ziweiwang's blog

I've had the opportunity to work with mice over the summer. It was really interesting to me because I've only ever worked with cell culture before. one of the things that I noticed while working with mice over working with cells is how variable the data was. There were some major differences between the highest number and the lowest number. I asked the postdoc about it and he told me that this is actually quite common. It interested me that there was so much to work with mice, especially working with mice that often didn't live until the reproductive age and as a result, the mut mice could only be produced by two het parents and praying for lots of pups. I've learned what to do and what not to do when making mice lines, and what is possible but still not a good idea.  I've also been quite good at handling the mice and doing oral gavages while the mice are still awake, although I was not quite as good as to do collect the blood without anesthesia. On the other hand, I did manage to collect blood without killing the mice I'm proud. 

I've also learned the reason why mice work was not for everyone. I ended up having my fingers bitten, and because a permanent mice line could not be produced, many of the mice had to be euthanized. This was quite a bit painful to watch and the way of being euthanized was not always framed in a way that looked painful but was not as direct as dislocating the spine. I think I will work with mice again, and it was a really good learning experience, but I think I will be less judgy when people are unwilling to work with mice. 

The goal of the research is to use the bioinformatics and homology model to obtain three dimensional native and mutated PMP22 models, and show how it is anchored to the cell membrane to determine how L16P and T118M mutation affect the conformational behavior of PMP22.

The conclusion obtained in the experiment tells that there is less stability with mutated protein compared to the normal proteins, resulting specifically from residue 16 and in alpha-helix h1-h2. The data obtained in the experiment also concluded that there was less hydrogen bonding at the side near the mutated site and as a result of this deviation from the normal protein, the mutated protein has a harder time getting out of the ER.   

The impact on the disease is that when the structure and how the protein complex interacts, it would make the mechanism of the disease easier to understand, and by understanding the mechanism of the disease at a molecular level, a relatively noninvasive treatment can be created to combat the effect of mutated proteins. 

The data from anchoring the protein complexes into the membrane tells that there is no spacial difference between the converged area per lipid values (table1).  In addition, there is a higher delta g binding in the mutated protein compared to the normal protein(table 2,3). Another difference includes the delta g total was higher in the mutated protein compared to the normal proteins (table 2,3). The results of the experiment also showed that residue 16 was crucial for changes that are induced by the mutation, and both of the mutations alter the structure and the interaction of the proteins by changing the binding properties. In the PCA, it’s revealed that PMP L16P hsd more negative eigenvalues on the first PC and PMP22 T118M had a more positive eigenvalue (figure 2). Revealing that the mutants are more flexible compared to that of the mon mutated protein, which suggests a higher number of conformational states.  The data that the mutated system is more flexible is supported by the data that the mutated systems have are less stable than the normal protein. The PCA also showed that the highest fluctuation was concentrated along the alpha helix 3 loop and the alpha-helix 4 loop in normal protein and in the mutated protein, the fluctuation was highest at lppo alpha-helix 1-2. In both of the mutations, there was a reduced number of hydrophobic contact and less hydrogen bond around the mutated parts of the protein (figure 2,4,5). The mutated complexes also have a higher delta g binding, leading to the inability of the protein to leave the ER(table3).

The first step of the experiment that they did is to obtain a 3D model of PMP22 and RER1. The three-dimensional structure was determined by homology modeling using mouse PMP22 protein which shares 29% of its PMP22 genes with humans. The 3D model was then mutated at L16P and T118M to generate the mutant protein. RER1 was then docked to the constructed protein to simulate its behavior in the ER. The PMP22 system was then anchored to POPC bilayer membrane and submitted for .5ul MD simulations, which mimics what happens in real cell membranes.  

    The second step was the analysis that was done with the data that was obtained in the MD simulations (figure 1). The researchers did Principal component analysis, which reduces the complexity of the data and extracts the relevant motions of the atoms examined (figure 2, 6). In this analysis, eigenvectors, which describes the motion of the protein and eigenvalues, which in this case describes the total mobility associated with each eigenvector, were identified. The protein-protein docking calculations were also performed (figure 3,4,5). This calculation generates a detailed model that indicates where each atom is at any time. The effective binding energy was also obtained through the MMGBSA approach.  This allows for the estimation of effective binding free energy, which is the energy needed to disassemble a protein or a molecule. The researchers then calculated the effective binding free energy for each of the PMP22- RER1, which is a measure of total free energy differences in the molecules that are involved (table 2,3). Clustering calculation was also performed to identify the most popular conformations. 

Phytophagy means eating of plants. It usually describes insect behavior. However, it can also be interpreted as any animal eating a plant. In this project, I took two pictures of evidence for autophagy and organized it into a figure. A map of where the evidence was found was also included.  I wrote a method on how to take that picture and someone else followed my method to make a similar figure. The result was a similar figure that had some differences in formatting, the object in the photo, and the photo quality. The biggest difference that changed how the figure looked was differences in formatting, whereas photo quality contributed to the difference but did not cause the figure to change drastically. The difference in the figure that is described in the result is due to a differing camera,  method of image export, object, and hand that was taken. Due to these differences, the two figures were similar but not completely the same.

