Consumer perception of astringency in clear acidic whey protein beverages.

Acidic whey protein beverages are a growing component of the functional food and beverage market. These beverages are also astringent, but astringency is an expected and desirable attribute of many beverages (red wine, tea, coffee) and may not necessarily be a negative attribute of acidic whey protein beverages. The goal of this study was to define the consumer perception of astringency in clear acidic whey protein beverages. Six focus groups (n=49) were held to gain understanding of consumer knowledge of astringency. Consumers were presented with beverages and asked to map them based on astringent mouthfeel and liking. Orthonasal thresholds for whey protein isolate (WPI) in water and flavored model beverages were determined using a 7-series ascending forced choice method. Mouthfeel/basic taste thresholds were determined for WPI in water. Acceptance tests on model beverages were conducted using consumers (n=120) with and without wearing nose clips. Consumers in focus groups were able to identify astringency in beverages. Astringency intensity was not directly related to dislike. The orthonasal threshold for WPI in water was lower (P < 0.05) than the mouthfeel/basic taste threshold of WPI in water. Consumer acceptance of beverages containing WPI was lower (P < 0.05) when consumers were not wearing nose clips compared to acceptance scores of beverages when consumers were wearing nose clips. These results suggest that flavors contributed by WPI in acidic beverages are more objectionable than the astringent mouthfeel and that both flavor and astringency should be the focus of ongoing studies to improve the palatability of these products.

Sensory properties and consumer perception of wet and dry cheese sauces.

Flavor and texture lexicons and consumer perception for 2 cheese sauce categories, wet and dry, were determined and compared. Commercial and prototype, as well as homemade, wet (n = 24) and dry cheese sauces (n = 14) were evaluated by a trained descriptive panel (n = 9). Consumer acceptance testing was conducted on representative wet sauces (10) and dry sauces (8) on different days (n = 122 consumers each day). Cheese sauces were served over pasta for consumer testing. Univariate and multivariate statistical analyses were used to evaluate the collected data. Flavor and cheese flavor liking were highly correlated with overall liking for both wet and dry sauces. Salty taste was a key driver of liking for both cheese sauce categories. Flavor attributes of wet sauces that contributed most to higher acceptance were beefy/brothy, sweet/caramelized, and free fatty acid. Liking of dry sauces was driven by Alfredo sauce specific flavors such as onion/garlic and herbal for 2 of the consumer clusters, but beefy/brothy and free fatty acid were drivers for the traditional macaroni and cheese consumers. The impact of color/appearance and texture attributes had only a minor influence on consumer liking. By knowing what drives liking in wet and dry cheese sauces, researchers and product developers can more easily develop cheese sauces that appeal to all categories of consumers.

Hoechst 33258 selectively inhibits group I intron self-splicing by affecting RNA folding.

Fungal pathogens are increasing in prevalence due to an increase in resistant strains and the number of immunocompromised humans. Candida albicans is one of these pathogens, and approximately 40% of strains contain a group I self-splicing intron, which is a potential RNA drug target, in their large subunit rRNA precursor. Here, we report that Hoechst 33258 and derivatives thereof are selective inhibitors of C. albicans group I intron self-splicing with an IC50 of 17 microM in 2 mM Mg2+. Chemical probing of the intron in the presence of Hoechst 33258 reveals that the folding of several nucleotides in the P4/P6 region of the intron is affected. A nucleotide near the J4/5 region is protected from chemical modification in the presence of Hoechst 33258 and several nearby are more reactive; this suggests that this region is the molecule's binding site. These results expand the available information on small-molecule targeting of RNA and suggest that the RNA-targeting scaffold provided by Hoechst may prove valuable in designing compounds that inhibit the functions of RNA.

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

Secondary structure models of the 3' untranslated regions of diverse R2 RNAs.

The RNA structure of the 3' untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure-function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3' UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3' UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with beta-ethoxy-alpha-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function.

New approaches to targeting RNA with oligonucleotides: inhibition of group I intron self-splicing.

RNA is one class of relatively unexplored drug targets. Since RNAs play a myriad of essential roles, it is likely that new drugs can be developed that target RNA. There are several factors that make targeting RNA particularly attractive. First, the amount of information about the roles of RNA in essential biological processes is currently being expanded. Second, sequence information about targetable RNA is pouring out of genome sequencing efforts at unprecedented levels. Third, designing and screening potential oligonucleotide therapeutics to target RNA is relatively simple. The use of oligonucleotides in cell culture, however, presents several challenges such as oligonucleotide uptake and stability, and selective targeting of genes of interest. Here, we review investigations aimed at targeting RNA with oligonucleotides that can circumvent several of these potential problems. The hallmark of the strategies discussed is the use of short oligonucleotides, which may have the advantage of higher cellular uptake and improved binding selectivity compared to longer oligonucleotides. These strategies have been applied to Group I introns from the mammalian pathogens Pneumocystis carinii and Candida albicans. Both are examples of fungal infections that are increasing in number and prevalence.

Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA.

Oligonucleotide directed misfolding of RNA (ODMiR) uses short oligonucleotides to inhibit RNA function by exploiting the ability of RNA to fold into different structures with similar free energies. It is shown that the 2'-O-methyl oligonucleotide, m(CAGCCUACCCGG), can trap Escherichia coli RNase P RNA (M1 RNA) in a nonfunctional structure in a transcription mixture containing RNase P protein (C5 protein). At about 200 nM, the 12-mer thus inhibits 50% of pre-tRNA processing by RNase P. Roughly 10-fold more 12-mer is required to inhibit RNase P containing full-length, renatured RNase P RNA. Diethyl pyrocarbonate modification in the presence of 12-mer reveals increased modification of sites in and interacting with P4, suggesting a structural rearrangement of a large pseudoknot important for catalytic activity. Thus, the ODMiR method can be applied to RNAs even when folding is facilitated by a cognate protein.

Oligonucleotide directed misfolding of RNA inhibits Candida albicans group I intron splicing.

RNA is becoming an important therapeutic target. Many potential RNA targets require secondary or tertiary structure for function. Examples include ribosomal RNAs, RNase P RNAs, mRNAs with untranslated regions that regulate translation, and group I and group II introns. Here, a method is described to inhibit RNA function by exploiting the propensity of RNA to adopt multiple folded states that are of similar free energy. This method, called oligonucleotide directed misfolding of RNA (ODMiR), uses short oligonucleotides to stabilize inactive structures. The ODMiR method is demonstrated with the group I intron from Candida albicans, a human pathogen. The oligonucleotides, (L)(TACCTTTC) and T(L)CT(L)AC(L)GA(L)CG(L)GC(L)C, with L denoting a locked nucleic acid residue, inhibit 50% of group I intron splicing in a transcription mixture at about 150 and 30 nM oligonucleotide concentration, respectively. Both oligonucleotides induce misfolds as determined by native gel electrophoresis and diethyl pyrocarbonate modification. The ODMiR approach provides a potential therapeutic strategy applicable to RNAs with secondary or tertiary structures required for function.