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Project 3 starting ground

Submitted by abnguyen on Thu, 04/27/2017 - 08:49
  • PD1 background

  • T-cell activation background:

    • CD28/B7

    • MHC/TCR

    • Suppressors of this (CTLA4)

  • Tumor infiltrating cells: how to select for them, advantages to using them

 

  • Prior drugs downfalls targeting the same stuff

 

GOALS:

  • Edit patient white blood cells using CRISPR to:

    • knock out PD-1

    • CD28 superagonist that won’t cause organ failure????

  • T cell activation and deactivation

    • Keep T cells constitutively active

  • Find antigens specific to non small cell lung cancer

“Immune system ablation - getting rid of the regulatory t cells and myeloid something suppressor cells - engineer the tcells to be unresponsive to those suppressive effects: another advantage to hyperactivated t-cells, you better make sure those t-cells only target specific neoantigens to prevent autoimmune disorders - must only target t-cells that exclusively bind to antigens on the tumor :D  “ - randy

 

Keytruda® is a highly selective humanized monoclonal IgG4 antibody directed against the PD-1 receptor on the cell surface. The drug blocks the PD-1 receptor, preventing binding and activation of PD-L1 and PD-L2. This mechanism causes the activation of T-cell mediated immune responses against tumor cells.

 

T-cell activation:

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/cell-analysis-resource-library/ebioscience-resources/t-cell-activation-functional-assay.html

History of T cells of the past 25 years

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740335/

 

Upregulation of EGFR is one of the main suspects that inhibit immune response.  

    Erlotinib, Tagrisso from project 1

https://www.nature.com/news/first-crispr-clinical-trial-gets-green-light-from-us-panel-1.20137

  • CRISPR

http://biosky.co/clinical-trial-using-crispr-edited-white-blood-cells-to-treat-lung-cancer/

  • More CRISPR

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730182/

More and More CRISPR

 

Warburg effect

Submitted by abnguyen on Sun, 04/23/2017 - 21:40

The warburg effect is the observation that most cancer cells mainly produce energy through glycolysis followed by lactic acid fermentation in the cytosol.  This differs from normal cells where there is a low rate of glycolysis and oxidation of pyruvates in the mitochondria.  A possible explanation for the warburg effect is that mitochondria was damaged during the process of tumor proliferation and a new means of energy production is required.  Another possible explanation is that glycolysis provides most of the building blocks for cell proliferation and used even in the presence of oxygen.  As such, glycolysis in the presence of oxygen is highly inefficient in energy production but makes up for it in the proliferation factor.

Script draft 2

Submitted by abnguyen on Sun, 04/23/2017 - 21:40

 

Slide 1

           Hello everybody and welcome to our Biochem 275 final presentation.  The focus of our presentation is Huntington’s disease.

 

Slide 2

           Like we just said, our project is on the Huntington’s disease.  This is a neurodegenerative disorder that is inherited dominantly on autosomal chromosomes.  Some symptoms of the disease are behavioral changes, involuntary movement, and intellectual disabilities.  However, there is no guaranteed way to predict when the symptoms will appear.  As shown by the bar graph, the most common age of Huntington’s onset ranges from 30-50 years old.  There is no treatment that currently detects the age a person will develop the disease, only an estimate from previous data.

 

Slide 3

           Currently, there are no treatments that can 100% cure huntington’s.  On the bright side, there are hundreds of scientists testing and trying to develop a cure.  There are 2 ongoing treatments that have a high chance of helping HTT patients.  The first is AUTEN-99.  This drug is a potent neuroprotective candidate for preventing and treating neurodegenerative disorder.  AUTEN-99 works to degrade excess detrimental material in the cytoplasm.  This will clear up cell space and provide room and nutrients for functioning proteins.  This has been tested in flies and has been shown to slow/stop neurodegeneration.  AUTEN-99 has been known to work for other diseases such as alzheimer's and parkinson's.   

 

Slide 4 (I think we should break up the treatment slide, we can add pictures of the drug, molecule or pathway and it will prevent the powerpoint slide from looking like a block of text.)

    Another treatment for Huntington’s is the regulation of HSF1.  This works to control protein quality.  Possible ways to regulate this transcription can lead to cures for huntington’s.  Some ways that this can be regulated are to inject more HSF1 TF directly into the patient or we can use CRISPR technology.  With CRISPR we can look for the gene that creates HSF1 and upregulate that promoter.     

    Alternatively, with CRISPR we can have it attach itself to parts of the CAG codon to prevent it from transcribing and creating proteins.  This method might help reduce the about of repetitions and restore normal function within the body.

