Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0

<p>Abstract</p> <p>Background</p> <p>Mutations in the mitochondrial genome (mtgenome) have been associated with many disorders, including breast cancer. Nipple aspirate fluid (NAF) from symptomatic women could potentially serve as a minimally invasive sample for breast cancer screening by detecting somatic mutations in this biofluid. This study is aimed at 1) demonstrating the feasibility of NAF recovery from symptomatic women, 2) examining the feasibility of sequencing the entire mitochondrial genome from NAF samples, 3) cross validation of the Human mitochondrial resequencing array 2.0 (MCv2), and 4) assessing the somatic mtDNA mutation rate in benign breast diseases as a potential tool for monitoring early somatic mutations associated with breast cancer.</p> <p>Methods</p> <p>NAF and blood were obtained from women with symptomatic benign breast conditions, and we successfully assessed the mutation load in the entire mitochondrial genome of 19 of these women. DNA extracts from NAF were sequenced using the mitochondrial resequencing array MCv2 and by capillary electrophoresis (CE) methods as a quality comparison. Sequencing was performed independently at two institutions and the results compared. The germline mtDNA sequence determined using DNA isolated from the patient's blood (control) was compared to the mutations present in cellular mtDNA recovered from patient's NAF.</p> <p>Results</p> <p>From the cohort of 28 women recruited for this study, NAF was successfully recovered from 23 participants (82%). Twenty two (96%) of the women produced fluids from both breasts. Twenty NAF samples and corresponding blood were chosen for this study. Except for one NAF sample, the whole mtgenome was successfully amplified using a single primer pair, or three pairs of overlapping primers. Comparison of MCv2 data from the two institutions demonstrates 99.200% concordance. Moreover, MCv2 data was 99.999% identical to CE sequencing, indicating that MCv2 is a reliable method to rapidly sequence the entire mtgenome. Four NAF samples contained somatic mutations.</p> <p>Conclusion</p> <p>We have demonstrated that NAF is a suitable material for mtDNA sequence analysis using the rapid and reliable MCv2. Somatic mtDNA mutations present in NAF of women with benign breast diseases could potentially be used as risk factors for progression to breast cancer, but this will require a much larger study with clinical follow up.</p

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

This work was supported by the Leverhulme Trust (Grant number RL2012-025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1 , a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3 , a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1 , to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.Publisher PDFPeer reviewe

Optimal barrier zones for stopping the invasion of Aedes aegypti mosquitoes via transgenic or sterile insect techniques

Biological invasions have dramatically altered the natural world by threatening native species and their communities. Moreover, when the invading species is a vector for human disease, there are further substantive public health and economic impacts. The development of transgenic technologies is being explored in relation to new approaches for the biological control of insect pests. We investigate the use of two control strategies, classical sterile insect techniques and transgenic late-acting bisex lethality (Release of Insects carrying a Dominant Lethal), for controlling invasion of the mosquito Aedes aegypti using a spatial stage-structured mathematical model. In particular, we explore the use of a barrier zone of sterile/transgenic insects to prevent or impede the invasion of mosquitoes. We show that the level of control required is not only highly sensitive to the rate at which the sterile/transgenic males are released in the barrier zone but also to the spatial range of release. Our models characterise how the distribution of sterile/transgenic mosquitoes in the barrier zone can be controlled so as to minimise the number of mass-produced insects required for the arrest of species invasion. We predict that, given unknown rates of mosquito dispersal, management strategies should concentrate on larger release areas rather than more intense release rates for optimal control

An Efficient Heuristic for Pooled Repair Shop Designs

An effective spare part supply system planning is essential to achieve a high capital asset availability. We investigate the design problem of a repair shop in a single echelon repairable multi-item spare parts supply system. The repair shop usually consists of several servers with different skill sets. Once a failure occurs in the system, the failed part is queued to be served by a suitable server that has the required skill. We model the repair shop as a collection of independent sub-systems, where each sub-system is responsible for repairing certain types of failed parts. The procedure of partitioning a repair shop into sub-systems is known as pooling, and the repair shop formed by the union of independent sub-systems is called a pooled repair shop. Identifying the best partition is a challenging combinatorial optimization problem. In this direction, we formulate the problem as a stochastic nonlinear integer programming model and propose a sequential solution heuristic to find the best-pooled design by considering inventory allocation and capacity level designation of the repair shop. We conduct numerical experiments to quantify the value of the pooled repair shop designs. Our analysis shows that pooled designs can yield cost reductions by 25% to 45% compared to full flexible and dedicated designs. The proposed heuristic also achieves a lower average total system cost than that generated by a Genetic Algorithm (GA)-based solution algorithm. - 2019, Springer Nature Switzerland AG.Acknowledgement. This research was made possible by the NPRP award [NPRP 7-308-2-128] from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the author[s].Scopu

Heteroplasmy in the mitochondrial genomes of human lice and ticks revealed by high throughput sequencing

The typical mitochondrial (mt) genomes of bilateral animals consist of 37 genes on a single circular chromosome. The mt genomes of the human body louse, Pediculus humanus, and the human head louse, Pediculus capitis, however, are extensively fragmented and contain 20 minichromosomes, with one to three genes on each minichromosome. Heteroplasmy, i.e. nucleotide polymorphisms in the mt genome within individuals, has been shown to be significantly higher in the mt cox1 gene of human lice than in humans and other animals that have the typical mt genomes. To understand whether the extent of heteroplasmy in human lice is associated with mt genome fragmentation, we sequenced the entire coding regions of all of the mt minichromosomes of six human body lice and six human head lice from Ethiopia, China and France with an Illumina HiSeq platform. For comparison, we also sequenced the entire coding regions of the mt genomes of seven species of ticks, which have the typical mitochondrial genome organization of bilateral animals. We found that the level of heteroplasmy varies significantly both among the human lice and among the ticks. The human lice from Ethiopia have significantly higher level of heteroplasmy than those from China and France (