Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq.

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use

Fructose transport-deficient Staphylococcus aureus reveals important role of epithelial glucose transporters in limiting sugar-driven bacterial growth in airway surface liquid.

Hyperglycaemia as a result of diabetes mellitus or acute illness is associated with increased susceptibility to respiratory infection with Staphylococcus aureus. Hyperglycaemia increases the concentration of glucose in airway surface liquid (ASL) and promotes the growth of S. aureus in vitro and in vivo. Whether elevation of other sugars in the blood, such as fructose, also results in increased concentrations in ASL is unknown and whether sugars in ASL are directly utilised by S. aureus for growth has not been investigated. We obtained mutant S. aureus JE2 strains with transposon disrupted sugar transport genes. NE768(fruA) exhibited restricted growth in 10 mM fructose. In H441 airway epithelial-bacterial co-culture, elevation of basolateral sugar concentration (5-20 mM) increased the apical growth of JE2. However, sugar-induced growth of NE768(fruA) was significantly less when basolateral fructose rather than glucose was elevated. This is the first experimental evidence to show that S. aureus directly utilises sugars present in the ASL for growth. Interestingly, JE2 growth was promoted less by glucose than fructose. Net transepithelial flux of D-glucose was lower than D-fructose. However, uptake of D-glucose was higher than D-fructose across both apical and basolateral membranes consistent with the presence of GLUT1/10 in the airway epithelium. Therefore, we propose that the preferential uptake of glucose (compared to fructose) limits its accumulation in ASL. Pre-treatment with metformin increased transepithelial resistance and reduced the sugar-dependent growth of S. aureus. Thus, epithelial paracellular permeability and glucose transport mechanisms are vital to maintain low glucose concentration in ASL and limit bacterial nutrient sources as a defence against infection

Tools for delivering entomopathogenic fungi to malaria mosquitoes: effects of delivery surfaces on fungal efficacy and persistence.

BACKGROUND\ud \ud Entomopathogenic fungi infection on malaria vectors increases daily mortality rates and thus represents a control measure that could be used in integrated programmes alongside insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS). Before entomopathogenic fungi can be integrated into control programmes, an effective delivery system must be developed.\ud \ud METHODS\ud \ud The efficacy of Metarhizium anisopliae ICIPE-30 and Beauveria bassiana I93-825 (IMI 391510) (2 × 10(10) conidia m(-2)) applied on mud panels (simulating walls of traditional Tanzanian houses), black cotton cloth and polyester netting was evaluated against adult Anopheles gambiae sensu stricto. Mosquitoes were exposed to the treated surfaces 2, 14 and 28 d after conidia were applied. Survival of mosquitoes was monitored daily.\ud \ud RESULTS\ud \ud All fungal treatments caused a significantly increased mortality in the exposed mosquitoes, descending with time since fungal application. Mosquitoes exposed to M. anisopliae conidia on mud panels had a greater daily risk of dying compared to those exposed to conidia on either netting or cotton cloth (p < 0.001). Mosquitoes exposed to B. bassiana conidia on mud panels or cotton cloth had similar daily risk of death (p = 0.14), and a higher risk than those exposed to treated polyester netting (p < 0.001). Residual activity of fungi declined over time; however, conidia remained pathogenic at 28 d post application, and were able to infect and kill 73 - 82% of mosquitoes within 14 d.\ud \ud CONCLUSION\ud \ud Both fungal isolates reduced mosquito survival on immediate exposure and up to 28 d after application. Conidia were more effective when applied on mud panels and cotton cloth compared with polyester netting. Cotton cloth and mud, therefore, represent potential substrates for delivering fungi to mosquitoes in the field

Challenges to the development of antigen-specific breast cancer vaccines

Continued progress in the development of antigen-specific breast cancer vaccines depends on the identification of appropriate target antigens, the establishment of effective immunization strategies, and the ability to circumvent immune escape mechanisms. Methods such as T cell epitope cloning and serological expression cloning (SEREX) have led to the identification of a number target antigens expressed in breast cancer. Improved immunization strategies, such as using dendritic cells to present tumor-associated antigens to T lymphocytes, have been shown to induce antigen-specific T cell responses in vivo and, in some cases, objective clinical responses. An outcome of successful tumor immunity is the evolution of antigen-loss tumor variants. The development of a polyvalent breast cancer vaccine, directed against a panel of tumor-associated antigens, may counteract this form of immune escape

