ROMK channels are regulated by internal pH (pH(i)) and extracellular K(+) (K(+)(o)). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H(+), increasing the apparent pKa for inhibition by approximately 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K(+)(o), consistent with coupling between the responses to pH(i) and K(+)(o). The ion selectivity sequence of the activation of the channel by cations was K(+) congruent with Rb(+) > NH(4)(+) >> Na(+), similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K(+) from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K(+)(o). We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K(+)(o) between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K(+) to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba(2+) block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H(+). We conclude that decreasing pH(I) increases the sensitivity of ROMK2 channels to K(+)(o) by altering the properties of the selectivity filter.

The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for antivirals, cell-based replication assays can be performed, but these are often labor intensive and have limited throughput.

We demonstrated previously that the incorporation of a membrane-anchored form of flagellin into influenza virus-like particles (VLPs) improved the immunogenicity of VLPs significantly, inducing partially protective heterosubtypic immunity by intramuscular immunization. Because the efficacy of mucosal vaccination is highly dependent on an adjuvant, and is particularly effective for preventing mucosal infections such as influenza, we determined whether the membrane-anchored flagellin is an efficient adjuvant for VLP vaccines by a mucosal immunization route. We compared the adjuvant effect of membrane-anchored and soluble flagellins for immunization with influenza A/PR8 (H1N1) VLPs by the intranasal route in a mouse model. The results demonstrate that membrane-anchored flagellin is an effective adjuvant for intranasal (IN) immunization, inducing enhanced systemic and mucosal antibody responses. High cellular responses were also observed as shown by cytokine production in splenocyte cultures when stimulated with viral antigens. All mice immunized with flagellin-containing VLPs survived challenge with a high lethal dose of homologous virus as well as a high dose heterosubtypic virus challenge (40 LD(50) of A/Philippines/82, H3N2). In contrast, no protection was observed with a standard HA/M1 VLP group upon heterosubtypic challenge. Soluble flagellin exhibited a moderate adjuvant effect when co-administered with VLPs by the mucosal route, as indicated by enhanced systemic and mucosal responses and partial heterosubtypic protection. The membrane-anchored form of flagellin incorporated together with antigen into influenza VLPs is effective as an adjuvant by the mucosal route and unlike standard VLPs, immunization with such chimeric VLPs elicits protective immunity to challenge with a distantly related influenza A virus.

The increasing availability of large-scale protein-protein interaction data has made it possible to understand the basic components and organization of cell machinery from the network level. The arising challenge is how to analyze such complex interacting data to reveal the principles of cellular organization, processes and functions. Many studies have shown that clustering protein interaction network is an effective approach for identifying protein complexes or functional modules, which has become a major research topic in systems biology. In this review, recent advances in clustering methods for protein interaction networks will be presented in detail. The predictions of protein functions and interactions based on modules will be covered. Finally, the performance of different clustering methods will be compared and the directions for future research will be discussed.

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

Small heterodimer partner (SHP, NR0B2) is a unique member of the nuclear receptor (NR) superfamily that contains the dimerization and ligand-binding domain found in other family members, but lacks the conserved DNA-binding domain. The ability of SHP to bind directly to multiple NRs is crucial for its physiological function as a transcriptional inhibitor of gene expression. A wide variety of interacting partners for SHP have been identified, indicating the potential for SHP to regulate an array of genes in different biological pathways. In this review, we summarize studies concerning the structure and target genes of SHP and discuss recent progress in understanding the function of SHP in bile acid, cholesterol, triglyceride, glucose, and drug metabolism. In addition, we review the regulatory role of SHP in microRNA (miRNA) regulation, liver fibrosis and cancer progression. The fact that SHP controls a complex set of genes in multiple metabolic pathways suggests the intriguing possibility of developing new therapeutics for metabolic diseases, including fatty liver, dyslipidemia and obesity, by regulating SHP with small molecules. To achieve this goal, more progress regarding SHP ligands and protein structure will be required. Besides its metabolic regulatory function, studies by us and other groups provide strong evidence that SHP plays a critical role in the development of cancer, particularly liver and breast cancer. An increased understanding of the fundamental mechanisms by which SHP regulates the development of cancers will be critical in applying knowledge of SHP in diagnostic, therapeutic or preventive strategies for specific cancers. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

It has been proved that chronic administration and pre-treatment with atorvastatin could protect brain tissue against ischemic injury. However, little is known regarding the effect of atorvastatin in the acute phase of ischemic stroke. This study investigated the potential neuroprotective effects of atorvastatin and underlying mechanisms in vivo.

