E-Cadherin Is Transcriptionally Activated via Suppression of ZEB1 Transcriptional Repressor by Small RNA-Mediated Gene Silencing

RNA activation has been reported to be induced by small interfering RNAs (siRNAs) that act on the promoters of several genes containing E-cadherin. In this study, we present an alternative mechanism of E-cadherin activation in human PC-3 cells by siRNAs previously reported to possess perfect-complementary sequences to E-cadherin promoter. We found that activation of E-cadherin can be also induced via suppression of ZEB1, which is a transcriptional repressor of E-cadherin, by seed-dependent silencing mechanism of these siRNAs. The functional seed-complementary sites of the siRNAs were found in the coding region in addition to the 3′ untranslated region of ZEB1 mRNA. Promoter analyses indicated that E-boxes, which are ZEB1-binding sites, in the upstream promoter region are indispensable for E-cadherin transcription by the siRNAs. Thus, the results caution against ignoring siRNA seed-dependent silencing effects in genome-wide transcriptional regulation. In addition, members of miR-302/372/373/520 family, which have the same seed sequences with one of the siRNAs containing perfect-complementarity to E-cadherin promoter, are also found to activate E-cadherin transcription. Thus, E-cadherin could be upregulated by the suppression of ZEB1 transcriptional repressor by miRNAs in vivo

Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells

Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets

Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.This research was funded by the Spanish Ministry of Science and Innovation and Ministry of Economy and Competitiveness and FEDER (EU): BIO2009-13261-C02-01/02 (ACA); BIO2012-39922-C02-01/02 (ACA); CTQ2013-4493 (JLN) and CSD2008-00005 (JLN); Andalusian Regional Government (Spain) and FEDER (EU): P09-CVI-5063 (ACA); and Valentian Regional Government (Spain) and FEDER (EU): Prometeo 2013/018 (JLN). Data collection was supported by European Synchrotron Radiation Facility (ESRF), Grenoble, France: BAG proposals MX-1406 (ACA) and MX-1541 (ACA); and ALBA (Barcelona, Spain) proposals 2012010072 (ACA) and 2012100378 (ACA)

Exploring Coping and Social Support with Gender and Education Among People Living with HIV in China

Social support promotes positive coping strategies among people living with HIV (PLH); however, little is known about the various aspects of social support and their distinct effects on coping. The present study investigates the specific links between coping and perceived social support with respect to gender and education among PLH. A total of 522 PLH in Anhui, China, participated in an assessment that collected data on demographics, perceived tangible and emotional support, and cognitive and behavioral coping. The assessment was conducted using the computer-assisted personal interviewing method. The data were analyzed using linear mixed models. Emotional support was significantly associated with both cognitive and behavioral coping. Tangible support was significantly associated with behavioral coping but not with emotional coping. Women reported significantly lower levels of emotional support, cognitive coping, and behavioral coping than men did. Significant associations between tangible support and coping were found only among illiterate males. Women living with HIV are in greater need of social support and coping strategies. Future interventions should be gender specific, with targeted support for women with lower education levels to enhance their coping strategies

X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystals

Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals. In the absence of rigid-body motions and lattice defects, B-factors are thought to accurately reflect the structural heterogeneity of such ensembles. In order to study the effects of averaging on B-factors, we employ molecular dynamics simulations to controllably manipulate microscopic heterogeneity of a crystal containing 216 copies of villin headpiece. Using average structure factors derived from simulation, we analyse how well this heterogeneity is captured by high-resolution molecular-replacement-based model refinement. We find that both isotropic and anisotropic refined B-factors often significantly deviate from their actual values known from simulation: even at high 1.0 Å resolution and R(free) of 5.9%, B-factors of some well-resolved atoms underestimate their actual values even sixfold. Our results suggest that conformational averaging and inadequate treatment of correlated motion considerably influence estimation of microscopic heterogeneity via B-factors, and invite caution in their interpretation