Reliability of Bayesian posterior probabilities and bootstrap frequencies in phylogenetics

Many empirical studies have revealed considerable differences between nonparametric bootstrapping and Bayesian posterior probabilities in terms of the support values for branches, despite claimed predictions about their approximate equivalence. We investigated this problem by simulating data, which were then analyzed by maximum likelihood bootstrapping and Bayesian phylogenetic analysis using identical models and reoptimization of parameter values. We show that Bayesian posterior probabilities are significantly higher than corresponding nonparametric bootstrap frequencies for true clades, but also that erroneous conclusions will be made more often. These errors are strongly accentuated when the models used for analyses are underparameterized. When data are analyzed under the correct model, nonparametric bootstrapping is conservative. Bayesian posterior probabilities are also conservative in this respect, but less so.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.     

Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.     

Osmotic stress leads to decreased intracellular pH of Listeria monocytogenes as determined by fluorescence ratio-imaging microscopy

Intracellular pH (pH(i)) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH(i), and recovery on solid medium was impaired compared to that in liquid medium. N,N'-dicyclohexylcarbodiimide abolished pH(i) recovery, and lowering a(w) with glycerol showed no effect on pH(i).     

Direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples by integrated cell concentration and DNA purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5'-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry

AIMS: To investigate the antibacterial effect of five essential oils (EO) extracted from aromatic plants (Cymbopogon citratus, Ocimumbasilicum, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale) of Cameroon against strains of Listeria monocytogenes, L. innocua and Staphylococcus aureus. The ability of selected EO to permeabilize the cytoplasmic membrane of L. innocua was also examined. METHODS AND RESULTS: The antibacterial activity of the EO determined by the agar diffusion method showed that T. vulgaris had the highest activity followed by O. gratissimum and C. citratus. Lowest activity was recorded from Z. officinale and O. basilicum. Significant differences in sensitivity between strains of Listeria and S. aureus were observed. Flow cytometry of L. innocua stained with carboxy-fluorescein diacetate showed that the fluorescence intensity of cells exposed to EO decreased faster than nonexposed cells, indicating that EO permeabilized the cytoplasmic membrane with the leakage of carboxy-fluorescein. CONCLUSIONS: Almost all the EO tested showed antibacterial activity to a different extent. The antibacterial effect was due to permeabilization of the cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified the preservative potential of the EO examined. The use of sensitive method, such as flow cytometry, is advantageous for quick generation of data on the antibacterial effect of EO.     

Cyanogenic glucosides and plant-insect interactions

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are considered to have an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their predator defense. A few species of Arthropoda (within Diplopoda, Chilopoda, Insecta) are able to de novo synthesize cyanogenic glucosides and, in addition, some of these species are able to sequester cyanogenic glucosides from their host plant (Zygaenidae). Evolutionary aspects of these unique plant-insect interactions with focus on the enzyme systems involved in synthesis and degradation of cyanogenic glucosides are discussed.