vvikhrev's blog

This is just a paragraph on why one would use whole exome sequencing instead of whole genome sequencing to find a variant of Leber's congenital amaurosis. Leber's congenital amaurosis is characterized by atrophic macular lesions, pale optic disk, reduction of retinal blood vessels, pigment disruption, and scattered pigment clumping in the peripheral retina. It is an inherited retinal disorder and the most common variants are at the gene NMNAT1. Why would you use whole exome sequencing to find a variant in a family that has been affected by this disease?
exome: part of the genome formed by exons, coding portions of genes,
- because most known mutations that cause disease occur in exons,
- cheaper than whole-genome tests,
- in this example, good for testing already present mutations, proven to be successful
- most alleles known to underlie Mendelian disorders disrupt protein-coding sequences
- a lot of rare, protein-altering variants (missense, non-sense single-base substitutions, INDELs) are predicted to have functional
consequences and/or be deleterious
- the exome presents a highly enriched subset of the genome in which to search for variants w/ large effect sizes

FIG 1 - before the actual experiment, they needed to make sure that the tau_lacZ would be transported down the OSN axons and be visualized
- replace the exon OMP (unknown function) with tau_lacZ during homologous recombination
- use cre Recombinase to cut at the loxP sites to removes tkneo
- before they began this experiment, what was known? what kind of topographic mapping? the unknown?
- receptor activation in the bulb evidence derives from ISH experiments that detect the presence of specific receptor mRNAs at
convergent loci in the OB, but this doesn’t permit the visualization of the projection pathways but only identifies sites of axonal
convergence, also w/ this technique, can’t see single axons
FIG 2 – since OMP is expressed in all OSNs, you see intense X-gal staining in OSN axons and in OB glomeruli
- conclusion = tau_lacZ fusion protein is efficiently transported down axons
FIG3 – generate P2 tau_lacZ mice, restrict tau_lacZ to subset of OSNs and use those that express the P2 receptor
- if you just replaced P2 coding sequence w/ tau_lacZ then P2 expression would be eliminated from the modified allele but OSNs express a receptor from only one of the 2 alleles therefore cells expressing the modified P2 allele would express tau_lacZ but not the receptor and cells expressing the WT P2 allele would express the receptor but not tau_lacZ
- to assure that cells expressing tau_lacZ also express a fnuctional P2 receptor, they designed a targeting vector that would result in a
bicistronic mRNA
FIG 4 - see expression of lacZ in P2-IRES-tau_lacZ mice as early as E12.5, also no wandering axons (they saw that these results matched w/ the ISH experiment mentioned above), only a subset of OSNs express tau_lacZ = two distinct glomeruli receive projections from P2-tau_lacZ OSNs
FIG 5 – do P2-tau_lacZ OSNs project to the same glomeruli as WT P2 neurons?
- b/c in the P2-tau_lacZ mouse the other allele is WT
- 50% of P2 OSNs express the WT and the P2-tau_lacZ = monoallelic
FIG 7 – Receptor Swap replace P2 w/ M12 receptor (into P2 locus)
- model 1: OR is sole determinant of axon targeting therefore replacing P2 w/ M12 should target these OSN axons to the “M12 glomeruli”
- model 2: OR plays no role in targeting therefore replacing P2 w/ M12 won’t effect OSN targeting and the axons will go to “P2 glomeruli”
Results: project v. close to P2 (not M12) glomeruli (true in heter- and homo-)
- M12 to P2 projections about 200 micrometers posterior to WT P2 projections
- this suggest that OR is not the sole determinant of axonal targeting but OR is an important factor
So what is the role of OR in axon targeting? - data suggest that OR plays an instructive role in guidance process
- OR expressed in noth axons and OR may recognize guidances cues presented by OB cells independently of cilia OR activation
- activity is important – synchronous firing of subset of OSN expressing a specific OR could result in the segregation of M12P2
OSNs at sites close to but distant from P2 OSNs

source: (Mombaerts, Peter et al. "Visualizing an Olfactory Sensory Map." Cell, Vol. 87, 675–686, 15 Nov. 1996.)

Proposal

      This was an interesting project because it made me think outside the box in terms of the entire scientific publication and writing process. I was excited to do this particular group project because it felt as if I was writing an actual research proposal for a grant. Reflecting back on this project, I learned a lot of new things. Even though this was a “simulation,” I still got an idea of what it would be like to work with other colleagues on a research project, the various protocols required beforehand and how to construct a proposal that will hopefully be accepted.

