269 research outputs found
An ensemble of AMIP simulations with prescribed land surface temperatures
General circulation models (GCMs) are routinely run under
Atmospheric Modelling Intercomparison Project (AMIP) conditions with
prescribed sea surface temperatures (SSTs) and sea ice concentrations (SICs)
from observations. These AMIP simulations are often used to evaluate the role
of the land and/or atmosphere in causing the development of systematic errors
in such GCMs. Extensions to the original AMIP experiment have also been
developed to evaluate the response of the global climate to increased SSTs
(prescribed) and carbon dioxide (CO2) as part of the Cloud Feedback
Model Intercomparison Project (CFMIP). None of these international modelling
initiatives has undertaken a set of experiments where the land conditions are
also prescribed, which is the focus of the work presented in this paper.
Experiments are performed initially with freely varying land conditions
(surface temperature, and soil temperature and moisture) under five different
configurations (AMIP, AMIP with uniform 4 K added to SSTs, AMIP SST with
quadrupled CO2, AMIP SST and quadrupled CO2 without the plant
stomata response, and increasing the solar constant by 3.3 %). Then, the
land surface temperatures from the free land experiments are used to perform
a set of AMIP prescribed land (PL) simulations, which are evaluated
against their free land counterparts. The PL simulations agree well with the
free land experiments, which indicates that the land surface is prescribed in
a way that is consistent with the original free land configuration. Further
experiments are also performed with different combinations of SSTs,
CO2 concentrations, solar constant and land conditions. For example,
SST and land conditions are used from the AMIP simulation with quadrupled
CO2 in order to simulate the atmospheric response to increased
CO2 concentrations without the surface temperature changing. The
results of all these experiments have been made publicly available for
further analysis. The main aims of this paper are to provide a description of
the method used and an initial validation of these AMIP prescribed land
experiments.</p
The Use of Silver Nitrate Staining and Backscattered Electron Imaging to Visualize Nematode Sensory Structures
Parasitic nematodes of the species Cosmocercoides variabilis were stained with silver nitrate and examined with backscattered electron imaging (BEI). Sensory papillae were selectively highlighted in backscatter images. Silver stain deposited on papillae was located on the papillary surface as well as on the underlying dendritic process. Portions of the body cuticle were also stained. Some cuticular staining was attributed to non-specific deposition of silver but, consistent patterns of cuticular staining were noted in the anterior and posterior regions. This observation suggests that some staining of the cuticle was specific. Results of this preliminary work suggest that BEI is a technique useful to the study of nematode form
Using E. coli NfsA as a model to improve our understanding of enzyme engineering
There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to the difficulty for step-wise evolutionary approaches in effectively retaining mutations that are beneficial in combination with one another, but on an individual basis are neutral or deleterious (i.e., exhibit positive epistasis). We sought to determine whether a superior enzyme identified using a simultaneous mass site directed mutagenesis approach could have been identified using a step-wise approach. We conducted simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 394 million unique variants. We then applied a powerful positive selection using chloramphenicol which is toxic to E. coli but can be detoxified via nitro-reduction. This has enabled us to recover a diverse range of highly active nitroreductase variants. For two of the most active variants, we have created all possible combinations of single mutations. This allowed us to examine whether a step-wise mutagenesis pathway could have also yielded these enzymes. As anticipated, we identified complex epistatic interactions between residues in these enzyme variants. We have also investigated the âblack-boxâ effect of enzyme engineering, examining the consequences that evolving NfsA towards one specialist activity had on the other promiscuous activities of NfsA. Variants generated in this study have also had practical applications, in particular for targeted cell ablation in zebrafish. We have identified NfsA variants that are highly active with nil-bystander prodrugs that can selectively ablate nitroreductase expressing cells without harm to adjacent cells. In ongoing work, our lead variants are being evaluated for their utility in transgenic zebrafish models of degenerative disease
An intra-neural microstimulation system for ultra-high field magnetic resonance imaging and magnetoencephalography.
