An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level.

RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates.

Infantile neuroblastoma of the urinary bladder detected by hematuria.

Malignant tumors of the urinary bladder in infants are extremely rare. Rhabdomyosarcoma is the most likely tumor in this site, whereas neuroblastoma of the urinary bladder is exceedingly uncommon and is not listed as a differential diagnosis for tumors of this site. We present a case of neuroblastoma arising from the dome of the bladder wall, detected by hematuria.

Pre and post-operative evaluation of gastroesophageal reflux and esophageal motility in neurologically impaired children using combined pH-multichannel intraluminal impedance measurements.

Background: Gastroesophageal reflux disease (GERD) in patients with neurological impairment (NI) has not been fully studied before and after fundoplication procedure because their characteristics such as generalized gastrointestinal dysmotility, non-acid reflux, and the proximal reflux due to feeding of enteral nutrition via a nasogastric tube prevent their GERD from being detected by 24 h pH monitoring. The aim of this study was to elucidate whether multichannel impedance-pH measurement (pH/MII) is able to detect the subtypes of GERD and the differences in the reflux episodes of the severity of GERD, the ingestion pathway, and before and after fundoplication. The second aim was to determine whether a trial evaluation of dry swallows was able to be used to assess the esophageal motility of NI patients as an alternative examination.

Plakoglobin rescues adhesive defects induced by ectodomain truncation of the desmosomal cadherin desmoglein 1: implications for exfoliative toxin-mediated skin blistering.

Desmoglein 1 (Dsg1) is a desmosomal cadherin that is essential to epidermal integrity. In the blistering diseases bullous impetigo and staphylococcal scalded-skin syndrome, pathogenesis depends on cleavage of Dsg1 by a bacterial protease, exfoliative toxin A, which removes residues 1 to 381 of the Dsg1 ectodomain. However, the cellular responses to Dsg1 cleavage that precipitate keratinocyte separation to induce blister formation are unknown.

Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition.

Vimentin is used widely as a marker of the epithelial to mesenchymal transitions (EMTs) that take place during embryogenesis and metastasis, yet the functional implications of the expression of this type III intermediate filament (IF) protein are poorly understood. Using form factor analysis and quantitative Western blotting of normal, metastatic, and vimentin-null cell lines, we show that the level of expression of vimentin IFs (VIFs) correlates with mesenchymal cell shape and motile behavior. The reorganization of VIFs caused by expressing a dominant-negative mutant or by silencing vimentin with shRNA (neither of which alter microtubule or microfilament assembly) causes mesenchymal cells to adopt epithelial shapes.

RNA interference in keratinocytes and an organotypic model of human epidermis.

Gene silencing approaches afford investigators the ability to gain important insight into the normal functional requirements of specific epidermal proteins and promise to yield a powerful therapeutic means to dampen the level of proteins that are mutated or frequently overexpressed in skin disease. The efficient and tractable delivery of siRNAs into epidermal keratinocytes is seminal to this process. Here, we describe techniques for transient and long-term silencing of a representative gene product, namely desmoglein 1, in primary human epidermal keratinocytes maintained as submerged cultures or three-dimensional organotypic raft cultures.

Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation.

The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription.

The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A- and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS).

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells.

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches.

Ena/VASP proteins have an anti-capping independent function in filopodia formation.

Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation.

Role of fascin in filopodial protrusion.

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization.

Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end.

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia.

Improved silencing vector co-expressing GFP and small hairpin RNA.

Small interfering RNA (siRNA) is a powerful tool for the specific silencing of gene expression. We developed an improved vector, pG-SUPER, that co-expresses green fluorescent protein (GFP) and small hairpin RNA simultaneously to facilitate analysis of silencing at the level of individual cells. As a test system, we analyzed lamin A/C knockdown in HeLa cells.

p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions.

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell-cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain.

Mechanism of filopodia initiation by reorganization of a dendritic network.

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells.

Cytoplasmic linker proteins promote microtubule rescue in vivo.

The role of plus end-tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold.