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result conclusion and the impact of the study

Submitted by ziweiwang on Thu, 11/07/2019 - 23:08

The second experiment established that in the N15S, one glycosylation site is missing, which caused a change of molecular weight, allowing it to have a lower molecular weight than normal. After PNGase F treatment, however, the protein appears more similar to the wild type indicating that it is more like the wildtype compared to when it is not treated. In the P23H, the mutation that the group is interested in, there was also a lower molecular weight, which indicates that there is degradation. Compared to the control, both of the dimers were had a heavier molecular weight compared to that of the wild type which indicates that aggregation is more severe in the mutants, with the P23H being more severe compared to N15S(figure 3)


In the 3rd experiment, the total fluorescence was lower than the wild type in both of the mutants. The researchers hypothesized that this may be due to the aggregation of the mutant proteins. There was a reduction in the stability in N15S, compared to WT, and P23H was much less stable compared to both. However, because there was a total decrease of fluorescence signal, which indicates that the properties of that are expressed in this experiment causes some of the mechanism of pathogenesis of the mutants. 


In the fourth experiment, the experiment indicated that in N15S, there was a decrease in alpha helix, an increase in beta-sheet, random coils and turns compared to the wild type. In the P23H, there were even more differences compared to that of wild type with an increase in random coil and decrease in alpha-helix, beta-sheet and turns. This indicates that when the P23H mutation happens, there is a larger structural change compared to the N15S mutation. (figure 4, table1)


In the fifth experiment, the rescue experiment was conducted. The proteins were denatured, in the presence and absence of chlorin e6.  The proteins were then put through circular dichroism spectroscopy. In the resulting graph, the % helix content was lower in P23H with chlorin e6, but not in N15S. This indicates that the compound restored some of the stability to the protein with a mutation of P23H.  This implies that compound chlorin e6 may be a viable treatment for patients with a mutation of P23H but not with N15S.

The experiment provided the answer that when N15S mutation and P23H mutation was compared, in general, the P23H mutation was less stable, and had a higher aggregation compared to N15S. However, despite the more serious effect of the mutation, P23H was also able to be resued by a compound choline e6, indicating that while the symptom of those with the mutation of P23H is more severe, there is also a compound that is likely to be able to rescue the mutation and restore the stability of the protein, and preventing it from aggregating.
The paper impacts the disease by indicating why the mutation of P23H has a more impact compared to patients with a mutation in S15N. This study also indicates which secondary structures of the protein may be the reason for the illness, and a possible compound can be tested in animal studies, for their ability to stabilize the alpha helix of the P23H protein mutations.

research goal and methods

Submitted by ziweiwang on Thu, 11/07/2019 - 23:07

The goal of the research is to compare the two mutants of the rhodopsin mutation, P23H, and S15N. Specifically, the researchers were interested in how the mutations in the N terminus domain affect their invitro biochemical properties, their UV visible absorption, Fourier transform infrared, circular dichroism and metarhodopsin II fluorescence spectroscopy properties.  While this was not a formal goal of the research, the study also found that chlorin e6have the ability to rescue mutated rhodopsin protein that had its stability affected.

In the experiment, the proteins were first prepared through PCR mutagenesis technique. The genes were then placed into HEK293S cell lines and the P23H and V15S opsins were established. The cells were harvested and the proteins were purified using immunoaffinity chromatography. 


In the first experiment, the proteins were subjected to UV spectrophotometry, which would establish the degree of misfolding of the mutated protein. For the second experiment, both of the mutant proteins had their oligosaccharide chain from rhodopsin glycosylation sites cleaved using PNGase F. The resulting immunoblot was used for densitometric analysis of the immunoblot was done using Image J, then the Box plot was used to summarize the result of the analysis. This method would establish the amount of aggregation that may occur in cells, which would disrupt their protein function. For the third experiment, MetaII fluorescence was measured for three proteins to measure the amount of aggregation that may occur in the cell. For the fourth experiment, Fourier transforms infrared spectra were collected for COS-1 cell rhodopsin to gain structural insight into the degree of misfolding in the two mutants. For the fourth experiment, circular dichroism spectra were measured with the samples of rhodopsin and Ce6 at 2.5 and 100 micro M this would indicate the stability of the presence and absence of Chlorin e6. 


From experiment 1, the result of the experiment determined to be that the absorbance of the UV light was increased at the wavelength of 510nm compared to the wild type, and there was a decrease in fluorescence intensity and both of the mutated proteins had a lower percentage of the proteins that are intact compared to the wild type. More interestingly, there was a higher amount of P23H that was not intact compared to N15S. (figure 2)


what is not known about HOX genes

Submitted by ziweiwang on Thu, 11/07/2019 - 20:40

Now, while there have been many studies that are done on hox genes, there have been relatively few studies that were done on HOXC genes specifically. Also, the scope of the study has been quite narrow, with the study often limited to studying a few well known HOX gene in one specific family of an organism or how the mutations in hox genes often affect disease, specifically cancer. In both cases, the studies were mostly conducted through studies where specific hox genes are mutated in a model organism, and the resulting developmental structures are looked at or studying HOX genes in specific animals and using genetic data analysis using specific cancer cells. Because these two studies are the most common studies done on HOX genes, there are very few experiments done on how the hox genes differ between a wide range of species, and how they interact with each other. This experiment would aim to fill some of the gaps in the knowledge through the use of online genomic analysis, which would make it possible to generate a large quantity of predictive data that can be the basis of other experiments which can establish a more concrete interaction between different protein interaction that hox gene would have with other transcription factors.

