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.

Can we measure terrestrial photosynthesis from space directly, using spectral reflectance and fluorescence?

Attempts to estimate photosynthetic rate or gross primary productivity from remotely sensed absorbed solar radiation depend on knowledge of the light use efficiency (LUE). Early models assumed LUE to be constant, but now most researchers try to adjust it for variations in temperature and moisture stress. However, more exact methods are now required. Hyperspectral remote sensing offers the possibility of sensing the changes in the xanthophyll cycle, which is closely coupled to photosynthesis. Several studies have shown that an index (the photochemical reflectance index) based on the reflectance at 531 nm is strongly correlated with the LUE over hours, days and months. A second hyperspectral approach relies on the remote detection of fluorescence, which is a directly related to the efficiency of photosynthesis. We discuss the state of the art of the two approaches. Both have been demonstrated to be effective, but we specify seven conditions required before the methods can become operational.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

Background. The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. Results. Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. Conclusions. The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

The influence of powdery mildew infection on photosynthesis, chlorophyll fluorescence, leaf chlorophyll and carotenoid content of three woody plant species

The effect of powdery mildew development on photosynthesis, chlorophyll fluorescence, leaf chlorophyll and carotenoid concentrations on three woody plants frequently planted in urban environments was studied. Rates of photosynthetic CO2 fixation were rapidly reduced in two of the three genotypes tested prior to visible signs of infection. Effects on chlorophyll fluorescence (Fo, Fv/Fo, Fv/Fm), leaf chlorophyll and carotenoid content were not manifest until >25 per cent of the leaf area was observed to be covered by mycelial growth indicating reduced photo-synthetic rates during the early stages of infection were not due to degradation of the leaf chloroplast structure. Observation of the fluorescence transient (OJIP curves) showed powdery mildew infection impairs photosynthetic electron transport system by reducing the size but not heterogeneity of the plastoquninone pool, effecting both the acceptor and donor side of photosystem II. Impairment of the photosynthetic electron transport system was reflected by reduced values of a performance index used in this investigation as a measure of photochemical events within photosystem II electron transport. In addition interpretation of the fluorescence data indicated powdery mildew infection may impair the photo-protective process that facilitates the dissipation of excess energy within leaf tissue.

Measurement of the salinity and freezing tolerance of Crataegus genotypes using chlorophyll fluorescence

The effect of increasing salinity and freezing stress singly and in combination on a range of chlorophyll fluorescence parameters in foliar tissue of six Crataegus genotypes was examined. In general, increased stress reduced fluorescence values and absorption, trapping and electron transport energy fluxes per leaf reaction center and cross section, with decreased sigmoidicity of OJIP curves as a measure of the plastoquinone pool, reflecting decreased energy fluxes. Based on percentage reduction in a performance index from controls compared to stress-treated values, plants were ranked in order of tolerant > intermediate > sensitive. Use of this PIp ranking criteria enabled the distinguishing of marked differences in foliar salt/freezing hardiness between the Crataegus species used. Interpretation of the photochemical data showed that salinity and freezing affects both the acceptor and donor side of Photosystem II, while OJIP observations provided information regarding structural and functional changes in the leaf photosynthetic apparatus of the test species. It is concluded that chlorophyll fluorescence offers a rapid screening technique for assessing foliar salinity and freezing tolerance of woody perennials

Foliar salt tolerance of Acer genotypes using chlorophyll fluorescence

The effect of increasing salinity on a range of chlorophyll fluorescence parameters in foliar tissue of 30 Acer genotypes was examined. The magnitude of the fluorescence responses differed among genotypes ranging from minor effects to substantial leaf tissue damage. Interpretation of the fluorescence expressions provided an insight into mechanisms of salt damage and resilience among genotypes. Based on reductions in a performance index (PIp) following salinity, genotypes were ranked in order from tolerant to sensitive. Based on this ranking criterion, marked differences in salt tolerance among genotypes were distinguished. It is concluded that chlorophyll fluorescence offers a rapid screening technique for assessing the foliar salinity tolerance of urban trees.

Correlation between Fourier series expansion and Hückel orbital theory

The Fourier series can be used to describe periodic phenomena such as the one-dimensional crystal wave function. By the trigonometric treatements in Hückel theory it is shown that Hückel theory is a special case of Fourier series theory. Thus, the conjugated π system is in fact a periodic system. Therefore, it can be explained why such a simple theorem as Hückel theory can be so powerful in organic chemistry. Although it only considers the immediate neighboring interactions, it implicitly takes account of the periodicity in the complete picture where all the interactions are considered. Furthermore, the success of the trigonometric methods in Hückel theory is not accidental, as it based on the fact that Hückel theory is a specific example of the more general method of Fourier series expansion. It is also important for education purposes to expand a specific approach such as Hückel theory into a more general method such as Fourier series expansion.

The correlation of ULF waves and auroral intensity before, during and after substorm expansion phase onset

We present case studies of the evolution of magnetic wave amplitudes and auroral intensity through the late growth phase and the expansion phase of the substorm cycle. We present strong evidence that substorm-related auroral enhancements are clearly and demonstrably linked to ULF wave amplitudes observed at the same location. In most cases, we find that the highest correlations are observed when the magnetometer time series is advanced in time, indicating that the ULF wave amplitudes start to grow before measured auroral intensities, though interestingly this is not always the case. Further we discuss the four possible reasons that may be able to explain both the timing and the high correlations between these two phenomena, including: a simple coincidence, an artifact of instrumental effects, the response of the ionosphere to magnetic waves and auroral particle precipitation, and finally that ULF waves and auroral particle precipitation are physically linked. We discount coincidence and instrumental effects since in the studies presented here they are unlikely or in general will contribute negligible effects, and we find that the ionospheric response to waves and precipitation can explain some, but not all of the results contained within this paper. Specifically, ionospheric response to substorm waves and auroral precipitation cannot explain that the result that previous studies have shown, that onset of ULF wave activity and the onset of auroral particle precipitation occur at the same time and in the same location. This leaves the possibility that ULF waves and auroral particles are physically linked.