 

Slides 5-7 (new 7)

    The Huntington’s gene is located on chromosome 4 for humans.  The nucleotide is 13,498 base pairs long and it codes for roughly 3144 amino acids.  The main protein produced is characterized by a single codon repetition.  The CAG codon, also known as the glutamine codon, is repeated 40-120 times.  A normal human will have between 10-35 repetitions.  An individual with 35-39 repetitions may not develop the disease while someone with 40+ repeated glutamine proteins will almost always develop huntington's.  The protein created from this string of codons are associated with the nervous system and interact with the neurons within the brain.  The exact function of this protein is not fully understood.  However, what we do know about this protein is that it undergoes alternative splicing.  The splice variants are not related to the disease; the disease is caused by a different mutation.

 

Slide 7:

The HTT gene, as you can tell is relatively huge, but even so, we must amplify it to be able to study it better. First, we heat up the environment so that the hydrogen bonds between the two strands break and allow for the strands to separate. Next, we cool the DNA strands to allow the PCR primers to anneal to the 3’ ends of the gene segment on each strand. If you look in the middle, here, you can see that these two are our left and right primers. Now that the primers are annealed, this allows us to have our heat-withstanding Taq polymerase to start at the primers and elongate the complementary strands. More DNA copies are being made, so when this process repeats, our gene gets amplified enough to study.

 

Slide 8: Now we move onto cloning, expressing, and purifying the HTT gene, and you may be wondering why we would want to clone the gene. Well, cloning the gene allows us to study the gene’s function and determine how mutating it causes Huntington’s Disease. It can allow the specific HTT gene to be studied and the protein to be produced. Many variables affecting the gene and protein expression can be tested, as well as comparing proteins with the mutations to those without.

Slide 9: With that in mind, we must first chemically cut the open reading frame of the HTT gene from the strand of a DNA sample. Then, attach the cut gene in between the restriction enzymes Not1 and Sbf1, in the multiple cloning sequence for the plasmid. Next, insert the plasmid into the BL21 derivative NEB Express™ host strain of E. Coli. Using IPTG as a promoter, the gene can be expressed in the bacterial cells. A chaotrope is used to destabilize and collect the proteins. The specific protein can be purified by affinity chromatography and further analyzed by gel electrophoresis on an SDS PAGE gel.

Draft script for biochem

Submitted by abnguyen on Fri, 04/21/2017 - 10:24

Video Presentation Script

Introduction??(not sure how we want to set up the video)

  • What is your disease?

The disease that we chose to research is Huntington's disease(HD). Huntington’s disease is a dominant hereditary disease that causes neurodegeneration. Symptoms of the disease include behavioral changes, involuntary movements, and cognitive degradation. These symptoms typically occur when a patient is between the ages of 30-50, however, as you can see based on the graph, symptoms could potentially pop up at any age. Because symptoms aren’t seen until later in life and this disease is dominant, many people who think they are at risk will get tested, to see if their children are at risk.

Currently, there are no treatments for this disease, but there is a lot of research occurring to find a drug or therapy for HD patients. One potential drug that we read about is AUTEN-99, which is a protein that can degrade excess junk material in the cytoplasm that can sometimes hinder neurological function. In HD, nonfunctioning proteins can build up and getting rid of them could potentially slow or stop neurodegeneration. A potential therapy could be to work on regulating the protein HSF1 which functions as a protein quality control protein. HSF1 levels have been seen to be low in HD patients, increasing the levels of HSF1 could work as a therapy.

  • What is your gene?

The gene that controls HD is the HTT gene

  • What size, in nucleotide and amino acids, is your gene?

This gene is very large, it has 13498 base pairs and 3,144 amino acids

  • Does your gene undergo alternative splicing?

    • How do these different splice variants function in the body? Are they relevant to your disease?

Yes this gene undergoes alternative splicing, but only one form or the protein is produced in the body. The splice variants are not related to the disease, the disease is caused by a different mutation.

  • What is the function of your protein?

The exact function of this protein is not known yet, however, it is typically associated with the nerves in the brain. This makes since since the symptoms of huntington’s ( when this protein doesn’t function) are variation of neurological degradation.

  • How is your gene associated with your disease? Include information from your journal articles.

  • Explain how you cloned, expressed, and purified your protein.

  • Why did you clone your gene?

    • How can this be used to study your gene, protein, or disease?

 

Reading Reflection

Submitted by abnguyen on Tue, 04/18/2017 - 11:42

After reading this article, I was surprised that children born by C-section have less mother -child similarity in gut microbiota than infants born vaginally.  I figured that since births involved the child having the mother's genes and receiving nutrients from the mother for 37-42 weeks, it would not make a difference the way the baby was delivered.  A questions that I have is, are there any studies using different races of humans?  Each race has a different percentage of genetic abnormalities; Sweden has 4% (from parent to offspring) while the US has 9.8% as of 1982.  Doing this study on different races might reveal differing results from this one. 

