Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells

The antimetabolite mercaptopurine (MP) is widely used to treat childhood acute lymphoblastic leukaemia (ALL). To study the dynamics of MP on the cell cycle, we incubated human T-cell leukaemia cell lines (Molt-4 sensitive and resistant subline and P12 resistant) with 10 μM MP and measured total cell count, cell cycle distribution, percent viable, percent apoptotic, and percent dead cells serially over 72 h. We developed a mathematical model of the cell cycle dynamics after treatment with MP and used it to show that the Molt-4 sensitive controls had a significantly higher rate of cells entering apoptosis (2.7-fold, P<0.00001) relative to the resistant cell lines. Additionally, when treated with MP, the sensitive cell line showed a significant increase in the rate at which cells enter apoptosis compared to its controls (2.4-fold, P<0.00001). Of note, the resistant cell lines had a higher rate of antimetabolite incorporation into the DNA of viable cells (>1.4-fold, P<0.01). Lastly, in contrast to the other cell lines, the Molt-4 resistant subline continued to cycle, though at a rate slower relative to its control, rather than proceed to apoptosis. This led to a larger S-phase block in the Molt-4 resistant cell line, but not a higher rate of cell death. Gene expression of apoptosis, cell cycle, and repair genes were consistent with mechanistic dynamics described by the model. In summary, the mathematical model provides a quantitative assessment to compare the cell cycle effects of MP in cells with varying degrees of MP resistance

A preliminary study of genetic factors that influence susceptibility to bovine tuberculosis in the British cattle herd

Associations between specific host genes and susceptibility to Mycobacterial infections such as tuberculosis have been reported in several species. Bovine tuberculosis (bTB) impacts greatly the UK cattle industry, yet genetic predispositions have yet to be identified. We therefore used a candidate gene approach to study 384 cattle of which 160 had reacted positively to an antigenic skin test (‘reactors’). Our approach was unusual in that it used microsatellite markers, embraced high breed diversity and focused particularly on detecting genes showing heterozygote advantage, a mode of action often overlooked in SNP-based studies. A panel of neutral markers was used to control for population substructure and using a general linear model-based approach we were also able to control for age. We found that substructure was surprisingly weak and identified two genomic regions that were strongly associated with reactor status, identified by markers INRA111 and BMS2753. In general the strength of association detected tended to vary depending on whether age was included in the model. At INRA111 a single genotype appears strongly protective with an overall odds ratio of 2.2, the effect being consistent across nine diverse breeds. Our results suggest that breeding strategies could be devised that would appreciably increase genetic resistance of cattle to bTB (strictly, reduce the frequency of incidence of reactors) with implications for the current debate concerning badger-culling

MPHASYS: a mouse phenotype analysis system

<p>Abstract</p> <p>Background</p> <p>Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery.</p> <p>Results</p> <p>Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd.</p> <p>Conclusion</p> <p>MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Background: The genome of a number of species of malaria parasites ( Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo. Results: We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated. Conclusion: These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in viv

The "Statinth" wonder of the world: a panacea for all illnesses or a bubble about to burst

After the introduction of statins in the market as effective lipid lowering agents, they were shown to have effects other than lipid lowering. These actions were collectively referred to as 'pleiotropic actions of statins.' Pleiotropism of statins formed the basis for evaluating statins for several indications other than lipid lowering. Evidence both in favour and against is available for several of these indications. The current review attempts to critically summarise the available data for each of these indications

A collaborative approach to adopting/adapting guidelines - the Australian 24-hour Movement Guidelines for the early years (birth to 5 years): an integration of physical activity, sedentary behavior, and sleep

BACKGROUND: In 2017, the Australian Government funded the update of the National Physical Activity Recommendations for Children 0-5&nbsp;years, with the intention that they be an integration of movement behaviours across the 24-h period. The benefit for Australia was that it could leverage research in Canada in the development of their 24-h guidelines for the early years. Concurrently, the Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group published a model to produce guidelines based on adoption, adaption and/or de novo development using the GRADE evidence-to-decision framework. Referred to as the GRADE-ADOLOPMENT approach, it allows guideline developers to follow a structured and transparent process in a more efficient manner, potentially avoiding the need to unnecessarily repeat costly tasks such as conducting systematic reviews. The purpose of this paper is to outline the process and outcomes for adapting the Canadian 24-Hour Movement Guidelines for the Early Years to develop the Australian 24-Hour Movement Guidelines for the Early Years guided by the GRADE-ADOLOPMENT framework. METHODS: The development process was guided by the GRADE-ADOLOPMENT approach. A Leadership Group and Consensus Panel were formed and existing credible guidelines identified. The draft Canadian 24-h integrated movement guidelines for the early years best met the criteria established by the Panel. These were evaluated based on the evidence in the GRADE tables, summaries of findings tables and draft recommendations from the Canadian Draft Guidelines. Updates to each of the Canadian systematic reviews were conducted and the Consensus Panel reviewed the evidence for each behaviour separately and made a decision to adopt or adapt the Canadian recommendations for each behaviour or create de novo recommendations. An online survey was then conducted (n&nbsp;=&nbsp;302) along with five focus groups (n&nbsp;=&nbsp;30) and five key informant interviews (n&nbsp;=&nbsp;5) to obtain feedback from stakeholders on the draft guidelines. RESULTS: Based on the evidence from the Canadian systematic reviews and the updated systematic reviews in Australia, the Consensus Panel agreed to adopt the Canadian recommendations and, apart from some minor changes to the wording of good practice statements, keep the wording of the guidelines, preamble and title of the Canadian Guidelines. The Australian Guidelines provide evidence-informed recommendations for a healthy day (24-h), integrating physical activity, sedentary behaviour (including limits to screen time), and sleep for infants (&lt;1&nbsp;year), toddlers (1-2&nbsp;years) and preschoolers (3-5&nbsp;years). CONCLUSIONS: To our knowledge, this is only the second time the GRADE-ADOLOPMENT approach has been used. Following this approach, the judgments of the Australian Consensus Panel did not differ sufficiently to change the directions and strength of the recommendations and as such, the Canadian recommendations were adopted with very minor alterations. This allowed the Guidelines to be developed much faster and at lower cost. As such, we would recommend the GRADE-ADOLOPMENT approach, especially if a credible set of guidelines, with all supporting materials and developed using a transparent process, is available. Other countries may consider using this approach when developing and/or revising national movement guidelines