LouAnn Bierwert

Development and standardization of a rapid, PCR-based method for the detection of Wuchereria bancrofti in mosquitoes, for xenomonitoring the human prevalence of bancroftian filariasis.

PCR has recently been studied as a promising tool for monitoring the progress of efforts to eliminate lymphatic filariasis. PCR can be used to test concurrently at least 30 pools, with as many as 40 mosquitoes in each pool, for the presence of filarial larvae. The SspI PCR assay for the detection of Wuchereria bancrofti DNA in pools of mosquitoes has been used since 1994 in a variety of laboratories worldwide. During that time, the original assay has been modified in these different laboratories and no standardized assay currently exists. In an effort to standardize and improve the assay, a meeting was held on 15-16 November 2001, at Emory University in Atlanta, with representatives from most of the laboratories currently using the assay. The first round of testing was designed to test the four most promising methods for DNA extraction from pools of mosquitoes. Two of the four methods stood out as clearly the best and these will be now optimised and evaluated in two further rounds of testing.

Helminth genome analysis: The current status of the filarial and schistosome genome projects

Genome projects for the parasitic helminths Brugia malayi (a representative filarial nematode) and Schistosoma were initiated in 1995 by the World Health Organization with the ultimate objectives of identifying new vaccine candidates and drug targets and of developing low resolution genome maps. Because no genetic maps are available, and very few genes have been characterized from either parasite group, the first goal of both Initiatives has been to catalogue new genes for future placement on chromosome and physical maps. These genes have been identified by the expressed sequence tag (EST) approach, utilising cDNA libraries constructed from diverse life cycle stages. To date, the Initiatives have deposited over 16,000 Brugia ESTs and nearly 8000 Schistosoma ESTs in Genbanks dbEST database, corresponding to 6000 and over 3600 genes respectively (33% of Brugias estimated gene compliment, 18-24% of that of Schistosoma). Large fragment, genomic libraries have been constructed in BAC and YAC vectors for studies of genomic organization and for physical and chromosome mapping, and public, hypertext genomic databases have been established to facilitate data access. We present a summary of progress within the helminth genome initiatives and give several examples of important gene discoveries and future applications of these data.

Sexual dimorphism in the effect of GDF-6 deficiency on murine tendon.

Three members of the growth/differentiation factor (GDF) subfamily of bone morphogenetic proteins (BMPs), GDFs‐5, ‐6, and ‐7, have demonstrated the potential to augment tendon and ligament repair. To gain further insight into the in vivo role of these molecules, previous studies have characterized intact and healing tendons in mice with functional null mutations in GDF‐5 and ‐7. The primary goal of the present study was to perform a detailed characterization of the intact tendon phenotype in 4‐ and 16‐week‐old male and female GDF6−/− mice and their +/+ littermates. The results demonstrate that GDF6 deficiency was associated with an altered tendon phenotype that persisted into adulthood. Among males, GDF6−/− tail tendon fascicles had significantly less collagen and glycosaminoglycan content, and these compositional differences were associated with compromised material properties. The effect of GDF6 deficiency on tendon was sexually dimorphic, however, for among female GDF6−/− mice, neither differences in tendon composition nor in material properties were detected. The tendon phenotype that was observed in males appeared to be stronger in the tail site than in the Achilles tendon site, where some compositional differences were present, but no material property differences were detected. These data support existing in vitro studies, which suggest a potential role for BMP‐13 (the human homologue to GDF‐6) in tendon matrix modeling and/or remodeling.

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.

Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

Effect of GDF-7 Deficiency on Tail Tendon Phenotype in Mice

The subfamily of growth/differentiation factors (GDFs) known as GDFs 5, 6, and 7 appears to be involved in tendon maintenance and repair, although the precise nature of this role has yet to be elucidated. The aim of the present study was to examine the role of GDF‐7 in tendon maintenance by studying tail tendon fascicle gene expression, composition, and material property strain rate dependency in 16‐week‐old male and female GDF‐7 deficient mice. GDF‐7 deficiency did not affect the biochemical composition of tail tendon fascicles, nor did it significantly affect the tensile material properties obtained at either slow (5%/s) or fast (50%/s) strain rates. Further, no difference was found between genotypes in the strain rate sensitivity of any tensile material property. Consistent with the compositional analyses, QRT‐PCR data did not reveal any differences of twofold or greater in the gene expression levels of collagens I, III, V, nor in the proteoglycans decorin, fibromodulin, lumican, biglycan, versican, or aggrecan. Gdf5 expression was upregulated twofold in GDF‐7 deficient tail tendons, and Bmp7 expression was downregulated twofold. No notable differences in expression levels for Bmp1‐6 or Gdf6 were detected. GDF‐5 protein levels were 50% higher in GDF‐7 deficient tail tendon compared to wild type tail tendon. The results of this study support the intriguing possibility that compensation by Gdf‐5 may be at least in part responsible for the absence of a strong phenotype in GDF‐7 deficient mice.

