Effects of altered expression of the sumo conjugating enzyme, UBC9 on mitosis, meiosis and conjugation in Tetrahymena thermophila

SUMOylation is a critical posttranslational modification in eukaryotic species. Ubc9p is the E2-conjugating enzyme for SUMOylation and consequently it influences multiple cellular pathways. Nuclear proteins are common targets of SUMOylation and regulate nuclear events such as transcription, DNA repair and mitosis. The segregation of the Tetrahymena thermophila genome into two different nuclear compartments provides an unusual context for the analysis of SUMOylation. Each cell contains a transcriptionally silent, diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid transcriptionally active somatic macronucleus (MAC) that divides by an amitotic mechanism. With the long-term goal to exploit these opportunities we initiated studies of Ubc9p and therefore indirectly SUMOylation, on the functionally distinct nuclei in T. thermophila using genetic analysis combined with proteomics study. We found that complete deletion of the UBC9 gene is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with a slow growth phenotype in the absence of CdCl 2-dependent expression of GFP-Ubc9p. Altered expression of Ubc9p resulted in differential effects in MICs and MACs. MICs were lost from cells during vegetative growth but MACs were capable of division. Interestingly, cells expressing a catalytically inactive dominant negative Ubc9p (DN-Ubc9p) accumulated multiple MICs. Ubc9p depleted cells were hypersensitive to DNA damaging agents that promote double-strand DNA breaks. Additional studies point to critical roles for Ubc9p during the sexual life cycle of Tetrahymena. Crosses between cell lines expressing the dominant negative Ubc9p were delayed in meiosis and produced fewer exconjugant progeny who successfully completed genetic exchange and conjugation than from wild-type controls. In contrast, cell lines that were depleted for Ubc9p did not form pairs and therefore could not complete any of the subsequent stages of conjugation including meiosis and macronuclear development. The results are consistent with roles for Ubc9p in mitosis, meiosis and double strand break repair. A proteomics-based approach generated an unbiased spectrum of Ubc9p interacting proteins during Tetrahymena vegetative growth and conjugation. We identified 128 high-confidence Ubc9p interacting proteins duringTetrahymena vegetative growth and 106 proteins during conjugation, among which 58 are conjugation-specific. Seven proteins with homologs in other species have been reported previously as SUMO substrates, or Ubc9p interacting proteins. The Ubc9p interactome covers a wide range of cellular processes, including chromatin remodeling, cell cycle progression, stress response, gene transcription and Tetrahymena macronuclear development, which further support our observations from phenotypic analysis. The findings provide evidence for distinct roles for SUMOylation in ciliate nuclei and provide opportunities for future studies of SUMOylated substrates in a context specific for gene expression (MAC) or mitotic and meiotic division (MIC)

Characterization of small molecules inhibiting the pro-angiogenic activity of the zinc finger transcription factor Vezf1

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 μM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases

U.S. Monetary Policy Announcements and Foreign Exchange Market Behavior

This paper examines the time-varying and currency-dependency nature of exchange rate responses following the U.S. monetary policy announcements. Using high frequency exchange rate data in the past decade, we find that the exchange rates of most major currencies against the US Dollar respond negatively to the monetary surprises in the 2001 recession, while the response becomes positive during the 2008 recession. Meanwhile, the JPY has the opposite response than the other major currencies in the 2008 recession. These results further confirm the nonlinearity in the relationship between exchange rate dynamics and fundamental news announcements

Summary of the Researches on the Influence of Investor Sentiment on Stock Returns Under the Background of Big Data

In order to analyze the promoting effect of big data technology on research results, and to explore the impact of investor sentiment on stock returns, this paper combs and summarizes the research results of domestic and foreign scholars on the impact of investor sentiment on stock returns under the background of big data. This paper defines the concepts of big data, investor sentiment and stock returns, analyzes the measurement methods of investor sentiment, and deeply analyzes the overall effect and cross-sectional effect of investor sentiment on stock returns under the background of living alone. The results show that big data technology plays a strong role in promoting the research results, can comprehensively analyze various influencing factors, and investor sentiment has a great impact on stock returns

Approaches for Identifying Consumer Preferences for the Design of Technology Products: A Case Study of Residential Solar Panels

This paper investigates ways to obtain consumer preferences for technology products to help designers identify the key attributes that contribute to a product's market success. A case study of residential photovoltaic panels is performed in the context of the California, USA, market within the 2007–2011 time span. First, interviews are conducted with solar panel installers to gain a better understanding of the solar industry. Second, a revealed preference method is implemented using actual market data and technical specifications to extract preferences. The approach is explored with three machine learning methods: Artificial neural networks (ANN), Random Forest decision trees, and Gradient Boosted regression. Finally, a stated preference self-explicated survey is conducted, and the results using the two methods compared. Three common critical attributes are identified from a pool of 34 technical attributes: power warranty, panel efficiency, and time on market. From the survey, additional nontechnical attributes are identified: panel manufacturer's reputation, name recognition, and aesthetics. The work shows that a combination of revealed and stated preference methods may be valuable for identifying both technical and nontechnical attributes to guide design priorities.Center for Scalable and Integrated Nanomanufacturin

Propagating Uncertainty in Solar Panel Performance for Life Cycle Modeling in Early Stage Design

One of the challenges in accurately applying metrics for life cycle assessment lies in accounting for both irreducible and inherent uncertainties in how a design will perform under real world conditions. This paper presents a preliminary study that compares two strategies, one simulation-based and one set-based, for propagating uncertainty in a system. These strategies for uncertainty propagation are then aggregated. This work is conducted in the context of an amorphous photovoltaic (PV) panel, using data gathered from the National Solar Radiation Database, as well as realistic data collected from an experimental hardware setup specifically for this study. Results show that the influence of various sources of uncertainty can vary widely, and in particular that solar radiation intensity is a more significant source of uncertainty than the efficiency of a PV panel. This work also shows both set-based and simulation-based approaches have limitations and must be applied thoughtfully to prevent unrealistic results. Finally, it was found that aggregation of the two uncertainty propagation methods provided faster results than either method alone.Center for Scalable and Integrated NanomanufacturingNational Science Foundation (U.S.) (Nanoscale Science and Engineering Center

Sumoylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle

High-throughput discovery of chemical structure-polarity relationships combining automation and machine learning techniques

As an essential attribute of organic compounds, polarity has a profound influence on many molecular properties such as solubility and phase transition temperature. Thin layer chromatography (TLC) represents a commonly used technique for polarity measurement. However, current TLC analysis presents several problems, including the need for a large number of attempts to obtain suitable conditions, as well as irreproducibility due to non-standardization. Herein, we describe an automated experiment system for TLC analysis. This system is designed to conduct TLC analysis automatically, facilitating high-throughput experimentation by collecting large experimental data under standardized conditions. Using these datasets, machine learning (ML) methods are employed to construct surrogate models correlating organic compounds' structures and their polarity using retardation factor (Rf). The trained ML models are able to predict the Rf value curve of organic compounds with high accuracy. Furthermore, the constitutive relationship between the compound and its polarity can also be discovered through these modeling methods, and the underlying mechanism is rationalized through adsorption theories. The trained ML models not only reduce the need for empirical optimization currently required for TLC analysis, but also provide general guidelines for the selection of conditions, making TLC an easily accessible tool for the broad scientific community

Gab2 facilitates epithelial-to-mesenchymal transition via the MEK/ERK/MMP signaling in colorectal cancer

Clinicopathologic factors and Gab2 expression in 35 CRC patients. (DOCX 22 kb