cultivation-methods-spirochetes-from-borrelia-burgdorferi-sensu-lato

Cultivation Methods of Spirochetes from <em>Borrelia burgdorferi</em> Sensu Lato Complex and Relapsing Fever <em>Borrelia</em>

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently ...

Mt. Allison University

Analysis of Drosophila eye-colour mutations has made seminal contributions to the fields of genetics and biochemistry. Recent findings suggest that a subset of eye-colour genes is crucial for vesicular transport of proteins to pigment granules, specialized lysosomes of eye-pigment cells. Thus, classical work describing more than 85 eye-colour mutations and their genetic interactions offers a remarkable, untapped resource for the genetic analysis of protein delivery to lysosomes.

Not just pretty eyes: Drosophila eye-colour mutations and lysosomal delivery.

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.

The white gene of Drosophila melanogaster has been extensively studied, yet it is still not understood how its ectopic overexpression induces male-male courtship. To investigate the cellular basis of this behavior, we examined the sexual behavior of several classes of mutants. We find that male-male courtship is seen not only in flies overexpressing the white gene, but also in mutants expected to have mislocalized White protein. This finding confirms that mislocalizing White transporter in the cells in which it is normally expressed will produce male-male courtship behaviors; the courtship behavior is not an indirect consequence of aberrant physiological changes elsewhere in the body. Male-male courtship is also seen in some mutants with altered monoamine metabolism and deficits in learning and memory, but can be distinguished from that produced by White mislocalization by its reduced intensity and locomotor activity. Double mutants overexpressing white and with mutations in genes for serotonergic neurons suggest that male-male courtship produced by mislocalizing White may not be mediated exclusively by serotonergic neurons. We also find decreased olfactory learning in white mutants and in individuals with mutations in the genes for White's binding partners, brown and scarlet. Finally, in cultured Drosophila and mammalian cells, the White transporter is found in the endosomal compartment. The additional genes identified here as being involved in male-male courtship increase the repertoire of mutations available to study sexual behavior in Drosophila.

The white gene of Drosophila melanogaster encodes a protein with a role in courtship behavior.

Genomic imprinting is a process that marks DNA, causing a change in gene or chromosome behavior, depending on the sex of the transmitting parent. In mammals, most examples of genomic imprinting affect the transcription of individual or small clusters of genes whereas in insects, genomic imprinting tends to silence entire chromosomes. This has been interpreted as evidence of independent evolutionary origins for imprinting. To investigate how these types of imprinting are related, we performed a phenotypic, molecular, and cytological analysis of an imprinted chromosome in Drosophila melanogaster. Analysis of this chromosome reveals that the imprint results in transcriptional silencing. Yet, the domain of transcriptional silencing is very large, extending at least 1.2 Mb and encompassing over 100 genes, and is associated with decreased somatic polytenization of the entire chromosome. We propose that repression of somatic replication in polytenized cells, as a secondary response to the imprint, acts to extend the size of the imprinted domain to an entire chromosome. Thus, imprinting in Drosophila has properties of both typical mammalian and insect imprinting which suggests that genomic imprinting in Drosophila and mammals is not fundamentally different; imprinting is manifest as transcriptional silencing of a few genes or silencing of an entire chromosome depending on secondary processes such as differences in gene density and polytenization.

Genomic imprinting in Drosophila has properties of both mammalian and insect imprinting.

CTCF is a versatile zinc finger DNA-binding protein that functions as a highly conserved epigenetic transcriptional regulator. CTCF is known to act as a chromosomal insulator, bind promoter regions, and facilitate long-range chromatin interactions. In mammals, CTCF is active in the regulatory regions of some genes that exhibit genomic imprinting, acting as insulator on only one parental allele to facilitate parent-specific expression. In Drosophila, CTCF acts as a chromatin insulator and is thought to be actively involved in the global organization of the genome.

The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster.

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.

Ixodes scapularis, a known vector of Borrelia burgdorferi sensu stricto (Bbss), is undergoing range expansion in many parts of Canada. The province of New Brunswick, which borders jurisdictions with established populations of I. scapularis, constitutes a range expansion zone for this species. To better understand the current and potential future distribution of this tick under climate change projections, this study applied occupancy modelling to distributional records of adult ticks that successfully overwintered, obtained through passive surveillance. This study indicates that I. scapularis occurs throughout the southern-most portion of the province, in close proximity to coastlines and major waterways. Milder winter conditions, as indicated by the number of degree days <0 °C, was determined to be a strong predictor of tick occurrence, as was, to a lesser degree, rising levels of annual precipitation, leading to a final model with a predictive accuracy of 0.845 (range: 0.828-0.893). Both RCP 4.5 and RCP 8.5 climate projections predict that a significant proportion of the province (roughly a quarter to a third) will be highly suitable for I. scapularis by the 2080s. Comparison with cases of canine infection show good spatial agreement with baseline model predictions, but the presence of canine Borrelia infections beyond the climate envelope, defined by the highest probabilities of tick occurrence, suggest the presence of Bbss-carrying ticks distributed by long-range dispersal events. This research demonstrates that predictive statistical modelling of multi-year surveillance information is an efficient way to identify areas where I. scapularis is most likely to occur, and can be used to guide subsequent active sampling efforts in order to better understand fine scale species distributional patterns.

Combining public participatory surveillance and occupancy modelling to predict the distributional response of Ixodes scapularis to climate change.

Lyme disease is the most common tick-borne disease in North America and Europe, and on-going surveillance is required to monitor the spread of the tick vectors as their populations expand under the influence of climate change. Active surveillance involves teams of researchers collecting ticks from field locations with the potential to be sites of establishing tick populations. This process is labor- and time-intensive, limiting the number of sites monitored and the frequency of monitoring. Citizen science initiatives are ideally suited to address this logistical problem and generate high-density and complex data from sites of community importance. In 2014, the same region was monitored by academic researchers, public health workers, and citizen scientists, allowing a comparison of the strengths and weaknesses of each type of surveillance effort. Four community members persisted with tick collections over several years, collectively recovering several hundred ticks. Although deviations from standard surveillance protocols and the choice of tick surveillance sites makes the incorporation of community-generated data into conventional surveillance analyses more complex, this citizen science data remains useful in providing high-density longitudinal tick surveillance of a small area in which detailed ecological observations can be made. Most importantly, partnership between community members and researchers has proven a powerful tool in educating communities about of the risk of tick-vectored diseases and in encouraging tick bite prevention.

Citizen Science and Community Engagement in Tick Surveillance-A Canadian Case Study.

Lyme disease arises from infection with pathogenic  species. In Canada, current case definition for confirmed Lyme disease requires serological confirmation by both a positive first tier ELISA and confirmatory second tier immunoblot (western blot). For surveillance and research initiatives, this requirement is intentionally conservative to exclude false positive results. Consequently, this approach is prone to false negative results that lead to underestimation of the number of people with Lyme disease. The province of New Brunswick (NB), Canada, can be used to quantify under-detection of the disease as three independent data sets are available to generate an estimate of the true human disease prevalence and incidence. First, detailed human disease incidence is available for the US states and counties bordering Canada, which can be compared with Canadian disease incidence. Second, published national serology results and well-described sensitivity and specificity values for these tests are available and deductive reasoning can be used to query for discrepancies. Third, high-density tick and canine surveillance data are available for the province, which can be used to predict expected human Lyme prevalence. Comparison of cross-border disease incidence suggests a minimum of 10.2 to 28-fold under-detection of Lyme disease (3.6% to 9.8% cases detected). Analysis of serological testing predicts the surveillance criteria generate 10.4-fold under-diagnosis (9.6% cases detected) in New Brunswick for 2014 due to serology alone. Calculation of expected human Lyme disease cases based on tick and canine infections in New Brunswick indicates a minimum of 12.1 to 58.2-fold underestimation (1.7% to 8.3% cases detected). All of these considerations apply generally across the country and strongly suggest that public health information is significantly under-detecting and under-reporting human Lyme cases across Canada. Causes of the discrepancies between reported cases and predicted actual cases may include undetected genetic diversity of  in Canada leading to failed serological detection of infection, failure to consider and initiate serological testing of patients, and failure to report clinically diagnosed acute cases. As these surveillance criteria are used to inform clinical and public health decisions, this under-detection will impact diagnosis and treatment of Canadian Lyme disease patients.

Article	Year
Not just pretty eyes: Drosophila eye-colour mutations and lysosomal delivery. Trends in cell biology\| PubMed ID: 9714595	1998
Genome and chromosome structure: Twelve dynamic and evolving genomes. Fly\| PubMed ID: 18820460	2008
The white gene of Drosophila melanogaster encodes a protein with a role in courtship behavior. Journal of neurogenetics\| PubMed ID: 19012054	2008
Genomic imprinting in Drosophila has properties of both mammalian and insect imprinting. Development genes and evolution\| PubMed ID: 19031081	2009
The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster. BMC biology\| PubMed ID: 20673338	2010
¹H NMR metabolomics analysis of the effect of dichloroacetate and allopurinol on breast cancers. Journal of pharmaceutical and biomedical analysis\| PubMed ID: 24074721	2014
Combining public participatory surveillance and occupancy modelling to predict the distributional response of Ixodes scapularis to climate change. Ticks and tick-borne diseases\| PubMed ID: 29478885	2018
Citizen Science and Community Engagement in Tick Surveillance-A Canadian Case Study. Healthcare (Basel, Switzerland)\| PubMed ID: 29498648	2018
Under-Detection of Lyme Disease in Canada. Healthcare (Basel, Switzerland)\| PubMed ID: 30326576	2018

Vett Lloyd

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