Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation

Mid-cervical spinal cord contusion causes robust deficits in respiratory parameters and pattern variability

Mid-cervical spinal cord contusion disrupts both the pathways and motoneurons vital to the activity of inspiratory muscles. The present study was designed to determine if a rat contusion model could result in a measurable deficit to both ventilatory and respiratory motor function under “normal” breathing conditions at acute to chronic stages post trauma. Through whole body plethysmography and electromyography we assessed respiratory output from three days to twelve weeks after a cervical level 3 (C3) contusion. Contused animals showed significant deficits in both tidal and minute volumes which were sustained from acute to chronic time points. We also examined the degree to which the contusion injury impacted ventilatory pattern variability through assessment of Mutual Information and Sample Entropy. Mid-cervical contusion significantly and robustly decreased the variability of ventilatory patterns. The enduring deficit to the respiratory motor system caused by contusion was further confirmed through electromyography recordings in multiple respiratory muscles. When isolated via a lesion, these contused pathways were insufficient to maintain respiratory activity at all time points post injury. Collectively these data illustrate that, counter to the prevailing literature, a profound and lasting ventilatory and respiratory motor deficit may be modelled and measured through multiple physiological assessments at all time points after cervical contusion injury

Numerical Methods for Flow in Fractured Porous Media

In this work we present the mathematical models for single-phase flow in fractured porous media. An overview of the most common approaches is considered, which includes continuous fracture models and discrete fracture models. For the latter, we discuss strategies that are developed in literature for its numerical solution mainly related to the geometrical relation between the fractures and porous media grids

Oxygen transport kinetics underpin rapid and robust diaphragm recovery following chronic spinal cord injury

Months after spinal cord injury (SCI), respiratory deficits remain the primary cause of morbidity and mortality for patients. It is possible to induce partial respiratory motor functional recovery in chronic SCI following 2 weeks of spinal neuroplasticity. However, the peripheral mechanisms underpinning this recovery are largely unknown, limiting development of new clinical treatments with potential for complete functional restoration. Utilizing a rat hemisection model, diaphragm function and paralysis was assessed and recovered at chronic time points following trauma through chondroitinase ABC induced neuroplasticity. We simulated the diaphragm's in vivo cyclical length change and activity patterns using the work loop technique at the same time as assessing global and local measures of the muscles histology to quantify changes in muscle phenotype, microvascular composition, and oxidative capacity following injury and recovery. These data were fed into a physiologically informed model of tissue oxygen transport. We demonstrate that hemidiaphragm paralysis causes muscle fibre hypertrophy, maintaining global oxygen supply, although it alters isolated muscle kinetics, limiting respiratory function. Treatment induced recovery of respiratory activity normalized these effects, increasing oxygen supply, restoring optimal diaphragm functional properties. However, metabolic demands of the diaphragm were significantly reduced following both injury and recovery, potentially limiting restoration of normal muscle performance. The mechanism of rapid respiratory muscle recovery following spinal trauma occurs through oxygen transport, metabolic demand and functional dynamics of striated muscle. Overall, these data support a systems‐wide approach to the treatment of SCI, and identify new targets to mediate complete respiratory recovery

Continuous synthesis of block copolymer nanoparticles via telescoped RAFT solution and dispersion polymerisation in a miniature CSTR cascade

Miniaturised continuous-flow reactors offer a safe, economical, and scalable route to explore the synthesis of high-value chemical products. In the context of polymer synthesis, precisely defined and tuneable products can be prepared via reversible de-activation radical polymerisation (RDRP) techniques such as reversible addition-fragmentation chain-transfer (RAFT), for which tubular reactors are commonly reported. Herein, we present a miniature continuous stirred-tank reactor (CSTR) cascade for continuous-flow RAFT polymerisation with active mixing throughout, which is found to perform close to a theoretical CSTR cascade for the polymerisations considered in this study. The performance of the reactor is evaluated for both the aqueous solution RAFT polymerisation of N,N-dimethylacrylamide (DMAm) and the RAFT dispersion polymerisation of diacetone acrylamide using a poly(DMAm) macromolecular chain transfer agent (macro-CTA). It was determined that the residence time distribution (RTD) is important for informing the properties of the resulting polymers, with more CSTRs resulting in a narrower molar mass distribution. For particle synthesis by polymerisation-induced self-assembly (PISA), a series of block copolymers were prepared in separate batch and flow experiments for which the particles obtained were found to vary despite comparable molecular weights. Towards the development of a high throughput screening platform, a multi-stage, telescoped tubular-CSTR cascade reactor configuration was applied for inline macro-CTA synthesis and subsequent block extension. Differences in product properties between the processing methods used supports the idea that polymers are so-called ‘products-by-process’; indeed different polymer products can be accessed from the same chemistry through the application of alternative synthesis approaches

