Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

Assimilation of RAPID array observations into an ocean model

Experiments assimilating the RAPID dataset of deep temperature and salinity profiles at 26.5°N on the western and eastern Atlantic boundaries into a 1° global NEMO ocean model have been performed. The meridional overturning circulation (MOC) is then assessed against the transports calculated directly from observations. The best initialization found for this short period was obtained by assimilating the EN3 upper-ocean hydrography database prior to 2004, after which different methods of assimilating 5-day average RAPID profiles at the western boundary were tested. The model MOC is strengthened by ∼ 2 Sv giving closer agreement with the RAPID array transports, when the western boundary profiles are assimilated only below 900 m (the approximate depth of the Florida Straits, which are not well resolved) and when the T,S observations are spread meridionally from 10 to 35°N along the deep western boundary. The use of boundary-focused covariances has the largest impact on the assimilation results, otherwise using more conventional Gaussian covariances has a very local impact on the MOC at 26°N with strong adverse impacts on the MOC stream function at higher and lower latitudes. Even using boundary-focused covariances only enables the MOC to be strengthened for ∼ 2 years, after which the increased transport of warm waters leads to a negative feedback on water formation in the subpolar gyre which then reduces the MOC. This negative feedback can be mitigated if EN3 hydrography data continue to be assimilated along with the RAPID array boundary data. Copyright © 2012 Royal Meteorological Society and Crown in the right of Canada.

Keeping the world at hand: rapid visuomotor processing for hand-object interactions

The existence of hand-centred visual processing has long been established in the macaque premotor cortex. These hand-centred mechanisms have been thought to play some general role in the sensory guidance of movements towards objects, or, more recently, in the sensory guidance of object avoidance movements. We suggest that these hand-centred mechanisms play a specific and prominent role in the rapid selection and control of manual actions following sudden changes in the properties of the objects relevant for hand-object interactions. We discuss recent anatomical and physiological evidence from human and non-human primates, which indicates the existence of rapid processing of visual information for hand-object interactions. This new evidence demonstrates how several stages of the hierarchical visual processing system may be bypassed, feeding the motor system with hand-related visual inputs within just 70 ms following a sudden event. This time window is early enough, and this processing rapid enough, to allow the generation and control of rapid hand-centred avoidance and acquisitive actions, for aversive and desired objects, respectively

Aerosol contribution to the rapid warming of near-term climate under RCP 2.6

The importance of aerosol emissions for near term climate projections is investigated by analysing simulations with the HadGEM2-ES model under two different emissions scenarios: RCP2.6 and RCP4.5. It is shown that the near term warming projected under RCP2.6 is greater than under RCP4.5, even though the greenhouse gas forcing is lower. Rapid and substantial reductions in sulphate aerosol emissions due to a reduction of coal burning in RCP2.6 lead to a reduction in the negative shortwave forcing due to aerosol direct and indirect effects. Indirect effects play an important role over the northern hemisphere oceans, especially the subtropical northeastern Pacific where an anomaly of 5-10\,Wm$^{-2}$ develops. The pattern of surface temperature change is consistent with the expected response to this surface radiation anomaly, whilst also exhibiting features that reflect redistribution of energy, and feedbacks, within the climate system. These results demonstrate the importance of aerosol emissions as a key source of uncertainty in near term projections of global and regional climate.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Initialized decadal predictions of the rapid warming of the North Atlantic Ocean in the mid 1990s

In the mid 1990s the North Atlantic subpolar gyre (SPG) warmed rapidly, with sea surface temperatures (SST) increasing by 1°C in just a few years. By examining initialized hindcasts made with the UK Met Office Decadal Prediction System (DePreSys), it is shown that the warming could have been predicted. Conversely, hindcasts that only consider changes in radiative forcings are not able to capture the rapid warming. Heat budget analysis shows that the success of the DePreSys hindcasts is due to the initialization of anomalously strong northward ocean heat transport. Furthermore, it is found that initializing a strong Atlantic circulation, and in particular a strong Atlantic Meridional Overturning Circulation, is key for successful predictions. Finally, we show that DePreSys is able to predict significant changes in SST and other surface climate variables related to the North Atlantic warming.

Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype typhimurium DT104 isolates

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC --> AAC) mutation, an Asp-87-to-Gly (GAC --> GGC) mutation, and a Ser-83-to-Phe (TCC --> TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC --> TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC --> TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Atlantic meridional heat transports in two ocean reanalyes evaluated against the RAPID array

The Atlantic meridional overturning circulation in two versions of the NEMO ¼° global ocean model has been compared with the RAPID transport array at 26oN. Both model versions reproduce the mean MOC strength well although the Florida Straits flows differ because the pathway of the Gulf Stream is not strongly constrained at this resolution. Both models however have a mean meridional heat transport of 1.07PW, much lower than the 1.35PW from RAPID observations in Apr04-Oct07. Much of the heat transport discrepancy is due to lower transports in summer across the MidOcean (Bahamas-Africa) section, due to stronger southward geostrophic flows in the top 100m where the water is warmest. Seasonal thermocline changes increase temperature differences across the basin driving stronger geostrophic shear, but this effect is much weaker in the top 100m of the RAPID velocity data. The effect accounts for a reduction of 1.1Sv in MOC and 0.1PW in heat transports. The rest of the discrepancy comes from lower Ekman transports from using ERAInterim winds instead of QuikSCAT, a smaller zonally-varying “Eddy” heat transport component, estimated from repeat XBT sections in the observations, and the southward throughflow in the model. Other differences in depth structure of the model MOC and RAPID observations are described but have much less impact on heat transports.

