ziweiwang's blog

People who have RP4 goes through unique challenges that result from their vision loss. In patients with RP, the disease usually starts in childhood when patients find that their eyesight takes a long time to adjust to the dark. This is followed by eventual loss of night vision altogether, which the patients often describe as though they have walked into a dark room with sunglasses on.  This is followed by the loss of peripheral vision, where the field of vision becomes narrower and narrower until complete blindness. Despite the devastating effect of this disease on the quality of life in patients, RP is not fatal, and as a result, the lifespan of those who suffer from the disease is not affected. People who have RP generally know that they may also have RP because their family members have RP. Because RP is a degenerative disorder, people with RP find it harder to accept and adjust to being blind than people who were born blind. However, patients of RP also tend to adjust better than people who lose their sight suddenly as they have time to prepare for the eventual sight loss. Some of the adjustments that come with the loss of sight are, learning to use a walking stick and braille. Other challenges that are posed by the disease are there is a need for adjustment in jobs and loss of ability to drive. However, despite these challenges and adjustments, people who have advanced RP also have said that the disease was not as bad as they thought and that even though they are blind, they are still able to participate in society, which was not something that they expected.

 I can't say I didn't enjoy the poster making the process. Something about having a graphic to make was calming. I'm good at giving presentations, unlike writing, so that fueled my concentration and willingness to make the poster. I think that I actually did quite well on this particular project. Especially since I was able to look it over and make sure nothing was missing and those were things that I can actually check for unlike things like wordiness where I think I know what to check for, but the paragraph ends up getting wordy anyway. 

    For the drafts that the class was asked to post every week, it wasn’t as hard to do as I thought it would be possible because I had so many classes that required me to write this semester. When I didn’t really have any classes I usually wrote about the experience of being in a lab and how it was different from the classroom and how procedures were done in different settings. I think that this sort of forced me to write relatively consistently, even though that wasn’t every day. I think I can handle it if it was every week but I don’t think I can do it well if it was every day. I also think that if I didn’t have a class that required me to write every week, I would do it because of this, it would be a good exercise, especially since I think that the key to being good is not the quality but the quantity. I personally thought that having to do drafts was a good idea to have everyone write more in a stress-free environment was a pretty good idea and I enjoyed it, especially because it was something that I had to be doing anyway.

I think that the biggest problem is that I was kind of vague about the project and I tested the project constantly and switched the project method constantly, so it made the group members a bit difficult to keep up what is actually happening. Of course, I also did a run-through of the project to make sure that it is actually feasible which it was, but it took me 8 hours to do so, and that was without the research. I kind of regretted this because I just wasted 8 hours on things that wouldn't be graded on when I could have been working on grammar, which was something that needed desperate attention. I think that this project that I did the worst one, especially since I was so burned out by the end, I forgot to do a final run-through of editing process, and while both of my group members told me that they have done a run-through by themselves, and this is going to sound terrible, I don't necessarily trust them to get things done. 

The third project went surprisingly alright. It, however, started terrible, with group members going missing at a crucial time point when data needed to be gathered. I was actually quite angry at this because group members have failed to show up before and I‌ think at least an apology was warranted, as the plan went off-course and other people's time was wasted, but I never got any, which was frustrating, because it just shows how much the group members only care about themselves. After the Thanksgiving break was when the real hell started. First, no one answers my emails. Then my concerns about who is going to work on what has brushed aside, and only when I absolutely refused to do more work at the night before when the poster was supposed to be printed at around 1 am everyone got to work. Most of the things that were needed for the poster was done at 10 am. We ended up meeting to print the poster at 6\.

The negative side of this is that it's time-consuming, and combined with my procrastination I usually end up only able to revise one or two times before handing it in. When I first did the first draft of the method section, it was the actual first draft, so it was not the best it could be and while I understand that the points were taken off, I wish I'd have known that I would not be allowed to edit after. I don't actually remember much about this project because it was an individual project, and while I was really frustrated, it was mostly about my own inability to write well. I know where I have problems with, and I try to make sure that I get better, but I also can't improve my writing significantly in only a few short months. I know that it's important, which is why it's so frustrating that I am not able to. Knowing that I'm not good at writing, and knowing that that will hurt me grade-wise, I probably spent more time catatonically awake at night than working on the actual project. But if I'm honest, I think this might the best project that I did for this course. It was both simple and the writing was at least somewhat more organized than the others. 

With the proposal project, I began working with two other classmates on this project. I think that the most frustrating thing wasn't that the classmates were bad at writing, but that we had some trouble lining up our work habits together. I tend to procrastinate, but I also tend to get at least the first draft done in advance and then wait a few days then redo them and hand them in literally at the last minute. I think the classmates that I worked with like to do things last minute this did have some trouble with the scheduling but at least for this project, it mostly worked out.

This year, I worked on a total of three projects for writing in biology class. These projects included methods project, where I was asked to create a method, and someone else would follow it, and I would be then asked to analyze the results and why there were differences. The second project that I‌worked on was the proposal project, where were were asked to propose a project that can be done in the class. The third project was the poster project, where we would follow the method proposed by the proposal to make a poster around the result. Throughout the year, I was asked to write some drafts every week write a perfect paragraph every week and write a reflection essay at the end of semester In the end, I think that in these three projects, method, proposal poster, drafts, perfect paragraph and reflection I did learn many things, however, I also face some unique challenges such as working in a group, dealing with my own mental health, and learning to ask for help. 

For the method project, because the project was done individually, the biggest challenge that I faced was my own problems, namely my less than a stable mental state, my insecurities with writing, and my tendencies to mess up the first draft significantly. When I first started the project, the biggest thing that I thought was less what place to put the camera or where to take it, but how can I make it so that the result would be the most similar and I approached it from there I also did write the method section of the project and hand it in, but it was somewhat incomplete. This was a major problem because usually when I first submit a draft, as most professors can tell, it is terrible. I usually generally rewrite for every single draft. While this is time-consuming, by rewriting it, the paragraph structure would sound less forced and more natural compared to just being edited part by part.

The result indicates that because the experiment was done in vivo, the results of the experiment suggest a high chance that the method would work in an organism as long as the two organisms are similar. In the third experiment that was done the ONL thickness in electroporated eyes and the non electroporated eyes were assessed through histological analysis. The result of this experiment indicated that the retina is able to undergo phototransduction. However, the researchers also observed that there was damage to the retina from the electroporation treatment. The results suggest that the treatment works in restoring vision, However, electroporation is not a viable method for use on humans in rescuing vision. The fourth experiment tested the efficiency of using AAV mediated delivery by injecting the virus with therapy that was used in previous experiments and indel frequency and TIDE analysis was done to evaluate the efficiency of this delivery method. Using this delivery method, all retinal cell types were targeted but to a differing extent. The P23H mutation had a moderate level of cleavage while wt was unaffected the P23H also had a 5 times higher amount of indel compared to wt. This indicates that the viral delivery of the treatment was as effective as electroporation and usable for human trials. In the fifth experiment that was done the NGS analysis of the edited retina was performed through a sequencing analysis. The result is that plus one and plus two insertions was the most common mutation which caused a deletion codon, indicating that it would be unlikely that there would be an inframe mutation. This indicates that the treatment method is highly selective and is effective at the silencing of the mutated protein.

 In the sixth experiment, Human cells were treated with the therapy to induce mutations into the cells using sgRNA-hWT and hMUT. The results show that both guides showed different cleavage efficiency in different analysis and there was a high specificity in targeting the complementary genes.  In addition, because most of the indel inframe mutation was very low there was a higher chance that the mutation would have no effect on the gene. Because of this result, there is a high chance that this method of delivery would also work well in the human retina. This paper allows the field of disease research to move forward by showing a method of treatment that has worked in the human cells and mice cells this would mean that this treatment system and the delivery combination is likely to work in the human eye and that the treatment can be moved into clinical trials. 

