Elucidation of Substrate Binding Interactions for Human Organic Cation Transporters 1 (SLC22A1) and 2 (SLC22A2) Using In Silico Homology Modeling in Conjunction with In Vitro Site-Directed Mutagenesis and Kinetic Analysis

The organic cation transporters (OCTs) play a critical role in the absorption, distribution and elimination of many drugs, hormones, herbal medicines, and environmental toxins. Given the broad substrate specificity of OCTs, they fall victim to the high susceptibility for contributing to harmful drug-drug interactions. Further defining how human (h)OCTs mechanistically bind to its broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCTs. The goal of the current study was to elucidate the critical amino acid residues for transporter-substrate binding interactions on human (h)OCT1 and 2 utilizing in silico molecular modeling techniques (homology modeling and automated docking), as well as in vitro mutagenesis and kinetic transport experiments. Three-dimensional homology models were generated for hOCT1 and 2 using Piriformospora indica phosphate transporter (PiPT) serving as template. A putative binding pocket was identified and used to dock the prototypical substrate MPP+. Docking studies revealed five residues for each transporter (hOCT1 and hOCT2) that may be critical for substrate-transporter interactions. The in silico data was used to guide subsequent in vitro site-directed mutagenesis and kinetic analysis. Four hOCT1 mutants (Gln241Lys, Thr245Lys, Tyr361Ala, and Glu447Lys) and three hOCT2 mutants (Gln242Lys, Tyr362Phe, and Tyr362Ala) showed complete loss of MPP+ transporter activity. Decreased affinity for MPP+ was observed for Phe244Ser and Thr245Ser in hOCT1, and Tyr245Ala in hOCT2. All amino acid residues highlighted in the in vitro experiments may be potentially critical for substrate-transporter interactions particularly Tyr361, Phe244 and Thr245 in hOCT1; and Tyr362 and Tyr245 in hOCT2. Docking of known structurally divergent hOCT1 and hOCT2 substrates revealed similar binding interactions as that identified for MPP+, albeit with some unique residues, suggesting the presence of a large central cavity within both transporters. Through the combination of in silico and in vitro experiments, a putative binding pocket was defined and several residues important for substrate-transporter interaction were identified and verified for hOCT1 and hOCT2. Further defining how OCTs biochemically interact with their broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCT1 and OCT2

Automatic domain ontology extraction for context-sensitive opinion mining

Automated analysis of the sentiments presented in online consumer feedbacks can facilitate both organizations’ business strategy development and individual consumers’ comparison shopping. Nevertheless, existing opinion mining methods either adopt a context-free sentiment classification approach or rely on a large number of manually annotated training examples to perform context sensitive sentiment classification. Guided by the design science research methodology, we illustrate the design, development, and evaluation of a novel fuzzy domain ontology based contextsensitive opinion mining system. Our novel ontology extraction mechanism underpinned by a variant of Kullback-Leibler divergence can automatically acquire contextual sentiment knowledge across various product domains to improve the sentiment analysis processes. Evaluated based on a benchmark dataset and real consumer reviews collected from Amazon.com, our system shows remarkable performance improvement over the context-free baseline

Information Granulation for the Design of Granular Information Retrieval Systems

With the explosive growth of the amount of information stored on computer networks such as the Internet, it is increasingly more difficult for information seekers to retrieve relevant information. Traditional document ranking functions employed by Internet search engines can be enhanced to improve the effectiveness of information retrieval (IR). This paper illustrates the design and development of a granular IR system to facilitate domain specific search. In particular, a novel computational model is designed to rank documents according the searchers’ specific granularity requirements. The initial experiments confirm that our granular IR system outperforms a classical vector-based IR system. In addition, user-based evaluations also demonstrate that our granular IR system is effective when compared with a well-known Internet search engine. Our research work opens the door to the design and development of the next generation of Internet search engines to alleviate the problem of information overload

China's Gains from WTO Accession: Imports versus Exports

We examine the gains from Chinese accession to the WTO. Using Arkolakis, Costinot, and Rodríguez-Clare (2012) we provide a new quantitative welfare measure by dividing the manufacturing sector into import and export sub-sectors. We then evaluate how the increased openness caused by China’s accession to the WTO effects the importing and exporting sectors. We find surprisingly, that the gains to the import sector are larger than the gains to the export sector. Moreover, the size and the dynamic pattern of such gains are different across sectors

Exploring the Functional Interaction Between CaMK-II and p53

Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is a multifunctional member of a family of Ca2+/calmodulin-dependent serine/threonine protein kinases that respond to transient intracellular calcium signaling. CaMK-II has been reported to be involved with transcription regulation, cell motility, neuronal development, cell cycle regulation, and more recently early development of vertebrates (Easley et al., 2008; Rothschild et al., 2009; Francescatto et al., 2010). Through previous work in the lab using tandem mass spectrometry and “substrate-trapping mutants”, tumor suppressor protein 53 (p53) was identified as a novel CaMK-II binding partner in tissue culture. In this study, I sought to provide characterization of the functional interaction of p53 and CaMK-II. First, a stable p53 knockdown human cell line (HEK) was established through lentiviral transduction of p53 shRNA and verified with immunoblots and immunostaining assays. Next, the localization of CaMK-II and the cell growth rate in these cells was determined. In wild type HEK cells, catalytically inactive CaMK-II inhibited cell growth, which is consistent with previous studies in mouse fibroblasts with pharmacological inhibition. p53-deficient cells were less sensitive to CaMK-II deficiencies using dominant negative CAMK-II, but not pharmacological disruption. The overall results of this study have provided significant clues to the mechanism between CaMK-II and p53 in the control of cell cycle progression

Gene Methylation and Silencing of WIF1 Is a Frequent Genetic Abnormality in Mantle Cell Lymphoma