CCR6(+) subset of CD4(+) regulatory T cells, a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6(+) Tregs are present in tumor environment and their relation to the prognosis of tumor remain to be elucidated. In this study, we found that CCR6(+) CD4(+) CD25(high) Tregs, expressing high levels of CD45RO, are dominantly enriched in tumor mass from patients with breast cancer. Furthermore, the frequency of CCR6(+) Tregs, but not CCR6(-) Tregs in tumor infiltrating lymphocytes (TILs), significantly increased in patients during tumor progression, which reversely correlated with decreased frequency of the IFN-gamma(+)CD8(+)T cells in TILs. Most importantly, the frequency of CCR6(+) Tregs, but not CCR6(-) Tregs, reversely correlated to the survival of patients with breast cancer. This study suggested that a new subset of tumor-resident Tregs, CCR6(+) Tregs, may be dominantly responsible for the immunosuppression in tumor immunity and a potential predictor of the poor prognosis of breast cancer.

A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.

Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

To identify the pathogenic gene mutation in a Chinese family with autosomal dominant congenital nuclear cataract.

Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell-type specificities and their functional interactions. Focusing on cell-type-specific patterns of promoters and enhancers, we define multicell activity profiles for chromatin state, gene expression, regulatory motif enrichment and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell-type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for the interpretation of genome-wide association studies. Top-scoring disease single nucleotide polymorphisms are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus suggesting a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.

The tumor suppressor candidate gene Ras association domain family 1, isoform A (RASSF1A) encodes a microtubule-associated protein that is implicated in the regulation of cell proliferation, migration, and apoptosis. Several studies indicate that down-regulation of RASSF1A resulting from promoter hypermethylation is a frequent epigenetic abnormality in malignant melanoma. In this study, we report that compared with melanocytes in normal skins or benign skin lesions, RASSF1A is down-regulated in melanoma tissues as well as cell lines, and its expression negatively correlates with lymph node metastasis. Following ectopic expression in RASSF1A-deficient melanoma A375 cell line, RASSF1A reduces cell viability, suppresses cell-cycle progression but enhances apoptotic cell death. In vivo, RASSF1A expression inhibits the tumorigenic potential of A375 cells in nude mice, which also correlates with decreased cell proliferation and increased apoptosis. On the molecular level, ectopic RASSF1A expression leads to differential expression of 209 genes, including 26 down-regulated and 183 up-regulated ones. Among different signaling pathways, activation of the apoptosis signal-regulating kinase 1 (ASK1)/p38 MAP kinase signaling is essential for RASSF1A-induced mitochondrial apoptosis, and the inhibition of the Akt/p70S6 kinase/eIF4E signaling is also important for RASSF1A-mediated apoptosis and cell-cycle arrest. This is the first study exploring the biological functions and the underlying mechanisms of RASSF1A during melanoma development. It also identifies potential targets for further diagnosis and clinical therapy.

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 microg/l) and nonanal (0.15 ng/microl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture.

The low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Frizzled and transmit signals from the plasma membrane to the cytosol. However, the mechanism for LRP5/6 signal transmission remains undefined. Here, we identify cytoplasmic activation/proliferation-associated protein 2 (Caprin-2) as a LRP5/6-binding protein. Our data show that Caprin-2 stabilizes cytosolic beta-catenin and enhances lymphoid enhancer-binding factor 1/T cell factor-dependent reporter gene activity as well as the expression of Wnt target genes in mammalian cells. Morpholino-mediated knockdown of Caprin-2 in zebrafish embryos inhibits Wnt/beta-catenin signaling and results in a dorsalized phenotype. Moreover, Caprin-2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3, and thus enhances the interaction between Axin and LRP5/6. Therefore, Caprin-2 promotes activation of the canonical Wnt signaling pathway by regulating LRP5/6 phosphorylation.

Centromere protein A (CENP-A) plays important roles in cell-cycle regulation and genetic stability. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC).

A recessive, adult-onset neuronal ceroid-lipofuscinosis (NCL) occurs in Tibetan terriers. A genome-wide association study restricted this NCL locus to a 1.3Mb region of canine chromosome 2 which contains canine ATP13A2. NCL-affected dogs were homozygous for a single-base deletion in ATP13A2, predicted to produce a frameshift and premature termination codon. Homozygous truncating mutations in human ATP13A2 have been shown by others to cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disease. These findings suggest that KRS is also an NCL, although analysis of KRS brain tissue will be needed to confirm this prediction. Generalized brain atrophy, behavioral changes, and cognitive decline occur in both people and dogs with ATP13A2 mutations; however, other clinical features differ between the species. For example, Tibetan terriers with NCL develop cerebellar ataxia not reported in KRS patients and KRS patients exhibit parkinsonism and pyramidal dysfunction not observed in affected Tibetan terriers. To see if ATP13A2 mutations could be responsible for some cases of human adult-onset NCL (Kufs disease), we resequenced ATP13A2 from 28 Kufs disease patients. None of these patients had ATP13A2 sequence variants likely to be causal for their disease, suggesting that mutations in this gene are not common causes of Kufs disease.

The aim of this study was to explore the clinical and other associated laboratory features of chronic lymphocytic leukemia (CLL) patients with immunoglobulin (Ig) paraproteinemia. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to measure serum Ig paraprotein. The correlations between serum Ig paraprotein and other prognostic factors were analyzed. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. In 133 Chinese CLL patients, 27 (20.3%) patients occurred Ig paraproteinemia at diagnosis. According to the correlation analysis, advanced Binet stage (r=0.314, P<0.001), direct antiglobulin test (DAT)-positive (r=0.366, P<0.001), high level of serum β2-microglobulin (β2-MG) (r=0.296, P=0.001) and thymidine kinase (TK) 1 (r=0.227, P=0.037), unmutated immunoglobulin heavy chain variable gene (IGHV) status (r=0.284, P=0.002), ZAP-70-positive (r=0.305, P=0.001), CD38-positive (r=0.284, P=0.002), and cytogenetic abnormalities of del(17p13) or del(11q22.3) (r=0.208, P=0.032) emerged as factors significantly related to the occurrence of Ig paraproteinemia. Survival analysis showed that the patients with Ig paraproteinemia had significantly shorter survival times than the patients without serum Ig paraprotein (P=0.013). Binet stage (P=0.028), high levels of lactate dehydrogenase (LDH) (P=0.004), IgG paraproteinemia (P=0.048), IgM paraproteinemia (P=0.001), ZAP-70-positive (P=0.003), DAT-positive (P=0.013), unmutated IGHV status (P=0.009), and del(17p13) (P=0.001) were the adverse factors in determining overall survival (OS). Del(17p13) (P=0.006), ZAP-70 (P=0.030), and IgM paraproteinemia (P=0.040) were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that serum Ig paraprotein might be applied for the assessment of prognosis in patients with CLL.

MicroRNAs play a critical role in many essential cellular functions in the mammalian species. However, limited information is available regarding the regulation of miRNAs gene transcription. Microarray profiling and real-time PCR analysis revealed a marked down-regulation of miR-206 in nuclear receptor SHP(-/-) mice. To understand the regulatory function of SHP with regard to miR-206 gene expression, we determined the putative transcriptional initiation site of miR-206 and also its full length primary transcript using a database mining approach and RACE. We identified the transcription factor AP1 binding sites on the miR-206 promoter and further showed that AP1 (c-Jun and c-Fos) induced miR-206 promoter transactivity and expression which was repressed by YY1. ChIP analysis confirmed the physical association of AP1 (c-Jun) and YY1 with the endogenous miR-206 promoter. In addition, we also identified nuclear receptor ERRgamma (NR3B3) binding site on the YY1 promoter and showed that YY1 promoter was transactivated by ERRgamma, which was inhibited by SHP (NROB2). ChIP analysis confirmed the ERRgamma binding to the YY1 promoter. Forced expression of SHP and AP1 induced miR-206 expression while overexpression of ERRgamma and YY1 reduced its expression. The effects of AP1, ERRgamma, and YY1 on miR-206 expression were reversed by siRNA knockdown of each gene, respectively. Thus, we propose a novel cascade "dual inhibitory" mechanism governing miR-206 gene transcription by SHP: SHP inhibition of ERRgamma led to decreased YY1 expression and the de-repression of YY1 on AP1 activity, ultimately leading to the activation of miR-206. This is the first report to elucidate a cascade regulatory mechanism governing miRNAs gene transcription.