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cancer genetics project 3 PP

Submitted by jdantonio on Fri, 04/28/2017 - 13:12

The first step in our treatment process is the identification of our cancer’s neoantigens. These would vary from patient to patient and would have to be Identified experimentally. Identification of neoantigens begins by first sequencing the genome of the patient's tumors using massive parallel sequencing(MPS). MPS is a system which readily identifies tumor cell mutations by comparison of the genome of the cancer cells to the genome of a somatic cell (Gubin et al 2015).  This method of genome analysis has been shown to be an effective means to identify cancer cell gene mutations (Shiraishi et al 2011).We will use a hybrid exome sequencing technique which allows for the analysis of only genes which encode proteins and allow sequencing on a time scale that is relevant to clinical treatment (Hodges et al 2009). Once the tumor genome has been sequenced and analyzed to identify mutations we will then determine which of the tumors mutations are in oncogenes capable of binding to the MHC protein within the cell (Gubin et al 2015). This will be accomplished by utilizing bioinformatic databases and softwares, specifically the  NetMHCpan algorithm system which identifies a wide range probable MHC binding sequences in Human and nonhuman primates (Nielsen et al 2007).We will then harvest lymphocytes from the patient and test them for neoantigen binding specificity in vitro and select T-cells with tumor suppressing ability that possess the receptor for one of the neoantigens we derive from or cancer cell genome analysis. Following this we would grow these cells in culture to create a large amount of tumor infiltrating lymphocytes (TIL) (Perica et al 2012). These TIL’s will be further modified to improving their binding specificity and resistance to T-cell suppression.

 

Gubin MM, Artyomov MN, Mardis ER,and Schreiber RD. 2015. Tumor neoantigens: building a framework for personalized cancer immunotherapy. The Journal of Clinical Investigation 125(9): 3413–3421. National Center for Biotechnology Information[NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588307/>. Accessed 2017 April 24.

 

Hodges E, Rooks M, Xuan Z, Bhattacharjee A, Gordon DB, Brizuela L, McCombie WR, and Hannon GJ. 2009. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nature Protocol 4(6): 960-974. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990409/>. Accessed 2017 April 24.

 

Nielsen M, Lundegaard C, Blicher T, Lamberth K, Harndahl M, Justesen S, Røder G, Peters B, Sette A, Lund O, Buus S. 2007. NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence. PLOS one 2(8): e796. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949492/>. Accessed 2017 April 24.   

Shiraishi T,Terada N,  Zeng Y, Suyama T, Luo J, Trock B,  Kulkarni P, and Getzenberg RH. 2011. Cancer/Testis antigens as potential predictors of biochemical recurrence of prostate cancer following radical prostatectomy. Journal of Translational Medicine 9: 153. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184272/>. Accessed 2017 April 24.

Cancer genetics project 3 excerpt 2

Submitted by jdantonio on Fri, 04/28/2017 - 12:06

One of the major problems that the immune system faces when fighting cancer is a lack of T-cells that are able to recognize the cancer as a pathogenic entity and begin an immune response to the cancer cells.(Drake et al 2014)We propose that we create adapted T-cells that are capable of recognising and binding to tumor cells via tumor specific neoantigens (Rosenberg 2014). We will accomplish this with a multi step T-cell adaptation which will include selection for T-cells with endogenous neoantigen bind receptors and an addition of a chimeric neoantigen specific receptor via gene transfer to those cells already selected for endogenous receptors. These double neoantigen  receptor possessing T-cells will then be further genetically modified to remove T-cell inactivation pathways thus preventing tumor cell inhibition of T-cell activity. Specifically we will be be down regulating the PD1 receptor which stimulates T-cell death and the CTLA-4  which inhibits T-cell activation.(Rosenburg 2014)This will lead to T-cells that have a very high binding specificity to cancer cells and that are resistant to T-cell suppression mechanisms thus allowing them to activate the cell death pathway in cancer cells and elicit a greater immune response against the cancer cells.

