Evaluation of the environmental specificity of fluorescence in situ hybridization (FISH) using fluorescence-activated cell sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNAtargeted fluorescence in situ hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridised population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted (FACS) -recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz® method for the extraction of bacterial cells from soil.

Interactions of surfactants (edge activators) and skin penetration enhancers with liposomes

Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with ipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (Tm). Surfactants also removed the lipid pre-transition but reduced Tm and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower Tm. The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced Tm and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The Tm and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2 mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.

A time-domain probe method for three-dimensional rough surface reconstructions

The task of this paper is to develop a Time-Domain Probe Method for the reconstruction of impenetrable scatterers. The basic idea of the method is to use pulses in the time domain and the time-dependent response of the scatterer to reconstruct its location and shape. The method is based on the basic causality principle of timedependent scattering. The method is independent of the boundary condition and is applicable for limited aperture scattering data. In particular, we discuss the reconstruction of the shape of a rough surface in three dimensions from time-domain measurements of the scattered field. In practise, measurement data is collected where the incident field is given by a pulse. We formulate the time-domain fieeld reconstruction problem equivalently via frequency-domain integral equations or via a retarded boundary integral equation based on results of Bamberger, Ha-Duong, Lubich. In contrast to pure frequency domain methods here we use a time-domain characterization of the unknown shape for its reconstruction. Our paper will describe the Time-Domain Probe Method and relate it to previous frequency-domain approaches on sampling and probe methods by Colton, Kirsch, Ikehata, Potthast, Luke, Sylvester et al. The approach significantly extends recent work of Chandler-Wilde and Lines (2005) and Luke and Potthast (2006) on the timedomain point source method. We provide a complete convergence analysis for the method for the rough surface scattering case and provide numerical simulations and examples.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.

An iterative contractive framework for probe methods: LASSO

We present a new iterative approach called Line Adaptation for the Singular Sources Objective (LASSO) to object or shape reconstruction based on the singular sources method (or probe method) for the reconstruction of scatterers from the far-field pattern of scattered acoustic or electromagnetic waves. The scheme is based on the construction of an indicator function given by the scattered field for incident point sources in its source point from the given far-field patterns for plane waves. The indicator function is then used to drive the contraction of a surface which surrounds the unknown scatterers. A stopping criterion for those parts of the surfaces that touch the unknown scatterers is formulated. A splitting approach for the contracting surfaces is formulated, such that scatterers consisting of several separate components can be reconstructed. Convergence of the scheme is shown, and its feasibility is demonstrated using a numerical study with several examples.

A twenty-year independent record of sea surface temperature for climate from Along-track Scanning Radiometers

A new record of sea surface temperature (SST) for climate applications is described. This record provides independent corroboration of global variations estimated from SST measurements made in situ. Infrared imagery from Along-Track Scanning Radiometers (ATSRs) is used to create a 20 year time series of SST at 0.1° latitude-longitude resolution, in the ATSR Reprocessing for Climate (ARC) project. A very high degree of independence of in situ measurements is achieved via physics-based techniques. Skin SST and SST estimated for 20 cm depth are provided, with grid cell uncertainty estimates. Comparison with in situ data sets establishes that ARC SSTs generally have bias of order 0.1 K or smaller. The precision of the ARC SSTs is 0.14 K during 2003 to 2009, from three-way error analysis. Over the period 1994 to 2010, ARC SSTs are stable, with better than 95% confidence, to within 0.005 K yr−1(demonstrated for tropical regions). The data set appears useful for cleanly quantifying interannual variability in SST and major SST anomalies. The ARC SST global anomaly time series is compared to the in situ-based Hadley Centre SST data set version 3 (HadSST3). Within known uncertainties in bias adjustments applied to in situ measurements, the independent ARC record and HadSST3 present the same variations in global marine temperature since 1996. Since the in situ observing system evolved significantly in its mix of measurement platforms and techniques over this period, ARC SSTs provide an important corroboration that HadSST3 accurately represents recent variability and change in this essential climate variable.