Identification of a Tendon Phenotype in GDF6 Deficient Mice

Increasing evidence suggests that the growth/differentiation factors, GDFs 5, 6, and 7 in particular, may play a role in tendon and ligament biology. Mice with genetic mutations in Gdf5 have altered tendon composition and mechanical behavior, whereas animals with functional null mutations in Gdf7 have a more subtle tendon phenotype. The present study demonstrates for the first time that a null mutation in Gdf6 is associated with substantially lower levels of tail tendon collagen content (−33%) in 4‐week‐old male mice, which has direct functional consequences for the mechanical integrity of the tissue (45–50% reduction in material properties). These data support a role for GDF6 in tendon matrix modeling. Anat Rec, 292:396–400, 2009.

The River Blindness Genome Project

We wish to give special thanks to TIGR for the cluster analysis of the EST data. The authors would also like to recognize the financial support of the Edna McConnell Clark Foundation, the Blakeslee Fund for Genetics Research at Smith College and the generous support to Smith College of the A.K. Watson Trust.

Sex matters in the establishment of murine tendon composition and material properties during growth

The existence of sex‐based differences in tendon and ligament injury rates has led investigators to test the hypothesis that sex plays a significant role in modulating tendon and ligament composition and material properties. To date, no studies have attempted to characterize how such differences develop during the course of normal tissue maturation and growth. Thus, the primary aim of the present study was to use a murine model to test the hypothesis that sex‐based differences in the normal age‐related development of tendon composition and material properties exist by assessing these parameters in the Achilles and tail tendons from 4‐, 6‐, 9‐, 12‐, and 15‐week‐old male and female C57Bl/6J mice. Despite significantly lower levels of total collagen content in females subsequent to sexual maturity (p < 0.0001), as well as a significant effect of sex on glycosaminoglycan content (p < 0.0001), Achilles tendon elastic modulus was not compromised in females. Female Achilles tendons did exhibit a significantly higher failure strain (p = 0.0201) and strain energy density (p = 0.0004) than did males, as well as a trend toward higher ultimate strength (p = 0.0556). In contrast to the high load‐bearing environment of the Achilles tendon site, sex did not have a statistically significant effect on any compositional or material property in the low load‐bearing tendon fascicles of the tail. These data support recent studies by others, which suggest that male and female tendons have a differential adaptational response to their local mechanical loading environment.

Tasting Phenylthiocarbamide (PTC): A New Integrative Genetics Lab with an Old Flavor

Author(s): Robert B. Merritt, Lou Ann Bierwert, Barton Slatko, Michael P. Weiner, Jessica Ingram, Kristianna Sciarra, and Evan Weiner Source: The American Biology Teacher, 70(5):e23-e28. 2008. Published By: National Association of Biology Teachers DOI: http://dx.doi.org/10.1662/0002-7685(2008)70[23:TPPANI]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.1662/0002-7685%282008%2970%5B23%3ATPPANI %5D2.0.CO%3B2

Human Xq28 Inversion Polymorphism: From Sex Linkage to Genomics--A Genetic Mother Lode.

An inversion polymorphism of the filamin and emerin genes at the tip of the long arm of the human X‐chromosome serves as the basis of an investigative laboratory in which students learn something new about their own genomes. Long, nearly identical inverted repeats flanking the filamin and emerin genes illustrate how repetitive elements can lead to alterations in genome structure (inversions) through nonallelic homologous recombination. The near identity of the inverted repeats is an example of concerted evolution through gene conversion. While the laboratory in its entirety is designed for college level genetics courses, portions of the laboratory are appropriate for courses at other levels. Because the polymorphism is on the X‐chromosome, the laboratory can be used in introductory biology courses to enhance understanding of sex‐linkage and to test for Hardy‐Weinberg equilibrium in females. More advanced topics, such as chromosome interference, the molecular model for recombination, and inversion heterozygosity suppression of recombination can be explored in upper‐level genetics and evolution courses. DNA isolation, restriction digests, ligation, long PCR, and iPCR provide experience with techniques in molecular biology. This investigative laboratory weaves together topics stretching from molecular genetics to cytogenetics and sex‐linkage, population genetics and evolutionary genetics.