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With regard to the carnivorans, the character "tail" has two states. In this phylogenetic analysis, an elongated tail is the ancestral character state (scored with a 0) and a short tail is the derived character state (scored with a 1). In phylogeny A, a short tail is hypothesized to have evolved after the split between otters and the taxa of bears, sea lions, walrus, and seals. This relationship proposes that a short tail is the synapomorphy for the monophyletic group of bears, sea lions, walrus, and seals. In phylogeny B, a short tail is hypothesized to have evolved twice, exhibiting homoplasy. A short tail here is a derived trait for the seals, but it is also a shared derived trait for bears, sea lions, and walrus. However, there are a few separate divergences between seals and this group, and the common ancestor is hypothesized to have an elongated tail. In phylogeny C, a short tail is hypothesized to have evolved twice as well, but then was lost in one lineage branch. A short tail is a derived trait for the bears, but it also initially evolved as a shared derived trait for taxa of sea lions, walrus, seals, civets, hyenas, and cats. Cats, hyenas, and civets then lost this short tail, demonstrating an evolutionary reversal. In phylogeny D, a short tail evolved once in the lineage to include the monophyletic group branching from seals to dogs, but it was lost later in the taxa of otters, raccoons, and dogs, exhibiting another evolutionary reversal. Based on this trait and the parsimony principle, phylogeny A is the most likely evolutionary hypothesis as the tail trait only evolved once in the lineage and was not subsequently lost. The parsimony principle guides us to the evolutionary tree with the fewest character-state changes, which is usually regarded as the best.

Each resulting immunogenic neoantigen sequence from this extensive screening process will then be incorporated into a single gene with the fascin promoter and edited into the genome of differentiated dendritic cells via CRISPR (Population 1). The fascin gene promoter is highly active in mature dendritic cells, making it an ideal element of the researchers' neoantigen gene construct (Bros et al. 2003). A second gene construct identical to the first but including a signal sequence directed to the endoplasmic reticulum (ER) will be edited via CRISPR into a second population (Population 2) of differentiated dendritic cells. These two populations will be kept together in vitro and all dendritic cells will be treated in solution with Poly(I:C), a toll-like receptor 3 (TLR3) agonist shown to induce activation of dendritic cells (Garzon-Muvdi et al. 2018). Since the fascin gene promoter is active in mature dendritic cells, expression of these genes in both dendritic cell populations will result in expression of both endogenous and exogenous neoantigen peptides. In population 1, the expressed endogenous neoantigen peptides will be processed by the proteasome and subsequently move to the ER, bind to MHC class I, and display on the plasma membrane. In population 2, the addition of the signal sequence to the ER will result in the secretion of neoantigen peptides, which then can be recognized by population 1 dendritic cells as “exogenous.” While displaying these same neoantigens on MHC class I, these dendritic cells will also phagocytose the neoantigens secreted by population 2. Since these neoantigens are exogenous relative to population 1, they will be displayed via MHC class II on the plasma membrane.

While the PBMCs are differentiating and through the use of comparative DNA isolated from PBMCs, the researchers will identify all nonsynonymous mutations through whole exome sequencing of biopsied pancreatic cancer cells (Cullinan et al. 2018). Comparing normal exons to those found in pancreatic tumors not only allows for scrutinization of potential neoantigens, but also allows for more treatment flexibility as whole exome sequencing is less expensive than whole genome sequencing. Whole exome sequencing also allows for the inference of HLA types from exon sequences (Cullinan et al. 2018). Expression of identified nonsynonymous mutations can then be verified with cDNA capture. From these expressed mutations, potential neoantigen sequences will be further scrutinized through multiple MHC class I epitope prediction algorithms. The NetMHC algorithm has been previously utilized in analysis of Panc02 tumor cells to predict neoantigens (Kinkead et al. 2018). A Random Forest-based computational algorithm has also demonstrated an immunogenicity prediction accuracy of 83% and an HLA binding prediction accuracy of 97.4% (Wilson et al. 2018). Therefore, the researchers will use the NetMHC and Random Forest algorithms to screen for tumor-specific neoantigens in individual patients. Additional filters can be applied to these algorithms to eliminate epitopes predicted to be poorly processed by the immunoproteasome and those with lower binding affinities than the corresponding wild-type sequences (Gubin et al. 2014). These filters will discern any potential neoantigens that would not be broken down effectively to be displayed via MHC complexes. These filters also eliminate any neoantigens outcompeted to bind to MHC complexes by the wild-type sequences and assure that resulting candidate neoantigens are sufficiently different from self-antigens (Hopkins and Jaffee, 2018). A common method for evaluating immunogenicity and further scrutinizing final candidate neoantigens is interferon-gamma enzyme-linked immunospot assay (Cullinan et al. 2018). This assay allows for the quantification of cytokine secretion [interferon-gamma] within a specific cell. The researchers will perform this assay in undifferentiated PBMCs taken from the patient with the candidate neoantigens to determine immunogenicity.

