Thirty-eight fruit salad samples including cantaloupe, citrus fruits, honeydew, pineapple, cut strawberries and mixed fruit salads, and 65 pasteurized fruit juice samples (apple, carrot, grapefruit, grape and orange juices, apple cider, and soy milk) were purchased from local supermarkets in the Washington, DC area and tested for fungal contamination. The majority of fruit salad samples (97%) were contaminated with yeasts at levels ranging from o2.0 to 9.72 log 10 of colony forming units per gram (cfu/g). Frequently encountered yeasts were Pichia spp., Candida pulcherrima, C. lambica, C. sake, Rhodotorula spp., and Debaryomyces polymorphus. Low numbers of Penicillium spp. were found in pineapple salads, whereas Cladosporium spp. were present in mixed fruit and cut strawberry salads. Twenty-two per cent of the fruit juice samples tested showed fungal contamination. Yeasts were the predominant contaminants ranging from o1.0 to 6.83 log 10 cfu/ml. Yeasts commonly found in fruit juices were C. lambica, C. sake, and Rhodotorula rubra. Geotrichum spp. and low numbers of Penicillium and Fusarium spp. (1.70 and 1.60 log 10 cfu/ml, respectively) were present in grapefruit juice. r

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Protein-DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein-DNA interactions. Here we show that photonic crystal (PC) technology can be utilized to detect protein-DNA interactions, and can be used in a high-throughput screening mode to identify compounds that prevent protein-DNA binding. The PC technology is used to detect binding between protein-DNA interactions that are DNA-sequencedependent (the bacterial toxin-antitoxin system MazEF) and those that are DNA-sequenceindependent (the human apoptosis inducing factor (AIF)). The PC technology was further utilized in a screen for inhibitors of the AIF-DNA interaction, and through this screen aurin tricarboxylic acid was identified as the first in vitro inhibitor of AIF. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein-DNA binding.High-throughput screening (HTS) of compound collections is now a staple of modern drug discovery. In the most common incarnation, in vitro enzyme inhibition screens of large (>100,000 members) compound libraries are conducted using substrates that provide an easily quantified chromogenic/ fluorescent readout. Such screens have led to the discovery of many novel enzyme inhibitors and drug leads (1,2). Unfortunately, many potential drug targets are not enzymes, and thus for these systems high-throughput methods are needed that go beyond enzyme inhibition assays and directly report on small molecule-protein binding events.One area in which small molecule ligands for nonenzyme proteins would be useful is in the disruption of protein-macromolecule interactions. The identification of compounds that perturb protein-protein or protein-nucleic acid interactions is extremely challenging (3-6), and this is partly due to the paucity of good high-throughput screens. Successes in modulating protein-protein and protein-nucleic acid interactions with small molecules fall into a few classes: surface receptor-ligand interactions (integrins (7-9), IL-1/2 (10,11), TNFα (12)), (47). Another limitation is the potential for false positives due to fluorescent compounds, which is an inherent limitation of any fluorescence-based HTS method. Given the largely unexplored pharmacological realm that is protein-nucleic acid interactions, HTS assays independent of fluorescent tags would be extremely useful, especially in those cases where fluorescence anisotropy is not possible. In this report we describe the first use of photonic crystal technology for the development of an assay capable of detecting protein-DNA binding and further apply it in a high-throughput screening mode fo...

Edited by F. Anne StephensonPrion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrP C ) into the infectious isoform (PrP Sc ). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (⌬105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrP Sc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrP Sc -induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrP C , and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrP Sc -seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RTQuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrP Sc metabolism and PrP C function.Prion diseases are fatal neurodegenerative disorders that are due to the conversion of a normal, neuronal glycoprotein (PrP C ) 3 into an infectious isoform (PrP Sc ) that propagates itself by an autocatalytic templating process (1, 2). In addition to their intrinsic interest to biologists, prion diseases are of enormous medical and public health concern. A global epidemic of bovine spongiform encephalopathy, a prion disease of cattle, emerged in the 1980s and 1990s, resulting in contamination of food supplies and transmission of the disease to a small number of human beings (3, 4). Prion contamination has also increased the risk of blood transfusions, and organ transplants (5). Most recently, a prion-like process has been found to play a role in the CNS dissemination of misfolded proteins in more common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and tauopathies, and there is even evidence that these diseases can be spread between individuals by iatrogenic means (6, 7). There are currently no cures for prion diseases. A great deal of effort has been invested over the past 25 years in identifying compounds that block the conversion of PrP C into PrP Sc as a therapeutic strategy. Most of these efforts have used as a readout a single kind of cellular assay system: N2a neuroblastoma cells that are chronically infected with scrapie prions (designated ScN2a cells). N2a cells are one of the few cell lines capable...

High-throughput screening (HTS) has played an integral role in the development of small molecule modulators of biological processes. These screens are typically developed for enzymes (such as kinases or proteases) or extracellular receptors, two classes of targets with well-established colorimetric or fluorimetric activity assays. In contrast, methods for detection of protein-protein interactions lack the simplicity inherent to enzyme and receptor assays. Technologies that facilitate the discovery of small molecule modulators of protein-protein interactions are essential to the exploitation of this important class of drug targets. As described in this critical review, photonic crystal (PC) biosensors and other emerging technologies can now be utilized in high-throughput screens for the identification of compounds that disrupt or enhance protein-protein interactions (167 references).