Integrated Cryptosporidium Assay To Determine Oocyst Density,Infectivity, and Genotype for Risk Assessment of Source and Reuse Water

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures.     

Evaluation and optimization of antifibrotic activity of cinnamoyl anthranilates

Tranilast is an anti-inflammatory drug in use for asthma and atopic dermatitis. In studies over the last decade it has been revealed that tranilast can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. We report a structure–activity study aimed at optimizing the antifibrotic activity of tranilast. A series of cinnamoyl anthranilates were prepared and assessed for their ability to prevent TGF-β-stimulated production of collagen in cultured renal mesangial cells. We reveal derivatives with improved potency and reduced cellular toxicity relative to tranilast. 3-Methoxy-4-propargyloxycinnamoyl anthranilate reduces albuminuria in a rat model of progressive diabetes, and thus has potential as an innovative treatment for diabetic nephropathy.     

Toward resolving deep neoaves phylogeny: data, signal enhancement, and priors

We report three developments toward resolving the challenge of the apparent basal polytomy of neoavian birds. First, we describe improved conditional down-weighting techniques to reduce noise relative to signal for deeper divergences and find increased agreement between data sets. Second, we present formulae for calculating the probabilities of finding predefined groupings in the optimal tree. Finally, we report a significant increase in data: nine new mitochondrial (mt) genomes (the dollarbird, New Zealand kingfisher, great potoo, Australian owlet-nightjar, white-tailed trogon, barn owl, a roadrunner [a ground cuckoo], New Zealand long-tailed cuckoo, and the peach-faced lovebird) and together they provide data for each of the six main groups of Neoaves proposed by Cracraft J (2001). We use his six main groups of modern birds as priors for evaluation of results. These include passerines, cuckoos, parrots, and three other groups termed "WoodKing" (woodpeckers/rollers/kingfishers), "SCA" (owls/potoos/owlet-nightjars/hummingbirds/swifts), and "Conglomerati." In general, the support is highly significant with just two exceptions, the owls move from the "SCA" group to the raptors, particularly accipitrids (buzzards/eagles) and the osprey, and the shorebirds may be an independent group from the rest of the "Conglomerati". Molecular dating mt genomes support a major diversification of at least 12 neoavian lineages in the Late Cretaceous. Our results form a basis for further testing with both nuclear-coding sequences and rare genomic changes.     

The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro

Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High-speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non-rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.     

Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein

The use of a temperature switch to control the growth and productivity of temperature-sensitive (ts) mutants was investigated to extend the productive life span of recombinant Chinese hamster ovary (CHO) cells in batch culture. Bromodeoxyuridine was used at 39 degrees C to select mutagenized CHO-K1 cells, which resulted in the isolation of 31 temperature-sensitive mutants that were growth inhibited at 39 degrees C. Two of these mutants were successfully transfected with the gene for tissue inhibitor of metalloproteinases (TIMP) using glutamine synthetase amplification, and a permanent recombinant cell line established (5G1-B1) that maintains the ts phenotype.Continuous exposure to the nonpermissive temperature (npt) of 39 degrees C led to a rapid decline in cell viability. However, a temperature regime using alternating incubations at 34 degrees C and 39 degrees C arrested the 5G1-B1 cells while retaining a high cell viability for up to 170 h in culture. The specific production rate of the growth-arrested cells was 3-4 times that of control cultures maintained at a constant 34 degrees C over the crucial 72-130-h period of culture, which resulted in a 35% increase in the maximum product yield. Glucose uptake and lactate production both decreased in arrested cells. Flow cytometric analysis indicated that 5G1-B1 cells arrested in the G(1) or G(0) phase of the cell cycle, and no major structural damage was caused to these cells by the alternating temperature regime.These results demonstrate that growth-arrested ts CHO cells have increased productivity compared to growing cultures and maintain viability for longer periods. The system offers the prospect of enhancing the productivity of recombinant mammalian cells grown in simple batch fermentors. (c) 1993 John Wiley & Sons, Inc.     

Mitochondrial DNA and allozyme analysis of Atlantic salmon, Salmo salar L., in England and Wales

Allozyme variation was examined in 429 Atlantic salmon, from seven sampling sites in England and Wales. Contingency tests for genetic homogeneity using three diagnostic loci revealed no significant differences among populations from the north-east and north-west of the U.K., but detected significant aspartate aminotransferase (AAT) differences between these samples and those from the R. Itchen in Hampshire (southern England). Mitochondrial DNA variation was analysed in 40 salmon from five spawning sites in the R. Itchen. Eight restriction endonucleases were used, of which three (AVAII, HAE III and HINFI) revealed polymorphisms. Six matriarchal lineages were discriminated. Two of the six mtDNA types observed were site-specific. These results suggest a reasonable expectation of discriminating spawning aggregations of Atlantic salmon.     

Family-level relationships among the Australasian marsupial "herbivores" (Diprotodontia: Koala, wombats, kangaroos and possums)

The marsupial order Diprotodontia includes 10 extant families, which occupy all terrestrial habitats across Australia and New Guinea and have evolved remarkable dietary and locomotory diversity. Despite considerable attention, the interrelations of these families have for the most part remained elusive. In this study, we separately model mitochondrial RNA and protein-coding sequences in addition to nuclear protein-coding sequences to provide near-complete resolution of diprotodontian family-level phylogeny. We show that alternative topologies inferred in some previous studies are likely to be artifactual, resulting from branch-length and compositional biases. Subordinal groupings resolved herein include Vombatiformes (wombats and koala) and Phalangerida, which in turn comprises Petauroidea (petaurid gliders and striped, feathertail, ringtail and honey possums) and a clade whose plesiomorphic members possess blade-like premolars (phalangerid possums, kangaroos and their allies and most likely, pygmy possums). The topology resolved reveals ecological niche structuring among diprotodontians that has likely been maintained for more than 40 million years.