Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Effect of creatine supplementation on intermittent sprint running performance in highly trained athletes

This study examined the impact of short-term (7-day), high-dose (0.35 g.kg(-1).d(-1)) oral creatine monohydrate supplementation (CrS) on single sprint running performance (40 m, <6 seconds) and on intermittent sprint performance in highly trained sprinters. Nine subjects completed the double-blind cross-over design with 2 supplementation periods (placebo and creatine) and a 7-week wash-out period. A test protocol consisting of 40-m sprint runs was performed, and running velocity was continuously recorded over the total distance. The maximal sprint performance, the relative degree of fatigue at the end of intermittent sprint exercise (6 x 40 m, 30-second rest interval), as well as the degree of recovery (120-second passive rest) remained unchanged following CrS. There were no significant changes related to CrS in absolute running velocity at any distance between start and finish (40 m). It was concluded that no ergogenic effect on single or repeated 40-m sprint times with varying rest periods was observed in highly trained athletes.     

Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria.     

Four differently chromatin‐associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific β-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their ‘extractability’ from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.     

Novel 16S rRNA gene analyses reveal new in vitro effects of insoluble barley fibres on the human faecal microbiota

Aims:  The aim of this work was to analyse the growth of human faecal microbiota on barley dietary fibres (DF). It is generally accepted that insoluble DF are health promoting, but the information is scarce about how these fibres affect the gastrointestinal (GI) microbiota. A major reason for the limited knowledge is that there are currently no proper tools to analyse the complete GI microbiota.
Methods and Results:  Here we present a novel 16S rRNA gene analytical approach that enables the analyses of the complete microbiota, including the part that has not yet been characterized. The basic principle of the method is use of 16S rRNA gene signature sequences to determine both the phylogenetic relatedness and the distribution of bacteria in the samples analysed.
Using this approach, we analysed the microbiota after in vitro fermentation of different barley fractions with human faeces. Our main finding was that groups of actinobacteria were selectively enriched by growth on the insoluble DF fractions.
Conclusions:  Our novel analytical approaches revealed new enrichment patterns in the taxa that respond to insoluble DF.
Significance and Impact of the Study:  Our results may have major implications for future understanding of insoluble DF health effects.     

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO₂ anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO₂ in Drosophila melanogaster. The impact of CO₂ anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.