015 (EST)
- Lab 2
- --Z3459996 (talk) 16:52, 19 March 2015 (EST)
- Lab 3
- --Z3459996 (talk) 16:08, 26 March 2015 (EST)
- Lab 4
- --Z3459996 (talk) 16:33, 2 April 2015 (EST)
- Lab 5
- --Z3459996 (talk) 16:03, 16 April 2015 (EST)
- Lab 6
- --Z3459996 (talk) 17:02, 23 April 2015 (EST)
- Lab 7
- --Z3459996 (talk) 17:14, 7 May 2015 (EST) I was here but too polite to log in to list attendance. z3459996 does not miss classes.
- Lab 8
- --Z3459996 (talk) 17:14, 7 May 2015 (EST)
- Lab 9
- --Z3459996 (talk) 16:09, 14 May 2015 (EST)
Spatial Covariance Reconstructive (SCORE) Super-Resolution Fluorescence Microscopy
Super-resolution microscopy overcomes the physical limitation of the diffraction of the objective aperture using a variety of techniques, each with their own restrictions. For example, Stimulated Emission Depletion (STED) microscopy uses a set of techniques which may damage some biological systems, while Stochastic Optical Reconstruction microscopy (STORM) requires 1 000 to 10 000 individual images to be taken (by necessity) over a period of minutes to produce a decent final composite, thereby rendering it a poor option for live-cell imaging. In this article, the authors propose an new algorithm for super-resolution microscopy, which they call the Spatial Covariance Reconstructive (SCORE) algorithm, which can achieve a lateral resolution of 100 nm in 5 to 7 seconds of imaging.
Like STORM, SCORE combines data on the fluorescent intensity covariance of each pixel with the shape of the overlapping point spread function (PSF) data to compile a composite image representing the probability distribution of each emitter. In less technical language, a series of images of the subject is taken, the difference in light intensity between corresponding pixels in different images is measured, and the resulting data run through a set of algorithms to compensate for optical phenomena in order to "pinpoint" the source of light emission. In this case, resolution is limited not by the diffraction of the objective lens, but by the quality of the fluorescent label being used. Several comparisons between SCORE and STORM are given, and it is concluded that SCORE produce superior quality images with less noise and closer resemblance to actual structure, but maintaining a similar processing time to existing techniques. A most incredible example is shown in 2(e), where simulated data depicting a sub-diffraction ellipse is parsed using both STORM and SCORE algorithms with various volumes of data and duty cycles.
Article 1: Small leucine-rich proteoglycans in the vertebrae of Atlantic salmon Salmo salar.
The distribution of small leucine-rich proteoglycans (SLRPs) across different tissues in various organisms other than humans is poorly-understood. This study analyses small leucine-rich proteoglycan distribution in S. salar vertebral columns. It had previously been demonstrated that decorin mRNA is present in S. salar vertebral columns, and this study established that decorin, biglycan and lumican are all present in regions of intermembranous and endochondral ossification, suggesting a role in mineralisation of bone. It was found that the developmental expression of SLRPs is similar to that of mammals, hinting at the evolutionary origin of proteoglycans as a biochemical family.
Article 2: A comparative evaluation of the small leucine-rich proteoglycans of pathological human intervertebral discs
In intervertebral discs, SLRPs have multiple functions. Decorin, fibromodulin and lumican have been reported as being present in intervertebral discs. These interact with fibrillar and other collagens, fibronectan, elastin, and various growth factors. This study finds that all SLRPs in pathological intervertebral discs exhibit fragmentation to some extent, which is contrasted to the findings of an earlier study indicating little fragmentation of normal post-mortem intervertebral tissue. Only lumican was left unfragmented, indicating that it does not undergo enzymic cleavage in disc pathology as other SLRPs do. This was noted to differ from osteoarthritis, where fragmentation of lumican does occur. The reverse effect is reported for fibromodulin: there is little fragmentation in osteoarthritis, but significant fragmentation in intravertebral disc pathology. Fragmentation of different SLRPs may be used clinically in the future to determine the pathway of joint degradation, therefore improving treatment of affected individuals.
