Novel multiplex assay platforms to detect influenza A hemagglutinin subtype-specific antibody responses for high-throughput and in-field applications.

Background: Detections of influenza A subtype-specific antibody responses are often complicated by the presence of cross-reactive antibodies. We developed two novel multiplex platforms for antibody detection. The multiplexed magnetic fluorescence microsphere immunoassay (MAGPIX) is a high-throughput laboratory-based assay.

A multicenter performance evaluation of the DPP(®) HIV-1/2 assay for the detection of HIV antibodies in various HIV testing algorithms.

Background: Multicenter studies were conducted to evaluate the DPP(®) HIV 1/2 Assay using oral fluid (OF) and fingerstick (FS) specimens in two different countries at the point of care (POC).

Objective: To evaluate the DPP(®) HIV 1/2 Assay using OF and FS specimens when compared to various worldwide algorithms for the detection of HIV.

Methods: At each testing center, each participant was tested using the DPP HIV 1/2 Assay using OF and FS specimens.

N-acetylglucosamine (GlcNAc) induction of hyphal morphogenesis and transcriptional responses in Candida albicans are not dependent on its metabolism.

N-acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling.

Identification of GIG1, a GlcNAc-induced gene in Candida albicans needed for normal sensitivity to the chitin synthase inhibitor nikkomycin Z.

The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc.

Nuclear magnetic resonance structural studies of a potassium channel-charybdotoxin complex.

Ion channels play critical roles in signaling processes and are attractive targets for treating various diseases. Here we describe an NMR-based strategy for structural analyses of potassium channel-ligand complexes using KcsA (residues 1-132, with six mutations to impart toxin binding and to mimic the eukaryotic hERG channel). Using this approach, we determined the solution structure of KcsA in complex with the high-affinity peptide antagonist charybdotoxin.

Development of a tightly regulated U6 promoter for shRNA expression.

Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Here, we report the development of a tightly regulated tetracycline-responsive human U6 promoter for shRNA expression. By engineering two copies of the tet operators flanking the TATA box of the human U6 promoter, we created a U6 promoter derivative (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters.

Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction.

A novel fluorescent substrate was devised for the sirtuin (SIRT) class of human protein deacetylases comprised of a peptide sequence containing a single acetyl-lysine residue, with a fluorescent group (tetramethylrhodamine-6-carboxylic acid, 6-TAMRA) near the carboxyl terminus and a nonfluorescent quenching group (QSY-7) near the amino terminus. The peptide sequence is modeled after the p53 acetylation site but is unreactive toward trypsin because all other lysine and arginine residues have been replaced by serine. However, the SIRT-deacetylated peptide is readily cleaved by trypsin, resulting in a maximal 30-fold enhancement of the 6-TAMRA fluorescence.

Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein.

The severe acute respiratory syndrome (SARS) virus belongs to the Coronaviridea family of viruses. Its virion encodes several proteins including a replicase and four structural proteins. Here we describe the three-dimensional structure of the N-terminal domain of the SARS coronavirus (CoV) nucleocapsid protein.

Defining the p53 DNA-binding domain/Bcl-x(L)-binding interface using NMR.

p53 exerts its tumor suppressor activity through both transcription-dependent and transcription-independent processes. Although the transcription-dependent activity of p53 has been extensively studied, the mechanism for transcription-independent p53-mediated tumor suppression is less well known. Recently, it was reported that p53 can directly induce mitochondrial permeabilization and promote apoptosis.

Characterization of protein kinase A phosphorylation: multi-technique approach to phosphate mapping.

A multi-technique approach to identification and mapping of phosphorylation on protein kinase A (PKA) is described. X-ray crystallography revealed phosphorylation at T197 and S338 while mass spectrometry (MS) on the intact protein suggested phosphorylation at three sites. Tryptic digestion, followed by MS, confirmed the presence of three phosphates.

Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents.

UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C.