The relationship between rapid naming and word spelling in English

A study of the concurrent relationships between naming speed, phonological awareness and spelling ability in 146 children in Year 3 and 4 of state funded school in SE England (equivalent to US Grades 2 and 3) is reported. Seventy-two children identified as having normal phonological awareness but reduced rapid automatized naming (RAN) performance (1 standard deviation below the mean) participated in the study. A group of 74 children were further identified. These children were matched on phonological awareness, verbal and non verbal IQ, and visual acuity but all members of this group showed normal rapid automatized naming performance. Rapid automatized naming made a significant unique contribution to spelling performance. Further analyses showed that the participants with low naming performance were significantly poorer spellers overall and had a specific difficulty in spelling irregular words. The findings support the view that rapid automatized naming may be indexing processes that are implicated in the establishment of fully specified orthographic representations.

Rapid-onset opioids and the management of episodic pain

This is the fourth in a short series of articles that focus on what GPs should consider when monitoring and prescribing specialist-initiated palliative-care medicines. Here, the authors summarise the key issues around the use of apid onset opioids in palliative care.

The resting-state neurovascular coupling relationship: rapid changes in spontaneous neural activity in the somatosensory cortex are associated with haemodynamic fluctuations that resemble stimulus-evoked haemodynamics

Although promise exists for patterns of resting-state blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) brain connectivity to be used as biomarkers of early brain pathology, a full understanding of the nature of the relationship between neural activity and spontaneous fMRI BOLD fluctuations is required before such data can be correctly interpreted. To investigate this issue, we combined electrophysiological recordings of rapid changes in multi-laminar local field potentials from the somatosensory cortex of anaesthetized rats with concurrent two-dimensional optical imaging spectroscopy measurements of resting-state haemodynamics that underlie fluctuations in the BOLD fMRI signal. After neural ‘events’ were identified, their time points served to indicate the start of an epoch in the accompanying haemodynamic fluctuations. Multiple epochs for both neural ‘events’ and the accompanying haemodynamic fluctuations were averaged. We found that the averaged epochs of resting-state haemodynamic fluctuations taken after neural ‘events’ closely resembled the temporal profile of stimulus-evoked cortical haemodynamics. Furthermore, we were able to demonstrate that averaged epochs of resting-state haemodynamic fluctuations resembling the temporal profile of stimulus-evoked haemodynamics could also be found after peaks in neural activity filtered into specific electroencephalographic frequency bands (theta, alpha, beta, and gamma). This technique allows investigation of resting-state neurovascular coupling using methodologies that are directly comparable to that developed for investigating stimulus-evoked neurovascular responses.

Infra-red laser ablative micromachining of parylene-C on SiO2substrates for rapid prototyping, high yield, human neuronal cell patterning

Cell patterning commonly employs photolithographic methods for the micro fabrication of structures on silicon chips. These require expensive photo-mask development and complex photolithographic processing. Laser based patterning of cells has been studied in vitro and laser ablation of polymers is an active area of research promising high aspect ratios. This paper disseminates how 800 nm femtosecond infrared (IR) laser radiation can be successfully used to perform laser ablative micromachining of parylene-C on SiO2 substrates for the patterning of human hNT astrocytes (derived from the human teratocarcinoma cell line (hNT)) whilst 248 nm nanosecond ultra-violet laser radiation produces photo-oxidization of the parylene-C and destroys cell patterning. In this work, we report the laser ablation methods used and the ablation characteristics of parylene-C for IR pulse fluences. Results follow that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells. We disseminate the variation in yield of patterned hNT astrocytes on parylene-C with laser pulse spacing, pulse number, pulse fluence and parylene-C strip width. The findings demonstrate how laser ablative micromachining of parylene-C on SiO2 substrates can offer an accessible alternative for rapid prototyping, high yield cell patterning with broad application to multi-electrode arrays, cellular micro-arrays and microfluidics.

Oxidised low-density lipoproteins induce rapid platelet activation and shape change through tyrosine kinase and Rho kinase-signaling pathways

Oxidized low-density lipoproteins (oxLDL) generated in the hyperlipidemic state may contribute to unregulated platelet activation during thrombosis. Although the ability of oxLDL to activate platelets is established, the underlying signaling mechanisms remain obscure. Weshow that oxLDL stimulate platelet activation through phosphorylation of the regulatory light chains of the contractile protein myosin IIa (MLC). oxLDL, but not native LDL, induced shape change, spreading, and phosphorylation of MLC (serine 19) through a pathway that was ablated under conditions that blocked CD36 ligation or inhibited Src kinases, suggesting a tyrosine kinase–dependent mechanism. Consistent with this, oxLDL induced tyrosine phosphorylation of a number of proteins including Syk and phospholipase C g2. Inhibition of Syk, Ca21 mobilization, and MLC kinase (MLCK) only partially inhibited MLC phosphorylation, suggesting the presence of a second pathway. oxLDL activated RhoA and RhoA kinase (ROCK) to induce inhibitory phosphorylation of MLC phosphatase (MLCP). Moreover, inhibition of Src kinases prevented the activation of RhoA and ROCK, indicating that oxLDL regulates contractile signaling through a tyrosine kinase–dependent pathway that induces MLC phosphorylation through the dual activation of MLCK and inhibition of MLCP. These data reveal new signaling events downstream of CD36 that are critical in promoting platelet aggregation by oxLDL.