A lipid mass spectrometry investigation on the effect of non-bilayer forming amphiphiles to the phospholipid metabolism of HeLa cells

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A multi-parametric assessment of decontamination protocols for the subglacial Lake Ellsworth probe

Direct measurement and sampling of pristine environments, such as subglacial lakes, without introducing contaminating microorganisms and biomolecules from the surface, represents a significant engineering and microbiological challenge. In this study, we compare methods for decontamination of titanium grade 5 surfaces, the material extensively used to construct a custom-made probe for reaching, measuring and sampling subglacial Lake Ellsworth in West Antarctica. Coupons of titanium were artificially contaminated with Pseudomonas fluorescens bacteria and then exposed to a number of decontamination procedures. The most effective sterilants were (i) hydrogen peroxide vapour, and (ii) Biocleanse™, a commercially available, detergent-based biocidal solution. After each decontamination procedure the bacteria were incapable of proliferation, and showed no evidence of metabolic activity based on the generation of adenosine triphosphate (ATP). The use of ultraviolet irradiation or ethyl alcohol solution was comparatively ineffective for sterilisation. Hydrogen peroxide vapour and ultraviolet irradiation, which directly damage nucleic acids, were the most effective methods for removing detectable DNA, which was measured using 16S rRNA gene copy number and fluorescence-based total DNA quantification. Our results have not only been used to tailor the Ellsworth probe decontamination process, but also hold value for subsequent engineering projects, where high standards of decontamination are required

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of clostridium difficile on a SlipChip platform

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 ?L. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of &lt;20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents

Reliability Case Notes No. 6. Risk assessment of the Ellsworth probe: development, design and deployment

The Natural Environment Research Council is partly funding the development and deployment of a scientific probe in the Antarctic subglacial Lake Ellsworth. The deployment will take place in 2013. This report aims to quantify the technical risks of the probe development, design and deployment. A high level representation of the entire probe deployment process is captured in a Markov chain – a form of graphical probabilistic model. The transition from one state, or phase of the deployment, to the next depends on several factors, including: reliability of components and reliability of processes. We use fault trees to quantify the probability of failure of the complex processes that must take place to facilitate the transition from one state to another. The Shelf expert judgment elicitation package was followed to elicit expert judgments for the probability of failure for each failure mode.The top two technical risks are:• Failure to clean Jacket of the hot water drill hose: 95% quantile at 0.96.• Probe electronic failure: 95% quantile at 0.11. Here, the top failure mode is optical connector failure.The Markov chain was used to estimate the availability of the probe from different phases of thedeployment.• From on-site probe cleaning: 95% confidence that the probability of success is higher than 0.7.• From on-site testing: 95% confidence that the probability of success is higher than 0.73.• From sheave deployment: 95% confidence that the probability of success if higher than 0.83.• From probe positioning: 95% confidence that the probability of success is higher than 0.90.Finally, the probability of a successful probe deployment following the probe pre-deployment isgreater than 0.87, with 95% confidence

Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification

We describe here a simple device for dielectrophoretic concentration of marine microalga Karenia brevis non-motile cells, followed by electric field-mediated lysis for RNA extraction. The lysate was purified using magnetic beads and pure RNA extracted. RNA quality was assessed off-chip by nucleic acid sequence-based amplification and the optimum conditions for lysis were determined. This procedure will form part of an integrated microfluidic system that is being developed with sub-systems for performing cell concentration and lysis, RNA extraction/purification and real-time quantitative RNA detection. The integrated system and its components could be used for a large range of applications including in situ harmful algal bloom detection, transcriptomics and point-of-care diagnostics

Thiamine deficiency disorders : diagnosis, prevalence, and a roadmap for global control programs

Thiamine is an essential micronutrient that plays a key role in energy metabolism. Many populations worldwide may be at risk of clinical or subclinical thiamine deficiencies, due to famine, reliance on staple crops with low thiamine content, or food preparation practices, such as milling grains and washing milled rice. Clinical manifestations of thiamine deficiency are variable; this, along with the lack of a readily accessible and widely agreed upon biomarker of thiamine status, complicates efforts to diagnose thiamine deficiency and assess its global prevalence. Strategies to identify regions at risk of thiamine deficiency through proxy measures, such as analysis of food balance sheet data and month-specific infant mortality rates, may be valuable for understanding the scope of thiamine deficiency. Urgent public health responses are warranted in high-risk regions, considering the contribution of thiamine deficiency to infant mortality and research suggesting that even subclinical thiamine deficiency in childhood may have lifelong neurodevelopmental consequences. Food fortification and maternal and/or infant thiamine supplementation have proven effective in raising thiamine status and reducing the incidence of infantile beriberi in regions where thiamine deficiency is prevalent, but trial data are limited. Efforts to determine culturally and environmentally appropriate food vehicles for thiamine fortification are ongoing

Probe Technology for the Direct Measurement and Sampling of Ellsworth Subglacial Lake

The direct measurement and sampling of Ellsworth Subglacial Lake is a multidisciplinary investigation of life in extreme environments and West Antarctic ice sheet history. The project's aims are (1) to determine whether, and in what form, microbial life exists in Antarctic subglacial lakes and (2) to reveal the post-Pliocene history of the West Antarctic Ice Sheet. A U.K. consortium has planned an extensive logistics and equipment development program that will deliver the necessary resources. This will include hot water drill technology for lake access through approximately 3.2 km of ice, a probe to make measurements with sensors and to collect water and sediment samples, and a percussion corer to acquire an ˜3–4 m sediment core. This chapter details the requirements and early stages of design and development of the probe system. This includes the instrumentation package, water samplers, and a mini gravity corer mounted on the front of the probe. Initial design concepts for supporting equipment required at the drill site to deploy and operate the probe are also described. A review of the literature describing relevant technology is presented. The project will implement environmental protection in line with principles set out by the Scientific Committee on Antarctic Research