Drake CG, Lipson EJ, and Brahmer JR. 2014. Breathing new life into immunotherapy: review of melanoma, lung and kidney cancer. Nature Review 11: 24-37.  <http://www.nature.com/nrclinonc/journal/v11/n1/full/nrclinonc.2013.208.h....



Rosenberg SA. 2014. Decade in review—cancer immunotherapy: Entering the mainstream of cancer treatment. Nature Review 11: 630-632. <http://www.nature.com/nrclinonc/journal/v11/n11/full/nrclinonc.2014.174.....

Cancer genetics project 3 excerpt

Submitted by jdantonio on Thu, 04/27/2017 - 20:00

The first step in our treatment process is the identification of our cancer’s neoantigens, this would vary from patient to patient would have to be experimentally Identified. This is done by first sequencing the genome of the cancer tumors using massive parallel sequencing a system which readily identifies tumor cell mutations by comparison of the genome of the cancer cells to the genome of a somatic cell (Gubin et al 2015).  This method of genome analysis has been shown to be an effective means to identify cancer cell gene mutations (Shiraishi et al 2011).We will use a hybrid exome sequencing technique which allows for the analysis of only genes included in the cells exomes and allows for faster sequencing in a time scale that is relevant to treatment (Hodges et al 2009). Once the tumor genome has been sequenced and analyzed to identify mutations we will then determine which of the tumors mutations would be indicative of an oncogene capable of binding to the MHC protein within the cell (Gubin et al 2015). This will be accomplished by utilizing bioinformatic databases and softwares, specifically the  NetMHCpan algorithm system which identifies a wide range probable MHC binding sequences in Human and nonhuman primates (Nielsen et al 2007). We will then harvest lymphocytes from the patient and test them for neoantigen binding specificity in vitro and select from among those harvest T-cells with tumor suppressing ability that posses the receptor for one of the neoantigens we derive from or cancer cell genome analysis. Following this we would grow these cells in culture to create a large amount of tumor infiltrating lymphocytes (TIL) (Perica et al 2012). These TIL’s will be further modified by the introduction of  chimeric receptor that binds one of the other  neoantigens which we identified.

 

Gubin MM, Artyomov MN, Mardis ER,and Schreiber RD. 2015. Tumor neoantigens: building a framework for personalized cancer immunotherapy. The Journal of Clinical Investigation 125(9): 3413–3421. National Center for Biotechnology Information[NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588307/>. Accessed 2017 April 24.

 

Hodges E, Rooks M, Xuan Z, Bhattacharjee A, Gordon DB, Brizuela L, McCombie WR, and Hannon GJ. 2009. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nature Protocol 4(6): 960-974. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990409/>. Accessed 2017 April 24.

 

Nielsen M, Lundegaard C, Blicher T, Lamberth K, Harndahl M, Justesen S, Røder G, Peters B, Sette A, Lund O, Buus S. 2007. NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence. PLOS one 2(8): e796. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949492/>. Accessed 2017 April 24.   

Shiraishi T,Terada N,  Zeng Y, Suyama T, Luo J, Trock B,  Kulkarni P, and Getzenberg RH. 2011. Cancer/Testis antigens as potential predictors of biochemical recurrence of prostate cancer following radical prostatectomy. Journal of Translational Medicine 9: 153. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184272/>. Accessed 2017 April 24.

Immunotherapy PP

Submitted by jdantonio on Fri, 04/21/2017 - 10:36

Immunotherapies seek to increase the activity or effectiveness of T-cells and increase T cell cancer targeting specificity (Drake et al 2014).  This can be done by the administering of a vaccine that contains a cancer specific antigen to the body, this in turn activates DC cells that recruit and activate T-cells that recognise the cancer specific antigens (Drake et al 2014). The affects of vaccines to induce more T-cell activity have been found to be effective in some cancer types but not all as the expression of immune checkpoints greatly reduce treatment effectiveness (Drake et al 2014). Antibody suppression of immune checkpoints, so they no longer interfere with endogenous T-cells,  has been shown to be an effective treatment in melanoma cancer in large scale clinical trials. (Drake et al 2014). Another method of immunotherapy is the genetic modification of existing T-cells to target cancer specific antigens, this is done by viral transfection of engineered antigen receptor genes to endogenous T-cells (Drake et al 2014). An alternative to this method is ACT treatment a process by which endogenous T-cells are harvested and selected in vitro for cancer cell binding specificity, then raised in  culture and reintroduced in vivo to the patient (Rosenberg 2014).These methods are necessary in many cancer types as relatively few endogenous T-cells have receptors for cancer specific antigens thus making other treatments like vaccines and immune checkpoint therapy ineffective (Drake et al 2014). While immunotherapies are a promising addition to traditional cancer treatments they face one major obstacle, the lack of Identified cancer specific antigens (Drake et al 2014). To further immunotherapy as a treatment option more cancer specific antigens must be discovered (Drake et al 2014).     

 

Drake CG, Lipson EJ, and Brahmer JR. 2014. Breathing new life into immunotherapy: review of melanoma, lung and kidney cancer. Nature Review 11: 24-37.  <http://www.nature.com/nrclinonc/journal/v11/n1/full/nrclinonc.2013.208.html>.

Rosenberg SA. 2014. Decade in review—cancer immunotherapy: Entering the mainstream of cancer treatment. Nature Review 11: 630-632. <http://www.nature.com/nrclinonc/journal/v11/n11/full/nrclinonc.2014.174.html>.  

research project post methods

Submitted by jdantonio on Fri, 04/21/2017 - 00:18

Methods

  • Moss samples were collected from the east, west, north and south side of a tree in front of French Hall on the University of Massachusetts campus

  • All 2 inch by 2 inch moss samples were placed on dry 60 mm filter paper in a single growth chamber

  • Moss samples were dried over a 72 hour period in a growth chamber at 20 ℃ and ambient humidity

  • Moss samples were rehydrated over a 24 hour period on wetted filter paper in 60 mm petri dishes

  • Moss condition was photographically recorded at times 0 min, 10 min, 20 min, 40 min, 60 min, and 24 h.

  • Moss rebound was analysed by color analysis of photographic record of moss photos using imagej software(should we put more detail here or is this fine?)

neurobio Paper review project excerpt 3

Submitted by jdantonio on Thu, 04/20/2017 - 22:54

The purpose of the of the paper by suida et al was to study the effects of general and specific noandrogenic signalling in the amygdala of mammals on stress and anxiety related response behaviors(siuda et al 2016). The hypothesis is that the activation of these pathways causes an increase in stress like behavior in mammalian species(Sidua et al 2016). Anxiety disorders are among the most common type of psychiatric disorder in the united states of america.(Vazey 2017) By better understanding the cellular mechanisms that cause the behavior doctors and clinicians can design novel ways to better treat or even cure these debilitating diseases. The researchers are using male mice as a model for this study. They chose this as a subject as the mice are complex enough to allow for the study of specific brain areas and simple enough to avoid ethical issues over the surgical manipulation of the brain, implantation of an LED and electrode to stimulate the rhodopsin channel receptor.

In order to further this study's finding you must perform several new experiments. The most obvious is to repeat the experiment as performed but with the addition of female mice in the treatment group this would increase the ability of the finding to be generalized as affecting all stress response in mammals, not just male mammals. In addition to this the experiment could be extended by including injection of viral vectors to the amygdala as a whole thus demonstrating the effects of the signalling pathways on the entire amedalaya to see if increased activity in the whole amygdala would increase. They could also do localized studies like the one performed but for other regions of the amygdala to see if specific areas have a greater effect on stress responses or types of stress response. They could also do similar studies with more higher order mammals such as primates to see if the effects are present with increased brain complexity. Finally they could use different types of controllable chimeric receptors to see if they can elicit event greater responses with greater pathway activity.

 

Vazey E. 2017. Lecture 14 Emotional brains.



    Siuda ER, Al-Hasani R, McCall JG, Bhatti DL, and Burchas MR. 2016. Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States. Neuropsychopharmacology 41: 2011-2023.

 

neurobio Paper review project excerpt 2

Submitted by jdantonio on Thu, 04/20/2017 - 22:03

The results of this experiment show that an increase in activity of the G-alpha-s and beta2AR pathways caused increased stress behavior in mice.(Siuda et al 2016) This Points to the role of these pathways in the control and generation of the emotion anxiety and lead to their possible role as targets of therapy for anxiety disorders.(siuda et al 2016) The major strengths of this experiment are the tight control of the experimenters methods. The experimental setup and data collection provided clear control groups and exclude any irrelevant information(siuda et al 2016). They only include data from the experimental group mice that were shown to have expression of the chimeric receptor to ensure that there results were not skewed by failure of gene transfection(siuda et al 2016). They also choose relatively inert viral vectors that would not elicit a major immune response that may have altered the results(siuda et al 2016).The major weakness of this study is the strange choice they made  in limiting their model organism. They used only male mice in the study which seems imprudent given that anxiety disorders are more common in females than males(Vazey 2017). This limits the generalisability of the findings as they may not hold true for female mammals. The mice are also not the most approximate model for the human brain an experient with a primate model may have been more insightful to the cause of anxiety in human disorders(Vazey 2017). They also limited the injection of viral vectors to only the basolateral amygdala excluding the possibility of the upper portions of the amygdala affecting the anxiety response via these same pathways.

 

Vazey E. 2017. Lecture 14 Emotional brains.

Siuda ER, Al-Hasani R, McCall JG, Bhatti DL, and Burchas MR. 2016. Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States. Neuropsychopharmacology 41: 2011-2023.

observations of greeough tree

Submitted by jdantonio on Thu, 04/20/2017 - 20:49

The tree is to the north of the greenough residential hall and is approximately 15 feet away from the building. The tree appears to be coniferous as it has pine needles and has 12 pine cones scattered about its base. In addition the tree has 15 pine cones growing on it in clusters of three. The needles on the tree are a dark green color and the base of the tree is layered with brown needles that have fallen off the tree presumably. The bark of the tree is a light brown color and has an irregular bumpy surface. The tree is approximately 40 feet tall and the trunk of the tree has a diameter of two and a half feet. The branches of tree first emanate from the tree approximately 10 feet up on the tree. The lower third of the tree's branches first grow down towards the ground then arch back up to reach an area roughly parallel to the area of the tree they emanate from , making them roughly “u” shaped. The upper two thirds of branches from the tree grow directly up from the tree form a shape like that of an exponential growth curve. As the tree ascends it branches grow progressively shorter causing the tree to have an overall conical shape. The tree has a ring on the west side of its trunk in the area where branching first begins which appears to be the remanence of a branch that was either cut away by university staff or snapped cleanly in some natural circumstance. The tree has several spots that appear to be lichen growing on it that are green and grey in color. The trees pine cones are light brown and are approximately 7 inch long and have a diameter of about one and a half inches. The branches of the tree range from about 6 feet long at the base to less than one foot at the top. There is a single branch at the bottom of the branches are of the tree that has no pine needles and it appears that the end of the branch has been damaged.