HOX gene back ground and roles in the body

Submitted by ziweiwang on Thu, 11/07/2019 - 20:40

Hox genes are a family of genes that are vital to an animal’s embryonic development. The HOX genes belong to the homeobox gene family, which is the second-largest gene family which all encodes for 61 amino acid sequence that forms a helix turn helix structure. The expression pattern of HOX genes dictates the body plans of organisms. In vertebrates, HOX genes are organized into clusters that are on different chromosomes named abcd which individually each contain 9 to 11 hox genes  In this study, we propose to study HOXC genes for this experiment. 

HOXC  is a gene cluster that is located on the chromosome. There are 9 HOXC genes;HOX 4,5,6,8,9,10,11,12, and 13.  All of the genes are located close to one another and in some cases share a noncoding exon. HOXC 4,5,6 shares an exon. HOXC4 is involved with the development and involved in the stimulation of androgen receptors. The function of HOXC5 is unclear. However, the gene is associated with Lymphoma. The function of HOXC6 is similarly unknown. HOXC8 is involved in cartilage differentiation and a malfunction of the gene gives rise to cartilage disorders.  HOXC9 is involved in the differentiation of white and brown adipose tissue. The function of HOXC10 is not clear but it is highly expressed in cell differentiation and proliferation. HOXC11 is involved in the development, specifically the mesodermal commitment pathway. The function of HOXC12 is not known but is associated with a club foot. HOXC13 is involved in the development of hair and nails.


retinitis pigmentosa disease inheritance and phenotype

Submitted by ziweiwang on Tue, 11/05/2019 - 20:13

According to the NIH genetic inheritance reference and OMIM, the mode of inheritance is autosomal dominant or recessive inheritance pattern. 

An attempt to discover the gene that is responsible for retinitis pigmentosa was made in the 1970s, which the discovered gene was named to be RP1. more concrete evidence that the gene causes the disease was made in 1999, where two different research confirmed the gene on chromosome 8. For a disease that we are studying, the mutation was first found in a large Irish family in 1989. The RHO gene was discovered in 1992, in a study that studied 12 families that had retinitis pigmentosa. In the 1989 paper, the researchers attempted to identify the mutation through linkage study with a gene in chromosome 3. In 1990, a paper established that the mutation is in pro23 to His mutation in the RHO gene. In 1992, another paper established that retinitis pigmentosa can also be caused by an arg207 to met mutation In RHO gene. So far, there have been 9 mutations in the RHO genes that cause retinitis pigmentosa.

The mutation that we are specifically studying is related to the mutation in the gene that codes for the RHO protein (3q22.1). However, there are other mutations that are associated with the disease, such as in the gene coding for RPGR, (xp11.4), CRB1 (1q31.3), and IDH3B(20p13).

    No, because retinitis pigmentosa can be caused by multiple genes, and many of the symptoms are similar and cannot be distinguished clinically. Because of this, there is no clear cut association between the genotype and phenotype.

retinitis pigmentosa

Submitted by ziweiwang on Tue, 11/05/2019 - 20:11

Clinically,  in a patient with retinitis pigmentosa, the eye, specifically, the cone cell is affected. In a patient with retinitis pigmentosa, the symptom starts with often  childhood, starting with the loss of night vision and gradual loss of peripheral vision and then the loss of central vision, ending in complete blindness usually around 30 to 40

    In a less clinical sense, the patients of retinitis pigmentosa are aware of their potential to become blind through one or both of their parents and other family members because they have other family members that have a similar disease, there is an expectation that the patient will go through life with a similar sight loss pattern as that for their parents. From the description that the patients give on how it feels to have the disease, the sight is mostly light and dark with very little details. Another description is that their vision is slowly being smudged by vaseline, or it’s like wearing a pair of sunglasses in the dark.  The onset of symptoms often start with childhood, but the symptoms can manifest at a later life stage. Similarly, while it is common that the patients would be completely blind by 40, it is not unheard of for people to retain vision until their 80s. The decrease in vision and later, complete loss of a vision is a major disability and often results in the patients unable to do certain jobs and for those who do not have complete blindness, patients often pause before telling their bosses and managers about their condition, fearing repercussion. While most patient’s vision will degrade to the point where they are unable to drive, because it is a progressive disease, the patients are aware that this will happen, and that they may have to use walking stick eventually. The patients also have a harder time adjusting their sense of self compared to those that were born blind.



