Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages

Burkholderia cenocepacia is an opportunistic pathogen causing serious infections in patients with cystic fibrosis. The widespread distribution of this bacterium in the environment suggests that it must adapt to stress to be able to survive. We identified in B. cenocepacia K56-2 a gene predicted to encode RpoE, the extra-cytoplasmic stress response regulator. The rpoE gene is the first gene of a predicted operon encoding proteins homologous to RseA, RseB, MucD and a protein of unknown function. The genomic organization and the co-transcription of these genes were confirmed by PCR and RT-PCR. The mucD and rpoE genes were mutated, giving rise to B. cenocepacia RSF24 and RSF25, respectively. While mutant RSF24 did not demonstrate any growth defects under the conditions tested, RSF25 was compromised for growth under temperature (44 degrees C) and osmotic stress (426 mM NaCl). Expression of RpoE in trans could complement the osmotic growth defect but exacerbated temperature sensitivity in both RSF25 and wild-type K56-2. Inactivation of rpoE altered the bacterial cell surface, as indicated by increased binding of the fluorescent dye calcofluor white and by an altered outer-membrane protein profile. These cell surface changes were restored by complementation with a plasmid encoding rpoE. Macrophage infections in which bacterial colocalization with fluorescent dextran was examined demonstrated that the rpoE mutant could not delay the fusion of B. cenocepacia-containing vacuoles with lysosomes, in contrast to the parental strain K56-2. These data show that B. cenocepacia rpoE is required for bacterial growth under certain stress conditions and for the ability of intracellular bacteria to delay phagolysosomal fusion in macrophages.

The mgtC gene of Burkholderia cenocepacia is required for growth under magnesium limitation conditions and intracellular survival in macrophages

Burkholderia cenocepacia, a bacterium commonly found in the environment, is an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known about the mechanisms by which B. cenocepacia causes disease, but chronic infection of the airways in CF patients may be associated, at least in part, with the ability of this bacterium to survive within epithelial cells and macrophages. Survival in macrophages occurs in a membrane-bound compartment that is distinct from the lysosome, suggesting that B. cenocepacia prevents phagolysosomal fusion. In a previous study, we employed signature-tagged mutagenesis and an agar bead model of chronic pulmonary infection in rats to identify B. cenocepacia genes that are required for bacterial survival in vivo. One of the most significantly attenuated mutants had an insertion in the mgtC gene. Here, we show that mgtC is also needed for growth of B. cenocepacia in magnesium-depleted medium and for bacterial survival within murine macrophages. Using fluorescence microscopy, we demonstrated that B. cenocepacia mgtC mutants, unlike the parental isolate, colocalize with the fluorescent acidotropic probe LysoTracker Red. At 4 h postinfection, mgtC mutants expressing monomeric red fluorescent protein cannot retain this protein within the bacterial cytoplasm. Together, these results demonstrate that, unlike the parental strain, an mgtC mutant does not induce a delay in phagolysosomal fusion and the bacterium-containing vacuoles are rapidly targeted to the lysosome, where bacteria are destroyed.

Antimicrobial activity of fosfomycin and tobramycin in combination against cystic fibrosis pathogens under aerobic and anaerobic conditions

There is a need for new antibiotics or combination of antibiotics that possess activity against increasingly resistant cystic fibrosis (CF) respiratory pathogens such as Pseudomonas aeruginosa and MRSA.

Micro-environmental conditions modulate protein secretion and infectivity of the Trichinella spiralis L1 larva

After digestion of infected meat the free L1 of Trichinella spp. penetrate the intestinal mucosa where they moult to the mature adult stage. We have used proteomics to identify changes in protein secretion during in vitro culture of free T. spiralis muscle larvae under different environmental conditions, and to correlate these changes with their infectivity in mice. Muscle larvae were cultured in different media (RPMI-1640, C-199 and HBSS) under conditions of anaerobiosis, microaerobiosis and in 5% CO(2) at 37 degrees C. Following incubation the larval excretory/secretory proteins were analysed by two-dimensional gel electrophoresis and the larvae were used to orally infect naïve CD1 mice. For all culture media tested, infectivity of the L1 was preserved following incubation in anaerobic conditions. In contrast, the infectivity of worms cultured in nutrient-rich media was almost completely abolished in both microaerobiosis and in the presence of 5% CO(2). Some infectivity was retained in poor or reduced culture media. Comparative analysis of larval infectivity and protein secretion showed that loss of infectivity correlated with the appearance of non-tyvelosylated proteins that in turn may be related to the onset of moulting.