Figure A shows a phylogenetic tree of metaOTUs (metagenomic operational taxonomic units), its not a standard tree where you see a bunch of branches coming off of 1 ancester, its a swirl.  This was constructed using the average distance between metaOTU pairs through the neighbor joining method.  The colored blocks are phyla (outer circle)  The inner circle is a heatmap of most common metaOTU

Figure 1B is a box plot based on metaOTU's principle coordinate analysis (unifrac distance).  Figure 1C is a scatterplot of the same data.  From this data, we see that infants of 12 months are the most similar to their mothers

Figure 1D shows a bar graph, over time alpha diversity increases while beta diversity decreases.  This indicates more complexity and increased numbers in microbial genomes in older infants

http://www.sciencedirect.com/science/article/pii/S1931312815001626

Additional MiR-200 Targets

Submitted by abnguyen on Fri, 04/14/2017 - 00:00

Along with targeting Snail, Zeb, Twist, Vimentin, and HIF1, the miR-200 family also targets other genes in the cell that aid cancer survival and propagation.  Noxa is a member of the of the Bcl-2 family and an apoptotic inhibitor.  When this is directly targeted by miR-200c, Noxa levels decrease and apoptosis levels increase to the same level of an apoptotic drug, bortezomib.  CD95 is a death receptor mediated apoptosis site that is inhibited by FAP-1.  FAP-1 is directly silenced by miR-200c; onced inhibited, CD95 surface expression increases.  Another target of the miR-200 family is PKCɑ.  PKCɑ is overexpresed in cancer cells and onced exposed to miR-200b these levels drop along with the decrease in cell migration.  This migration is independent of ZEB-1 migration and metastasis.  Finally, WAS protein family member 3 (WAVE3) is also directly inhibited by miR-200b.  WAVE3 is selected for its 3’UTR interaction with the miR family and will result in the cancer cell becoming less invasive.  When miR-200b was silenced an increase of WAVE3 dependent cellular invasion was observed (Humphries and Yang 2015).

 

Humphries B and Yang C.  “The microRNA-200 family: small molecules with novel roles in cancer development, progression and therapy.”  Oncotarget.  30 Jan 2015.  <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466628/>.  Accessed 10 Apr 2017.

PP research proposal methods

Submitted by abnguyen on Thu, 04/13/2017 - 15:16

For this experiment, each group will look at a different aspect that impacts moss phenology (life cycle).  Using the information gained from the methods project (moss location, type of moss, how to locate it, etc.), each team member will use their moss as the test subject and observe it over the course of a month.  During this month, their subject will be observed at least 10 times. This will vary depending on each individual’s schedule and availability to observe their moss.

Analyzing SNPS

Submitted by abnguyen on Thu, 04/13/2017 - 00:11

            For this lab, I looked into the SNPs of Promethease person 1 and 2 from the moodle website.  Promethease person 1 is a male and has a few notable risks on the main page.   This man is 1.6x times more likely to get prostate cancer and 1.4x more likely to develop colorectal cancer than individuals without the rs6983267 G;C SNP on chromosome 8.  Another notable risk is having gs191 where this individual has a high chance of gastrointestinal bleeding when taking some types of medication like ibuprofen and naproxen.  This man also has some contradicting SNPs of rs1800497 C;C, rs1421085 C;C and rs1121980 T;T.  The first SNP has the characteristic of lower obesity due to an increased pleasure response to food while the other 2 SNPs have a 1.7x and 2.76x risk for obesity respectively.  Some beneficial SNPs are rs1815739; a mix of muscle types and this man is probably a sprinter, rs738409; less live damage from alcohol and rs17070145; having a stronger memory than the average person.  Although this man has SNPs that are detrimental and could impact him later in life, his lifestyle should also be considered when considering future.  A healthy lifestyle can combat the negative effects that his SNPs are known for; knowing about a gene is not everything, a strong mental fortitude and attitude can overcome many obstacles.

            Promethease person 2 is a woman (SNP gs145) with lighter green, brown or hazel eyes (SNP gs241).  She has a 1.6x risk of breast cancer (SNP rs3803662 T;T) and increased risk of type-2 diabetes (SNP rs13266634 C;C).  Type 2 diabetes is usually adult-onset and can be delayed or prevented with a conscious lifestyle of maintaining a healthy weight, diet and exercise.  This woman has a 1:14.625 ratio when comparing bad repute to good.  Another polarizing SNP is rs3750817 where an individual has a .64x reduced risk for breast cancer.  This SNP is located on chromosome 10 and on FGFR2.  The FGFRs are critical in determining breast cancer status.  This receptor is mutated in almost all forms breast cancer.  Some beneficial SNPs that this individual possess are rs4988235; the ability to drink milk, rs53576; the ability to handle stress well and rs1815739; better muscle performance and likely a sprinter.  This woman, similar to the man, can overcome many of her genomic problems by having a healthy lifestyle.  Having a higher risk of a trait does not always guarantee a trait’s expression.  

