Stress situations induce cyanide-resistant respiration in spoilage yeasts

AIMS: To investigate the conditions that promote the expression of cyanide-resistant respiration (CRR) in the spoilage yeasts Pichia membranifaciens and Debaryomyces hansenii. METHODS AND RESULTS: CRR was detected by sensitivity of oxygen consumption to salicylhydroxamic acid. It was absent in both yeasts in the early exponential phase, but was triggered by several stress situations. Starvation under aerobic conditions, decreasing pH or incubation of the culture in a narrow temperature range below the maximum temperature for growth promoted the emergence of CRR in both yeasts. In D. hansenii, CRR was also induced by 1.5-2 mol l(-1) NaCl. Although the presence of H2O2 and menadione induced CRR, radical scavengers had no effect on the emergence of CRR. Also, the level of reactive oxygen species did not vary with the CRR activity. CONCLUSIONS: Under aerobic conditions, a respiratory pathway alternative to the cytochrome chain is triggered by stress conditions in P. membranifaciens and D. hansenii. SIGNIFICANCE AND IMPACT OF THE STUDY: The relationship between stress situations and CRR must be taken into account in studies on the performance of spoilage yeasts in the food processing environments where several forms of stress are common.     

Genetic variability of the frameshift region in IS911 transposable elements from Escherichia coli clinical isolates

The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.     

Overall survival in advanced breast cancer: relevance of progesterone receptor expression and DNA ploidy in fine-needle aspirates of 392 patients

Fine-needle aspiration cytology (FNAC) is essential for making a diagnosis in advanced breast cancer. The determination of hormone receptors in the material obtained is useful for predicting patient response to endocrine therapy, but the prognostic value of hormone receptor expression as well as the clinical utility of DNA flow cytometry are controversial. The aim of this prospective study with long-term follow-up (median: 81 months) was to evaluate these biomarkers in relation to overall survival in a series of 392 patients with advanced breast cancer (stage IIB, n=106; IIIA, n=66; IIIB, n=174; and IV, n=46) using FNAC. Estrogen and progesterone receptor expression was found in 65.1% and 46.1% of the tumors, respectively. Hormone receptors were not found to be associated with clinical staging. DNA aneuploidy was present in 70.9% of the cases and the median S-phase fraction (SPF) was 9.4%. There was a significant correlation of aneuploidy and high SPF with lack of hormone receptors. In univariate analysis, advanced disease stage, absence of hormone receptors, DNA aneuploidy and high SPF showed a statistically significant correlation with poor clinical outcome. In multivariate analysis, disease stage, progesterone receptors and DNA ploidy retained independent prognostic significance in relation to overall survival. These data indicate that progesterone receptor expression and DNA ploidy are independent prognostic factors in advanced breast cancer.     

Protein stability and plasticity of the hydrophobic cavity in wheat ns-LTP

Plant ns-LTPs display an original structure with four helices and a flexible C-terminus, maintained together by four disulphide bridges and delineating an elongated central hydrophobic cavity. In order to relate these structural features to the protein stability and plasticity, combined molecular mechanics and simulated annealing calculations were undertaken on a wheat ns-LTP "mutant" with Cys-Ala replacement and with the application of core inter-residue restraints up to 2 A, reducing the cross-section size of the hydrophobic cavity. Analysis of the energy-minimized structures shows that removal of the disulphide bridges results in structures with a lower total energy and a smaller cavity volume. A 1-ns MD simulation at 300K in water, underlines that, despite the absence of a well-packed hydrophobic core, the native structure is extremely stable at room temperature and the cavity is not hydrated. This confirms that the disulphide bridges are essential for the existence of the cavity, whereas its plasticity depends both on the hydrophobic chain lining the cavity and on the C-terminal flexibility. A high temperature (500K) MD simulation confirms the stability of the secondary structure elements and the flexibility of the loops and of the C-terminal segment. Two important structural transitions during this simulation are discussed and possible routes for the insertion and release of hydrophobic ligands are suggested.     

FTIR and UV spectroscopy studies of triplex formation between alpha-oligonucleotides with non-ionic phoshoramidate linkages and DNA targets

The triplexes formed by pyrimidine alpha-oligodeoxynucleotides, 15mers alpha dT(15) or 12mers alpha dCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non-ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester beta-oligodeoxynucleotides (beta-ODNs). Among the alpha-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (alpha dCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the alpha dCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All alpha dCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30 degrees C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65 degrees C). While unfavorable steric effects are overcome with alpha anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate alpha-thymidilate strands (alpha dT(15)PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to beta dA(15) either of alpha dT(15)PN as duplex strand and beta dT(15) as third strand, or of two hydrophobic alpha dT(15)PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the alpha dT(15)PN binds as Hoogsteen third strand.     