Low temperature conditions in building sandstone: the role of extreme events in temperate environments

Data on rock temperatures has previously been collected to characterise typical diurnal regimes, and more recently to describe short-term variability in extreme locations. However, there is also the case that little is understood concerning the impact of extreme events in otherwise temperate environments. Internal stone temperatures (5?cm) collected during the atypical cold extreme experienced, throughout the UK, in December 2010 show a difference between ambient air temperatures and aspect-related thermal differences, particularly concerning temperature lows and the influence of radiative heating. In this case, debris release was not visible; however, laboratory simulations have shown that under such conditions, surface loss does not necessarily negate the occurrence of internal stone modifications. This preparatory sequence of change demonstrates that surface loss is not the result of one process, but rather many operating over time to sufficiently decrease stone strength to facilitate obvious damage.

Methylation using dimethylcarbonate catalysed by ionic liquids under continuous flow conditions

The ionic liquid, tributylmethylammonium methylcarbonate, has been employed as a catalytic base for clean N-methylation of indole with dimethylcarbonate. The reaction conditions were optimised under microwave heating to give 100% conversion and 100% selectivity to N-methylindole, and subsequently transferred to a high temperature/high pressure (285 degrees C/150 bar) continuous flow process using a short (3 min) residence time and 2 mol% of the catalyst to efficiently methylate a variety of different amines, phenols, thiophenols and carboxylic acid substrates. The extremely short residence times, versatility, and high selectivity have significant implications for the synthesis of a wide range of pharmaceutical intermediates, as high product throughputs can be obtained via this scalable continuous flow protocol. It has also been shown that the ionic liquid can be generated in situ from tributylamine, which has the net effect of transforming an ineffective stoichiometric base into a highly efficient catalyst for this broad class of reactions.

Promotion of Ceria Catalysts by Precious Metals: Changes in Nature of the Interaction under Reducing and Oxidizing Conditions

By depositing ceria over supported precious metal (PM) catalysts and characterizing them with in situ diffuse reflectance UV (DR UV) and in situ Raman spectroscopy, we have been able to prove a direct correlation between a decrease in ceria band gap and the work function of the metal under reducing conditions. The PM ceria interaction results in changes on the ceria side of the metal ceria interface, such that the degree of oxygen vacancy formation on the ceria surface also correlates with the precious metal work function. Nevertheless, conclusive evidence for a purely electronic interaction could not be provided by X-ray photoelectron spectroscopy (XPS) analysis. On the contrary, the results highlight the complexity of the PM ceria interaction by supporting a spillover mechanism resulting from the electronic interaction under reducing conditions. Under oxidizing conditions, another effect has been observed; namely, a structural modification of ceria induced by the presence of PM cations. In particular, we have been able to demonstrate by in situ Raman spectroscopy that, depending on the PM ionic radius, it is possible to create PM ceria solid solutions. We observed that this structural modification prevails under an oxidizing atmosphere, whereas electronic and chemical interactions take place under reducing conditions.

Robust Audio-Visual Speech Recognition under Noisy Audio-Video Conditions

This paper presents the maximum weighted stream posterior (MWSP) model as a robust and efficient stream integration method for audio-visual speech recognition in environments, where the audio or video streams may be subjected to unknown and time-varying corruption. A significant advantage of MWSP is that it does not require any specific measurements of the signal in either stream to calculate appropriate stream weights during recognition, and as such it is modality-independent. This also means that MWSP complements and can be used alongside many of the other approaches that have been proposed in the literature for this problem. For evaluation we used the large XM2VTS database for speaker-independent audio-visual speech recognition. The extensive tests include both clean and corrupted utterances with corruption added in either/both the video and audio streams using a variety of types (e.g., MPEG-4 video compression) and levels of noise. The experiments show that this approach gives excellent performance in comparison to another well-known dynamic stream weighting approach and also compared to any fixed-weighted integration approach in both clean conditions or when noise is added to either stream. Furthermore, our experiments show that the MWSP approach dynamically selects suitable integration weights on a frame-by-frame basis according to the level of noise in the streams and also according to the naturally fluctuating relative reliability of the modalities even in clean conditions. The MWSP approach is shown to maintain robust recognition performance in all tested conditions, while requiring no prior knowledge about the type or level of noise.

