The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment.

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water.

Rapid method demonstration project at four New Jersey marine beaches using real time quantitative Polymerase Chain Reaction (qPCR).

Real time quantitative Polymerase Chain Reaction (qPCR) was used at four marine bathing beaches in New Jersey as part of a demonstration project to evaluate the potential for use of qPCR as part of a routine beach monitoring program. Split sample analyses for Enterococcus spp. using membrane filtration (MF) and qPCR were performed for 11weeks during the summer of 2011 using swimming advisories based on qPCR results.

Comparison of Enterococcus density estimates in marine beach and bay samples by real-time polymerase chain reaction, membrane filtration and defined substrate testing.

Currently, densities of Enterococcus in marine bathing beach samples are performed using conventional methods which require 24 h to obtain results. Real-time PCR methods are available which can measure results in as little as 3 h. The purpose of this study was to evaluate a more rapid test method for the determination of bacterial contamination in marine bathing beaches to better protect human health.

Toxicity of ammonia to surf clam (Spisula solidissima) larvae in saltwater and sediment elutriates.

Ammonia is a natural component of sediments and has been identified as a common contributor to toxicity in marine sediment, elutriate and porewater testing. In our study, the role of ammonia as a possible toxicant in sediment toxicity tests was evaluated using larvae of the surf clam, Spisula solidissima. Elutriates were prepared and tested using six baseline sediment samples.

Intramolecular interaction in the tail of Acanthamoeba myosin IC between the SH3 domain and a putative pleckstrin homology domain.

The 466-aa tail of the heavy chain of Acanthamoeba myosin IC (AMIC) comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue Src homology 3 (SH3) domain, and a C-terminal 135-residue Gly/Pro/Ala-rich region (GPA2). Cryo-electron microscopy of AMIC had shown previously that the AMIC tail is folded back on itself, suggesting the possibility of interactions between its N- and C-terminal regions. We now show specific differences between the NMR spectrum of bacterially expressed full-length tail and the sum of the spectra of individually expressed BR and GPA1-SH3-GPA2 (GSG) regions.

Physical and functional interactions between the prostate suppressor homeoprotein NKX3.1 and serum response factor.

The NKX3.1 transcription factor is an NK family homeodomain protein and a tumor suppressor gene that is haploinsufficient and down-regulated in the early phases of prostate cancer. Like its cardiac homolog, NKX2.

Cardiac-specific Nkx2.5 homeodomain: conformational stability and specific DNA binding of Nkx2.5(C56S).

The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.

Interlaboratory comparison of a reduced volume marine sediment toxicity test method using the amphipod Ampelisca abdita.

The U.S. Environmental Protection Agency has standardized methods for performing acute marine amphipod sediment toxicity tests.

Experimentally biased model structure of the Hsc70/auxilin complex: substrate transfer and interdomain structural change.

A model structure of the Hsc70/auxilin complex has been constructed to gain insight into interprotein substrate transfer and ATP hydrolysis induced conformational changes in the multidomain Hsc70 structure. The Hsc70/auxilin system, which is a member of the Hsp70/Hsp40 chaperone system family, uncoats clathrin-coated vesicles in an ATP hydrolysis-driven process. Incorporating previous results from NMR and mutant binding studies, the auxilin J-domain was docked into the Hsc70 ATPase domain lower cleft using rigid backbone/flexible side chain molecular dynamics, and the Hsc70 substrate binding domain was docked by a similar procedure.

Structure of the functional fragment of auxilin required for catalytic uncoating of clathrin-coated vesicles.

The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically.

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation.

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue.

Sequence of a conserved region of a new sea urchin homeobox gene from the NK family.

The partial sequence of a novel homeobox-containing gene from Paracentrotus lividus is described. Both cDNA and genomic DNA were screened using probes from the vnd/NK-2 homeobox gene found in Drosophila melanogaster. The new DNA sequence found in P.

Mutations that affect the ability of the vnd/NK-2 homeoprotein to regulate gene expression: transgenic alterations and tertiary structure.

The importance in downstream target regulation of tertiary structure and DNA binding specificity of the protein encoded by the vndNK-2 homeobox gene is analyzed. The ectopic expression patterns of WT and four mutant vndNK-2 genes are analyzed together with expression of two downstream target genes, ind and msh, which are down-regulated by vndNK-2. Three mutants are deletions of conserved regions (i.

Evaluation of reduced sediment volume toxicity test procedures using the marine amphipod Ampelisca abdita.

During the past 20 years, the role of sediment toxicity tests has expanded from testing of primarily dredged material to risk assessment, decontamination technologies, large-scale regional sediment-quality assessments, and toxicity identification evaluations. Sediment toxicity tests are needed that can provide reliable data using less sediment and smaller test chambers, thus utilizing resources more efficiently. We compared survival results from 10-d standard marine amphipod tests at 20 degrees C using 200 ml of sediment to reduced-volume tests containing 20 or 50 ml of sediment and found no significant differences.