Design of a Microsphere-Based High-Throughput Gene Expression Assay to Determine Estrogenic Potential

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 μg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed

Effect of a multiple-micronutrient-fortified fruit powder beverage on the nutrition status, physical fitness, and cognitive performance of schoolchildren in the Philippines

This study aimed to determine the effect of a multiple-micronutrient-fortified beverage on the micronutrient status, physical fitness, and cognitive performance of schoolchildren. The study was a randomized, double-blind, placebo-controlled trial of schoolchildren assigned to receive either the fortified or nonfortified beverage with or without anthelmintic therapy. Data on hemoglobin level, urinary iodine excretion (UIE) level, physical fitness, and cognitive performance were collected at baseline and at 16 weeks post-intervention. The fortified beverage significantly improved iron status among the subjects that had hemoglobin levels /dl at baseline. The proportion of children who remained moderately to severely anemic was significantly lower among those given the fortified beverage. In the groups that received the fortified product, the median UIE level increased, whereas among those who received the placebo beverage, the median UIE level was reduced significantly. Iron- and/or iodine-deficient subjects who received the fortified beverage showed significant improvements in fitness (post-exercise reduction of heart rate) and cognitive performance (nonverbal mental ability score). The study showed that consumption of a multiple-micronutrient-fortified beverage for 16 weeks had significant effects on iron status, iodine status, physical fitness, and cognitive performance among iron- and/or iodine-deficient Filipino schoolchildren. Anthelmintic therapy improved iron status of anemic children and iodine status of the iron-adequate children at baseline but it had no effect on physical fitness and cognitive performance. The results from the clinical study showed that a multiple-micronutrient-fortified beverage could play an important role in preventing and controlling micronutrient deficiencies

Gene expression profiles during in vivo human rhinovirus infection: insights into the host response. Am J Respir Crit Care Med

Rationale: Human rhinovirus infections cause colds and trigger exacerbations of lower airway diseases. Objectives: To define changes in gene expression profiles during in vivo rhinovirus infections. Methods: Nasal epithelial scrapings were obtained before and during experimental rhinovirus infection, and gene expression was evaluated by microarray. Naturally acquired rhinovirus infections, cultured human epithelial cells, and short interfering RNA knockdown were used to further evaluate the role of viperin in rhinovirus infections. Measurements and Main Results: Symptom scores and viral titers were measured in subjects inoculated with rhinovirus or sham control, and changes in gene expression were assessed 8 and 48 hours after inoculation. Real-time reverse transcription-polymerase chain reaction for viperin and rhinoviruses was used in naturally acquired infections, and viperin mRNA levels and viral titers were measure

Zinc Pyrithione Inhibits Yeast Growth through Copper Influx and Inactivation of Iron-Sulfur Proteins▿†

Zinc pyrithione (ZPT) is an antimicrobial material with widespread use in antidandruff shampoos and antifouling paints. Despite decades of commercial use, there is little understanding of its antimicrobial mechanism of action. We used a combination of genome-wide approaches (yeast deletion mutants and microarrays) and traditional methods (gene constructs and atomic emission) to characterize the activity of ZPT against a model yeast, Saccharomyces cerevisiae. ZPT acts through an increase in cellular copper levels that leads to loss of activity of iron-sulfur cluster-containing proteins. ZPT was also found to mediate growth inhibition through an increase in copper in the scalp fungus Malassezia globosa. A model is presented in which pyrithione acts as a copper ionophore, enabling copper to enter cells and distribute across intracellular membranes. This is the first report of a metal-ligand complex that inhibits fungal growth by increasing the cellular level of a different metal