While retinitis pigmentosa has unique genetics, protein structure, and tissue behavior that result in unique challenges for both treatment providers and patients, there have also been advances that are developing to solve the problem of the illness. However, there will be a long time until a cure is found. Because of this, funding and continuation of research will be crucial.

Despite the imperfect animal model and lack of specific treatment for RP, there have been certain methods that have been tested for retinitis pigmentosa, including gene therapy, small molecules that stabilize the protein and microchips that take place of retinas. However, microchips that are currently in development would only be viable for those who have lost their sight completely. Those who still retain their eyesight does not have many treatment options. One treatment option for those people who still retain some eyesight is gene therapy. Because the eye is immunologically isolated from the rest of the body and easily accessible, some complications from gene therapy is not a factor in the eye. In the research paper, Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery, the paper aimed at validating a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant while preserving the WT allele in vitro and translate the approach in vivo by delivering the CRISPR/Cas9 component in RHO mutant retina.  In the first experiment that was done, the researchers created various Cas 9 containing vectors to test with one disrupted the P23H mutation most efficiently. The cleavage efficiency was measured using T7El assay, TIDE, and Sanger sequencing. The result indicated that sgRNA-mMUT discriminated with the highest specificity of the mutant RHO sequence, and no off-target indel was detected. The result indicates that sgRNA-mMUT is the best candidate and should be used in further studies regarding the gene therapy of the retina. In the second experiment, to test whether the treatment would work in vivo, the vectors were inserted into the retina of the mice by in vivo electroporation and the product of the genes was assessed for the behavior of the RHO protein and mutation rate in vivo. The final result of the experiment is that Cas9-VQR/sgRNA-mMUT system enabled an efficient and selective targeting of the P23H mutant capable of a high degree of inactivation in the RHO allele in vivo.

To measure the effect that thyroid hormone (TH) has on neural stem cells, thyroid hormones in zebrafish were manipulated, fixed, treated with EdU, and imaged. TH levels in zebrafish were altered by adding either, T4, a precursor to the T3 thyroid hormone that is biologically active, or PTU, which blocks the conversion of T4 to T3, therefore significantly decreasing the amount of thyroid hormones available to the body. In the first trial of the experiment we discovered that our drug dosage was too high and would kill the fish, so we lowered the doses in the second trial to 300nM of PTU and .5mM of T4. The 5dpf zebrafish were treated with respective drugs for two days. At 7dpf, the fish were fixed in 4% PFA and the brains were dissected. The dissected brains then went through an EdU Click-it copper-catalyzed cycloaddition reaction where a thiamine analog is added to dividing cells and labels those cells with fluorescent dye. EdU is incorporated into actively dividing cells and is often used to label stem cells in a proliferative state. The brains were then divided into well reacted and poorly reacted and imaged in both bright field and fluorescent field and  merged. The representative images of each treatment in well reacted brain are shown in Figure 1. The poorly reacted brain are not shown because the poorly reacted brain could nor be imaged due to time constraints. Because of this, there are no poorly reacted brain EdU+ cell count data for this group.  

    The EdU positive cells in Lateral Recess and Posterior Recess were then counted and the number was recorded (table 1). The average and standard deviation was calculated for each condition. The result indicated that the  average EdU positive cells in Lateral recess in the control is about 35( standard deviation:22), in PTU, 27, and in T4, 44(.7). Meanwhile the EdU positive cells in posterior recess is about 11(7.37) in control 15, in PTU and 13(1.17) in T4 (figure 2, A). The standard deviation was not included due to low sample size.  While there are some differences between the different values, due to the low number of samples that are available, no significant conclusion can be reached with this result.

