Takes Dying Animals

The following collection represents three years of creative work in the Masters of Fine Arts-Fiction program at the University of Nevada Las Vegas. The majority of these stories operate in the realm of the surreal with comic and farcical leanings. All of the characters portrayed deal with death in some way--whether it a parent, a marriage, an animal, or a friend--and each cope their loss by pushing grief to the periphery, often masking it with humor or denial, until the story requires them to consciously confront it. These stories should not be considered finished work, but as a summation of the stylistic and craft choices I have developed and nurtured throughout my workshops, thesis hours, and personal writing work. I hope these stories entertain--which is the original instinct that drew me to fiction--but more importantly, like all great literature, speak to the human condition in a meaningful and memorable way. I believe most of these stories are still drafts away from completion, but proudly present them here, confident they are well on their way to becoming the best versions of themselves

Circadian Genes Are Expressed during Early Development in Xenopus laevis

Circadian oscillators are endogenous time-keeping mechanisms that drive twenty four hour rhythmic changes in gene expression, metabolism, hormone levels, and physical activity. We have examined the developmental expression of genes known to regulate circadian rhythms in order to better understand the ontogeny of the circadian clock in a vertebrate.In this study, genes known to function together in part of the core circadian oscillator mechanism (xPeriod1, xPeriod2, and xBmal1) as well as a rhythmic, clock-controlled gene (xNocturnin) were analyzed using in situ hybridization in embryos from neurula to late tailbud stages. Each transcript was present in the developing nervous system in the brain, eye, olfactory pit, otic vesicle and at lower levels in the spinal cord. These genes were also expressed in the developing somites and heart, but at different developmental times in peripheral tissues (pronephros, cement gland, and posterior mesoderm). No difference was observed in transcript levels or localization when similarly staged embryos maintained in cyclic light were compared at two times of day (dawn and dusk) by in situ hybridization. Quantitation of xBmal1 expression in embryonic eyes was also performed using qRT-PCR. Eyes were isolated at dawn, midday, dusk, and midnight (cylic light). No difference in expression level between time-points was found in stage 31 eyes (p = 0.176) but stage 40 eyes showed significantly increased levels of xBmal1 expression at midnight (RQ = 1.98+/-0.094) when compared to dawn (RQ = 1+/-0.133; p = 0.0004).We hypothesize that when circadian genes are not co-expressed in the same tissue during development that it may indicate pleiotropic functions of these genes that are separate from the timing of circadian rhythm. Our results show that all circadian genes analyzed thus far are present during early brain and eye development, but rhythmic gene expression in the eye is not observed until after stage 31 of development

Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

A comparison of somite staining in the posterior of late tailbud embryos (stage 36–38).

<p>Shown are in situ hybridization results depicting RNA expression in paired whole mount and sagittal sections of the posterior of embryos stained with <i>xPer1</i> (A–B), <i>xPer2</i> (C–D), <i>xBmal1</i> (E–F), and <i>Nocturnin</i> (G–H).</p

A temporal summary of the expression patterns of <i>xPer1</i>, <i>xPer2</i>, <i>xBmal1</i>, and <i>Nocturnin</i>.

<p>The approximate stages of development are represented on the horizontal axis of this figure while the particular tissues and organs are listed on the vertical axis. <i>xPer1</i> is represented by the blue lines, <i>xPer2</i> by the green lines, <i>xBmal1</i> by the red lines, and <i>Nocturnin</i> by the black lines. Dotted lines indicate times during development when a gene may be present, but was not confirmed through sectioning or additional whole mount in situ analysis.</p

<i>xPer2</i> is expressed from neural plate to late tailbud stages.

<p>Shown are in situ hybridization results depicting expression of <i>xPer2</i> mRNA. Panels A, B, D, and F show a dorsal view of each embryo. Panels C and E show side views. All embryos are oriented with the anterior to the left. G–I show transverse sections and J shows a sagittal section of late tailbud stage embryos. Sections shown in G, H, and J are oriented with the dorsal side at the top right of the panel. Neural plate staining is shown in panel A (stage 16) and B (stage 18). C and D depict early tailbud embryos with continued expression in the CNS as well as in the eye (black arrow), otic vesicle (black arrowhead), cement gland (blue arrow) and somites (red arrowheads). In late tailbud embryos (E and F), <i>xPer2</i> is expressed in the otic vesicle (E,I, black arrowhead), pineal (F,G, orange arrowhead), brain, retina, lens (G), and olfactory pit (I, green arrowhead), although cement gland staining was lost (E, blue arrow). <i>xPer2</i> was also present at low levels in the heart (H, blue arrowhead) and notochord (J, orange arrow). J also shows somite staining (red arrowheads).</p

<i>xNocturnin</i> is expressed from neural plate to late tailbud stages.

<p>Shown are in situ hybridization results depicting expression of <i>xNocturnin</i> mRNA. All embryos in this figure are shown with the anterior facing left. Side views of the embryos are depicted in panels A,C,E,G,I, and K and dorsal views in panels B,D,F, H, and J. Low levels of <i>xNocturnin</i> were first detected in the neural plate of stage 15/16 embryos A and B. C and D show neural plate staining in a stage 18 embryo. E and F show a neural tube stage embryo (stage 24) with <i>xNocturnin</i> expression in the eyes (black arrow), somites (red arrowhead), and cement gland (blue arrow). G and H show early tailbud stage embryos with staining in the otic vesicle (black arrowhead), pronephric tubules (green arrow), heart (blue arrowhead), olfactory pit (green arrowhead), pineal (orange arrowhead), cement gland (blue arrow) and somites (red arrowhead). Late tailbud stages (I and J; stage 39) show similar results but additional staining in the anus/blastopore (brown arrow) and cement gland staining is absent (blue arrow). Sagittal (L) and transverse sections (M–O) of late tailbud embryos confirm <i>xNocturnin</i> expression in the brain, retina and lens (M), otic vesicle (N, black arrowhead), olfactory pit (L, green arrow), pronephric tubules (N, green arrow), heart (M, blue arrowhead), notochord (O, orange arrow) and in the somites (O, red arrowheads). <i>xNocturnin</i> is absent from the cement gland at late tailbud stages (L, blue arrow). No expression was seen using a sense probe specific to <i>Nocturnin</i> (K).</p

Isolated eyes show rhythmic expression of <i>xBmal1</i> at stage 40 but not at stage 31.

<p>Eyes were dissected from embryos maintained in a 12L:12D cycle at different stages of development and different circadian times (ZT 0 (dawn), ZT6 (mid-day), ZT12 (dusk),and ZT18 (midnight)). The eyes were analyzed by qRT-PCR. The relative quantitation (RQ) of <i>xBmal1</i> for each sample was calculated with respect to EF1α. No difference in the levels of <i>xBmal1</i> expression was observed in stage 31 embryonic eyes at any time of day tested (ANOVA; df3, F = 1.77, p = 0.176; arrhythmic). A significant difference in <i>xBmal1</i> expression was observed when all ZTs were analyzed in stage 40 embryonic eyes (ANOVA; df3, F12.23, p = 0.00009). The asterisk shows that the level of <i>xBmal1</i> expression at ZT18 was significantly different from ZT0 (ANOVA, df1, F = 27.82, p = 0.0004). Bars in each graph denote standard error.</p