ziweiwang's blog

 In the third experiment that was done for the paper, FTIR spectra of reconstituted and eluted rhodopsin protein were measured. The experiment indicated that there is a decrease in alpha-helix and an increase in beta-pleated sheets in both mutations but the differences were stronger in P23H. The N15S mutation protein is more folded compared to P23H mutation. This indicates that there is a decreased amount of alpha-helix in misfolded fraction compared to wt. Circular dichroism spectra were recorded with samples of rhodopsin and clathrin e 6.  There is a loss of alpha-helix structure from wt to the mutants with a further loss in P23H. Clathrin e6 stabilizes the alpha helix of the wt and the P23H but not N15S.  This indicates that clathrin e6 causes stability in the alpha helix of the P23H mutated proteins. The whole paper impacts the disease by indicating why the mutation of P23H has a more impact compared to patients with a mutation in S15N, and suggest a possible therapy for the mutation P23H.  
The mutation affects the stability of the cone structure causing the protein to aggregate and to be instable. This, in turn, causes degeneration of cone cells, which directly leads to a decrease in vision. There are phenotypic differences between individuals with the same genotype; however, this is not well studied since many different genes cause retinitis pigmentosa, and P23H mutation in RHO gene is relatively rare in an already rare disease. The variability is due to the different mutation types that are present as over 40 different mutations can result in retinitis pigmentosa. It is also possible that there are modifiers. However, this is not studied very well.

The mutation in the gene affects the physiology of the cell in the tissue by causing degeneration of the rod photoreceptors, which in turn causes changes in the surrounding cell tissue. Without rod cells in the retina is unable to process vision completely. In this experiment, the researchers investigate the evolution of microglial changes during retinal degeneration in P23H rats. The cell density and morphology of the retinal degeneration were studied at different ages in normal and diseased retinas through immunocytochemical localization of GFAP. Astrocyte quantification showed that astrocyte density increased astrocyte density was lower in adults there was a significant increase in astrocyte numbers in P23H rat at P120 in all regions examined. In P23H rats the retina also showed a progressive disruption of blood vessels and there were tangles of blood vessels. There was also astrocyte hyperplasia and hypertrophy accompanied by increased GFAP activity. When the retina is undergoing a dramatic remodeling due to retinal degeneration, there is a major change in the astrocyte number the numbers change less when the degeneration process slows down.

Mutation of RP4 mostly affects the rhodopsin protein. The structure of the protein is a protein made of an n tail, c tail, 8 alpha-helix, and 5 turns and the protein is embedded in a membrane. The normal function of the protein is to activate the G protein to start a phototransduction cascade. When the light hits Rhodopsin, retinal that is attached to the protein isomerizes and causes rhodopsin to activate the G-protein, which in turn starts the phototransduction cascade.
 An experiment that aimed at testing changes that leads to more instability in rhodopsin structure causes severe disease in patients. Specifically, the experiment aimed at testing the function of the protein that has the P23H mutation and the N15S mutation. In the first experiment, A purified protein of the mutant and wt rhodopsin were recorded using a UV spectrometer, and a thermal denaturation experiment was done on the proteins. The result showed that P23H had a lower yield of correctly folded protein compared to N15S, and the thermal stability of P23H was75% less compared to the wild type protein. This indicates that the P23H is a less stable protein compared to N15S and wt. The mutation is a more serious mutation compared to N15S indicating that people with the P23H will have serious symptoms compared to people who have an N15S mutation. In the second experiment that was done, Meta II fluorescence was measured, and the data were analyzed using a sigma plot. The result showed that Total fluorescence after light activation was lower than wt for both P23H and N15S due to aggregation. The results imply that both of the mutations were more prone to aggregation compared to the wt. In the third experiment that was done, the protein was reacted with n-glycosidase F. An immunoblot was run and used for densitometric analysis linear regression was performed on the result of the immunoblot. The result showed that the proportion of unglycosylated species was much higher in P23H than in N15S and WT and lower intensity at lower molecular weight bands indicating that there is some degradation of P23H. P23H and N15S both have a higher intensity of Dimer and higher molecular weight which indicates that there was a higher rate of aggregation. The degree of aggregation and glycosylation was higher in P23H compared to N15S which was in turn higher what wt. This shows the differences in aggregation and glycosylation explains why the severity of the phenotype differs between the two mutations.