A reprocessing for climate of sea surface temperature from the Along-Track Scanning Radiometers: initial validation, accounting for skin and diurnal variability

An initial validation of the Along Track Scanning Radiometer (ATSR) Reprocessing for Climate (ARC) retrievals of sea surface temperature (SST) is presented. ATSR-2 and Advanced ATSR (AATSR) SST estimates are compared to drifting buoy and moored buoy observations over the period 1995 to 2008. The primary ATSR estimates are of skin SST, whereas buoys measure SST below the surface. Adjustment is therefore made for the skin effect, for diurnal stratification and for differences in buoy–satellite observation time. With such adjustments, satellite-in situ differences are consistent between day and night within ~ 0.01 K. Satellite-in situ differences are correlated with differences in observation time, because of the diurnal warming and cooling of the ocean. The data are used to verify the average behaviour of physical and empirical models of the warming/cooling rates. Systematic differences between adjusted AATSR and in-situ SSTs against latitude, total column water vapour (TCWV), and wind speed are less than 0.1 K, for all except the most extreme cases (TCWV < 5 kg m–2, TCWV > 60 kg m–2). For all types of retrieval except the nadir-only two-channel (N2), regional biases are less than 0.1 K for 80% of the ocean. Global comparison against drifting buoys shows night time dual-view two-channel (D2) SSTs are warm by 0.06 ± 0.23 K and dual-view three-channel (D3) SSTs are warm by 0.06 ± 0.21 K (day-time D2: 0.07 ± 0.23 K). Nadir-only results are N2: 0.03 ± 0.33 K and N3: 0.03 ± 0.19 K showing the improved inter-algorithm consistency to ~ 0.02 K. This represents a marked improvement from the existing operational retrieval algorithms for which inter-algorithm inconsistency is > 0.5 K. Comparison against tropical moored buoys, which are more accurate than drifting buoys, gives lower error estimates (N3: 0.02 ± 0.13 K, D2: 0.03 ± 0.18 K). Comparable results are obtained for ATSR-2, except that the ATSR-2 SSTs are around 0.1 K warm compared to AATSR

A reprocessing for climate of sea surface temperature from the Along-Track Scanning Radiometers: a new retrieval scheme

We present a new coefficient-based retrieval scheme for estimation of sea surface temperature (SST) from the Along Track Scanning Radiometer (ATSR) instruments. The new coefficients are banded by total column water vapour (TCWV), obtained from numerical weather prediction analyses. TCWV banding reduces simulated regional retrieval biases to < 0.1 K compared to biases ~ 0.2 K for global coefficients. Further, detailed treatment of the instrumental viewing geometry reduces simulated view-angle related biases from ~ 0.1 K down to < 0.005 K for dual-view retrievals using channels at 11 and 12 μm. A novel analysis of trade-offs related to the assumed noise level when defining coefficients is undertaken, and we conclude that adding a small nominal level of noise (0.01 K) is optimal for our purposes. When applied to ATSR observations, some inter-algorithm biases appear as TCWV-related differences in SSTs estimated from different channel combinations. The final step in coefficient determination is to adjust the offset coefficient in each TCWV band to match results from a reference algorithm. This reference uses the dual-view observations of 3.7 and 11 μm. The adjustment is independent of in situ measurements, preserving independence of the retrievals. The choice of reference is partly motivated by uncertainty in the calibration of the 12 μm of Advanced ATSR. Lastly, we model the sensitivities of the new retrievals to changes to TCWV and changes in true SST, confirming that dual-view SSTs are most appropriate for climatological applications

A reprocessing for climate of sea surface temperature from the Along-Track Scanning Radiometers: basis in radiative transfer

We present new radiative transfer simulations to support determination of sea surface temperature (SST) from Along Track Scanning Radiometer (ATSR) imagery. The simulations are to be used within the ATSR Reprocessing for Climate project. The simulations are based on the “Reference Forward Model” line-by-line model linked with a sea surface emissivity model that accounts for wind speed and temperature, and with a discrete ordinates scattering model (DISORT). Input to the forward model is a revised atmospheric profile dataset, based on full resolution ERA-40, with a wider range of high-latitude profiles to address known retrieval biases in those regions. Analysis of the radiative impacts of atmospheric trace gases shows that geographical and temporal variation of N2O, CH4, HNO3, and CFC-11 and CFC-12 have effects of order 0.05, 0.2, 0.1 K on the 3.7, 11, 12 μm channels respectively. In addition several trace gases, neglected in previous studies, are included using fixed profiles contributing ~ 0.04 K to top-of-atmosphere BTs. Comparison against observations for ATSR2 and AATSR indicates that forward model biases have been reduced from 0.2 to 0.5 K for previous simulations to ~ 0.1 K.