            Before doing this lab, I knew that SNPs and genes could tell a lot about a person.  However, I did not think that a single SNP could reveal in depth information about physical traits such as being a sprinter giving birth to a normal weight child.  In the future I am considering getting my information from 23andMe and looking into my own SNPs to see if I can find anything interesting about myself. 

Methods of Research Proposal

Submitted by abnguyen on Thu, 04/06/2017 - 22:40

Methods

Materials

The materials will vary from group to group depending on which conditions they were assigned to record.  At most, the materials needed will be:

An air thermometer to measure the atmospheric temperature surrounding the moss but not the moss itself.

A standard laboratory temperature to measure the temperature of the moss.

A humidity recording device to measure the temperature of the humidity of the air surrounding the moss.

    Moss located on the Umass Amherst campus; each student will find the moss that he or she used for the methods project.

Experiment

For this experiment, each group will be looking at a different aspect that impacts moss phenology.  Using the information gained from the methods project, each team member will use their moss as the test subject and observe it over the course of a month.  The frequency of observations will be at least 10 times over the course of a month.  This will vary depending on each individual’s schedule and availability to observe their moss.

If an individual is looking at the effects moss temperature then each person will need a thermometer.  The thermometer will be inserted into the itself.  To obtain an accurate reading, the thermometer will be placed into the moss and the observer will wait 5 minutes.  After the 5 minutes, the temperature will be recorded into the respective section on Table 1.

If an individual is looking at the effects of the air temperature on moss, they will require an air thermometer.  The thermometer will hover the moss by 2 inches and will be held in place for 5 minutes.  After the 5 minutes, the temperature will be recorded into the respective section on Table 1.

If an individual is looking at the amount of sunlight received, this task will be more time restrictive.  The moss will be observed between the times of 11 am to 1 pm, when the sun is at its highest point in the sky.  On table 1, the team will record whether or not the moss is in direct sunlight or if it is hidden behind a plant or other structures.

If an individual is looking at the effects of humidity on moss, they will need a digital humidity monitor.  These devices are small and should be easy to obtain, either from a lab on campus or purchasing one online for less than $10.  Similar to the other steps, the device will be held next to the moss for 5 minutes and the data will be recorded in its respective box in Table 1.  

Each person, regardless of which team they are in, will record whether or not gametophytes and sporophytes are present on their moss, the location of their moss, the species if known, the date that the moss was observed and what stage in the life cycle the moss is in.  All of the data will be recorded onto their respective boxes in Table 1.  

Team 1 will look at the effects of moss temperature.  

Team 2 will look at the effects of the air temperature.

Team 3 will look at the effects of sunlight and humidity.

Team 4 will look at the effects of moss temperature and air temperature.

Team 5 will look at the effects of moss temperature and the amount of sunlight.

Team 6 will look at the effects of air temperature and the amount of sunlight.

Team 7 will look at the effects of humidity.

Team 8 will look at the effects of humidity and moss temperature.

 
 

Introduction section revised

Submitted by abnguyen on Wed, 04/05/2017 - 23:30

Introduction

Purpose

The purpose of this research proposal is to find out how different conditions impact the growth of moss. We will be looking at various types of moss located within the Umass Campus and compare their growing conditions alongside the presence of gametophytes and sporophytes.

Background research

There are several main factors that play into moss energy production: moisture, sunlight, humidity and temperature.  Some species of moss have shown to have strong correlations between stem elongation and temperature while some other species showed maximum growth rates in shaded areas with high humidity (Zechmeister 1995).  In this project we will explore the wide range that are habitable for moss and compare to see which condition is the universal ideal condition if such thing exists.  

    Mosses prefer dark environments as opposed to other plants that require sunlight to create their nutrients.  Most plants can absorb moisture through their roots but most mosses cannot, because of this, moss needs to be located in a damp setting where water is abundant in the air as opposed to the ground.

The typical temperature that moss reside in range between 20-70 degrees C and energy production varies depending on how well they acclimated. Moss life cycle are unique compared to humans as they can flip between haploid and diploid generations.

For this project, each of the eight groups will be assigned  different conditions to record over the course of a month.  The moss that they will be using to survey are the moss that each member of the team chose for their methods project.  This is to ensure that everyone has a different type of moss so a variety of conditions will be observed and recorded.  Each individual will also know where to locate a species of moss since they have already found and taken pictures of it before.  

Zechmeister HG. Growth rates of five pleurocarpous moss species under various climatic conditions. Journal of Bryology [Internet]. 1995;1885108102578219941987199256354342(33392112):455 - 468.

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