Article 3: The role of small leucine-rich proteoglycans in osteoarthritis pathogenesis
As an important component of the extracellular matrix, SLRPs and the genes that encode them have been implicated in many the pathogenesis of many conditions, particularly degenerative contitions. These is emerging evidence that SLRP may play a role in osteoarthritis pathogenesis. This article proposes mechanisms by which this may occur. Some SLRPs have been implicated in differentiation of mesenchymal progenitor cells, which are important players in osteoarthritis pathogenesis. Single- and double-SLRP knockout mice have shown increased osteoarthritis biomarkers, and this is postulated to be a result of one or more of the following: changes in extracellular collagen network, TGF-β signalling, or roles in innate immune inflammation, muscle weakness, or subchondral bone.
Article 4: Lumican-derived peptides inhibit melanoma cell growth and migration
Just as underexpression of SLRPs seems to have a negative effect on degenerative disease processes within the organism, so too is overexpression correlated with positive effects. Lumican in particular exhibits potent anti-tumor properties which are explored in this article. Migration of tumor cells causes progression of melanoma by tissue invasion and metastasis. This study finds that lumican decreases melanoma progression in vivo, and elucidates that it prevents migration in vitro. A complex mechanism by which this occurs is postulated in the article, which ends by proposing two mechanisms of melanoma cell migration inhibition (inhibition of phosphorylation and decrease of MMP-14 activity).
Figure 2: Lumcorin inhibits melanoma motility. This is relevant as it not only indicates a pathology that may result from underexpression of a specific SLRP, but also because it indicates a potential therapeutic application of SLRP expression modulation.
Counts from the images here, compiled into a convenient graph:
- Monoclonal antibody 2B6 (anti-CD73) is an anti-chondroitin sulfate antibody that targets small leucine-rich proteoglycans such as biglycan.
- Raised in mice (Mus musculus; BALB/c).
- 1/100 for Western Blotting; 1/20 for Immunohistochemistry.
- Anti-Mouse IgG1 VHH Single Domain Antibody (ab193651)
- The following article uses 2B6 antibodies: <pubmed>23801333</pubmed>
Information for this section taken from Cosmo Bio Co., Ltd.'s 2B6 listing.
MSDS (Sigma-Aldrich Australia)
Cytological preservative that functions by cross-linking proteins.
- Harmful if swallowed.
- Causes skin irritation (potentially allergic skin reaction).
- Serious eye damage on contact.
- Respiratory irritation.
- Suspected to cause cancer.
Lab 9 Cell Lines
American Type Culture Collection
- A101D, a melanoma cell line from a 56 year old male caucasian. To be grown in Dulbecco's Modified Eagle's Medium.
- NMuMG, epithelial cells from the mammary gland of a mouse.
European Collection of Cell Cultures
- DD1076, a chorionic villus cell line from chromosome 14 a female foetus affected by cystic fibrosis. Culture medium is Ham's F10 + 20% FBS.
- 10T½, fibroblast-like mouse embryonic tissue cell line.
Growth Media Components
Ham's F10 + 20% FBS
- Various inorganic salts.
- Essential L-amino acids.
20% FBS means that the medium contains 20% fetal bovine serum.
Dulbeco's Modified Eagle's Medium
This is Minimal Eagle's Medium which has been modified to contain:
- 4 mM L-glutamine
- 4500 mg/L glucose
- 1 mM sodium pyruvate
- 1500 mg/L sodium bicarbonate
- Marcus Persicke, Andreas Albersmeier, Hanna Bednarz, Karsten Niehaur, Jörn Kalinowski, Christian Rückert Genome sequence of the soil bacterium Corynebacterium callunae type strain DSM 20147T. Standards in Genomic Sciences: 2015, 10(5) doi:10.1186/1944-3277-10-5
2015 Course Content
Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | 2015 Revision
Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Cell Knockout Methods | Cytoskeleton Exercise | Immunochemistry | Project Work | Confocal Microscopy | Tissue Culture | Stem Cells Lab | Microarray Visit
Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G