Identification of diagnostic biomarkers for infection in premature neonates.

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity.

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics.

Background: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries.

Cerebral palsy is characterized by protein mediators in cord serum.

Cerebral palsy (CP) is a major neurodevelopmental disability in childhood. An association between intrauterine infection and CP has been reported. We examined the relationship between inflammatory mediators in cord serum and CP in term and preterm children.

Multiplexed protein profiling on microarrays by rolling-circle amplification.

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose.

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.

Background: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA.

Comparison of the expression patterns of genes coding for wheat gluten proteins and proteins involved in the secretory pathway in developing caryopses of wheat.

The synthesis of gluten proteins in the developing caryopsis of wheat is highly coordinated, with mRNAs for the various groups being detected from 11 days after anthesis, and the proteins from about 14 days. In contrast, the levels of transcripts for BiP, PDI and PPI are highest at earlier stages of development. The levels of transcripts for two small GTP binding proteins involved in the secretory pathway (Rab1 and Rab5) are also highest early in development, which is consistent with the retention of most of the gluten proteins within the ER to form protein bodies.

Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae.

We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified.

Physical and functional definition of the Drosophila Notch locus by P element transformation.

Notch is a developmentally regulated locus which controls the differentiation of various Drosophila tissues, among them the embryonic nervous system. Molecular analysis has suggested that Notch is defined by an approximately 40-kb transcription unit which is spliced into a 10.2-kb mRNA composed of nine exonic regions and coding for a 2703-amino acid long transmembrane protein that shows homology to the mammalian epidermal growth factor.

The molecular genetics of the Notch locus in Drosophila melanogaster.

The Notch locus of Drosophila melanogaster profoundly affects differentiation of the central nervous system in the early embryo. Previous molecular studies suggested that the locus spans 40 kb of DNA and encodes a 10.5-kb poly(A)+ RNA.

Selection of viable cells with known DNA content.

Cells doubly stained with Hoechst 33342 (H-33342) for DNA content and fluorescein diacetate for viability can be selected on the basis of both criteria using a single UV laser flow sorter. The selection is made possible due to resonance energy transfer occurring between the H-33342 and fluorescein fluorophores. Both a static fluorescence microscope and a dual laser flow sorter were used to demonstrate that energy transfer occurs in the doubly stained cells and that the sensitized emission in conjunction with the DNA emission can be used to select populations of cells with known DNA content and viability.

A dual laser flow sorter utilizing a CW pumped dye laser.

A dual laser flow sorter has been constructed from an existing single laser system by incorporating a dye laser as the second laser source. The argon ion laser emission was used both as a pump laser and as a source by beam splitting before entrance to the dye laser. The emissions of the dye laser and pump laser beams were recombined and focused with the same optical train used in the single laser system.

Fluorescence polarization and pulse width analysis of chromosomes by a flow system.

Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.