The reason why the formatting of the figures was off is that the person who made the replica had a different default than I did, so the replica and the original is different. One of the specific ones is the big border on either side of the map. This happened because the image was exported in a different way, and as a result, the figure was different. another reason why the formatting is off because I had a different idea of what an arrow was compared to the person who did the replica. 

The second category of differences was mainly that pictures of the object in the two figures were different, as was the person holding it. because of this, there were some differences in the leaf and in the hand holding it. In addition, there was also some different background because of this there were some fundamental changes that could not be reconciled when the picture was taken. 

The third category is the difference in photo quality and lightening. This is due to the camera being used is vastly different compared between the camera that was used to take the original compared to those that were used to take the replica. 

The biggest difference in the two seems to be the formatting of the figure. The major difference of the figure being the map. the first difference is two big borders on the map, which I didn't have. the second thing about the formatting is that the replica had an arrow that was pointing up rather than down. the arrow was longer in the original figure compared to the replica, and the arrow was more narrow. The third difference is the size of the label. It appears that the original label is quite a bit larger than the replica. 

Another difference is the image itself. The two leaves are significantly different whereas the leaf in the original is more rounded, the leaf in the replica is more oval-shaped and has a lighter color compared to the darker colored leaf in the original. The hand was also different. the third thing that was different is that the original had a background that had a slight slash across the back that the replica did not have. 

The least noticeable category of difference in image quality. The image quality on the original is clearly better than that of the original, and because of this, the original is noticeably blurrier than that of the replica. In addition, the original has a more warmed toned hue compared to that of the replica. 

During my planning process when I was thinking of what I wanted to take pictures of, I immediately thought of a salad bar or a dish with salad on the plate. However, one of the major disadvantages of that would be that it would make replication almost impossible. it's hard to describe a mass of things like salad. Then I would have to describe things like how many leaves there are on the plate and which leaf faces which direction. The way that this would be the easiest to replicate is to make this as simple as possible so that there is a limited amount of variables. In addition, the way that the method is interpreted may also be different depending on the person following it, so there should be as little misunderstanding as possible. to limit those variables, rather than taking a picture of an entire salad in which each leaf would be a variable, or taking the picture of the salad bar, which would cause wide variation in angle and color, I think that having a single leaf would be the most advantageous to me because describing one leaf would be much easier than describing multiple leaves, and if I just describe taking the picture from the front of the leaf, I think that most people would understand what I mean and would take a similar picture to how I'm imagining. To make sure that the background is not a factor it would be best if it was made as descriptive as possible, like a gray background. For the stylization of the figure, it would be best if the figure was made in such a way so that everything would be the default except for the things that are changed. this would ensure that the figure is more likely to be similar, although if the person following the method does not use Inkscape or have other ideas in mind, that would change. However, there is a limit to things that I can specify, and it would be confusing if I just listed all of them. It should be enough that I list the things that I changed and just assume that the rest will be the default. 

Phytophagy means eating of plants. This is often said regarding insects eating plants. However, it can also be interpreted as any animal eating a plant. I took a picture of evidence for autophagy in plants and presented it as a figure. I then wrote the method on how to take that picture and someone else followed my method to make a similar figure.  The result was a similar figure that had a differing light composition, sizing, and formatting of the map and different size and shape of the leaf. The difference in the figure that is described in the result is due to a differing camera, a differing preferred method of image export and differing leaf and hand that was taken. due to these differences, the two figures were similar but not completely the same. 

In doing research on the food allergy model in mice, transgenetic mice such as il4 gain of function mice are used as a model to do food allergy research. Mice with il4 gain of function are more susceptible to allergens, which makes sensitizing them for food allergy studies easier. In the case of the study that I've done over the summer, the procedure for sensitizing mice is to slowly expose the mice to the allergen along with SEB and then cause an anaphylactic shock by introducing them to a large amount of allergen. This would cause the mice's temperature to drop, which can be measured using an internal probe. The challenge to this model is the high variability. Because this is an animal model, the failure rate can be quite high, and the amount of temperature drop can vary wildly. This, I found out when I had to present the results of what I did over the summer to my PI who is an organic chemistry professor. The professor asked me why the error margin was so high, and I tried to explain that in these organismal models, the error margin is always relatively high compared to models with cells or proteins. Another problem with this model is that it is hard to do with many sample sizes. Because the measurement occurs in less than an hour, this would mean that the limit of how many samples is determined by how quickly oral gavage can be performed on a conscious mouse. This limits the size to about 25 mice at most, because oral gavages can take quite a long time to perform, especially if the mice are not cooperating. However, the benefit of doing this mouse model is that it is standardized across the field and it is relatively easy and inexpensive to do, especially for a lab that already works extensively with mice.