Technical note: Organics-induced fluorescence in Raman studies of sulfuric acid aerosols

Fluorescence is a troublesome side effect in laboratory Raman studies on sulfuric acid solutions and aerosol particles. We performed experiments showing that organic matter induces fluorescence in H2SO4/H2O solutions. The intensity of the fluorescence signal appears to be almost independent of the concentration of the organic substances, but depends strongly on the sulfuric acid concentration. The ubiquity of organic substances in the atmosphere, their relatively high abundance, and the insensitivity of the fluorescence with respect to their concentrations will render most acidic natural aerosols subject to absorption and fluorescence, possibly influencing climate forcing. We show that, while fluorescence may in the future become a valuable tool of aerosol diagnostics, the concurrent absorption is too small to significantly affect the atmosphere's radiative balance.

An evaluation of the correlation between open solar flux and total solar irradiance

The correlation between the coronal source flux F_{S} and the total solar irradiance I_{TS} is re-evaluated in the light of an additional 5 years' data from the rising phase of solar cycle 23 and also by using cosmic ray fluxes detected at Earth. Tests on monthly averages show that the correlation with F_{S} deduced from the interplanetary magnetic field (correlation coefficient, r = 0.62) is highly significant (99.999%), but that there is insufficient data for the higher correlation with annual means (r = 0.80) to be considered significant. Anti-correlations between I_{TS} and cosmic ray fluxes are found in monthly data for all stations and geomagnetic rigidity cut-offs (r ranging from −0.63 to −0.74) and these have significance levels between 85% and 98%. In all cases, the t is poorest for the earliest data (i.e., prior to 1982). Excluding these data improves the anticorrelation with cosmic rays to r = −0:93 for one-year running means. Both the interplanetary magnetic field data and the cosmic ray fluxes indicate that the total solar irradiance lags behind the open solar flux with a delay that is estimated to have an optimum value of 2.8 months (and is within the uncertainty range 0.8-8.0 months at the 90% level).

The dependence of high-latitude dayside ionospheric flows on the North-South component of the IMF: A high time resolution correlation analysis using EISCAT “Polar” and AMPTE UKS and IRM data

In 1984 and 1985 a series of experiments was undertaken in which dayside ionospheric flows were measured by the EISCAT “Polar” experiment, while observations of the solar wind and interplanetary magnetic field (IMF) were made by the AMPTE UKS and IRM spacecraft upstream from the Earth's bow shock. As a result, 40 h of simultaneous data were acquired, which are analysed in this paper to investigate the relationship between the ionospheric flow and the North-South (Bz) component of the IMF. The ionospheric flow data have 2.5 min resolution, and cover the dayside local time sector from ∼ 09:30 to ∼ 18:30 M.L.T. and the latitude range from 70.8° to 74.3°. Using cross-correlation analysis it is shown that clear relationships do exist between the ionospheric flow and IMF Bz, but that the form of the relations depends strongly on latitude and local time. These dependencies are readily interpreted in terms of a twinvortex flow pattern in which the magnitude and latitudinal extent of the flows become successively larger as Bz becomes successively more negative. Detailed maps of the flow are derived for a range of Bz values (between ± 4 nT) which clearly demonstrate the presence of these effects in the data. The data also suggest that the morning reversal in the East-West component of flow moves to earlier local times as Bz, declines in value and becomes negative. The correlation analysis also provides information on the ionospheric response time to changes in IMF Bz, it being found that the response is very rapid indeed. The most rapid response occurs in the noon to mid-afternoon sector, where the westward flows of the dusk cell respond with a delay of 3.9 ± 2.2 min to changes in the North-South field at the subsolar magnetopause. The flows appear to evolve in form over the subsequent ~ 5 min interval, however, as indicated by the longer response times found for the northward component of flow in this sector (6.7 ±2.2 min), and in data from earlier and later local times. No evidence is found for a latitudinal gradient in response time; changes in flow take place coherently in time across the entire radar field-of-view.

1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans-flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

Studies with a diverse array of 22 purified condensed tannin (CT) samples from nine plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans-flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR spectroscopy. The method was developed from samples containing 44 to ~100% CT, PC/PD ratios ranging from 0/100 to 99/1, and cis/trans ratios from 58/42 to 95/5 as determined by thiolysis with benzyl mercaptan. Integration of cross-peak contours of H/C-6' signals from PC and of H/C-2',6' signals from PD yielded nuclei adjusted estimates that were highly correlated with PC/PD ratios obtained by thiolysis (R2 = 0.99). Cis/trans-flavan-3-ol ratios, obtained by integration of the respective H/C-4 cross-peak contours, were also related to determinations made by thiolysis (R2 = 0.89). Overall, 1H-13C HSQC NMR spectroscopy appears to be a viable alternative to thiolysis for estimating PC/PD and cis/trans ratios of CT, if precautions are taken to avoid integration of cross-peak contours of contaminants.