Palaeoglacial and palaeoclimatic conditions in the NW Pacific, as revealed by a morphometric analysis of cirques upon the Kamchatka Peninsula

The distribution of glacial cirques upon the Kamchatka peninsula, Far Eastern Russia, is systematically mapped from satellite images and digital elevation model data. A total of 3,758 cirques are identified, 238 of which are occupied by active glaciers. The morphometry of the remaining 3,520 cirques is analysed. These cirques are found to show a very strong N bias in their azimuth (orientation), likely resulting from aspect-related variations in insolation. The strength of this N bias is considered to indicate that former glaciation upon the peninsula was often ‘marginal’, and mainly of cirque-type, with peaks extending little above regional equilibrium-line altitudes. This is supported by the fact that S and SE-facing cirques are the highest in the dataset, suggesting that glacier-cover was rarely sufficient to allow S and SE-facing glaciers to develop at low altitudes. The strength of these azimuth-related variations in cirque altitude is thought to reflect comparatively cloud-free conditions during former periods of glaciation. It is suggested that these characteristics, of marginal glaciation and comparatively cloud-free conditions, reflect the region’s former aridity, which was likely intensified at the global Last Glacial Maximum, and during earlier periods of ice advance, as a result of the development of negative pressure anomalies over the North Pacific (driven by the growth of the Laurentide Ice Sheet), combined with other factors, including an increase in the extent and duration of sea ice, a reduction in global sea levels, cooler sea surface temperatures, and the localised growth of mountain glaciers. There is published evidence to suggest extensive glaciation of the Kamchatka Peninsula at times during the Late Quaternary, yet the data presented here appears to suggest that such phases were comparatively short-lived, and that smaller cirque-type glaciers were generally more characteristic of the period.

Optimising sorting and washing of home-grown maize to reduce fumonisin contamination under laboratory-controlled conditions

Subsistence farming communities with low socio-economic status reliant on a mono cereal maize diet are exposed to fumonisin levels that exceed the provisional maximum tolerable daily intake of 2 mu g kg(-1) body weight day(-1) recommended by the Joint FAO/WHO Expert Committee on Food Additives. In the rural Centane magisterial district, Eastern Cape Province, South Africa, it is customary during food preparation to sort visibly infected maize kernels from good maize kernels and to wash the good kernels prior to cooking. However, this customary practice seems not to sufficiently reduce the fumonisin levels. This is the first study to optimise the reduction of fumonisin mycotoxins in home-grown maize based on customary methods of a rural population, under laboratory-controlled conditions. Maize obtained from subsistence farmers was analysed for the major naturally occurring fumonisins (FB1, FB2 and FB3) by fluorescence HPLC. Large variations were observed in the unsorted and the experimental maize batches attributable to the non-homogeneous distribution of fumonisin contamination in maize kernels. Optimised hand-sorting of maize kernels by removing the visibly infected/damaged kernels (fumonisins, 53.7 +/- 15.0 mg kg(-1), 2.5% by weight) reduced the mean fumonisins from 2.32 +/- 1.16 mg kg(-1) to 0.68 +/- 0.42 mg kg(-1). Hand washing of the sorted good maize kernels for a period of 10 min at 25 degrees C resulted in optimal reduction with no additional improvement for wash periods up to 15 h. The laboratory optimised sorting reduced the fumonisins by 71 +/- 18% and an additional 13 +/- 12% with the 10 min wash. Based on these results and on local practices and practicalities the protocol that would be recommended to subsistence farmers consists of the removal of the infected/damaged kernels from the maize followed by a 10 min ambient temperature water wash. (C) 2010 Elsevier Ltd. All rights reserved.

A material model for multiaxial stretching and stress relaxation of polypropylene under process conditions

Polypropylene sheets have been stretched at 160 °C to a state of large biaxial strain of extension ratio 3, and the stresses then allowed to relax at constant strain. The state of strain is reached via a path consisting of two sequential planar extensions, the second perpendicular to the first, under plane stress conditions with zero stress acting normal to the sheet. This strain path is highly relevant to solid phase deformation processes such as stretch blow moulding and thermoforming, and also reveals fundamental aspects of the flow rule required in the constitutive behaviour of the material. The rate of decay of stress is rapid, and such as to be highly significant in the modelling of processes that include stages of constant strain. A constitutive equation is developed that includes Eyring processes to model both the stress relaxation and strain rate dependence of the stress. The axial and transverse stresses observed during loading show that the use of a conventional Levy-Mises flow rule is ineffective, and instead a flow rule is used that takes account of the anisotropic state of the material via a power law function of the principal extension ratios. Finally the constitutive model is demonstrated to give quantitatively useful representation of the stresses both in loading and in stress relaxation.

Field observation of sequestration of uranium in iron-rich sediments under sequential reduction-oxidation conditions at the DOE Oak Ridge IFRC

At the U.S. DOE Oak Ridge Integrated Field Research Challenge (ORIFRC) site, the iron content of shallow subsurface materials (i.e. weathered saprolite) is relatively high (up to 5-6% as w/w), and therefore, the forms of the iron species present plays a critical role in the long-term sequestration of uranium. A long term pilot-scale study of the bioreduction and reoxidation of uranium conducted at the ORIFRC area 3 site, adjacent to the former S-3 disposal ponds (source zone), has provided us with the opportunity to study the impact of iron species on the sequestration of U(VI). The aqueous U(VI) concentrations at the site were decreased to below the EPA MCL through the intermittent injection of ethanol as the electron donor. Previous field tests indicated that both oxygen and nitrate could oxidize the bioreduced U(IV) and cause a short-term rebound of aqueous phase uranium concentration after the oxidative agents were delivered directly to the bioreduced zone.

A field test has been conducted to examine the long-term effect of exposure of bioreduced sediments to nitrate in contaminated groundwater for more than 1,380 days at the Area 3 site. Contaminated groundwater was allowed to invade the previously bioreduced zone via the natural groundwater gradient after an extended period in which reducing conditions were maintained and the bioreduced zone was protected from the influx of upgradient contaminated groundwater. The geochemical response to the invasion of contaminated groundwater was dependent on whether the monitoring location is in the middle or the fringe of the previously bioreduced zone. In general, the nitrate concentrations in the previously bioreduced area, increased gradually from near zero to ~50-300 mM within 200 days and then stabilized. The pH declined from bioreduced levels of 6.2-6.7 to below 5.0. Uranium concentrations rebounded in all monitoring wells but at different rates. At most locations U concentrations rebounded, declined and then rebounded again. Methane gas disappeared while a significant level (20,000 to 44,000 ppmv) N2O was found in the groundwater of monitoring wells after three years of reoxidization.

The U(IV) in sediments was mainly reoxidized to U(VI) species. Based on XANES analysis, the predominate uranium in all samples after re-oxidation was similar to a uranyl nitrate form. But the U content in the sediment remained as high as that determined after bioreduction activates were completed, indicating that much of the U is still sequestrated in situ. SEM observations of surged fine sediments revealed that clusters of colloidal-sized (200-500nm) U-containing precipitates appeared to have formed in situ, regardless from sample of FW106 in non-bioactivity control area or of pre-bioreduced FW101-2 and FW102-3. Additionally, SEM-EDS and microprobe analysis, showed that the U-containing precipitates (~1% U) in FW106 are notably higher in Fe, compared to the precipitates (~1-2.5% U) from FW101-2 and FW102-3. However, XRF analysis indicated that the U content was remained as high as 2180 and 1810 mg/kg with U/Fe ratio at 0.077 and 0.055 vs 0.037 g/g, respectively in pre-bioreduced FW101-2 and FW102-3, suggesting more U sequestrated by Fe in pre-bioreduced sediments.