Response Inhibition and Error Monitoring during a Visual Go/No-Go Task in Inuit Children Exposed to Lead, Polychlorinated Biphenyls, and Methylmercury

Background: Lead (Pb) and polychlorinated biphenyls (PCBs) are neurotoxic contaminants that have been related to impairment in response inhibition

Using technology to deliver cancer follow-up : a systematic review

Peer reviewedPublisher PD

Incorporating prior knowledge improves detection of differences in bacterial growth rate

BACKGROUND: Robust statistical detection of differences in the bacterial growth rate can be challenging, particularly when dealing with small differences or noisy data. The Bayesian approach provides a consistent framework for inferring model parameters and comparing hypotheses. The method captures the full uncertainty of parameter values, whilst making effective use of prior knowledge about a given system to improve estimation. RESULTS: We demonstrated the application of Bayesian analysis to bacterial growth curve comparison. Following extensive testing of the method, the analysis was applied to the large dataset of bacterial responses which are freely available at the web-resource, ComBase. Detection was found to be improved by using prior knowledge from clusters of previously analysed experimental results at similar environmental conditions. A comparison was also made to a more traditional statistical testing method, the F-test, and Bayesian analysis was found to perform more conclusively and to be capable of attributing significance to more subtle differences in growth rate. CONCLUSIONS: We have demonstrated that by making use of existing experimental knowledge, it is possible to significantly improve detection of differences in bacterial growth rate

Comparative Population Genetics of the Immunity Gene, Relish: Is Adaptive Evolution Idiosyncratic?

The frequency of adaptive evolution acting on common loci in distant lineages remains an outstanding question in evolutionary biology. We asked whether the immunity factor, Relish, a gene with a history of directional selection in Drosophila simulans, shows evidence of a similar selective history in other Drosophila species. We found only weak evidence of recurrent adaptive protein evolution at the Relish locus in three sister species pairs, suggesting that this key component of the insect immune system has an idiosyncratic evolutionary history in Drosophila

Vascular responses of the extremities to transdermal application of vasoactive agents in Caucasian and African descent individuals

This is an accepted manuscript of an article published by Springer in European Journal of Applied Physiology on 04/04/2015, available online: https://doi.org/10.1007/s00421-015-3164-2 The accepted version of the publication may differ from the final published version.© 2015, Springer-Verlag Berlin Heidelberg. Purpose: Individuals of African descent (AFD) are more susceptible to non-freezing cold injury than Caucasians (CAU) which may be due, in part, to differences in the control of skin blood flow. We investigated the skin blood flow responses to transdermal application of vasoactive agents. Methods: Twenty-four young males (12 CAU and 12 AFD) undertook three tests in which iontophoresis was used to apply acetylcholine (ACh 1 w/v %), sodium nitroprusside (SNP 0.01 w/v %) and noradrenaline (NA 0.5 mM) to the skin. The skin sites tested were: volar forearm, non-glabrous finger and toe, and glabrous finger (pad) and toe (pad). Results: In response to SNP on the forearm, AFD had less vasodilatation for a given current application than CAU (P = 0.027–0.004). ACh evoked less vasodilatation in AFD for a given application current in the non-glabrous finger and toe compared with CAU (P = 0.043–0.014) with a lower maximum vasodilatation in the non-glabrous finger (median [interquartile], AFD n = 11, 41[234] %, CAU n = 12, 351[451] %, P = 0.011) and non-glabrous toe (median [interquartile], AFD n = 9, 116[318] %, CAU n = 12, 484[720] %, P = 0.018). ACh and SNP did not elicit vasodilatation in the glabrous skin sites of either group. There were no ethnic differences in response to NA. Conclusion: AFD have an attenuated endothelium-dependent vasodilatation in non-glabrous sites of the fingers and toes compared with CAU. This may contribute to lower skin temperature following cold exposure and the increased risk of cold injuries experienced by AFD.Published versio

Quantitative cross-species extrapolation between humans and fish: The case of the anti-depressant fluoxetine

This article has been made available through the Brunel Open Access Publishing Fund.Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 μg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation