Lab 6: ABSENT
Lab Assessment 1
Scanning Electron Micrographs of S. aureus (fig. A and B) and B. subtilis (fig. C and D) that were treated with a cationic defensin (SibaDef) so as to reveal morphological changes.
Lab Assessment 2
Isotropic 3D Super-resolution Imaging with a Self-bending Point Spread Function
A self-bending point spread function (SB-PSF) was adapted using existing Airy beam technology with the aim of creating super-resolution fluorescence imaging in three dimensions (3D). This method produced a side-lobe-free SB-PSF, decreasing diffraction and lateral bending and allowing for super-resolution imaging with accurate 3D localisation of molecules over an increased image depth and higher localisation precision. This means that the SB-PSF method can image thicker samples with a much higher resolution then other current models.
- The optical set up included taking measurements with custom inverted microscope that was calibrated for internal reflection and oblique incidence excitation.
- Filtered fluorescence light was then passed through activated dye samples.
- The images were aligned between two channels and imposed with markers. They were then displaced in 100nm increments over a distance of roughly 3 microns.
- Images of ten regions were superimposed to create an image with a concentration of markers in 3D.
- The fiducial markers were then illuminated with a laser and were recorded as STORM movies.
- Single molecule and STORM movies were separated and analysed and any images that did not meet the set criteria were rejected.
- Results were conducted on the basis of localisation precision, image depth and error rates.
RESULTS and CONCLUSION:
The SB-PSF model can create super-resolution fluorescence images of depths 1.5-10 times larger than other current methods and 2-3x higher localisation precision. The main advantages of this method are the ability to scan thicker samples at higher resolution whilst preventing the effects of photo bleaching.
One downside of this new adaption is that the larger scanning area results in a small decline in imaging velocity due to the reduction in the number of fluorophores per frame. Another drawback is the lower density of activated fluorophores revealed by larger depth scans. In order to optimise the resolution, combining SB-PSF with ultra-bright photoactive fluorophores could help to distinguish a greater amount of photons and create images with incredibly high resolutions in nanoscale.
Commercial MSDS: Paraformaldehyde MSDS - Sigma-Aldrich
Search term: Collagen Type II
<pubmed limit=5>Collagen type II</pubmed>
Lab 3 Assessment
The effect of estrogen on the expression of cartilage-specific genes in the chondrogenesis process of adipose-derived stem cells.
SUMMARY: The aim of this study was to determine the effect of oestrogen on genes pertaining to chondrogenesis. Adipose-derived stem cells (ADSCs) were differentiated into cartilage and then treated with oestrogen in order to determine the genetic markers associated with the expression of type II collagen.
- Cell culture: Human adipose-derived stem cells were sourced from patients aged 25-55 years using enzymatic digestion of subcutaneous tissues.
- The cells were then cultured in a modified medium that was changed two times a week allowing for differentiation into cartilaginous tissues.
- Pellet culture: Pellets of chondrogenic APSCs were formed using a centrifuge and then suspended in a treated chondrogenic medium.
- The pellets were then incubated for two weeks and the control group was left untreated whilst the experimental group was treated with oestrogen E2.
- At this point in the procedure the medium was changed every three days.
- Reverse transcription polymerase chain reaction (RT-PCR): Gene expression of cartilage-specific markers was determined using the RT-PCR method.
- RNX-plus kits were used to extract the cellular RNA and any unwanted genomic DNA was removed through the use of DNase.
- Concentrations of RNA were assessed using a spectrophotometer and cDNA was synthesised from the identified RNA and used for PCR.
RESULTS and CONCLUSION: Type II collagen was found in the control group but no type II collagen was observed in the experimental group. Aggrecan was detected in both groups with a significant decrease in aggrecan expression in the experimental group.
The study demonstrated that oestrogen has an inhibitory effect on the expression of type II collagen and also leads to a significant reduction in aggrecan gene expression. This means that oestrogen is not suitable for use in the chondrogenesis of type II cartilage from ADSCs.
Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis.
SUMMARY: The aim of this study was to assess the potential of collagen type II regulatory T cells (Col-Treg) for treatment of rheumatoid arthritis (RA).
- Ethical guidelines were followed and approved of by appropriate ethic committees.
- BALB/c and DBA/1 mice were obtained and separated into transgenic groups of rearranged T-cell receptors (TCRs) and type II collagen specific T-cell hybrids.
- Col-Treg clones were generated from Col II–specific transgenic mice.
- Ova-Treg clones were generated were also generated.
- IL-10 was added to both groups on day 2 and the cells were then cloned.
- Cytokines IL-4 and interferon γ (IFN- γ) were counted using an enzyme-linked immunosorbent assay (ELISA) after two days of stimulation.
- Cytokine secretions were stained for using fluorescent dye and then analysed using fluorescence-activated cell sorting.
- The immunosuppressive function of Col-Treg clones was then assessed.
- Arthritogenic antibodies specific for collagen type II were injected intraperitoneally into 9 week old DBA/1 mice.
- Col-Treg cells were delivered via intravenous injection into the mice hours later.
- Beginning from 3 days post initial injection, the disease severity was scored using a scale:
0 = normal 1 = weak swelling 2 = significant swelling associated with redness 3 = intermediate swelling associated, or not, with redness 4 = maximal swelling and/or redness in all inflamed digits
- The number of inflamed digits were taken into account and scored appropriately (0 = no inflamed digits, 0.5 = zero to five inflamed digits, 1 = six to ten inflamed digits, 1.5 = one to fifteen inflamed digits and 2 = 16 or more inflamed digits).
- The mice were also weighed to determine any lose in body weight.
- 9-12 week old DBA/1 mice were immunised at the base of the tail with bovine type II collagen.
- 21 days after the arthritis tests, a booster shot was given.
- Col-Treg cells were injected intravenously into the mice on day 20, 22 or 28 after arthritis induction.
- The thickness of each hind paw was measured consistently after day 21 and the severity of arthritis was graded.
- Samples were collected and stained.
- Leukocyte infiltration and erosion were scored according to appropriate scales.
- Immunoglobins were measured.
- Detection of Col-Treg cells was carried out using PCR.
- 9 week old BALB/c mice were injected with ova-specific CD4 cells.
- This group was then immunised the following day with IFA.
- On day 5 one paw was injected with ovalbumin/phosphate-buffered and PBS was injected into another.
- Specific T regulatory cells were injected intravenously and the mice were killed after 2 days. Samples were then collected and stained.
RESULTS and CONCLUSION:
The results indicate that introduction of Col-Treg cells reduces the incidence and clinical symptoms of arthritis in both preventive and curative settings. There was a significant impact on collagen type II antibodies and there was a noticeable decrease in antigen-specific effecter T cells. These results indicate that collagen type II T regulatory cells could be an effective treatment from patients suffering from RA.
Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells.
SUMMARY: This study provided a new therapeutic strategy for autoimmune inflammatory diseases such as Rheumatoid Arthritis particularly with the effect on collage type II.
- 36 male Sprague-Dawley (SD) rats were used for the experiment and ethical guidelines were followed to keep suffering minimal.
- The SD rats were divided into for groups: CON, BLA, GEL and BMC. *All groups were injected subcutaneously with collagen type II emulsion. *Booster shots were given at d=21 with half the dose.
- All groups received operations one week after booster shots.
- In the BLA, GEL, and BMC groups a hole was drilled into the tibial plateau of the left knee and then the wound was closed.
- The CON group received a sham operation with no drilling performed. *The rats were then allowed to move freely after their respective operations and were monitored.
- Bone marrow was harvested from the tibia and femur of a 3 week old SD rat and the mononuclear cells were isolated. The bone marrow mesenchymal stem cells (BMMSCs) were prepared for use.
- The groups all received different treatments 3 days post surgery.
- BMC: PLGA-b-PEG-b-PLGA (BMMSCs); GEL: thermogel ; BLA (Blank group): phosphate-buffered saline (PBS); CON (control group): PBS
- The rats were then scored in terms of arthritis onset according to level of erythema, swelling or joint rigidity and oedema. An average of the limbs was calculated to determine scores.
- The rats were killed and both distal femurs were examined and photographed for macroscopic evaluation.
- The distal femurs were then fixed and stained in order to perform microscopic of the knee cartilage and surrounding synovium.
RESULTS and CONCLUSION: The results indicated that the majority of SD rats developed some form of irreversible bone or cartilage degradation with the exception of the BMC group. The BMC group displayed scores significantly lower than the non-treatment groups but were still possessed higher degrees of disease than normal rats. This means that BMMSC therapy can reverse synovial hyperplasia to an extent but cannot offer a full recovery.
TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells. 
SUMMARY: The purpose of this study was to test the effectiveness of a biofunctional hydrogel consisting of collagen type II nanofibers and transforming growth factor β1 (TGF-β1) in the regeneration of cartilage.
- Photocrosslinkable hydrogels were prepared.
- TGF-β1 was added to the hydrogels at a concentration of 10 μg/mL.
- Human synovium-derived mesenchymal stem cells (hSMSCs) were suspended in hydrogels and then cultured in chondrogenic medium.
- Growth of hSMSCs was observed with a light microscope.
- Cell viability was observed by staining the samples after washing with PBS and viewing them via fluorescent microscopy. Viability was determined by the ratio of live cells to total cells.
RESULTS and CONCLUSION: The results indicate that collagen type II impregnation and TGF-β1 delivery significantly promoted chondrogenesis. This hydrogel system could be an effective treatment for cartilage defects and the results support the hypothesis that collagen type II impregnation in conjunction with TGF-β1, promote chondrogenesis in hSMSCs.
Macroscopic Comparison of Rat Knee Articular Cartilages After Treatment with BMMSCs.
Lab 5 Assessment
This graph shows the data collected after the actin cytoskeleton of B35 neuro-epithelial cells was manipulated by Tropomyosin 4 (Tm4). The cells were categorised into five phenotypes: fan, broken fan, stumped, pronged, stringed, and pygnotic. Increased numbers of 'pronged' and 'stringed' phenotypes after Tm4 expression shows that Tm4 is involved in promoting the formation of neutrites. Tm4 overexpression stabilises actin filaments and thus facilitates the formation of neutrites.
Lab 6 Assessment
Collagen II Antibody MA5-37493 MA5-37493
Working concentration: Western Blot: 1-2 µg/ml
Imunofluorescence: Assay Dependent
Immunohistochemistry: 1-2 µg/ml
Flow Cytometry: Assay Dependent
Secondary antibody: Biotinylated secondary antibody to rabbit IgG
Paper that used Collagen Antibody MA5-37493: Articular cartilage increases transition zone regeneration in bone-tendon junction healing.
Mouse Cell Line: L Cells (ATCC® CRL-2648™)
Culture Media: Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Human Cell Line: 20B8 (ATCC® CRL-12582™)
Culture Media: RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Penicillin-Streptomycin 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.
Group 1: Proteoglycans
The introduction is really well written and you have made an effort to point out that you page will be focusing on SLRPs. At this point in your page there is already two images with plenty of colour and it is a great way to catch the eye of your audience. I like the little segue into the history section – it helped the page flow. The history section is well sourced and full of really interesting information.
The function section really shows how effective selecting a few SLRPs is because you were able to give a detailed description for each one. Perhaps if you formatted this section a little better (e.g putting the descriptions of each individual SLRP into a table or under their own headings) might make it look neater. The abnormalities section is really well organised and the three subheadings (eyes,kidneys and musculoskeletal) make it really easy to read through. You have recognised that you need to add images and this will make this section more interactive and stand out more.
Your group has done a good job of referencing most of your images (as someone has already mentioned there is one with an issue). Your student drawn images are excellent but they seem a little smudged (it is not a major issue). I like how you presented your glossary in a table format, it is neat and helpful but maybe it could include more definitions. You have used a wide variety of references to support your work and someone has mentioned how to fix the formatting error with repeating references.
It is clear that you have all out a lot of effort into your page and when all the finishing touches are applied it will definitely be something to be proud of. Well done.
Group 2: Integrins
Your introduction is a nice way to welcome people to your page and will tie in well with your history section when more information is added to that part of your page. The timeline and future research sections will be a great addition and will make your page seem more relevant. You could talk about important discoveries and medical applications of future research.
So far your structure section is well written although it is clear that it still needs work. The image in the really works well with the text and makes the concepts easy to understand. This section definitely needs more references to support your text.
No think the function section is the best part of your page so far. It is concise and really well written. You linked to other EM components and i think this is an opportunity for you to use links to the pages of other groups to make your page more interactive. My only issues with this page are surrounding he images and video. They seem to be just placed in amongst the text and although they are interesting they look out of place without some sort of reference to them in your text. Perhaps you could include a brief overview of the video underneath it or within your text.
The abnormalities section could be really improved by including more images but it is still a well written and informative section. You should be really proud of this effort so far because you must have really worked hard to find information pertaining to integrins. Well done.
People have mentioned problems with your referencing for you images so I won't harp on about that but I really like how you have included text to describe the images. I noticed that some of your abbreviations have no explanation so perhaps you could include a small table at bottom of your page to explain them (e.g for CSAT and JG-22). It is clear you have used a variety of references to source your work and someone has already mentioned how to fix he problem with repeating references in your formatting. Good work, your page will be an excellent resource when you fix all the issues and develop the sections that are lacking.
Group 3: Elastic fibres
Good work with your introduction. The structure and components section could would benefit with the inclusion of images to help the audience understand the concepts. Apart from this minor issue it is clear that the people behind his section have put a lot of effort into presenting a well written and extensively sourced section. The student drawn image is great and is well described.
The function section follows the organisation of the page in describing the roles of elastin with regards to different organs in the body - this is a great way of presenting your information as it makes it easier to understand. You have used images which is good but there needs to be some sort of reference to them in your text to help tie them into the section otherwise they just seem to be placed there for the sake of it. You could do this by adding captions under the images and then giving a concise description in the image description do that your audience could see this when they click on the images.
The diseases described in he clinical significance are interesting but they could use some images to support them. Perhaps you could go into more details about elastins exact role in some of these diseases. Great work all of you and I look forward to seeing your competed project.
Group 4: Fibronectin
You have a nice introduction to your page that is obviously well sourced. Your history section contains plenty of detail and this makes it both informative and interesting. The structure section is clear and informative but could benefit from a diagram or schematic drawing to help the audience understand the structure in conjunction with your text. People have mentioned that the isoform part of your text might be presented a little better in a tabulated format and I agree. It would help break up the larger slabs of text and make it look nicer. The breakdown of the function section makes it easy to understand and it includes plenty of relevant information in functions and the specific roles of fibronectin.
The abnormalities section contains lots of great information. Each disease has a good description but could use images to break up the text. I like his page so far and I think the most important change you can make is to fix your subheadings so that they appear as headings with a line underneath them. This will make it easier to see the sections on the page. Good work.
Group 5: Laminin
Good introduction to your page. I noticed right off the bat that you need to fix the formatting of your references and include them at the bottom of your page. This history section is well written and provides some interesting information.
The structure section is also well written and detailed but it you have used some images to breakup a large body of text hear. It would be neater if you were consistent with the caption formatting between the two images. The function section has obviously has a lot of research behind it. Well done on creating such a meticulously researched body of text. I am aware that you were told to include all 20-something types of laminin so maybe you could come up with a more creative way of presenting such a huge slab of information. I think you might benefit from selecting a few key laminins to leave in the body of the text and then place all the others in a hidden table so that if your audience wants to see more they can click on it to reveal the rest of the text. Maybe the inclusion of some images might help break your text up some more. Nevertheless, you should be proud of your effort to provide such a detailed resource.
The same can be said for the abnormalities section as it appears as another large body of text. Regardless of this fact, you should be proud of the effort you put in to creating a very detailed descriptions of your abnormalities. You may be able to fix this by including more images and then adding some of the more detailed text to the image descriptions so that people can click on the image to see more. It is obvious hat you have all done a lot of research in order to create these detailed blocks of text and when you add the finishing touches to make the page more visually appealing during the editing phase your page will be something to be proud of. Well done and I look forward to seeing your final product.
Group 7: Basement Membrane
Well done on your introduction and history sections. The links with the other components of EM in the formation section provide you with the opportunity to provide links to pages from other groups which could add an 'interactive' component to your page. This section has obviously been well referenced. Perhaps it could be made better with a schematic drawing or diagram illustrating the process of basement membrane formation. The function section is detailed and contains lots of references. You may need to come up with some creative ways of breaking up these large chunks of texts (e.g a table for any additional information and some images).
Your page has some good images ambit there are some referencing issues that have been mentioned already. Someone also mentioned how to fix your references because some of them have been repeated. You may need to may a little more attention to detail because there appears to be some typos on your work. Well done and I look forward to seeing the finished product.
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