Treatment of infections caused by multidrug-resistant Gram-negative bacteria: report of the British Society for Antimicrobial Chemotherapy/Healthcare Infection Society/British Infection Association Joint Working Party

The Working Party makes more than 100 tabulated recommendations in antimicrobial prescribing for the treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria (GNB) and suggest further research, and algorithms for hospital and community antimicrobial usage in urinary infection. The international definition of MDR is complex, unsatisfactory and hinders the setting and monitoring of improvement programmes. We give a new definition of multiresistance. The background information on the mechanisms, global spread and UK prevalence of antibiotic prescribing and resistance has been systematically reviewed. The treatment options available in hospitals using intravenous antibiotics and in primary care using oral agents have been reviewed, ending with a consideration of antibiotic stewardship and recommendations. The guidance has been derived from current peer-reviewed publications and expert opinion with open consultation. Methods for systematic review were NICE compliant and in accordance with the SIGN 50 Handbook; critical appraisal was applied using AGREE II. Published guidelines were used as part of the evidence base and to support expert consensus. The guidance includes recommendations for stakeholders (including prescribers) and antibiotic-specific recommendations. The clinical efficacy of different agents is critically reviewed. We found there are very few good-quality comparative randomized clinical trials to support treatment regimens, particularly for licensed older agents. Susceptibility testing of MDR GNB causing infection to guide treatment needs critical enhancements. Meropenem- or imipenem-resistant Enterobacteriaceae should have their carbapenem MICs tested urgently, and any carbapenemase class should be identified: mandatory reporting of these isolates from all anatomical sites and specimens would improve risk assessments. Broth microdilution methods should be adopted for colistin susceptibility testing. Antimicrobial stewardship programmes should be instituted in all care settings, based on resistance rates and audit of compliance with guidelines, but should be augmented by improved surveillance of outcome in Gram-negative bacteraemia, and feedback to prescribers. Local and national surveillance of antibiotic use, resistance and outcomes should be supported and antibiotic prescribing guidelines should be informed by these data. The diagnosis and treatment of both presumptive and confirmed cases of infection by GNB should be improved. This guidance, with infection control to arrest increases in MDR, should be used to improve the outcome of infections with such strains. Anticipated users include medical, scientific, nursing, antimicrobial pharmacy and paramedical staff where they can be adapted for local use

Muscle fiber conduction velocity is more affected after eccentric than concentric exercise

It has been shown that mean muscle fiber conduction velocity (CV) can be acutely impaired after eccentric exercise. However, it is not known whether this applies to other exercise modes. Therefore, the purpose of this experiment was to compare the effects of eccentric and concentric exercises on CV, and amplitude and frequency content of surface electromyography (sEMG) signals up to 24 h post-exercise. Multichannel sEMG signals were recorded from biceps brachii muscle of the exercised arm during isometric maximal voluntary contraction (MVC) and electrically evoked contractions induced by motor-point stimulation before, immediately after and 2 h after maximal eccentric (ECC group, N = 12) and concentric (CON group, N = 12) elbow flexor exercises. Isometric MVC decreased in CON by 21.7 ± 12.0% (± SD, p < 0.01) and by 30.0 ± 17.7% (p < 0.001) in ECC immediately post-exercise when compared to baseline. At 2 h post-exercise, ECC showed a reduction in isometric MVC by 24.7 ± 13.7% (p < 0.01) when compared to baseline, while no significant reduction (by 8.0 ± 17.0%, ns) was observed in CON. Similarly, reduction in CV was observed only in ECC both during the isometric MVC (from baseline of 4.16 ± 0.3 to 3.43 ± 0.4 m/s, p < 0.001) and the electrically evoked contractions (from baseline of 4.33 ± 0.4 to 3.82 ± 0.3 m/s, p < 0.001). In conclusion, eccentric exercise can induce a greater and more prolonged reduction in muscle force production capability and CV than concentric exercis

