Predicting the academic achievement of Lehigh freshmen

This thesis is the second in a series intended to improve prediction (in particular for admission) from knowledge of the applicant\u27s past and the institution\u27s past. It describes a rudimentary search for a model among sixteen factors related to the applicant\u27s past and representing four previously delimited categories: achievements, aptitudes, interests and family

High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs

TCR signal strength controls thymic differentiation of iNKT cell subsets.

During development in the thymus, invariant natural killer T (iNKT) cells commit to one of three major functionally different subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell antigen receptor (TCR) signal strength governs the development of iNKT cell subsets, with strong signaling promoting iNKT2 and iNKT17 development. Altering TCR diversity or signaling diminishes iNKT2 and iNKT17 cell subset development in a cell-intrinsic manner. Decreased TCR signaling affects the persistence of Egr2 expression and the upregulation of PLZF. By genome-wide comparison of chromatin accessibility, we identify a subset of iNKT2-specific regulatory elements containing NFAT and Egr binding motifs that is less accessible in iNKT2 cells that develop from reduced TCR signaling. These data suggest that variable TCR signaling modulates regulatory element activity at NFAT and Egr binding sites exerting a determinative influence on the dynamics of gene enhancer accessibility and the developmental fate of iNKT cells

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction

EPEN-11. TUMOR DIFFERENTIATION IMPACTS THE BIOLOGY OF RECURRENCE IN CHILDHOOD POSTERIOR FOSSA EPENDYMOMA

Ependymoma (EPN) of childhood is curable in only 50% of cases, with recurrences in the remainder that are refractory to treatment. In recent years significant advances have been made in understanding the molecular and cellular biology of EPN. Recent studies show that PFA subgroup EPN are comprised of multiple neoplastic subpopulations that show undifferentiated, differentiated and mesenchymal characteristics. These studies focused on tumor at presentation, with recurrent EPN being less well understood. In the present longitudinal study we examine changes in neoplastic cell heterogeneity in serial presentations of PFA EPN using deconvolution (Cibersort) of bulk RNAseq data. Analysis of a cohort of 48 PFA EPN presenting at Children’s Colorado showed survival and PFA1/PFA2 subtype assignment was associated with the proportion of individual neoplastic subpopulations as determined by deconvolution. Tumors that subsequently regrew had a significantly higher estimated proportion of undifferentiated EPN cells (UEC) at presentation, than those that were non-recurrent after 5 years follow-up. This outcome association potentially age related, as UEC proportions are significantly higher in PFA arising in children < 1 year old who have a particularly poor prognosis. Changes in PFA neoplastic subpopulations at recurrence was performed in two cohorts of patients from Children’s Colorado (n=23) and Nottingham, UK (n=15). As a whole, no subpopulation proportion was significantly changed at recurrence. However, separation of PFA into subtypes PFA1 and PFA2 revealed an increase in the proportion of the cilia-differentiated EPN cell subpopulation is more frequent event in PFA1 (15/24), and rare in PFA2 (2/11). Changes in other neoplastic subpopulations at recurrence were smaller and only seen in PFA1, both UEC and mesenchymal subpopulations being lower at recurrence. In summary, only PFA1 showed dynamic changes in neoplastic subpopulation proportions at recurrence, with potential impacts on transcriptomic based-subgroup assignment, whereas PFA2 proportions remained largely stable

Epen-22. Single-cell RNA sequencing identifies upregulation of IKZF1 in PFA2 myeloid subpopulation driving an anti-tumor phenotype

We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype and have a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples and used advanced machine learning techniques to identify distinct myeloid and lymphoid subpopulations. The myeloid compartment was difficult to interrupt as the data shows a continuum of gene expression profiles exist within PFA1 and PFA2. Through lineage tracing, we were able to tease out that PFA2 myeloid cells expressed more genes associated with an anti-viral response (MHC II, TNF-a, interferon-gamma signaling); while PFA1 myeloid cells had genes associated with an immune suppressive phenotype (angiogenesis, wound healing, IL-10). Specifically, we found expression of IKZF1 was upregulated in PFA2 myeloid cells. IKZF1 regulates differentiation of myeloid cells toward M1 or M2 phenotype through upregulation of either IRF5 or IRF4 respectively. IRF5 expression correlated with IKZF1, being predominately expressed in the PFA2 myeloid cell subset. IKZF1 is also involved in T-cell activation. While we have not completed our characterization of the T-cell subpopulation, we did find significantly more T-cell infiltration in PFA2 than PFA1. Moving forward these studies will provide us with valuable information regarding the molecular switches involved in the tumor-immune microenvironment and to better develop immunotherapy for PFA ependymoma

EPEN-07. SINGLE-CELL RNA SEQUENCING IDENTIFIES A UNIQUE MYELOID SUBPOPULATION ASSOCIATED WITH MESENCHYMAL TUMOR SUBPOPULATION IN POOR OUTCOME PEDIATRIC EPENDYMOMA

We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which induces secretion of myeloid cell IL-8 and pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype associated with a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples. Using advanced machine learning, we identified eight myeloid cell subpopulations with unique gene expression profiles. Interestingly, only one subpopulation was significantly enriched in PFA1 tumors. This subpopulation, denoted as the hypoxia myeloid subpopulation, was defined by genes associated with angiogenesis, response to hypoxia, wound healing, cell migration, neutrophil activation and response to oxygen levels. These myeloid cells also share similar gene expression profile to a mesenchymal tumor subpopulation (MEC) enriched in PFA1 and associated with poor outcome in EPN patients. This tumor subpopulation was the only population expressing IL-6. Using immunohistochemistry, we found the hypoxia myeloid located in regions of tumor necrosis and perivascular niches. The MEC cells were also more abundant in these regions. In an independent single-cell cytokine release assay, we identified eight subpopulations of functional myeloid cells. One subpopulation significantly secreted IL-8, which represented the hypoxia subpopulation based on IL-8 gene expression in the scRNAseq dataset. This data suggests the tumor necrosis resulting in the development of MEC tumor subpopulation is driving the immune suppressive myeloid phenotype in PFA1 tumors through polarization of myeloid cells to the hypoxia subpopulation. Further studies are needed to determine how these myeloid cells interact with the lymphocyte subpopulation

EPEN-07. SINGLE-CELL RNA SEQUENCING IDENTIFIES A UNIQUE MYELOID SUBPOPULATION ASSOCIATED WITH MESENCHYMAL TUMOR SUBPOPULATION IN POOR OUTCOME PEDIATRIC EPENDYMOMA

We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which induces secretion of myeloid cell IL-8 and pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype associated with a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples. Using advanced machine learning, we identified eight myeloid cell subpopulations with unique gene expression profiles. Interestingly, only one subpopulation was significantly enriched in PFA1 tumors. This subpopulation, denoted as the hypoxia myeloid subpopulation, was defined by genes associated with angiogenesis, response to hypoxia, wound healing, cell migration, neutrophil activation and response to oxygen levels. These myeloid cells also share similar gene expression profile to a mesenchymal tumor subpopulation (MEC) enriched in PFA1 and associated with poor outcome in EPN patients. This tumor subpopulation was the only population expressing IL-6. Using immunohistochemistry, we found the hypoxia myeloid located in regions of tumor necrosis and perivascular niches. The MEC cells were also more abundant in these regions. In an independent single-cell cytokine release assay, we identified eight subpopulations of functional myeloid cells. One subpopulation significantly secreted IL-8, which represented the hypoxia subpopulation based on IL-8 gene expression in the scRNAseq dataset. This data suggests the tumor necrosis resulting in the development of MEC tumor subpopulation is driving the immune suppressive myeloid phenotype in PFA1 tumors through polarization of myeloid cells to the hypoxia subpopulation. Further studies are needed to determine how these myeloid cells interact with the lymphocyte subpopulation

EPEN-07. SINGLE-CELL RNA SEQUENCING IDENTIFIES A UNIQUE MYELOID SUBPOPULATION ASSOCIATED WITH MESENCHYMAL TUMOR SUBPOPULATION IN POOR OUTCOME PEDIATRIC EPENDYMOMA

We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which induces secretion of myeloid cell IL-8 and pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype associated with a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples. Using advanced machine learning, we identified eight myeloid cell subpopulations with unique gene expression profiles. Interestingly, only one subpopulation was significantly enriched in PFA1 tumors. This subpopulation, denoted as the hypoxia myeloid subpopulation, was defined by genes associated with angiogenesis, response to hypoxia, wound healing, cell migration, neutrophil activation and response to oxygen levels. These myeloid cells also share similar gene expression profile to a mesenchymal tumor subpopulation (MEC) enriched in PFA1 and associated with poor outcome in EPN patients. This tumor subpopulation was the only population expressing IL-6. Using immunohistochemistry, we found the hypoxia myeloid located in regions of tumor necrosis and perivascular niches. The MEC cells were also more abundant in these regions. In an independent single-cell cytokine release assay, we identified eight subpopulations of functional myeloid cells. One subpopulation significantly secreted IL-8, which represented the hypoxia subpopulation based on IL-8 gene expression in the scRNAseq dataset. This data suggests the tumor necrosis resulting in the development of MEC tumor subpopulation is driving the immune suppressive myeloid phenotype in PFA1 tumors through polarization of myeloid cells to the hypoxia subpopulation. Further studies are needed to determine how these myeloid cells interact with the lymphocyte subpopulation

EPEN-07. SINGLE-CELL RNA SEQUENCING IDENTIFIES A UNIQUE MYELOID SUBPOPULATION ASSOCIATED WITH MESENCHYMAL TUMOR SUBPOPULATION IN POOR OUTCOME PEDIATRIC EPENDYMOMA

We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which induces secretion of myeloid cell IL-8 and pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype associated with a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples. Using advanced machine learning, we identified eight myeloid cell subpopulations with unique gene expression profiles. Interestingly, only one subpopulation was significantly enriched in PFA1 tumors. This subpopulation, denoted as the hypoxia myeloid subpopulation, was defined by genes associated with angiogenesis, response to hypoxia, wound healing, cell migration, neutrophil activation and response to oxygen levels. These myeloid cells also share similar gene expression profile to a mesenchymal tumor subpopulation (MEC) enriched in PFA1 and associated with poor outcome in EPN patients. This tumor subpopulation was the only population expressing IL-6. Using immunohistochemistry, we found the hypoxia myeloid located in regions of tumor necrosis and perivascular niches. The MEC cells were also more abundant in these regions. In an independent single-cell cytokine release assay, we identified eight subpopulations of functional myeloid cells. One subpopulation significantly secreted IL-8, which represented the hypoxia subpopulation based on IL-8 gene expression in the scRNAseq dataset. This data suggests the tumor necrosis resulting in the development of MEC tumor subpopulation is driving the immune suppressive myeloid phenotype in PFA1 tumors through polarization of myeloid cells to the hypoxia subpopulation. Further studies are needed to determine how these myeloid cells interact with the lymphocyte subpopulation