Circular dichroism and fluorescence spectroscopy of cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 demonstrates that group I cations are particularly effective in providing structure and stability to this halophilic protein.

Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein.

Optimization of a high-throughput whole blood expression profiling methodology and its application to assess the pharmacodynamics of interferon (IFN) beta-1a or polyethylene glycol-conjugated IFN beta-1a in healthy clinical trial subjects.

Background: Clinical trials offer a unique opportunity to study human disease and response to therapy in a highly controlled setting. The application of high-throughput expression profiling to peripheral blood from clinical trial subjects could facilitate the identification of transcripts that function as prognostic or diagnostic markers of disease or treatment. The paramount issue for these methods is the ability to produce robust, reproducible, and timely mRNA expression profiles from peripheral blood.

Serum IL-17F does not predict poor response to IM IFNβ-1a in relapsing-remitting MS.

Objective: There is a significant unmet need for serum biomarkers in relapsing-remitting multiple sclerosis (RRMS) that are predictive of therapeutic response to disease-modifying therapies. Following a recent Stanford study which reported that pretreatment levels of serum interleukin (IL)-17F could predict poor response to interferon-β (IFNβ) therapy, we sought to validate the finding using samples from a large clinical trial.

Methods: The validation cohort included 54 good responders (GR) and 64 poor responders (PR) selected from 762 subjects with RRMS from the IM IFNβ-1a dose comparison study (Avonex study C94-805).

Sequencing and analysis of JC virus DNA from natalizumab-treated PML patients.

Background: Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients.

GECKO: a complete large-scale gene expression analysis platform.

Background: Gecko (Gene Expression: Computation and Knowledge Organization) is a complete, high-capacity centralized gene expression analysis system, developed in response to the needs of a distributed user community.

Results: Based on a client-server architecture, with a centralized repository of typically many tens of thousands of Affymetrix scans, Gecko includes automatic processing pipelines for uploading data from remote sites, a data base, a computational engine implementing approximately 50 different analysis tools, and a client application. Among available analysis tools are clustering methods, principal component analysis, supervised classification including feature selection and cross-validation, multi-factorial ANOVA, statistical contrast calculations, and various post-processing tools for extracting data at given error rates or significance levels.

Identification of genes regulated during osteoblastic differentiation by genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Although several independent studies of gene expression patterns during osteoblast differentiation in cultures from calvaria and other in vitro models have been reported, only a small portion of the mRNAs expressed in osteoblasts have been characterized. We have previously analyzed the behavior of several known markers in osteoblasts, using Affymetrix GeneChip murine probe arrays (27,000 genes). In the present study we report larger groups of transcripts displaying significant expression modulation during the culture of osteoblasts isolated from mice calvaria.

Behavior of osteoblast, adipocyte, and myoblast markers in genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Several genes, such as alkaline phosphatase, osteocalcin, and Cbfa1/Osf2, are known to be regulated during osteoblastic differentiation and are commonly used as "osteoblast markers" for in vitro or in vivo studies. The number of these genes is very limited, however, and it is of major interest to identify new genes that are activated or repressed during the process of osteoblast differentiation and bone formation as well as to extend the available information on gene families relevant to this particular differentiation pathway. To identify such genes, we have implemented a genome-wide analysis by determining changes in expression levels of 27,000 genes during in vitro differentiation of primary osteoblasts isolated from mouse calvaria.

Finding genes in the C2C12 osteogenic pathway by k-nearest-neighbor classification of expression data.

A supervised classification scheme for analyzing microarray expression data, based on the k-nearest-neighbor method coupled to noise-reduction filters, has been used to find genes involved in the osteogenic pathway of the mouse C2C12 cell line studied here as a model for in vivo osteogenesis. The scheme uses as input a training set embodying expert biological knowledge, and provides internal estimates of its own misclassification errors, which furthermore enables systematic optimization of the classifier parameters. On the basis of the C2C12-generated expression data set with 34,130 expression profiles across 2 time courses, each comprised of 6 points, and a training set containing known members of the osteogenic, myoblastic, and adipocytic pathways, 176 new genes in addition to 28 originally in the training set are selected as relevant to osteogenesis.

Bayesian estimation of fold-changes in the analysis of gene expression: the PFOLD algorithm.

A general and detailed noise model for the DNA microarray measurement of gene expression is presented and used to derive a Bayesian estimation scheme for expression ratios, implemented in a program called PFOLD, which provides not only an estimate of the fold-change in gene expression, but also confidence limits for the change and a P-value quantifying the significance of the change. Although the focus is on oligonucleotide microarray technologies, the scheme can also be applied to cDNA based technologies if parameters for the noise model are provided. The model unifies estimation for all signals in that it provides a seamless transition from very low to very high signal-to-noise ratios, an essential feature for current microarray technologies for which the median signal-to-noise ratios are always moderate.

ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.

Motivation: The sensitivity and specificity of branched DNA (bDNA) assays are derived in part through the judicious design of the capture and label extender probes. To minimize non-specific hybridization (NSH) events, which elevate assay background, candidate probes must be computer screened for complementarity with generic sequences present in the assay.

Results: We present a software application which allows for rapid and flexible design of bDNA probesets for novel targets.

A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification.

Breast reconstruction after mastectomy.

This chapter includes brief descriptions of the major methods of reconstructive surgery, along with guidelines as to the optimal approach in a number of clinical situations. Considerations of the candidates for breast reconstruction and the psychologic benefits of reconstruction are also discussed.

The development and standardization of an enzyme-linked immunosorbent assay for the detection of antibodies to bovine herpesvirus 2.

A single dilution enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine herpesvirus 2 has been developed, standardized and compared with the virus neutralization test. The results of the two tests correlated well. A positive/negative threshold was established for the ELISA.

Identifying health care stakeholders: a key to strategic implementation.

As the number and complexity of stakeholders for health care organizations has increased, health care managers have become more aware of the ability of these groups to thwart or facilitate the implementation of strategic plans. Most stakeholder models have focused on identification of groups within the usual, global definition of affecting or being affected by an organization's actions. The authors argue that stakeholders management is critical to the implementation of strategic plans.

Establishment of a statistical base for use of ELISA in diagnostic serology for infectious bovine rhinotracheitis.

The critical statistical parameters of an enzyme-linked immunosorbent assay were determined to enable quantitation of antibody responses in cattle affected with infectious bovine rhinotracheitis. A system of controlling well-to-well variations in optical density reading across a microtitre plate was evolved and dose--response assays were carried out to determine the dilution of serum which gave the greatest discrimination between acute and convalescent sera from an infected animal. Use of a standard serum was studied in further assays.

Detection of bovine leukosis virus in bronchoalveolar lung washings and nasal secretions.

Cattle and sheep persistently infected with bovine leukosis virus (BLV) were studied for the presence of the virus in bronchoalveolar lung washings and nasal secretions. The virus was demonstrated in the cellular fraction of the lung washings in six out of nine cattle and in one out of six sheep. In no instance was bovine leukosis isolated from the cell-free bronchoalveolar lung washings.