Poster Project

        This was not only an enjoyable project, but it was also very educational. Before starting this project, I was nervous because I didn’t know where to begin. For the most part, this was because other groups chose the same hypothesis (but different variables) as us, therefore we had to communicate with them to make sure we all had to the same goal and ideas in mind. This project required the combination of everything we’ve learned in the semester. The Writing in the Biological Sciences textbook was the most helpful for me. It was also an exciting challenge to create the posters using Scribus however once I saw the posters, I was very proud of our group as well as the other groups. In regards to the future, this project has especially spiked my interest in the scientific research field and I hope to one day use the knowledge from this class in a clinical lab setting.

Drafts
When first introduced to this assignment, I thought about how much of a strain it would put on my week. I began with expectation that the weekly drafts would be something I looked forward to doing in order to improve my writing skills. Certain hectic weeks prevented me from building up this ability and accomplishing my goals. However, reflecting back on this and thinking about how I wasn’t the only one that shared these worries, has moved me in the right direction towards improvement. It has taught me how to gather my thoughts about a certain subject and write them down in other classes and I can see a difference in my writing from the beginning of the semester compared to my current writing skills.

Perfect Paragraphs
I was excited to write erfect paragraphs each week. It seemed very easy and not as time-consuming. However, I did not think that I would learn anything from it. Creating perfect paragraphs was a very interesting assignment because it included input from other students. This is important to me because when I write, I always wonder if other people (especially my peers) can understand what I am trying to express. With this assignment and the integration of the draft assignment, I now pay more attention to spelling and sentence structure. I used to struggle with tenses in my writing but I feel I have a better understanding of it now.

After making the correct diagnosis, the patient can participate in several types of therapeutic treatments and clinical trials. There are several ongoing and completed trials for treating retinitis pigmentosa with gene therapy, drugs, oxygen therapy, stem cell transplantation and even acupuncture, that can be found on ClinicalTrials.gov. For instance, in a study performed by Rubens C. Siqueira, patients with severe retinitis pigmentosa were treated with an “intravitreal injection of autologous bone marrow stem cells” and evaluated monthly for an entire year with OCT and ERG. Results revealed a “1-line improvement in best-corrected visual acuity was measured in 4 patients 1 week after injection and was maintained throughout follow-up” and no detectable ERG responses. They concluded that since there were no adverse/toxic effects, it would be probable to conduct and investigate more types of autologous bone marrow-derived mononuclear cell therapies (Siqueira, 2011).
In another 2011 study, a RHO suppression and RHO replacement gene therapy was administered on a mouse model (P347S) with RHO-linked autosomal dominant retinitis pigmentosa. Both 5-day-old and adult mice were injected with adeno-associated virus (AAV) vectors that were used to “deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site” (Millington, 2011). By suppressing and replacing the mutated photoreceptors, the researchers predicted that they should function similarly to wild-type photoreceptors. They found that the ONL completely disappeared in untreated mice (the control group with the disease) and mice that were treated rhodopsin was expressed in the ONL. ERG comparisons between both groups showed significant improved responses when both types of vectors were administered. They also concluded that this type of approach can possibly pertain to any patient with RHO-linked regardless of the mode of action of a particular RHO mutation in future clinical studies (Millington, 2011). Consequently, retinitis pigmentosa is uncurable, however it seems that gene therapy could provide some type of solution.

Siqueira, R C, et al. “Intravitreal Injection of Autologous Bone Marrow-Derived Mononuclear Cells for Hereditary Retinal Dystrophy: a Phase I Trial.” Retina (Philadelphia, Pa.)., U.S. National Library of Medicine, 31 June 2011, www.ncbi.nlm.nih.gov/pubmed/21293313?dopt=Abstract.

Millington-Ward, Sophia, et al. “Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa.” Molecular Therapy, vol. 19, no. 4, 11 Jan. 2011, pp. 642–649. NCBI, doi:10.1038/mt.2010.293.

We were interested in determining if there was any correlation between how many arthropods were in rooms and how far away the Morrill Greenhouses are. We found a very small insignificant correlation and a lot of our quantitative data deviated from the average. This probably means that there are other variables that play a role in how many arthropods (and spider webs) you can find in various windowsils. However, this proved to be a very interesting study because we can now compare our results with the results of the other groups that looked at other variables besides distance from the greenhouses such as temperature and distance to the reptile rooms. If we had more time, we would be interested to look at how outside temperaure compared to indoor temperature affects the arthropod count indoors. Also it would be helpful to know if room use also has any correlation with arthropod count in windowsills.

We were interested in determining if there was any correlation between how many arthropods were in rooms and how far away the Morrill Greenhouses are. We found barely any correlation and a lot of our quantitative data deviated from the mean. This probably means that there are other variables that play a role in how many arthropods (and spider webs) you can find in various classes. However, this proved to be a very interesting study because we can now compare our results with the results of the other groups that looked at other variables besides distance from the greenhouses such as temperature and distance to the reptile rooms. If we had more time, we would be interested in looking at how outside temperaure compared to indoor temperature affects the arthropod count indoors. Also it would be helpful to know if room use also has any correlation with arthropod count in windowsills.

After making the correct diagnosis, the patient can participate in several types of therapeutic treatments and clinical trials. There are several ongoing and completed trials for treating retinitis pigmentosa with gene therapy, drugs, oxygen therapy, stem cell transplantation and even acupuncture, that can be found on ClinicalTrials.gov. For instance, in a study performed by Rubens C. Siqueira, patients with severe retinitis pigmentosa were treated with an “intravitreal injection of autologous bone marrow stem cells” and evaluated monthly for an entire year with OCT and ERG. Results revealed a “1-line improvement in best-corrected visual acuity was measured in 4 patients 1 week after injection and was maintained throughout follow-up” and no detectable ERG responses. They concluded that since there were no adverse/toxic effects, it would be probable to conduct and investigate more types autologous bone marrow-derived mononuclear cell therapies (Siqueira, 2011).
In another 2011 study, a RHO suppression and RHO replacement gene therapy was administered on a mouse model (P347S) with RHO-linked autosomal dominant retinitis pigmentosa. Both 5-day-old and adult mice were injected with adeno-associated virus (AAV) vectors that were used to “deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site” (Millington, 2011). By suppressing and replacing the mutated photoreceptors, the researchers predicted that they should function similarly to wild-type photoreceptors. They found that the ONL completely disappeared in untreated mice (the control group with the disease) and mice that were treated rhodopsin was expressed in the ONL. ERG comparisons between both groups showed significant improved responses when both types of vectors were administered. They also concluded that this type of approach can possibly pertain to any patient with RHO-linked regardless of the mode of action of a particular RHO mutation in future clinical studies (Millington, 2011). Consequently, retinitis pigmentosa is uncurable, however it seems that gene therapy could provide some type of solution.

Siqueira, R C, et al. “Intravitreal Injection of Autologous Bone Marrow-Derived Mononuclear Cells for Hereditary Retinal Dystrophy: a Phase I Trial.” Retina (Philadelphia, Pa.)., U.S. National Library of Medicine, 31 June 2011, www.ncbi.nlm.nih.gov/pubmed/21293313?dopt=Abstract.

Millington-Ward, Sophia, et al. “Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa.” Molecular Therapy, vol. 19, no. 4, 11 Jan. 2011, pp. 642–649. NCBI, doi:10.1038/mt.2010.293.

There are several barriers to finding cures and treatments of retinitis pigmentosa. Based on the information presented above, this is true because retinitis pigmentosa comes in many specific forms of inheritance, however there are possible modern treatments that could overcome these barriers. Diagnosis would begin with genetic tests (highly recommended) such as applying and sending a DNA sample to “eyeGENE” to find the genes associated with the disease, severity and the progression of the disease (https://eyegene.nih.gov/). Doing so also allows the researchers to provide the patient with possible recruitment into clinical trials. An ERG would measure the retinal electrical activity in response to light (Facts About, 2014). An optical coherence tomography (OCT) would be used to take detailed images of the retina in order to see how much of the retina is affected and to provide a better case in treating the patient’s retinitis pigmentosa.

Title: Effect of Proximity to Greenhouse on Arthropod Presence in Morrill Windowsills
Introduction:
The University of Massachusetts Amherst Morrill Greenhouses have a variety of plant species and arthropods that can have an effect on the presence of arthropods in rooms inside the adjacent Morrill science buildings. Having more knowledge on the indoor arthropod microinvertebrate climate can influence future environmental or health implications. There are few similar studies that have been done on residential microclimates. In this study we collected the quantitative data on spiders, flies, insects and spider webs in window sills/ledges to examine if the distance from the greenhouses has an effect on arthropod presence. The further the room is from the greenhouses, the less arthropods will be present.
Methods:
Collect quantitative data on arthropods (spiders, insects and flies) and spider webs once per week for two weeks.
Find distances between rooms observed and Morrill Greenhouses using Google Earth Pro.
Record and tabulate results.