Quantification of the Effects of Salt Stress and Physiological State on Thermotolerance of Bacillus cereus ATCC 10987 and ATCC 14579

The food-borne pathogen Bacillus cereus can acquire enhanced thermal resistance through multiple mechanisms. Two Bacillus cereus strains, ATCC 10987 and ATCC 14579, were used to quantify the effects of salt stress and physiological state on thermotolerance. Cultures were exposed to increasing concentrations of sodium chloride for 30 min, after which their thermotolerance was assessed at 50°C. Linear and nonlinear microbial survival models, which cover a wide range of known inactivation curvatures for vegetative cells, were fitted to the inactivation data and evaluated. Based on statistical indices and model characteristics, biphasic models with a shoulder were selected and used for quantification. Each model parameter reflected a survival characteristic, and both models were flexible, allowing a reduction of parameters when certain phenomena were not present. Both strains showed enhanced thermotolerance after preexposure to (non)lethal salt stress conditions in the exponential phase. The maximum adaptive stress response due to salt preexposure demonstrated for exponential-phase cells was comparable to the effect of physiological state on thermotolerance in both strains. However, the adaptive salt stress response was less pronounced for transition- and stationary-phase cells. The distinct tailing of strain ATCC 10987 was attributed to the presence of a subpopulation of spores. The existence of a stable heat-resistant subpopulation of vegetative cells could not be demonstrated for either of the strains. Quantification of the adaptive stress response might be instrumental in understanding adaptation mechanisms and will allow the food industry to develop more accurate and reliable stress-integrated predictive modeling to optimize minimal processing conditions.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> MW005: lambda Red-mediated recombineering and copy-number induction of <Emphasis Type="Italic">oriV</Emphasis>-equipped constructs in a single host

Background  

Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans -replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV -equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a P _BAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.     

Insect oviposition behavior affects the evolution of adaptation to <Emphasis Type="Italic">Bt</Emphasis> crops: consequences for refuge policies

The major lepidopteran insect pests of cotton and maize harbor intra-specific variation for behavior determining the selection of host plants for oviposition. Yet, the consequences of behavioral adaptation for fitness have neither been modeled nor monitored for Bt cotton and maize crops, the most widely grown transgenic herbivore-resistant plants. Here, we present a general two-locus heuristic model to examine potential outcomes of natural selection when pest populations initially have low frequencies of alleles for both physiological and behavioral adaptation to Bt crops. We demonstrate that certain ecological conditions allow for the evolution of behavioral choices favoring alternative oviposition hosts that limit the increase in resistance alleles, even when they are phenotypically dominant. These results have implications for current refuge policies, which should be adapted to promote the evolution of certain behavioral choices for alternative oviposition hosts in addition to dilution of physiological resistance alleles. Collection of data on oviposition host preference as a component of monitoring schemes will provide important insights into mechanisms underlying the durability of Bt-transgenic host-plant resistance.     

Bidirectional Secretions from Glandular Trichomes of Pyrethrum Enable Immunization of Seedlings

Glandular trichomes are currently known only to store mono- and sesquiterpene compounds in the subcuticular cavity just above the apical cells of trichomes or emit them into the headspace. We demonstrate that basipetal secretions can also occur, by addressing the organization of the biosynthesis and storage of pyrethrins in pyrethrum (Tanacetum cinerariifolium) flowers. Pyrethrum produces a diverse array of pyrethrins and sesquiterpene lactones for plant defense. The highest concentrations accumulate in the flower achenes, which are densely covered by glandular trichomes. The trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. However, during achene maturation, the key pyrethrin biosynthetic pathway enzyme chrysanthemyl diphosphate synthase is expressed only in glandular trichomes. We show evidence that chrysanthemic acid is translocated from trichomes to pericarp, where it is esterified into pyrethrins that accumulate in intercellular spaces. During seed maturation, pyrethrins are then absorbed by the embryo, and during seed germination, the embryo-stored pyrethrins are recruited by seedling tissues, which, for lack of trichomes, cannot produce pyrethrins themselves. The findings demonstrate that plant glandular trichomes can selectively secrete in a basipetal direction monoterpenoids, which can reach distant tissues, participate in chemical conversions, and immunize seedlings against insects and fungi.     

Mitotic remodeling of the replicon and chromosome structure

Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.     

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.     

Flight altitude of trans-Sahara migrants in autumn: a comparison of radar observations with predictions from meteorological conditions and water and energy balance models

Radar observations on the altitude of bird migration and altitudinal profiles of meteorological conditions over the Sahara desert are presented for the autumn migratory period. Migratory birds fly at an average altitude of 1016 m (a.s.l.) during the day and 571 m during the night. Weather data served to calculate flight range using two models: an energy model (EM) and an energy-and-water model (EWM). The EM assumes that fuel supply limits flight range whereas the EWM assumes that both fuel and water may limit flight range. Flight ranges estimated with the EM were generally longer than those with the EWM. This indicates that trans-Sahara migrants might have more problems balancing their water than their energy budget. However, if we assume fuel stores to consist of 70% instead of 100% fat (the remainder consisting of 9% protein and 21% water), predicted flight ranges of the EM and EWM largely overlap. Increased oxygen extraction, reduced flight costs, reduced exhaled air temperature, reduced cutaneous water loss and increased tolerance to water loss are potential physiological adaptations that would improve the water budget in migrants. Both the EM and EWM predict optimal flight altitudes in agreement with radar observations in autumn. Optimal flight altitudes are differently predicted by the EM and EWM for nocturnal spring migration. During spring, the EWM predicts moderately higher and the EM substantially higher flight altitudes than during autumn. EWM predictions are therefore in better agreement with radar observations on flight altitude of migrants over the Negev desert in spring than EM predictions.     

Correlation of Standardized Uptake Value and Apparent Diffusion Coefficient in Integrated Whole-Body PET/MRI of Primary and Recurrent Cervical Cancer

Background

To evaluate a potential correlation of the maximum standard uptake value (SUV_max) and the minimum apparent diffusion coefficient (ADC_min) in primary and recurrent cervical cancer based on integrated PET/MRI examinations.

Methods

19 consecutive patients (mean age 51.6 years; range 30–72 years) with histopathologically confirmed primary cervical cancer (n = 9) or suspected tumor recurrence (n = 10) were prospectively enrolled for an integrated PET/MRI examination. Two radiologists performed a consensus reading in random order, using a dedicated post-processing software. Polygonal regions of interest (ROI) covering the entire tumor lesions were drawn into PET/MR images to assess SUV_max and into ADC parameter maps to determine ADC_min values. Pearson’s correlation coefficients were calculated to assess a potential correlation between the mean values of ADC_min and SUV_max.

Results

In 15 out of 19 patients cervical cancer lesions (n = 12) or lymph node metastases (n = 42) were detected. Mean SUV_max (12.5±6.5) and ADC_min (644.5±179.7×10⁻⁵ mm²/s) values for all assessed tumor lesions showed a significant but weak inverse correlation (R = −0.342, p<0.05). When subdivided in primary and recurrent tumors, primary tumors and associated primary lymph node metastases revealed a significant and strong inverse correlation between SUV_max and ADC_min (R = −0.692, p<0.001), whereas recurrent cancer lesions did not show a significant correlation.

Conclusions

These initial results of this emerging hybrid imaging technique demonstrate the high diagnostic potential of simultaneous PET/MR imaging for the assessment of functional biomarkers, revealing a significant and strong correlation of tumor metabolism and higher cellularity in cervical cancer lesions.     

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.