In order to acquire a personalized pancreatic cancer treatment profile, sequences of immunologically active tumor neoantigens must first be identified. An MRI will be performed on the patient to detect locations of pancreatic cancer throughout the body (Raman and Fishman, 2018). Although CT scans are also used for the same purpose, the MRI is safer for the patient’s long-term health as it is not irradiative and carcinogenic. To obtain a representative sample of cancerous tissue with as many neoantigens as possible, the PancreAss Kickers will biopsy regions of high tumor cellularity (Cullinan et al. 2018). PBMCs will be isolated from a blood sample, plated, and differentiated into dendritic cells through treatment with interleukin 4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). This process takes approximately a week (Geissmann et al. 2010). CD4+ and CD8+ T cells will also be isolated in this initial blood sample and set aside for later.

The PancreAss Kickers will take a personalized adoptive cell transfer and cancer vaccination immunotherapy approach to eradicating pancreatic cancer. Identification of expressed and immunologically active tumor-specific neoantigens in each patient will be performed in vitro and resulting neoantigens will be engineered into two genes. One gene construct will be incorporated into dendritic cells grown from peripheral blood mononuclear cells (PBMCs) and will express the neoantigens endogenously, while the other will be incorporated into eosinophils and will result in the secretion of neoantigens into the extracellular environment due to a signal sequence. After dendritic cell activation, presentation of neoantigens via MHC classes I and II will lead to subsequent activation of both CD4+ and CD8+ T cells. Both types of T cells along with dendritic cells and PBMCs will be incorporated into a vaccine administered to the patient, and the resulting immune response to the pancreatic cancer will abolish it.  

Based on the data for characters 1, 2, and 3, the evolutionary relationships among cats, hyenas, and civets cannot be resolved. This is because none of the taxa share traits 1 and 3, and too many of the taxa share trait 2. There needs to be more of a discrepancy between the taxa for a certain trait or traits that allows their relations to be hypothesized. Of the remaining nine traits, 4 and 7 are the only useful ones in reconstructing a phylogeny between these three taxa. This is because these allow the cat and the hyena to be put into a hypothesized relation as opposed to the civet. Traits 5, 6, 10, 11, and 12 are not useful because none of the taxa share them. Trait 8 is not useful because only the cat has it, so the other taxa cannot be compared alongside it. Trait 9 is not useful because all of the taxa in the carnivoran group have this trait, so it does not help distinguish them from each other.

Regarding the carnivorans, the character #6, tail, has two character states. In this phylogenetic analysis, an elongated tail is the ancestral character state (scored with a 0) and a short tail is the derived character state (scored with a 1). In phylogeny A, a short tail is hypothesized to have evolved after the split between otters and the group of bears, sea lions, walrus, and seals. This proposes that a short tail is the synapomorphy for the monophyletic group of bears, sea lions, walrus, and seals. In phylogeny B, a short tail is hypothesized to have evolved twice. This is an example of homoplasy. For example, a short tail here is a derived trait for the seals, but it is also a shared derived trait for bears, sea lions, and walrus. However, there are a few separate divergences between seals and this group, and the common ancestor is hypothesized to have an elongated tail. In phylogeny C, a short tail is hypothesized to have evolved twice as well, but then lost in one lineage branch. For example, a short tail is a derived trait for the bears, but it also initially evolved as a shared derived trait for the sea lions, walrus, seals, civets, hyenas, and cats taxa. Cats, hyenas, and civets then lost this short tail trait. This is an example of an evolutionary reversal. In phylogeny D, a short tail evolved once in the lineage to include its monophyletic group branching from seals to dogs, but then this trait was lost later in the phylogeny in otters, raccoons, and dogs. This is another example of an evolutionary reversal. Based on this trait and the parsimony principle, phylogeny A is the most likely. The parsimony principle guides us to the evolutionary tree with the fewest character-state changes, and this is the one usually regarded as the best. In phylogeny A, the tail trait only evolved once in the lineage and was not lost at any point.

There are two strategies to treating cancer through immunotherapy: passive and active. Passive immunotherapy involves treatments with monoclonal antibodies, adoptive T cell transfers, and genetically engineered T cells, whereas active immunotherapy involves vaccine-mediated immunity via the administration of tumor-associated antigens (Banerjee et al. 2018). Due to genetic alterations or post-translational modification of proteins, cancer cells can express and display proteins that differ from their normal cell counterparts or are overexpressed in the tumor phenotype (Battaglia et al. 2016). These proteins are known as tumor-associated antigens (TAA) and fail to be recognized by the immune system. As a result, cancer cells that display TAAs are able to evade the normal destructive response of active CD8+ T cells. Cancer vaccines serve as methods of active immunotherapy that can stimulate the CD8+ T cell response to these TAAs and hopefully eradicate all cancerous cells that display them (Banerjee et al. 2018).

A mixture of ground nutmeg (1.00 g) and tert-butyl methyl ether (3.0 mL) was added to a round-bottomed flask and gently boiled for ten minutes. After settling, the resulting liquid was cotton-filtered through a pipet, rinsed again with tert-butyl methyl ether (2.0 mL), and filtered as before into a tared 25 mL Erlenmeyer flask. The filtered solution was then evaporated gently to yield the crude product (0.763 g, 1.05 mmol, 76.3% recovery), which was subsequently recrystallized in acetone (15.26 mL) to yield a once-recrystallized product (0.147 g, 0.203 mmol, 19.3% recovery), isolated via vacuum filtration. Hydrolysis was performed in a clean round-bottomed flask through reflux of a solution of the trimyristin (0.060 g, 0.083 mmol), 6 M sodium hydroxide (2.0 mL), and ethanol (95%, 2.0 mL) gently for 45 minutes. The resulting contents of the flask were then poured into a 50 mL beaker containing 8 mL of water and concentrated hydrochloric acid (2.0 mL) was added dropwise while stirring. The beaker was then cooled in ice water for ten minutes while stirring, and vacuum filtration was used to collect the resulting myristic acid product (0.046 g, 0.201 mmol, 82.14% yield). During the hydrolysis portion of the experiment, the remaining trimyristin (0.086 g, 0.12 mmol) was recrystallized a second time to obtain a twice-recrystallized product (0.067 g, 0.093 mmol, 77.9% recovery), which was subsequently vacuum filtrated. The melting points of the once-recrystallized product (55 - 57 ℃), product of hydrolysis (54 - 55 ℃), and twice-recrystallized product (54 - 56 ℃) were then taken.

The researchers will take a personalized adoptive cell transfer and cancer vaccination immunotherapy approach to eradicating pancreatic cancer. Identification of expressed and immunologically active tumor-specific neoantigens in each patient will be performed in vitro and resulting neoantigens will be engineered into a gene incorporated into isolated peripheral blood mononuclear cells (PBMCs) and dendritic cells. Expression of endogenous antigens will be displayed via MHC class I, while a different cellular model will express an identical engineered gene with an additional signal sequence that will excrete the neoantigens subsequently displayed on the dendritic cells via MHC class II. CD4+ and CD8+ T cells will then be activated and incorporated into a vaccine delivered to the patient.