Submitted by ziweiwang on Thu, 10/31/2019 - 23:40

Genomic analysis is the identification, comparison of genetic features and their expression through the use of techniques such as DNA sequencing and bioinformatics. Genomic analysis is generally considered to be divided into two categories; structural genomics which identifies certain genomic structures in the genome and functional genomic analysis which analyses the expression of genes and their interactions often also called transcriptomes. Genomic analysis was made available through the prevalence and availability of gene sequencing. While the Human Genome Project sequenced the entire human genome most of the genomic analysis would not be possible without the further accessibility of sequencing using other techniques such as next-gen sequencing and whole-genome sequencing that does not depend on the isolation of cells in order to sequence the genes. Because these data that are generated from sequencing are generally put in online databases, these data are both available and able to be used for purposes that were not intended by the researchers who have originally read the sequence. The data set contains so many different information that a single sequencing can be the basis of several papers. Because of this, there is a data analysis bottleneck where there is so much data that needs to be analyzed but there is not enough time or computing power to analyze the entire genome has been a problem in the field.

neurobiology summary of the thyroid hormone.

Submitted by ziweiwang on Thu, 10/31/2019 - 23:13

The hypothalamus releases thyroid releasing hormone (TRH). The TRH then goes to the pituitary gland which in turn releases thyroid-stimulating hormone (TSH). TSH then goes to the thyroid gland which produces T4, a biologically active molecule and T4 with is a biologically inactive pre hormone. both are released into the blood some times attached to a protein other times not attached to a protein, They thyroid hormone comes out of the blood vessel and through the blood-brain barrier through a transporter, such as MCT8. Then when inside of the cell, T4 is turned into T3.  The T3 is then attached to the thyroid hormone receptor which attaches to the DNA along with coactivator or corepressor and causes the regulation of the gene. the thyroid hormone is important in changing the metabolism of the body and express developmental genes such as the notch signaling pathway. In the lab, the experiment that is trying to be done is how would changing the T3 concentration in zebrafish changes the number of neural stem cells. While this study have been done in mice before, the lab aims to do it on zebrafish, which is something that has never been studied before. In the lab the hypothesis is that the addition of T3 to the fish would cause an increase in thyroid hormone and cause an increase in notch signaling and as a result, there would be more cell differentiation and as a result, there would be less neural stem cells than there would be otherwise. 

experiment with large number of samples

Submitted by ziweiwang on Thu, 10/31/2019 - 22:28

I think that one of the most interesting things that I've encountered while working in a lab is how a large number of samples affect how the scientists behave around the samples. I have been in labs where the main experiment was to do PCR, and I've worked in labs where PCR is just a chore that no one else really wants to do. I think one of the biggest differences between the people who are still learning and the people who do this for a career is that people who do it as a career really does not care about the things that are not the most important things. for example, when teaching in the lab, one of the ways that the students do it is to make each master mix for a specific gene and then add to the DNA and everything is carefully calculated. This is different if a person does this for a living. They are much less likely to be careful and count the concentration of the cells. they are far more likely to just add enough that will give them the results that are needed to do the experiment that can actually be published because of this, when I worked in labs, I was told that I should have some idea of how much 1ul looks like in the pipette and be able to add that much instead of going back and forth between the DNA and the PCR tubes.  Being careful isn't as important as getting things done at work especially if there are a lot of resources and there is high pressure to get results like at a company.  Being careful is very good when starting out, but it quickly becomes a burden when there are over 100 PCR to do and that isn't even the main focus of the experiment. I have also taught people in trypsonizing the cell cultures that the amount that is added is whatever that will trypsinize the cells without killing the cells. I will usually just pour the reagent instead of taking careful measurements of it. this was a big change from how I did it at the beginning where I spent most of the time splitting cells and each time there was a new experience. now because the focus isn't n splitting the cells, I just do whatever is the fastest and still get the result that I want. I think that something like this is not really something that can be really be taught at a class but can only be learned through working in a lab. although, the idea of having a class where the main objective is to find better ways to do simple and common lab technique is interesting. 

cell culture

Submitted by ziweiwang on Thu, 10/31/2019 - 22:01

one of the techniques that I use in the lab the most often in culturing cells. this is also the technique that I encountered most often when analyzing studying biochemistry. it is also one of the hardest one to talk about because there are so many different variations on it. One of the biggest differences between different cell lines is the media used. Depending on the different cell lines used, there are different media used and different things that are added to cells. While immortalized cells need very few things, only FBS that provides nutrients and antibiotics to prevent contamination, primary normal cell lines can be incredibly fussy, with things like growth hormones and other things that are needed to keep them alive. Making the media itself can be a bit of a challenge because there is a need for many different things at specific concentrations. to cultivate a cell there is a need for a flask or a plate. while some cells are able to grow in a suspension, others must be grown in a container with a surface that the cells can adhere to. the cells are then left in the incubating, which is typically at 37 degrees to grow, however, if the cells are allowed to grow for too long, they will become too confluent, and as a result, the properties of the cell will change. This may or may not be desirable depending on the experiment that the cells are needed for. If the cells cannot be confluent, the cells would need to be split. to do this, the media is removed and the cell surface is washed with PBS and trypsinized so that the cells can detach (if the cell is an adherent cell). Then trypsin is neutralized using media and put into the centrifuge tube to be spun. after it is spun, the media is removed, and new media is added. then the cells are resuspended in the tube and a portion of it is put into a new flask to allow for growth along with the new media that is added. 


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