Retinitis pigmentosa is a degenerative monogenetic disorder of the retina that affects about 1 million people worldwide. People who are affected by the disease slowly lose their vision until they go blind. In retinitis pigmentosa, only the rod cells are affected, creating a unique characteristic at genetic, protein, cell, and tissue level, and have unique challenges in treating the disease. 
The disease usually progresses with prolonged time to adjust to the dark followed by the inability to see in the dark, and restriction in vision. While disease progression differs significantly between different people, most people lose their sight eventually. Because of this the patients often face unique challenges such as being aware that they may have the disease because there are family members that have the disease. Like many people who have a progressive vision illness that ends in blindness, people often have a harder time adjusting to blindness compared to those who were born blind. However, they also tend to be more accepting of the fact that they will be blind compared to those who lose their vision suddenly, with people indicating that things were not as bad as they thought. Other challenges that the people who have this illness must face is the challenge of learning braille, using walking sticks, inability to drive and fear of losing their job or not being hired due to their illness. People who have the blindness describe their sight as though they are walking into a dark room with sunglasses on. The peripheral vision fades first and when the disease advances enough, the vision appears to be narrowed, almost as though looking through a tube. Despite the challenges that the disease poses, it is not fatal, and only vision is lost. 
There are many different modes of inheritance in retinitis pigmentosa, including autosomal dominant, autosomal recessive, x linked and mitochondrial linked. However, RHO  P23H mutation on chromosome 3, also known as RP4 is autosomal dominant and is the most common form of the disease.  The disease was first discovered in the 1970s, with the mutation to be determined in the gene RP1. For the autosomal dominant RP caused by the mutation of P23H rhodopsin gene mutation, the genetic defect was discovered in a large Irish family that had early retinitis pigments for 5 generations in 1989. The paper also tried to establish that the mutation was on chromosome 3. In 1990, a paper established that the P23H mutation in the RHO gene was what caused the disease.

Hi, 
My name is Ziwei, and this is my poster on how the removal of the seed coat affects the seed germination rate. So, what is the seed coat? The seed coat is a protective covering that surrounds the seed and protect the seed from the environment that the adult plant may not be able to survive in. In addition to the protective role that the seed coat plays, the seed coat also plays a role in controlling germination and produces some compound that is beneficial to the seed. This indicates that while it may seem like the seed coat is not doing anything, the seed coat is actually really biologically active. One of the things that have been suggested recently is that the seed coat actually impedes seed growth. Of course, we can all see why that would be important. If the seed starts germinating, there is no going back. you can't turn time back so the seed has to be sure that the ideal condition is met. However, this becomes a problem in agriculture where the ideal environment is provided. the ideal, in this case, would be for the seed to germinate as fast as it can so that the time is not wasted. So, with that idea in mind, this project was done. We removed the seed coat of the seed, and allowed it to germinate, and measured the rate. Our result indicate that the seed germination is somewhat faster in certain types of seeds, however, we were not able to get a definite answer of whether removing the seed coat caused the seed to germinate faster. my personal theory is that because there are so many compounds that seed coat produces, there may be some compounds that are produced by the seed coat that is needed for germination. Our next experiment would be to remove half of the seed coat and see if that would make the seed germinate faster.  

Seed coat, also called a testa, is an outer covering of seed made from integuments that surround the ovule. Seed coats provide protection to the seed, allowing for the seed to survive conditions that they would otherwise not survive. Seed coat also has a role in controlling the growth, development of the embryo and create a compound that helps with the defense of the cell, with a large number of genes that are specifically expressed only in the seed coat, indicating that seed coat serves as more than that of a physical barrier. While the Seed coat serves an important protective role for seeds, there is evidence suggesting that the seed coat may also inhibit germination in some plants. The aim of this study is to see if the removal of the seed coat, which in turn removes all of the compound and protection offered by the seed coat result in faster germination compared to seeds that do not have their seed coat removed in the specific seeds studied.