Robot rights? Towards a social-relational justification of moral consideration \ud

Should we grant rights to artificially intelligent robots? Most current and near-future robots do not meet the hard criteria set by deontological and utilitarian theory. Virtue ethics can avoid this problem with its indirect approach. However, both direct and indirect arguments for moral consideration rest on ontological features of entities, an approach which incurs several problems. In response to these difficulties, this paper taps into a different conceptual resource in order to be able to grant some degree of moral consideration to some intelligent social robots: it sketches a novel argument for moral consideration based on social relations. It is shown that to further develop this argument we need to revise our existing ontological and social-political frameworks. It is suggested that we need a social ecology, which may be developed by engaging with Western ecology and Eastern worldviews. Although this relational turn raises many difficult issues and requires more work, this paper provides a rough outline of an alternative approach to moral consideration that can assist us in shaping our relations to intelligent robots and, by extension, to all artificial and biological entities that appear to us as more than instruments for our human purpose

Predicting Distribution of Aedes Aegypti and Culex Pipiens Complex, Potential Vectors of Rift Valley Fever Virus in Relation to Disease Epidemics in East Africa.

The East African region has experienced several Rift Valley fever (RVF) outbreaks since the 1930s. The objective of this study was to identify distributions of potential disease vectors in relation to disease epidemics. Understanding disease vector potential distributions is a major concern for disease transmission dynamics. DIVERSE ECOLOGICAL NICHE MODELLING TECHNIQUES HAVE BEEN DEVELOPED FOR THIS PURPOSE: we present a maximum entropy (Maxent) approach for estimating distributions of potential RVF vectors in un-sampled areas in East Africa. We modelled the distribution of two species of mosquitoes (Aedes aegypti and Culex pipiens complex) responsible for potential maintenance and amplification of the virus, respectively. Predicted distributions of environmentally suitable areas in East Africa were based on the presence-only occurrence data derived from our entomological study in Ngorongoro District in northern Tanzania. Our model predicted potential suitable areas with high success rates of 90.9% for A. aegypti and 91.6% for C. pipiens complex. Model performance was statistically significantly better than random for both species. Most suitable sites for the two vectors were predicted in central and northwestern Tanzania with previous disease epidemics. Other important risk areas include western Lake Victoria, northern parts of Lake Malawi, and the Rift Valley region of Kenya. Findings from this study show distributions of vectors had biological and epidemiological significance in relation to disease outbreak hotspots, and hence provide guidance for the selection of sampling areas for RVF vectors during inter-epidemic periods

Climacteric Lowers Plasma Levels of Platelet-Derived Microparticles: A Pilot Study in Pre-versus Postmenopausal Women

Background: Climacteric increases the risk of thrombotic events by alteration of plasmatic coagulation. Up to now, less is known about changes in platelet-(PMP) and endothelial cell-derived microparticles (EMP). Methods: In this prospective study, plasma levels of microparticles (MP) were compared in 21 premenopausal and 19 postmenopausal women. Results: No altered numbers of total MP or EMP were measured within the study groups. However, the plasma values of CD61-exposing MP from platelets/megakaryocytes were higher in premenopausal women (5,364 x 10(6)/l, range 4,384-17,167) as compared to postmenopausal women (3,808 x 10(6)/l, range 2,009-8,850; p = 0.020). This differentiation was also significant for the subgroup of premenopausal women without hormonal contraceptives (5,364 x 10(6)/l, range 4,223-15,916; p = 0.047; n = 15). Furthermore, in premenopausal women, higher plasma levels of PMP exposing CD62P were also present as compared to postmenopausal women (288 x 10(6)/l, range 139-462, vs. 121 x 10(6)/l, range 74-284; p = 0.024). This difference was also true for CD63+ PMP levels (281 x 10(6)/l, range 182-551, vs. 137 x 10(6)/l, range 64-432; p = 0.015). Conclusion: Climacteric lowers the level of PMP but has no impact on the number of EMP in women. These data suggest that PMP and EMP do not play a significant role in enhancing the risk of thrombotic events in healthy, postmenopausal women. Copyright (C) 2012 S. Karger AG, Base