An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more. Keywords: embryonic stem cells; imaging; live imaging; fluorescence; differentiation; pluripotency; GFP; microscopy; DNA damage; protein dynamicsNational Institutes of Health (U.S.) (Grant HD045022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant R01NS088538-01

Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Dynamic Shading: An Analysis

Thesis (Master's)--University of Washington, 2013Efficient use of energy is vital. Electric lighting contributes to a significant part of the total energy use in the US. Efficient use of daylighting offers a significant reduction in overall energy use. However, because the light available changes dynamically; the design of static shading systems adhering to both high and low levels of light is difficult. This thesis explores dynamic shading systems and analyses the benefits of an adaptive system when compared to a static system. The main goal of the thesis is to analyze a dynamic shading system in different conditions and compare it with a static system; in order to establish the advantages and disadvantages both quantitatively and qualitatively in terms of daylighting. Unfortunately, most of the daylighting metrics are not developed with a dynamic system in mind. So the thesis will also look to utilize a metric which takes account of the dynamism. The analysis process developed in this research involves building a series of simulation models in Ecotect. Each model represents one physical configuration of the system. Using Radiance and DaySim, Annual Illuminance Profiles are computed consisting of "snapshot" simulations at hourly intervals for a specific city. Custom software written in Java for individual static positions processes these profiles and computes the metrics adopted. Further, an hypothesized Dynamic system is computed by combining the individual static positions. Finally, the behavior and benefits of the Dynamic system is evaluated by comparing the Static and Dynamic system results for different latitudes

A Research Study on Waste Management and Waste Segregation Emperical Evidence on Metropolitian Cities of India

<p>Numerous studies and investigations have been done on the origins and properties of wastes, as well as the potential negative effects of improper treatment and best global practises. The definition of a waste, however, is still a matter of debate. How much do we actually know about what constitutes waste? What have waste management practises looked like historically? In order to provide answers from earlier studies, the current research paper aims to examine these crucial questions. To address the research aims, the report took a descriptive method. In particular, the work uses a descriptive approach to gather data from books, journal papers, and reports from environmental groups that have undergone peer review. Waste was determined to be a significant problem extent ambiguous, since a substance can only be considered garbage when the owner designates it as such. This is especially true given that one person might perceive a chemical as waste while another might see it as a resource. However, it was maintained that in order to properly regulate, it is necessary to define exactly what wastes are. Waste segregation is another crucial step in this research paper because it enables efficient Reuse, Recycling, and Recovery (RRR). Unfortunately, it has not received much attention and is often used informally in poor nations. It is also impacted by a lack of understanding, lax enforcement of regulations, a lack of financial incentive, and low planning priority. This study was carried out on metropolitan cities in India. The research will continue on issues like the reuse and recycling of plastics, electronics, and the selling chain for metals is informal, going from households to waste collectors to recycling facilities, and then to industries. Waste segregation in various industries and how would it advance the growth of the nation.</p><p>Keywords:- Waste management, waste segregation.</p&gt

Transcriptional competence in pluripotency

Embryonic stem (ES) cells possess a globally open, decondensed chromatin structure that, together with trans-acting factors, supports transcriptional competence of developmentally regulated genes. However, our understanding of the mechanisms that establish transcriptional competence of specific genes is limited. In this issue of Genes & Development, Xu and colleagues (pp. 2824–2838) show that tissue-specific enhancers are actively marked by an unmethylated window in ES cells and induced pluripotent stem (iPS) cells. They propose a model and present supporting evidence to demonstrate the active involvement of pioneer transcription factors in this process. This work marks an important step toward the understanding of the mechanisms that define and maintain pluripotency, and calls for the identification of the factors that participate in the establishment of transcriptional competence in pluripotent cells

Community education and engagement for Blue-Green Infrastructure (BGI) projects: Insights from Assam, India

The use of Blue-Green Infrastructure (BGI) for addressing problems of urban flood risk is increasingly being encouraged. However, effective and appropriate implementation of BGI requires local community involvement. This research explores attitudes towards BGI within communities in Guwahati, Assam. A participatory case study method was used to identify the role of community education and engagement in enabling BGI implementation. Through community workshops, physical models and creative media were used to help educate the communities regarding BGI solutions. Feedback and insights into local knowledge were then used to develop community-led BGI solutions. This study provides important evidence of the benefits of understanding diverse stakeholder needs and in utilising a co-creation process that can contribute towards removing potential social barriers and facilitate the design of scientifically informed, locally appropriate solutions to urban flood risk.</p

Integrator enforces the fidelity of transcriptional termination at protein-coding genes

Integrator regulates the 3′-end processing and termination of multiple classes of noncoding RNAs. Depletion of INTS11, the catalytic subunit of Integrator, or ectopic expression of its catalytic dead enzyme impairs the 3′-end processing and termination of a set of protein-coding transcripts termed Integrator-regulated termination (IRT) genes. This defect is manifested by increased RNA polymerase II (RNAPII) readthrough and occupancy of serine-2 phosphorylated RNAPII, de novo trimethylation of lysine-36 on histone H3, and a compensatory elevation of the cleavage and polyadenylation (CPA) complex beyond the canonical polyadenylation sites. 3′ RNA sequencing reveals that proximal polyadenylation site usage relies on the endonuclease activity of INTS11. The DNA sequence encompassing the transcription end sites of IRT genes features downstream polyadenylation motifs and an enrichment of GC content that permits the formation of secondary structures within the 3′UTR. Together, this study identifies a subset of protein-coding transcripts whose 3′ end processing requires the Integrator complex

Data from: Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1α, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Chad Tillberg READ Poster

Chad Tillberg, Assistant Professor of Biology, reading Cockroaches: Ecology, Behavior, and Natural History, by William J. Bell, Louis M. Roth, and Christine A. Nalepa.https://digitalcommons.linfield.edu/libraries_read/1067/thumbnail.jp

Data from: Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1α, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state