To study how the removal of the seed coat affects germination, the seed coat was removed from 6 species of seeds. To do this, 20 seeds of each species were soaked in water for 1 hour. Then the seeds were divided into two groups ten, one group being the control, the other being the seeds with the seed coat removed. The seed coat was removed using an Exacto knife and slicing the seed coat and then peeling it away. The groups of seed were then each placed onto a petri dish individually labeled with their type and experiment group that contains a wet paper towel. The petri dish was then closed and placed in a dark corner, with the Petri dish covered by the lid. The seeds were checked every 12 hours and the state of the seeds was recorded. The state was defined to be initial germination when there was a sign of germination.

The conclusion of the study is that the method of AAV9-PHPB delivery and CRISPR/Cas9 treatment combination resulted in the most effective disruption of the mutant gene, indicating that this method of the treatment may be the most effective and is likely to be successful in human studies. The study also indicates that the treatment was done successfully in a human cell, with high efficiency using the CRISPR/CAS9 treatment. The researchers also tested the method in mice, which indicates that not only does the treatment works on human cells, but it also works at a larger scale and is unlikely to affect other organ systems.  Specifically, of all of the treatments and delivery, it has been noted by the scientists that the mice that were given treatment through an injection into the eye, had a higher level of damage compared to the untreated eye, but had less damage compared to other methods of delivering the AAV virus. By indicating that a method of treatment that has worked in the human cell and mice. If the treatment has worked in both human cells and mice it is an indication that it is also likely to work in a human patient. Using data that is published in this paper it would be possible to go to human trial, which if it works, will be able to have permanent treatment for the retinitis prigmentosa, which takes away the sight of those that are affected. 

https://www.ncbi.nlm.nih.gov/pubmed/29281027

Figure 3 shows the same pattern as those seen in our experiment. The two markers that the researchers that were used to measure cell proliferation are Ki67 and BrdU. Ki67 is a protein that is highly expressed in cells that are proliferating. Ki67 labels cells that are in M, G1,S, and G2 phases. However, because the only cells that do not express the protein are cells in G0, there are many cells that would be positive for this marker. BrdU is extremely similar to EdU in how they mark cells that are proliferating. Unlike Ki67, BrdU and EdUmarkers only mark's cells that are undergoing S phase, as a result, it is more specific. However, BrdU is an antibody compared to EdU which is a click it reaction and incorporates thymidine analog into the DNA.  In hyperthyroid conditions, A2B5 was upregulated in the mitral cell layer compared to the control, and also had a positive cell in other layers of the olfactory bulb. There is a decrease in MBP protein expression in the optic nerve of hypothyroid rats. The conclusion that I can draw about the effect of thyroid hormone on oligodendrocyte progenitors and mature oligodendrocytes is thyroid hormones regulate something upstream of pre oligodendrocyte and mature oligodendrocyte. This conclusion can be drawn because the thyroid hormone is increasing preoligodendroctyes in hyperthyroid conditions which indicates that there are more preoligodendrocytes in hyperthyroid conditions. The decrease in MBP protein suggests that there is a decrease in mature oligodendrocytes.  From these two pieces of information, no conclusion can be drawn since they are in different parts of the brain and indicate two different types of cell regulation. However, since the paper mentions that there is an increase in MBP protein in hyperthyroid condition and a decrease or no change in hypothyroid condition, as well as that A2B5 is not observed in hypothyroid brains. Using this information, I can predict that the thyroid hormone regulates something that is upstream of pre oligodendrocyte. However, using the information that is just given, no conclusion can be reached because a decrease of thyroid hormone should have an opposite effect and that is not seen as clearly here.  

The goal of this study is to determine whether the effects of T3 on progenitor cell proliferation and oligodendrocyte maturation are causally related or instead are independent. O-2A are biopotential glial progenitor cells. A2B5+ marker for presence of O-2A (biopotential glial progenitor) cells. GC+ is a marker that is expressed by mature oligodendrocytes.Table 1 shows the percentage of glial progenitor cells and mature oligodendrocytes that are present in the cell cultures that has the T3 added and the control.  The control numbers mean that when there is no T3 present, there are around 75% glial progenitor cells and 6.8 mature dendrocytes 6 days after incubation. The T3 numbers means that 6 days after the T3 was added there were 24 percent glial progenitor cells and 57 mature oligodendrocytes, indicating that the presence of T3 causes the cells to mature into mature oligodendrocytes. Table 2 indicates that T3 blocks proliferation. The data is showing that T3 blocks the proliferation by marking the glial progenitor cells that have taken up BrdU. Because the percentage of BrdU in A2B5 labeled cell was twice as much in control as compared to the T3 treated cells. This indicates that the amount of A2B5 that are going under S phase, and as a result also indicates cell proliferation. Because the proliferation is twice as high in the control, the data is demonstrating that the proliferation is lower in the T3 cells. Because all other factors are kept constant, this table shows that T3 is what is causing the cells to stop proliferating.

The two main observations that are made in this paper are that there is a low level of thyroid hormone at the time of birth, with a sharp increase on day 6, peaking in day 15 and falling at day 25. This observation is shown in figure 1. The second observation is that in mice with induced hypothyroidism, the rate of mitosis increase compared to controls at day 2 and day 6. This observation is shown in figure 2. In figure 2, the researchers are measuring the cells that are going through DNA synthesis. This, in turn, indicates the cells that are going through mitosis. The difference between P2N and P6N indicates how many cells have been labeled with BrdU, and by extension, how cell division has increased or decreased in control mice brain on day 2 compared to day 6. The difference between P2N and P6N indicates how much cells have been labeled by BrdU and consequently, the amount of cell division has increased or decreased in PTU treated (induced hypothyroidism) mice in the brain at day 2 compared to day 6. P2N/P6N observation relates to the data in figure 1 because the thyroid level of the brain in P2N and P6N  which are normal levels are in figure 1. This indicates that at time of P2N and P6N, the level of thyroid hormone, which is not mentioned specifically, is found in figure 1. From figure 1, the conclusion that can be drawn is that because there was an increase in thyroid hormone from day 2 to day 6, the decrease in mitotic cells in P6N compared P2N indicates that thyroid hormone may be causing a decrease in cell division and DNA synthesis. The hypothesis of this paper hyperthyroidism causes decreased mitotic activity compared to control mice. In the paper, the researchers never explained the biological mechanism of how hyperthyroidism causes a decrease of mitotic activity, only that it does actually cause the decrease rather than just correlate with the decrease.

Figure 2 shows that the her2 is regulated by the delta notch pathway. The authors came to this conclusion by measuring the expression of her2 in notch deficient embryos. This shows that the her2 is somewhere in the notch pathway because everything else is kept constant. Because neurogenesis occurs in the first 3 days after fertilization, since the drug was added at day 5, her 2 is no longer regulated by Notch signaling at the time that our study is done. However, because the study that we are doing involves not only neurogenesis, but also oligogenesis, and how those two things that are influenced by the thyroid hormones affect stem cells. In the paper, while the paper mentions that her2 is not regulated by notch signaling after oligogenesis, it does not discount that her2 is not affected by the thyroid hormone through some other pathway. Because of this, it is possible that her2 does have an effect on the neural stem cell. However, the only thing that is known is that if her2 does affect neurogenesis and gliogenesis, it will not be through the notch pathway.  I think that her2 is definitely playing a role that can be seen in the class data. The paper mentions that her2 induces glial differentiation and that inhibits the neural differentiation, which was similar to how hypothyroidism affects the neural cells in the papers we have read. However, I am not sure whether her2 directly responds to TH. There is no evidence for me to think that the two are related, especially since her2 is not connected to notch signaling in fish that are past neurogenesis.  I think that her 2 is promoting the formation of glia because the study is involved in glial differentiation, this would imply that her2 is promoting the formation of the glia.