detecting-lyme-disease-spirochete-borrelia-burgdorferi-ticks-using

Detecting the Lyme Disease Spirochete, <em>Borrelia Burgdorferi</em>, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...

Mount Allison University

Chromosomes are not inert structures that haul the genome through cell division. The dynamic properties of chromosomes, during the cell cycle, the lifetime of the organism and across evolutionary time, featured prominently at the 49(th) Annual Drosophila Research Conference. Platform presentations, workshops and posters focused on many aspects of chromosome structure and function including chromosome interactions such as trans-silencing and pairing between homologous and non-homologous chromosomes, specialized portions of the chromosome including the centromere and telomeres, the structure, function and evolution of the large heterochromatic domains such as the Y and 4(th) chromosomes, centric heterochromatin and subtelomeric heterochromatin. The speed of evolutionary changes in these regions, and the consequences for speciation and hybrid-incompatibility, were recurring themes. Finally, there was considerable new insight offered into the mechanics by which chromosomes are rearranged and changes in the types of alterations occurring over the lifetime of the organism, which can result in novel genes and gene flow between chromosomes. The availability of the twelve sequenced Drosophila genomes has allowed new insights into the structure, function and evolutionary transformation of chromosomes and genomes that will continue to transform our view of the chromosome as a dynamic and flexible entity that houses and regulates the genome.

Genome and chromosome structure: Twelve dynamic and evolving genomes.

Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes.

Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency.

Daphnia offer a variety of benefits for the study of epigenetics. Daphnia's parthenogenetic life cycle allows the study of epigenetic effects in the absence of confounding genetic differences. Sex determination and sexual reproduction are epigenetically determined as are several other well-studied alternate phenotypes that arise in response to environmental stressors. Additionally, there is a large body of ecological literature available, recently complemented by the genome sequence of one species and transgenic technology. DNA methylation has been shown to be altered in response to toxicants and heavy metals, although investigation of other epigenetic mechanisms is only beginning. More thorough studies on DNA methylation as well as investigation of histone modifications and RNAi in sex determination and predator-induced defenses using this ecologically and evolutionarily important organism will contribute to our understanding of epigenetics.

Daphnia as an emerging epigenetic model organism.

Daphnids are fresh water microcrustaceans, many of which follow a cyclically parthenogenetic life cycle. Daphnia species have been well studied in the context of ecology, toxicology, and evolution, but their epigenetics remain largely unexamined even though sex determination, the production of sexual females and males, and distinct adult morphological phenotypes, are determined epigenetically. Here, we report on the characterization of histone modifications in Daphnia. We show that a number of histone H3 and H4 modifications are present in Daphnia embryos and histone H3 dimethylated at lysine 4 (H3K4me2) is present nonuniformly in the nucleus in a cell cycle-dependent manner. In addition, this histone modification, while present in blastula and gastrula cells as well as the somatic cells of adults, is absent or reduced in oocytes and nurse cells. Thus, the epigenetic repertoire of Daphnia includes modified histones and as these epigenetic forces act on a genetically homogeneous clonal population Daphnia offers an exceptional tool to investigate the mechanism and role of epigenetics in the life cycle and development of an ecologically important species.

The Epigenetic Repertoire of Daphnia magna Includes Modified Histones.

The epigenetics of emerging and nonmodel organisms.

Metabolomics analysis was used to determine the effect of two well known, non-proprietary metabolic modulators, dichloroacetate and allopurinol on breast cancer cell lines. Dichloroacetate, a pyruvate dehydrogenase kinase inhibitor and allopurinol, a xanthine oxidase/dehydrogenase inhibitor, have been previously explored as chemotherapeutics showing potential in some cancer subtypes while at the same time leading to unexpected increase in proliferation in others. In this work, metabolic effects of these drugs, applied singly and in combination, were explored in three different breast cell lines including cancer cells, MDA-MB-231 and MCF-7 and normal control cell line, MCF-10A. The metabolic changes induced by these drugs were monitored by (1)H NMR metabolic profiling. Analyses were performed on complete spectral data as well as quantified metabolic data in intracellular fractions and extracellular media leading to the determination of the most significantly affected metabolites. The effect of dichloroacetate and allopurinol is the most apparent in the metabolic profile of extracellular media. In MCF-7 cells, dichloroacetate treatment is dominant with only a minor observed influence of allopurinol in combined treatment. In MDA-MB-231 cells, both allopurinol and DCA lead to a metabolic shift with the allopurinol change dominating the effect of combined treatment. Results show the power of metabolomics as a tool for fast molecular profiling of drug effects in cells. In summary, treatments of breast cancer cells with DCA and allopurinol result in larger changes in metabolites found in extracellular medium than intracellular pools.

¹H NMR metabolomics analysis of the effect of dichloroacetate and allopurinol on breast cancers.

Mites are common ectoparasites of Drosophila and have been implicated in bacterial and mobile element invasion of Drosophila stocks. The obligate endobacterium, Wolbachia, has widespread effects on gene expression in their arthropod hosts and alters host reproduction to enhance its survival and propagation, often with deleterious effects in Drosophila hosts. To determine whether Wolbachia could be transferred between Drosophila melanogaster laboratory stocks by the mite Tyrophagus putrescentiae, mites were introduced to Wolbachia-infected Drosophila vials. These vials were kept adjacent to mite-free and Wolbachia-uninfected Drosophila stock vials. The Wolbachia infection statuses of the infected and uninfected flies were checked from generation 1 to 5. Results indicate that Wolbachia DNA could be amplified from mites infesting Wolbachia-infected fly stocks and infection in the previously uninfected stocks arose within generation 1 or 2, concomitant with invasion of mites from the Wolbachia-infected stock. A possible mechanism for the transfer of Wolbachia from flies to mites and vice versa, can be inferred from time-lapse photography of fly and mite interactions. We demonstrated that mites ingest Drosophila corpses, including Wolbachia-infected corpses, and Drosophila larva ingest mites, providing possible sources of Wolbachia infection and transfer. This research demonstrated that T. putrescentiae white mites can facilitate Wolbachia transfer between Drosophila stocks and that this may occur by ingestion of infected corpses. Mite-vectored Wolbachia transfer allows for rapid establishment of Wolbachia infection within a new population. This mode of Wolbachia introduction may be relevant in nature as well as in the laboratory, and could have a variety of biological consequences.

Evidence for horizontal transfer of Wolbachia by a Drosophila mite.

This study assessed the seroprevalence of anti-Borrelia burgdorferi antibodies in New Brunswick dogs. Testing of 699 serum samples from dogs across the province revealed a 6% province-wide seropositivity, more than 6 times higher than that found in 2008. The rapid increase in seropositivity indicates increased Lyme disease risk to both canine and human health.

Vett K. Lloyd

Department of Biology

Mount Allison University

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR