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.

Gingival transcriptome patterns during induction and resolution of experimental gingivitis in humans.

BACKGROUND: To our knowledge, changes in the patterns of whole-transcriptome gene expression that occur during the induction and resolution of experimental gingivitis in humans were not previously explored using bioinformatic tools. METHODS: Gingival biopsy samples collected from 14 subjects during a 28-day stent-induced experimental gingivitis model, followed by treatment, and resolution at days 28 through 35 were analyzed using gene-expression arrays. Biopsy samples were collected at different sites within each subject at baseline (day 0), at the peak of gingivitis (day 28), and at resolution (day 35) and processed using whole-transcriptome gene-expression arrays. Gene-expression data were analyzed to identify biologic themes and pathways associated with changes in gene-expression profiles that occur during the induction and resolution of experimental gingivitis using bioinformatic tools. RESULTS: During disease induction and resolution, the dominant expression pathway was the immune response, with 131 immune response genes significantly up- or downregulated during induction, during resolution, or during both at P <0.05. During induction, there was significant transient increase in the expression of inflammatory and oxidative stress mediators, including interleukin (IL)-1 alpha (IL1A), IL-1 beta (IL1B), IL8, RANTES, colony stimulating factor 3 (CSF3), and superoxide dismutase 2 (SOD2), and a decreased expression of IP10, interferon inducible T-cell alpha chemoattractant (ITAC), matrix metalloproteinase 10 (MMP10), and beta 4 defensin (DEFB4). These genes reversed expression patterns upon resolution in parallel with the reversal of gingival inflammation. CONCLUSIONS: A relatively small subset (11.9%) of the immune response genes analyzed by array was transiently activated in response to biofilm overgrowth, suggesting a degree of specificity in the transcriptome-expression response. The fact that this same subset demonstrates a reversal in expression patterns during clinical resolution implicates these genes as being critical for maintaining tissue homeostasis at the biofilm-gingival interface. In addition to the immune response pathway as the dominant response theme, new candidate genes and pathways were identified as being selectively modulated in experimental gingivitis, including neural processes, epithelial defenses, angiogenesis, and wound healing.

Gene expression profiles during in vivo human rhinovirus infection: insights into the host response.

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 measured in cultured cells. Rhinovirus-induced changes in gene expression were not observed 8 hours after viral infection, but 11,887 gene transcripts were significantly altered in scrapings obtained 2 days postinoculation. Major groups of up-regulated genes included chemokines, signaling molecules, interferon-responsive genes, and antivirals. Viperin expression was further examined and also was increased in naturally acquired rhinovirus infections, as well as in cultured human epithelial cells infected with intact, but not replication-deficient, rhinovirus. Knockdown of viperin with short interfering RNA increased rhinovirus replication in infected epithelial cells. CONCLUSIONS: Rhinovirus infection significantly alters the expression of many genes associated with the immune response, including chemokines and antivirals. The data obtained provide insights into the host response to rhinovirus infection and identify potential novel targets for further evaluation.

Genomic expression changes induced by topical N-acetyl glucosamine in skin equivalent cultures in vitro.

N-acetyl glucosamine (NAG) has been shown to be effective in reducing the appearance of hyperpigmented spots. From published in vitro mechanistic testing, glucosamine inhibits enzymatic glycosylation, a required processing step in converting inactive human pro-tyrosinase to the active tyrosinase, a key enzyme in the production of melanin. There is also published literature discussing the anti-inflammatory and antioxidant properties of glucosamine compounds. To identify additional mechanisms by which NAG might affect melanin production, an in vitro genomics experiment was conducted in SkinEthic skin equivalent cultures, which were topically dosed with NAG vs. a vehicle control. Relative to vehicle, NAG reduced melanin production, and the expression of several pigmentation-relevant genes were affected (down-regulated or up-regulated) by NAG treatment. Thus, there are several mechanisms that may be operative in the observed pigmentation effects.

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 microg 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.

Data requirements for protein identification using chemically-assisted fragmentation and tandem mass spectrometry.