We have previously shown that the Wnt canonical pathway (WCP) is constitutively active in most cases of mantle cell lymphoma (MCL). Here, we aimed to elucidate the mechanisms underlying this biochemical deregulation. We hypothesized that gene methylation/silencing of WIF1 (Wnt inhibitory factor-1), a physiologic inhibitor of WCP, contributes to the deregulation of WCP and promotes cell growth in MCL. In support of this hypothesis, we found that the expression of WIF1 was detectable in none of the 4 MCL cell lines, and in only 2 of 5 tumors (40%) examined. Using methylation-specific PCR, we found evidence of gene methylation of WIF1 in 4 of 5 cell lines (80%) and in 24 of 29 (82%) tumors. The addition of the demethylation agent 5-aza-2′-deoxycytidine to Mino and JeKo-1, two WIF1-negative cell lines, restored the expression of WIF1 mRNA in these cells. Gene transfection of WIF1 into JeKo-1 and Mino cells significantly reduced cell growth, and this finding correlated with substantial downregulations of various proteins in WCP, such as β-catenin and pGSK-3β. In conclusion, our results support the concept that gene methylation/silencing of WIF1 is a frequent event in MCL, and this abnormality contributes to the aberrant activation of WCP. These results have provided further evidence that aberrant Wnt signaling is pathogenetically important in MCL and it may represent a potential therapeutic target.publishedVersio

<em>Escherichia coli</em>: A Versatile Platform for Recombinant Protein Expression

Among the living organisms, Escherichia coli has been the most common choice employed for recombinant protein expression. In addition to its well-characterized genetics, E. coli is fast growing, relatively cheap, and easy to handle. These fine properties, in conjunction with the success achieved in transforming plasmid DNA into E. coli, as well as the advent of various genetic engineering techniques in the 1970s, have enabled E. coli to be considered as the most favorable host for genetic manipulations. The recent advances in better comprehension of regulatory controls of gene expression and the availability of various novel approaches, which include both intracellular, e.g., through intein-mediated expression and self-cleavages, and extracellular, e.g., through the use of secretion signals, to achieve successful expression of the target proteins in E. coli further support the view that E. coli is the most promising host choice for heterologous protein expression

YB-1 regulates Sox2 to coordinately sustain stemness and tumorigenic properties in a phenotypically distinct subset of breast cancer cells

BACKGROUND: Sox2, a transcription factor and an embryonic stem cell marker, has been implicated in the pathogenesis of breast cancer (BC). YB-1 is another transcription factor that has been shown to promote stemness in BC cells. METHODS: Western blotting, quantitative PCR, and siRNAs were used to query the regulatory relationships between YB-1, Sox2, and their downstream targets. Chromatin immunoprecipitation was used to detect YB-1 interactions at the Sox2 promoter. Mammosphere and soft agar assays were used to assess the phenotypic consequences of YB-1 knockdown. RESULTS: Here, we report that YB-1 regulates Sox2. YB-1 was found to bind to the SOX2 promoter and down-regulate its expression in MCF7 and ZR751. The regulatory interaction between YB-1 and Sox2 was drastically different between the two phenotypically distinct cell subsets, purified based on their differential response to a Sox2 reporter. They are referred to as the reporter unresponsive (RU) cells and the reporter responsive (RR) cells. Upon siRNA knockdown of YB-1, RU cells showed an increase in Sox2 expression but no change in Sox2 reporter activity; in contrast, RR cells exhibited increased expression and reporter activity of Sox2. Correlating with these findings, YB-1 knockdown induced a differential response in the expression of genes known to be regulated by both Sox2 and YB-1 (e.g. CCND1 and ITGA6). For instance, in response to YB-1 knockdown, CCND1 and ITGA6 expression were decreased or unchanged in RU cells but paradoxically increased in RR cells. Compared to RU cells, RR cells were significantly more resistant to the suppression of mammosphere formation due to YB-1 knockdown. Importantly, mammospheres derived from parental MCF7 cells treated with YB-1 siRNA knockdown exhibited higher expression levels of SOX2 and its downstream targets. CONCLUSIONS: To conclude, in a subset of BC cells, namely RR cells, YB-1 regulates Sox2 to coordinately maintain stemness and tumorigenic properties

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol

Murine Cytomegalovirus Is Regulated by a Discrete Subset of Natural Killer Cells Reactive with Monoclonal Antibody to Ly49h

Antiviral roles of natural killer (NK) cell subsets were examined in C57BL/6 mice infected with murine cytomegalovirus (MCMV) and other viruses, including lymphocytic choriomeningitis virus (LCMV), vaccinia virus (VV), and mouse hepatitis virus (MHV). Each virus vigorously induced an NK cell infiltrate into the peritoneal cavity and liver, causing some redistributions of NK cell subsets defined by monoclonal antibody (mAb) directed against Ly49A, C/I, D, and G2. Striking results were seen with a mAb (1F8) reactive with the positively signaling molecule Ly49H, present in MCMV-resistant C57BL/6 mice. mAb 1F8 also stains Ly49 C and I, but exclusion of those reactivities with mAb 5E6, which recognizes Ly49 C and I, indicated that Ly49H+ cells infiltrated the peritoneal cavity and liver and were particularly effective at synthesizing interferon γ. Depletion of 1F8+ but not 5E6+ cells in vivo by mAb injections enhanced MCMV titers by 20-1,000-fold in the spleen and approximately fivefold in the liver. Titers of LCMV or VV were not enhanced. These anti-MCMV effects were attributed to prototypical NK1.1+CD3− NK cells and not to NK1.1+CD3+ “NK/T” cells. This is the first evidence that control of a virus infection in vivo is mediated by a distinct NK cell subset