Immunotherapy

Submitted by jdantonio on Thu, 04/20/2017 - 01:45

One of the most promising fields of cancer treatment that are currently being studied is that of immunotherapy. Immunotherapy is a treatment where by the immune system of the patient is bolstered or trained to target the cancer cells within the patient's body (Rosenberg 2014). Immunotherapy was first pioneered in the 1980’s with the first effective cancer immunotherapy by adoptive cell therapy having been experimentally recorded in 1988 (Rosenberg 2014).  Immunotherapy was furthered in the early 2000’s with the advent of immune checkpoint suppression antibodies (Rosenberg 2014). Immune checkpoints are surface antigens that are expressed in cancer cell lines that interfere with T-cell activity and proliferation at the tumor site, thus preventing the immune system from attacking the tumor (Drake et al 2014).  The discovery that lymphodepletion prior to T-cell administration increased results also improved the effectiveness of treatment. (Rosenberg 2014).  In general these immunotherapies seek to increase the activity or effectiveness of a type of T-cell and have it target the cancer cells in the body. (Drake et al 2014).  This can be done by the administering of a vaccine that contains a cancer specific antigen to the body, this in turn activates DC cells that recruit and activate T-cells that recognise the cancer specific antigens. (Drake et al 2014). The affects of vaccines to induce more T-cell activity have been found to be effective in some cancer types but not all as the expression of immune checkpoints greatly reduce treatment effectiveness(Drake et al 2014). Antibody suppression of immune checkpoints, so they can not interfere with endogenous T-cells,  has been shown to be an effective treatment in melanoma cancer in large scale clinical trials. (Drake et al 2014). Another method of immunotherapy is the genetic modification of existing T-cells to target cancer specific antigens, this is done by viral transfection of engineered antigen receptor genes to endogenous T-cells (Drake et al 2014). This modification is necessary in many cancer types as relatively few endogenous T-cells have receptors for cancer specific antigens thus making other treatments like vaccines and immune checkpoint therapy ineffective (Drake et al 2014). While immunotherapies are a promising addition to traditional cancer treatments they face one major obstacle, the lack of Identified cancer specific antigens(Drake et al 2014). To further immunotherapy as a treatment option more antigens must be discovered.(Drake et al 2014).     

 

Drake CG, Lipson EJ, and Brahmer JR. 2014. Breathing new life into immunotherapy: review of melanoma, lung and kidney cancer. Nature Review 11: 24-37.  <http://www.nature.com/nrclinonc/journal/v11/n1/full/nrclinonc.2013.208.html>.

Rosenberg SA. 2014. Decade in review—cancer immunotherapy: Entering the mainstream of cancer treatment. Nature Review 11: 630-632. <http://www.nature.com/nrclinonc/journal/v11/n11/full/nrclinonc.2014.174.html>.

neurobio paper review project: paper methods excerpt draft

Submitted by jdantonio on Thu, 04/20/2017 - 00:21

In order to better study the function of the general G-alpha-s pathway and the specific beta2AR pathway the scientists conducted two separate sets of tests on two populations of treated mice. The first treatment group was administered an adeno associated viral vector which contained a CAMKIIa promoter and a Citrate tagged copy of the modified DREADD G-alpha-s receptor, they then allowed three weeks for transfection expression of the transgene and migration of the receptor protein to cellular surface.(siuda et al 2016) The mice were then administered the activating CNO drug that selectively activates the designed receptor 30 minutes before the start of behavioral testing. (siuda et al 2016)To assess whether the  mice were experiencing stress due to  G-alpha-s signalling pathway activation  they performed an open field test where in the mice were placed in a 50m by 50m plexiglass enclosed area and allowed to move around freely.(suida et al 2016) The mean time that the mice spent in the center was used as the proxy for anxiety response, more time in the center was considered less stressed.(siuda et al 2016) As a control for this experiment a second group of mice were injected with a viral vector with all the same elements as the treatment except for the receptor gene so they still produced GFP citrate but did not express the receptor ,this control group was tested in the same manner as the treatment group.(siuda et al 2016)

A second test was also conducted to assess the stress levels caused by the pathways activation. This test was termed the social approach by the experimenters, wherein they placed the mice in a chamber first containing a an empty overturned mesh pencil holder and tracked the movement of the mouse around the chamber.(siuda et al 2016) They then placed an age and gender matched mouse into the box under the overturned pencil holder cage and again tracked the movement of the mice around the box in relation to the pencil holder.(suida et al 2016) They compared the social and nonsocial behavior of the mouse and used social avoidance, actively avoiding going near the mouse in the pencil holder, as a proxy for stress response. (siuda et al 2016) Once again the GFP expressing mice were used as a control group for this experiment.

 

-(Bio 312 note for this neurobiology project we are asked to analyse and identify key parts of a single piece of primary literature we were assigned. In this part I describe the methods used in this paper, only one paper hence why there is only one source. Just wanted to clarify as you usually want two sources for journal entries but for this project we only use the one we are assigned)

 

Siuda ER, Al-Hasani R, McCall JG, Bhatti DL, and Burchas MR. 2016. Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States. Neuropsychopharmacology 41: 2011-2023.  

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