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence

Bayesian Markov chain Monte Carlo sampling has become increasingly popular in phylogenetics as a method for both estimating the maximum likelihood topology and for assessing nodal confidence. Despite the growing use of posterior probabilities, the relationship between the Bayesian measure of confidence and the most commonly used confidence measure in phylogenetics, the nonparametric bootstrap proportion, is poorly understood. We used computer simulation to investigate the behavior of three phylogenetic confidence methods: Bayesian posterior probabilities calculated via Markov chain Monte Carlo sampling (BMCMC-PP), maximum likelihood bootstrap proportion (ML-BP), and maximum parsimony bootstrap proportion (MP-BP). We simulated the evolution of DNA sequence on 17-taxon topologies under 18 evolutionary scenarios and examined the performance of these methods in assigning confidence to correct monophyletic and incorrect monophyletic groups, and we examined the effects of increasing character number on support value. BMCMC-PP and ML-BP were often strongly correlated with one another but could provide substantially different estimates of support on short internodes. In contrast, BMCMC-PP correlated poorly with MP-BP across most of the simulation conditions that we examined. For a given threshold value, more correct monophyletic groups were supported by BMCMC-PP than by either ML-BP or MP-BP. When threshold values were chosen that fixed the rate of accepting incorrect monophyletic relationship as true at 5%, all three methods recovered most of the correct relationships on the simulated topologies, although BMCMC-PP and ML-BP performed better than MP-BP. BMCMC-PP was usually a less biased predictor of phylogenetic accuracy than either bootstrapping method. BMCMC-PP provided high support values for correct topological bipartitions with fewer characters than was needed for nonparametric bootstrap.     

Extreme divergence of a homeotic gene: the bicoid case

Evolutionary developmental genetics (evo-devo) reveals that the plasticity of development is so important that every developmental biology project should carefully take this point into consideration. The example of bicoid, the first discovered morphogen, illustrates how an essential gene can change its function during evolution. The search for bicoid homologues showed that this gene is surprisingly specific to flies (cyclorraphan diptera) and absent in other insects. In fact, recent studies demonstrate that bicoid is a very derived Hox3 homeotic gene. During insect evolution, the ancestral Hox3 gene lost its homeotic function and acquired new roles in oocytes and embryonic annexes. Then, in the lineage leading to modern flies, a duplication of this new gene, followed by functional divergence, led to the formation of bicoid and zerknüllt. Both genes are located within the Drosophila Hox complex; however, they have no homeotic function. Thanks to the power of Drosophila genetics, it is possible to suggest that torso and hunchback may constitute the insect primitive anterior organizer. The bicoid evolutionary history reveals several fundamental mechanisms of the evolution of developmental genes, such as changes of gene regulation, modifications of protein sequences and gene duplication. It also shows the need for studying a wider range of model organisms before generalisations can be made from data obtained with one particular species.     

Influence of mutational and sampling factors on the estimation of demographic parameters in a "continuous" population under isolation by distance

In numerous species, individual dispersal is restricted in space so that "continuous" populations evolve under isolation by distance. A method based on individual genotypes assuming a lattice population model was recently developed to estimate the product Dsigma2, where D is the population density and sigma2 is the average squared parent-offspring distance. We evaluated the influence on this method of both mutation rate and mutation model, with a particular reference to microsatellite markers, as well as that of the spatial scale of sampling. Moreover, we developed and tested a nonparametric bootstrap procedure allowing the construction of confidence intervals for the estimation of Dsigma2. These two objectives prompted us to develop a computer simulation algorithm based on the coalescent theory giving individual genotypes for a continuous population under isolation by distance. Our results show that the characteristics of mutational processes at microsatellite loci, namely the allele size homoplasy generated by stepwise mutations, constraints on allele size, and change of slippage rate with repeat number, have little influence on the estimation of Dsigma2. In contrast, a high genetic diversity (approximately 0.7-0.8), as is commonly observed for microsatellite markers, substantially increases the precision of the estimation. However, very high levels of genetic diversity (>0.85) were found to bias the estimation. We also show that statistics taking into account allele size differences give unreliable estimations (i.e., high variance of Dsigma2 estimation) even under a strict stepwise mutation model. Finally, although we show that this method is reasonably robust with respect to the sampling scale, sampling individuals at a local geographical scale gives more precise estimations of Dsigma2.     

Role of vascularization in determining the time of hypoxic-ischemic encephalopathy in the neonate

OBJECTIVE: To examine the role of vascularization in determining the time of hypoxic-ischemic encephalopathy (HIEP). STUDY DESIGN: Brain sections of 126 neonatal autopsy cases were examined for edema, gliosis, congestion, inflammation and ischemia. Capillary vessels were examined with both reticulum stains and antibody against CD34. Vascular surface density (VSD) and number of vessels per stroma (NVES) were calculated by stereologic methods. RESULTS: Among 126 cases, 64 were male (50.8%) and 62 female (49.2%). In 25 cases HIEP was observed; 14 had a pregnancy history of hypertension, eclampsia or diabetes mellitus in the mother, with fetal distress or underdeveloped features. Statistically, NVES was strongly related to primary HIEP. However, the HIEP and non-HIEP cases revealed no differences in NVES and VSD means. CONCLUSION: Vascularization, especially NVES, helps in determining whether an HIEP case is pregnancy related or due to end-stage changes of dying, but is not an indicator of HIEP.