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.

A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

A survey on sampling and probe methods for inverse problems

The goal of the review is to provide a state-of-the-art survey on sampling and probe methods for the solution of inverse problems. Further, a configuration approach to some of the problems will be presented. We study the concepts and analytical results for several recent sampling and probe methods. We will give an introduction to the basic idea behind each method using a simple model problem and then provide some general formulation in terms of particular configurations to study the range of the arguments which are used to set up the method. This provides a novel way to present the algorithms and the analytic arguments for their investigation in a variety of different settings. In detail we investigate the probe method (Ikehata), linear sampling method (Colton-Kirsch) and the factorization method (Kirsch), singular sources Method (Potthast), no response test (Luke-Potthast), range test (Kusiak, Potthast and Sylvester) and the enclosure method (Ikehata) for the solution of inverse acoustic and electromagnetic scattering problems. The main ideas, approaches and convergence results of the methods are presented. For each method, we provide a historical survey about applications to different situations.

A numerical study of the probe method

The goal of this work is the numerical realization of the probe method suggested by Ikehata for the detection of an obstacle D in inverse scattering. The main idea of the method is to use probes in the form of point source (., z) with source point z to define an indicator function (I) over cap (z) which can be reconstructed from Cauchy data or far. eld data. The indicator function boolean AND (I) over cap (z) can be shown to blow off when the source point z tends to the boundary aD, and this behavior can be used to find D. To study the feasibility of the probe method we will use two equivalent formulations of the indicator function. We will carry out the numerical realization of the functional and show reconstructions of a sound-soft obstacle.

Unification of the probe and singular sources methods for the inverse boundary value problem by the no-response test

In this article, we use the no-response test idea, introduced in Luke and Potthast (2003) and Potthast (Preprint) and the inverse obstacle problem, to identify the interface of the discontinuity of the coefficient gamma of the equation del (.) gamma(x)del + c(x) with piecewise regular gamma and bounded function c(x). We use infinitely many Cauchy data as measurement and give a reconstructive method to localize the interface. We will base this multiwave version of the no-response test on two different proofs. The first one contains a pointwise estimate as used by the singular sources method. The second one is built on an energy (or an integral) estimate which is the basis of the probe method. As a conclusion of this, the probe and the singular sources methods are equivalent regarding their convergence and the no-response test can be seen as a unified framework for these methods. As a further contribution, we provide a formula to reconstruct the values of the jump of gamma(x), x is an element of partial derivative D at the boundary. A second consequence of this formula is that the blow-up rate of the indicator functions of the probe and singular sources methods at the interface is given by the order of the singularity of the fundamental solution.

Eddy current testing system with scanning probe head having parallel and normal sensing coils

An eddy current testing system consists of a multi-sensor probe, a computer and a special expansion card and software for data-collection and analysis. The probe incorporates an excitation coil, and sensor coils; at least one sensor coil is a lateral current-normal coil and at least one is a current perturbation coil.

Eddy current testing program with scanning probe head having parallel and normal sensing coils

An eddy current testing system consists of a multi-sensor probe, computer and a special expansion card and software for data collection and analysis. The probe incorporates an excitation coil, and sensor coils; at least one sensor coil is a lateral current-normal coil and at least one is a current perturbation coil.

Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study

The interaction between four flavonoids (catechin, epicatechin, rutin and quercetin) and bovine serum albumin (BSA) was investigated using tryptophan fluorescence quenching. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between the flavonoids and BSA. The binding affinity was found to be strongest for quercetin, and ranked in the order quercetin>rutin>epicatechin=catechin. The pH in the range of 5 to 7.4 does not affect significantly (p<0.05) the association of rutin, epicatechin and catechin with BSA, but quercetin exhibited a stronger affinity at pH 7.4 than at lower pH (p<0.05). Quercetin has a total quenching effect on BSA tryptophan fluorescence at a molar ratio of 10:1 and rutin at approximately 25:1. However, epicatechin and catechin did not fully quench tryptophan fluorescence over the concentration range studied. Furthermore, the data suggested that the association between flavonoids and BSA did not change molecular conformation of BSA and that hydrogen bonding, ionic and hydrophobic interaction are equally important driving forces for protein-flavonoid association.

Chlorophyll fluorescence technique as a rapid diagnostic test of the effects of the photosynthetic inhibitor chlorotoluron on two winter wheat cultivars

A modified chlorophyll fluorescence technique was evaluated as a rapid diagnostic test of the susceptibility of wheat cultivars to chlorotoluron. Two winter wheat cultivars (Maris Huntsman and Mercia) exhibited differential response to the herbicide. All of the parameters of chlorophyll fluorescence examined were strongly influenced by herbicide concentration. Additionally, the procedure adopted here for the examination of winter wheat cultivar sensitivity to herbicide indicated that the area above the fluorescence induction curve and the ratio F-V/F-M are appropriate chlorophyll fluorescence parameters for detection of differential herbicide response between wheat cultivars. The potential use of this technique as an alternative to traditional methods of screening new winter wheat cultivars for their response to photosynthetic inhibitor herbicide is demonstrated here.

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfprnut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT3, dsRedT4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

The effects of temperature and light integral on early vegetative growth and chlorophyll fluorescence of four contrasting genotypes of cacao (Theobroma cacao)

The effect of temperature on early vegetative growth, leaf chlorophyll fluorescence and chlorophyll content was examined on four genotypes of cacao (Amelonado, AMAZ 15–15, SCA 6 and SPEC 54/1). A controlled environment glasshouse was used to simulate the temperature conditions of three cacao-growing regions (Bahia, Brazil; Tafo, Ghana and Lower Perak, Malaysia) over the course of a year. Base temperatures calculated from increments in main stem growth varied from 18.6°C for AMAZ 15/15 to 20.8°C for SPEC 54/1. Temporal variation in Fv/Fm observed for two of the clones (SCA 6 and SPEC 54/1) in two of the compartments were correlated with temperature differences over time. Significant differences were also recorded between genotypes in leaf chlorophyll content. It was shown that variation over time in leaf chlorophyll content could be quantified accurately as a function of temperature and light integral. The results imply that genetic variability exists in cacao in response to temperature stress.