 In the data which was gathered by the entire class, the well reacted brain data indicates that in the lateral recess, there is an average of 56(32) EdU+ cells in control, 62(37) in PTU treated cells, and 67(32) in T4 treated cells.  This indicates that both PTU and T4 increases the amount of neural stem cell in the lateral recess. In the posterior recess., for controlling the average number of EdU+ cell is about 21(8), in PTU is about 21(5), and in T4 is 24(6) (figure 1, B). This indicates that while PTU does not have an effect on the number of neural stem cells, the addition of T4 increases the number of neural stem cells. However, the data may not be reliable since the standard deviation for the data is high. In the poorly reacted brains, the data indicates that there is an average of 38(21)for control, and 59 for PTU in the lateral region and 18 (7) for control and 23.5 for PTU in the posterior region (Figure 2, C). The standard deviation for PTU treated brains and the average and standard deviation of T4 treated cells were not included due to incomplete data.  However, very few things are able to be concluded from the pooly reacted data because it is unknown whether the poor reaction is due to the cells having low neural stem cells or not reing reacted completely to the click it reaction, and there are very small sample size.

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 protects 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 compounds that are beneficial to the seed. This indicates that while it may seem like the seed coat is not doing anything, the seed coat is actually biologically active. One of the things that have been suggested recently is that the seed coat actually impedes seed growth.  The reason why the timing of germination is important is if the seed starts germinating, there is no going back. There is no way for a germinated seed to become ungerminated. However, there are situations where a faster germination rate would be an advantage. To see if the removal of the seed coat increased the germination rate, we removed the seed coat of the seed, and allowed it to germinate, and measured the rate. Our result indicates 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. Due to the small sample size. 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. Because of this, my next experiment would be to remove half of the seed coat and see if that would make the seed germinate faster because if only half of the coat was removed, there would be a beneficial compound without the physical barrier of a seed coat. That's basically what this project is about. Do you have any questions?

 In the second experiment that was done for the paper, whole mounted retinas from control and P23H rats were labeled with Cx43 and GFAP.  The results indicated that the density of Cx43 IR puncta in GCL was greater in the P23H rats. Cx43 levels were increased 2 fold in the P23H rat retinas compared to the control. The results suggest that there is a role of Cx43 in the pathogenesis of degeneration. The expression observed in P23H rats correlated with astrocyte hypertrophy suggests an increment of Cx43 expression per astrocyte. Retinal immunofluorescence was done using the marker GFAP and vimentin. Immunofluorescence of GFAP protein was done. P23H rats showed intense GFAP immunoreactivity and revealed colocalization between intermediate filament proteins. The vimentin/GFAP positive Muller cells extended into the subretinal space and expanded, filling areas left by degenerated photoreceptors. There was a 7.8-5.3 fold increase in GFAp expression in the P23H rats compared to the control. This data suggest that the GFAP expression increased in retinal degeneration. The increased GFAP expression is also thought to stabilize newly formed terminal processes of Muller cells and provide resistance to the stress and is essential for the formation of glial scars neurite growth infiltration of monocytes neovascularization, and integration of cells in retinal transplants. GFAP immunoreactivity was analyzed in the ONL of whole mounted retinas from control and P23H rats.  In P23H rats, there was more loss of photoreceptors in the ONL which was linked to the appearance of hypertrophied side branches of muller cells into the outermost photo layer. Most of the cone cells also expressed a short morphology. The loss of rod cells also altered the cone mosaic in the ONL there is a correlation between the ring-like area of cone degeneration and the muller cell apical processes. The results suggest that the orientation of the cone cells are disrupted by cone degeneration with muller cell apical processes forming clusters.  This also indicates that the loss of photoreceptors induces changes in vascular tissue that activates muller cells. The overall research shows that the disease in the photoreceptor cells also affects the cells that are around those cells, and as a result, there is a need to take into account when discovering future research.

 To do the experiments  P23H-1 line rat is often used. To a certain extent. It presents a similar phenotype but not the genotype since the mutated rhodopsin protein gene sequence is inserted into the rats and the preexisting normal rhodopsin was not mutated. In addition, the rats that were used in the studies had 18 copies of the mutated gene, whereas in a human there would be one or two at most.  It added to the understanding of the disease by creating a model of the disease that can be studied, even though the model is an imperfect one.