An infrared desert dust index for the Along-Track Scanning Radiometers

A new aerosol index for the Along-Track Scanning Radiometers (ATSRs) is presented that provides a means to detect desert dust contamination in infrared SST retrievals. The ATSR Saharan dust index (ASDI) utilises only the thermal infrared channels and may therefore be applied consistently to the entire ATSR data record (1991 to present), for both day time and night time observations. The derivation of the ASDI is based on a principal component (PC) analysis (PCA) of two unique pairs of channel brightness temperature differences (BTDs). In 2-D space (i.e. BTD vs BTD), it is found that the loci of data unaffected by aerosol are confined to a single axis of variability. In contrast, the loci of aerosol-contaminated data fall off-axis, shifting in a direction that is approximately orthogonal to the clear-sky axis. The ASDI is therefore defined to be the second PC, where the first PC accounts for the clear-sky variability. The primary ASDI utilises the ATSR nadir and forward-view observations at 11 and 12 μm (ASDI2). A secondary, three-channel nadir-only ASDI (ASDI3) is also defined for situations where data from the forward view are not available. Empirical and theoretical analyses suggest that ASDI is well correlated with aerosol optical depth (AOD: correlation r is typically > 0.7) and provides an effective tool for detecting desert mineral dust. Overall, ASDI2 is found to be more effective than ASDI3, with the latter being sensitive only to very high dust loading. In addition, use of ASDI3 is confined to night time observations as it relies on data from the 3.7 μm channel, which is sensitive to reflected solar radiation. This highlights the benefits of having data from both a nadir- and a forward-view for this particular approach to aerosol detection.

3D cloud reconstructions: evaluation of scanning radar scan strategy with a view to surface shortwave radiation closure

The ability of six scanning cloud radar scan strategies to reconstruct cumulus cloud fields for radiation study is assessed. Utilizing snapshots of clean and polluted cloud fields from large eddy simulations, an analysis is undertaken of error in both the liquid water path and monochromatic downwelling surface irradiance at 870 nm of the reconstructed cloud fields. Error introduced by radar sensitivity, choice of radar scan strategy, retrieval of liquid water content (LWC), and reconstruction scheme is explored. Given an in␣nitely sensitive radar and perfect LWC retrieval, domain average surface irradiance biases are typically less than 3 W m␣2 ␣m␣1, corresponding to 5–10% of the cloud radiative effect (CRE). However, when using a realistic radar sensitivity of ␣37.5 dBZ at 1 km, optically thin areas and edges of clouds are dif␣cult to detect due to their low radar re-ectivity; in clean conditions, overestimates are of order 10 W m␣2 ␣m␣1 (~20% of the CRE), but in polluted conditions, where the droplets are smaller, this increases to 10–26 W m␣2 ␣m␣1 (~40–100% of the CRE). Drizzle drops are also problematic; if treated as cloud droplets, reconstructions are poor, leading to large underestimates of 20–46 W m␣2 ␣m␣1 in domain average surface irradiance (~40–80% of the CRE). Nevertheless, a synergistic retrieval approach combining the detailed cloud structure obtained from scanning radar with the droplet-size information and location of cloud base gained from other instruments would potentially make accurate solar radiative transfer calculations in broken cloud possible for the first time.

Deriving a sea surface temperature record suitable for climate change research from the along-track scanning radiometers

We describe the approach to be adopted for a major new initiative to derive a homogeneous record of sea surface temperature for 1991–2007 from the observations of the series of three along-track scanning radiometers (ATSRs). This initiative is called (A)RC: (Advanced) ATSR Re-analysis for Climate. The main objectives are to reduce regional biases in retrieved sea surface temperature (SST) to less than 0.1 K for all global oceans, while creating a very homogenous record that is stable in time to within 0.05 K decade−1, with maximum independence of the record from existing analyses of SST used in climate change research. If these stringent targets are achieved, this record will enable significantly improved estimates of surface temperature trends and variability of sufficient quality to advance questions of climate change attribution, climate sensitivity and historical reconstruction of surface temperature changes. The approach includes development of new, consistent estimators for SST for each of the ATSRs, and detailed analysis of overlap periods. Novel aspects of the approach include generation of multiple versions of the record using alternative channel sets and cloud detection techniques, to assess for the first time the effect of such choices. There will be extensive effort in quality control, validation and analysis of the impact on climate SST data sets. Evidence for the plausibility of the 0.1 K target for systematic error is reviewed, as is the need for alternative cloud screening methods in this context.

Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe- set, consisting of up to 16 probe-pairs. Signal intensities across probe- pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.

Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ webcite and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Scanning tunneling microscopy and molecular dynamics study of the Li2TiO3(001) surface

We have investigated the (001) surface structure of lithium titanate (Li2TiO3) using auger electron spectroscopy (AES), low-energy electron diffraction (LEED), and scanning tunneling microscopy (STM). Li2TiO3 is a potential fusion reactor blanket material. After annealing at 1200 K, LEED demonstrated that the Li2TiO3(001) surface was well ordered and not reconstructed. STM imaging showed that terraces are separated in height by about 0.3 nm suggesting a single termination layer. Moreover, hexagonal patterns with a periodicity of ∼0.4 nm are observed. On the basis of molecular dynamics (MD) simulations, these are interpreted as a dynamic arrangement of Li atoms.