Many laboratories identify proteins by searching tandem mass spectrometry data against genomic or protein sequence databases. These database searches typically use the measured peptide masses or the derived peptide sequence and, in this paper, we focus on the latter. We study the minimum peptide sequence data requirements for definitive protein identification from protein sequence databases. Accurate mass measurements are not needed for definitive protein identification, even when a limited amount of sequence data is available for searching. This information has implications for the mass spectrometry performance (and cost), data base search strategies and proteomics research.

Molecular factor analysis applied to collections of NMR spectra.

It is often useful to identify and quantify mixture components by analyzing collections of NMR spectra. Such collections arise in metabonomics and many other applications. Many mixtures studied by NMR can contain hundreds of compounds, and it is challenging to analyze the resulting complex spectra. We have approached the problem of separating signals from different molecules in complex mixtures by using self-modeling curve resolution as implemented by the alternating least-squares algorithm. Alternating least squares uses nonnegativity criteria to generate spectra and concentrations from a collection of mixture spectra. Compared to previous applications of alternating least squares, NMR spectra of complex mixtures possess unique features, such as large numbers of components and sample-to-sample variability in peak positions. To deal with these features, we developed a set of data preprocessing methods, and we made modifications to the alternating least-squares algorithm. We use the term "molecular factor analysis" to refer to the preprocessing and modified alternating least-squares methods. Molecular factor analysis was tested using an artificial data set and spectra from a metabonomics study. The results show that the tools can extract valuable information on sample composition from sets of NMR spectra.

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 < 11 g/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.

Hydration vs. skin permeability to nicotinates in man.

BACKGROUND/AIMS: Prolonged skin occlusion increases stratum corneum water content and often increases skin permeability and irritant dermatitis. As skin wetness from wearing diapers is considered an important factor favouring the onset of diaper dermatitis, optimal diapering might decrease skin hyperhydration and dermatitis. Our aim is to define the quantitative relationship between nicotinate ester (a model penetrant) skin permeability and hydration, as measured by water evaporation rate (WER), decay curves (at individual time points) and WER-area under the curve (WER-AUC); and also to determine the level of skin hydration and skin permeability to nicotinates following a diapering simulation. METHODS/RESULTS: Nine healthy Caucasian adult women were enrolled after a prescreening procedure (time to peak redness response to nicotinate); each received three wet occlusive patches for different exposure times (10 min, 30 min, and 3h) and two wet model diapers (3 and 8 h). Prior to patching or diapering of forearms, basal values of WER, skin blood flow volume (BFV), capacitance (Cap) and redness (a*) were measured on premarked sites (a, b, c and d). Immediately, following occlusive patch or diaper removal, 20 microL of each nicotinate (methyl and hexyl nicotinate) was applied to its respective site (a or b). The WER and Cap readings were recorded at designated sites (c and d) with the following intervals after nicotinate applications: 0, 5,10,15 and 20 min. The a* and BFV measurements were made on each nicotinate challenged site (a and b) with the following intervals after nicotinate applications: 5, 10, 15, 20, 30, 40, and 60 min. RESULTS: WER-AUC and thus, skin hyperhydration, increased with occlusive patch and diaper exposure time, but there was no statistical difference between 3 and 8h diaper sites. All patched sites had significantly (P<0.05) increased hydration in comparison to control sites (undiapered or unpatched skin). Cap increased with occlusion time with patches, but not with diapers. The degree and time-course of redness from nicotinates did not vary with extent of skin hydration, but was significantly increased compared to non-hydrated skin. BFV-AUC did not show a significant increase between diapers at 3 and 8h sites; the BFV-AUC values varied on the patched sites, but some were significantly (P<0.05) higher than control site. CONCLUSION: Wet patches and diapers increased skin hyperhydration proportional to exposure time. Permeation of nicotinates was increased for hydrated skin vs. control, even after only 10 min of patch exposure. For these model permeants, we found no evidence of increased permeation rates with increased hyperhydration, once a relatively low threshold of hyperhydration was achieved (e.g. that reached after a 10 min wet patch). The data showed no meaningful differences in permeation following either diapering simulation and also suggested that the WER-AUC method was superior to capacitance for measuring the absolute extent of hyperhydration. We believe this is a suitable model for evaluating the quality of diaper product performance, as well as in pharmacologic assays of occlusive therapy.