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Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment. [ABSTRACT FROM AUTHOR]

Research has been conducted on the Stachybotrys chartarum strain. The random polymorphic DNA analysis of the Houston strain for hemolytic activity, siderophore production and relatedness has been performed.

Sixteen strains of the indoor air, toxigenic fungus Stachybotrys chartarum isolated from case or control homes in Cleveland, OH, and 12 non-Cleveland strains from diverse geographic locations were examined for hemolytic activity, conidial toxicity and randomly amplified polymorphic DNA banding patterns. Only three of the 28 strains studied were both consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland, OH, where an infant had idiopathic pulmonary hemorrhage.

Two novel approaches for the synthesis of fluorinated fluoresceins were developed through the use of fluorinated nitrobenzenes. The nitro group directed the fluoride displacement by methoxy groups at positions ortho and para establishing the resorcinol as the framework. Hydrodediazoniation and catalytic hydrogenation removed the directing nitro group, while demethylation yielded the desired fluorinated resorcinols.

The binding of a fluorescent diltiazem analogue (3R,4S)-cis-1- 2- 3- 3- 4,4-difluoro-3a,4-dihydro-5,7-dimethyl-4-bora-3a,4 a-diaza-s-indacen-3-yl propionyl amino ethyl -1,3,4,5-tetrahydro-3-hydroxy-4 -(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepin-2-one to L-type Ca2+ channels in the presence of 1,4-dihydropyridines has been investigated. The results showed that the binding domains of these drugs are localized in close proximity to each other on the extracellular side of the channel.

Newly developed N-propylamino-substituted benzazepinones (NPSBs) enable the study of the benzothiazepinone (BTZ) binding domain of muscle L-type calcium channels (LTCCs). Drug binding causes the occurrence of fluorescence resonance energy transfer between tryptophan residues of the channel protein and the NPSB derivatives. The kinetic properties of these derivatives confirm the existence of a high-affinity BTZ binding domain on purified LTCCs.

Examination of the binding-stimulated fluorescence properties of a fluorescent 1,4-dihydropyridine (DHP) and its analogue DMBODIPY-DHP helps investigate the molecular mechanism of the interaction of DHPs with alpha1-subunit of skeletal muscle L-type Ca2+-channels. Fluorescence yield of DMBODIPY-DHP decreases to specific binding. Binding permits the fluorescence resonance energy transfer (FRET) from between ligand and tryptophan residues to the alpha1-subunit. FRET signal helps direct measurements of DHP binding of high resolution. Application of this technique reveals a step-wise mechanism associated with reversible DHP binding to L-type Ca2+ channels.