Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling

Apoptosis, mastocytosis, and diminished adipocytokine gene expression accompany reduced epididymal fat mass in long-standing diet-induced obese mice

<p>Abstract</p> <p>Background</p> <p>Obesity is characterized by increased cell death and inflammatory reactions in the adipose tissue. Here, we explored pathophysiological alterations taking place in the adipose tissue in long-standing obesity. In the epididymal fat of C57BL/6 mice fed a high-fat diet for 20 weeks, the prevalence and distribution of dead adipocytes (crown-like structures), mast cells (toluidine blue, mMCP6), macrophages (F4/80), and apoptotic cells (cleaved caspase-3) were measured. Moreover, gene and/or protein expression of several adipocytokines (leptin, adiponectin, TNF-α, IL-10, IL-6, MCP-1), F4/80, mMCP6, cleaved caspase-3 were determined.</p> <p>Results</p> <p>We observed that the epididymal fat mass was lower in obese than in lean mice. In obese mice, the epididymal fat mass correlated inversely with body weight and liver mass. Dead adipocytes, mast cells, macrophages, and apoptotic cells were abundant in the epididymal fat of obese mice, especially in the rostral vs. caudal zone. Accordingly, mMCP6, F4/80, and cleaved caspase-3 gene and/or protein expression was increased. Conversely, adiponectin, leptin, IL-6, and MCP-1 gene expression levels were lower in the epididymal fat of obese than lean mice. Although TNF-α and IL-10 gene expression was higher in the epididymal fat of obese mice, their expression relative to F4/80 and mMCP6 expression were lower in the heavily infiltrated rostral than caudal zone.</p> <p>Conclusions</p> <p>This study demonstrates that in mice with long-standing obesity diminished gene expression of several adipocytokines accompany apoptosis and reduced mass of the epididymal fat. Our findings suggest that this is due to both increased prevalence of dead adipocytes and altered immune cell activity. Differential distribution of metabolically challenged adipocytes is indicative of the presence of biologically diverse zones within the epididymal fat.</p

Investigating How Undergraduate Students Develop Scientific Reasoning Skills When Coordinating Data and Model Representations in Biology

There has been a call to reform science education to integrate scientific thinking practices, such as data interpretation and modeling, with learning content in science classrooms. This call to reform has taken place in both K-12 science education through Next Generation Science Standards and undergraduate education through AAAS initiative Vision and Change in Undergraduate Biology Education. This dissertation work examines undergraduate students' learning of multiple scientific thinking skills in a curricular format called Teaching Real data Interpretation with Models (TRIM) applied to a large-enrollment course in Cellular and Developmental Biology. In TRIM, students are provided worksheets in groups and tasked to interpret authentic biological data. Importantly, groups are tasked to relate their data interpretations to a 2D visual model representation of the relevant biological process. This dissertation work consists of two studies with the overarching question: How do students use model representations to interpret data interpretations? In the first study, we primarily describe how students learn to navigate and interpret discipline-based data representations. We found the majority of groups could construct quality written data interpretations. Qualitative coding analysis on group discourse found students relied on strategies such as decoding the data representation and noticing data patterns together to construct claims. Claims were refined through spontaneous collaborative argumentation. We also found groups used the provided model to connect their data inferences to a biological context. In the second study, we primarily target our analysis on how individual students relate their data interpretations to different modeling tasks, including student-generation of their own model drawing. I interviewed students one-on-one as they worked through TRIM-style worksheets. From iterative qualitative analysis of transcripts and collected video on hand movements, I characterize the forms of reasoning at play at the interface of data and model representations. I propose a model at the end of Study 2 describing three modes of reasoning in data abstraction into models. I found when relating between data and models, students needed to link signs in both representations to a common referent in the real-world phenomenon. Establishing this sign-referent relationship seemed to depend on bringing in outside mechanistic information about the phenomenon. Once a mechanism was established, students could fluidly move between data and model representations through mechanistic reasoning. Thus data abstraction seems to rely on mechanistic reasoning with models. The findings from this dissertation work support the feasibility of student development of multiple scientific thinking skills within a large lecture course, and provide targets for curriculum and assignment designs centered on teaching higher order reasoning skills.Release after 21-Jul-201

Through the Eyes of Faculty: Using Personas as a Tool for Learner-Centered Professional Development

College science instructors need continuous professional development (PD) to meet the call to evidence-based practice. New PD efforts need to focus on the nuanced blend of factors that influence instructors’ teaching practices. We used persona methodology to describe the diversity among instructors who were participating in a long-term PD initiative. Persona methodology originates from ethnography. It takes data from product users and compiles those data in the form of fictional characters. Personas facilitate user-centered design. We identified four personas among our participants: Emma the Expert views herself as the subject-matter expert in the classroom and values her hard-earned excellence in lecturing. Ray the Relater relates to students and focuses on their points of view about innovative pedagogies. Carmen the Coach coaches her students by setting goals for them and helping them develop skill in scientific practices. Beth the Burdened owns the responsibility for her students’ learning and feels overwhelmed that students still struggle despite her use of evidence-based practice. Each persona needs unique PD. We suggest ways that PD facilitators can use our personas as a reflection tool to determine how to approach the learners in their PD. We also suggest further avenues of research on learner-centered PD

pUL135 and pUL138 impact phosphorylation of EGFR.

<p>Fibroblasts were infected with WT, <i>UL135</i>_STOP, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 1. At 48 hpi, infected cells were pulsed with 10nM EGF for 15min and then lysed. (A) EGFR was immunoprecipitated with ms α-EGFR and both IP and lysate samples were separated by SDS-PAGE. Blots were stained with rb α-EGFR, rb α-EGFR phosphotyrosine 1068, ms α-phosphotyrosine, and ms α-IE1/2. (B) The quantification of phosphorylation over three experiments is shown. To control for the variation in EGFR levels in different infections, we normalized signals associated with pY1068 or pY to total EGFR. Statistical significance relative to WT was calculated by student t-test (* p-value ≤ 0.05). (C) Serum-starved or fed fibroblasts expressing EGFR_3XFLAG were infected with WT CMV at 20 hours post transduction. Cells were stained with ms α-EGFR, rb α-EGFR pY1068 at 48 hpi. A merge of all three images in shown to the right. (D) Serum-starved, infected fibroblasts were pulsed with Alexa Fluor 647-conjugated EGF ligand on ice, fixed 20 min after a shift to 37C and stained with rb α-EGFR. Cells were imaged by deconvolution microscopy. For C and D, nuclei are stained with DAPI.</p

Through the Eyes of Faculty: Using Personas as a Tool for Learner-Centered Professional Development

College science instructors need continuous professional development (PD) to meet the call to evidence-based practice. New PD efforts need to focus on the nuanced blend of factors that influence instructors’ teaching practices. We used persona methodology to describe the diversity among instructors who were participating in a long-term PD initiative. Persona methodology originates from ethnography. It takes data from product users and compiles those data in the form of fictional characters. Personas facilitate user-centered design. We identified four personas among our participants: Emma the Expert views herself as the subject-matter expert in the classroom and values her hard-earned excellence in lecturing. Ray the Relater relates to students and focuses on their points of view about innovative pedagogies. Carmen the Coach coaches her students by setting goals for them and helping them develop skill in scientific practices. Beth the Burdened owns the responsibility for her students’ learning and feels overwhelmed that students still struggle despite her use of evidence-based practice. Each persona needs unique PD. We suggest ways that PD facilitators can use our personas as a reflection tool to determine how to approach the learners in their PD. We also suggest further avenues of research on learner-centered PD

Antibody description and sources.

<p>Antibody description and sources.</p

pUL135 and pUL138 regulate EGFR trafficking.

<p>Fibroblasts were infected with WT, <i>UL135</i>_STOP, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 1. At 48hpi, cell were stimulated with EGF, collected over a time course of 0–180 minutes post pulse, and stained with BV421 conjugated ms α-EGFR. EGFR surface levels were measured in infected (GFP+) cells by FACS. (A and B) EGFR surface levels over the time course in mock- and WT-infected cells. The WT curve is replotted in panel B on a scale to better discern trafficking dynamics. (C-E) EGFR trafficking during <i>UL135</i>_<i>STOP</i> and <i>UL138</i>_<i>STOP</i> infection in fibroblasts relative to WT. Panel D expands the 0–60 minute time points and panel E expands 0–20 min. (F) Data from selected timepoints were chosen and compared to initial EGFR levels summarize the differences within each infection.</p