BACKGROUND: Intra-neural microstimulation (INMS) is a technique that allows the precise delivery of low-current electrical pulses into human peripheral nerves. Single unit INMS can be used to stimulate individual afferent nerve fibres during microneurography. Combining this with neuroimaging allows the unique monitoring of central nervous system activation in response to unitary, controlled tactile input, with functional magnetic resonance imaging (fMRI) providing exquisite spatial localisation of brain activity and magnetoencephalography (MEG) high temporal resolution. NEW METHOD: INMS systems suitable for use within electrophysiology laboratories have been available for many years. We describe an INMS system specifically designed to provide compatibility with both ultra-high field (7T) fMRI and MEG. Numerous technical and safety issues are addressed. The system is fully analogue, allowing for arbitrary frequency and amplitude INMS stimulation. RESULTS: Unitary recordings obtained within both the MRI and MEG screened-room environments are comparable with those obtained in 'clean' electrophysiology recording environments. Single unit INMS (current <7ÎŒA, 200ÎŒs pulses) of individual mechanoreceptive afferents produces appropriate and robust responses during fMRI and MEG. COMPARISON WITH EXISTING METHOD(S): This custom-built MRI- and MEG-compatible stimulator overcomes issues with existing INMS approaches; it allows well-controlled switching between recording and stimulus mode, prevents electrical shocks because of long cable lengths, permits unlimited patterns of stimulation, and provides a system with improved work-flow and participant comfort. CONCLUSIONS: We demonstrate that the requirements for an INMS-integrated system, which can be used with both fMRI and MEG imaging systems, have been fully met
Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation
There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to âjumpâ straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall
Effective radiative forcing in a GCM with fixed surface temperatures
Effective radiative forcing (ERF) is evaluated in the ACCESS1.0 General Circulation Model (GCM) with fixed land and seaâsurfaceâtemperatures as well as seaâice. The 4xCO2 ERF is 8.0 Wmâ2. In contrast, a typical ERF experiment with only fixed seaâsurfaceâtemperatures (SST) and seaâice gives rise to an ERF of only 7.0 Wmâ2. This difference arises due to the influence of land warming in the commonly used fixedâSST ERF experimental design, which results in: (i) increased emission of longwave radiation to space from the land surface (â0.45 Wmâ2) and troposphere (â0.90 Wmâ2), (ii) reduced land snowâcover and albedo (+0.17 Wmâ2), (iii) increased waterâvapour (+0.49 Wmâ2), and (iv) a cloud adjustment (â0.26 Wmâ2) due to reduced stability and cloudiness over land (positive ERF) counteracted by increased lower tropospheric stability and marine cloudiness over oceans (negative ERF) . The sum of these radiative adjustments to land warming is to reduce the 4xCO2 ERF in fixedâSST experiments by âŒ1.0 Wmâ2. CO2 stomatal effects are quantified and found to contribute just over half of the land warming effect and adjustments in the fixedâSST ERF experimental design in this model. The basic physical mechanisms in response to land warming are confirmed in a solar ERF experiment. We test various methods that have been proposed to account for land warming in fixedâSST ERFs against our GCM results and discuss their strengths and weaknesses
Organized crime and preventive justice
By comparison with the prevention of terrorism, the prevention of acts of organizedcrime might be thought easier to conceptualize precisely and less controversial to legislate against and police. This impression is correct up to a point, because it is possible to arrive at some general characteristics of organized crime, and because legislation against it is not obviously bedevilled by the risk of violating civil or political rights, as in the case of terrorism. But there is a significant residue of legal, moral and political difficulty: legislation against organized crime is hard to make effective; the harm of organized crime is not uniform, and so some preventive legislation seems too sweeping and potentially unjust. More fundamentally, the scale and rewards of organized crime are often dependent on mass public participation in markets for proscribed goods, which may point to a hidden public consensus in favour of some of what is criminalized. Preventive policing and legislation in both areas, then, are less easily justified than first appears
C-tactile afferent stimulating touch carries a positive affective value
The rewarding sensation of touch in affiliative interactions is hypothesized to be underpinned by a specialized system of nerve fibers called C-Tactile afferents (CTs), which respond optimally to slowly moving, gentle touch, typical of a caress. However, empirical evidence to support the theory that CTs encode socially relevant, rewarding tactile information in humans is currently limited. While in healthy participants, touch applied at CT optimal velocities (1-10cm/sec) is reliably rated as subjectively pleasant, neuronopathy patients lacking large myelinated afferents, but with intact C-fibres, report that the conscious sensation elicited by stimulation of CTs is rather vague. Given this weak perceptual impact the value of self-report measures for assessing the specific affective value of CT activating touch appears limited. Therefore, we combined subjective ratings of touch pleasantness with implicit measures of affective state (facial electromyography) and autonomic arousal (heart rate) to determine whether CT activation carries a positive affective value. We recorded the activity of two key emotion-relevant facial muscle sites (zygomaticus majorâsmile muscle, positive affect & corrugator superciliiâfrown muscle, negative affect) while participants evaluated the pleasantness of experimenter administered stroking touch, delivered using a soft brush, at two velocities (CT optimal 3cm/sec & CT non-optimal 30cm/sec), on two skin sites (CT innervated forearm & non-CT innervated palm). On both sites, 3cm/sec stroking touch was rated as more pleasant and produced greater heart rate deceleration than 30cm/sec stimulation. However, neither self-report ratings nor heart rate responses discriminated stimulation on the CT innervated arm from stroking of the non-CT innervated palm. In contrast, significantly greater activation of the zygomaticus major (smiling muscle) was seen specifically to CT optimal, 3cm/sec, stroking on the forearm in comparison to all other stimuli. These results offer the first empirical evidence in humans that tactile stimulation that optimally activates CTs carries a positive affective valence that can be measured implicitly
Accessing bacterial dark matter for improved enzyme discovery and engineering
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Restoring tumour selectivity of the bioreductive prodrug pr-104 by developing an analogue resistant to aerobic metabolism by human aldo-keto reductase 1c3
PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes âoff-targetâ two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC(50) ratios of 9 to 145, remains a substrate for POR and triggers ÎłH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5â3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials
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