Methylglyoxal and other carbohydrate metabolites induce lanthanum-sensitive Ca2+ transients and inhibit growth in E. coli

The results here are the first demonstration of a family of carbohydrate fermentation products opening Ca²⁺ channels in bacteria. Methylglyoxal, acetoin (acetyl methyl carbinol), diacetyl (2,3 butane dione), and butane 2,3 diol induced Ca²⁺ transients in Escherichia coli, monitored by aequorin, apparently by opening Ca²⁺ channels. Methylglyoxal was most potent (K_1/2 = 1 mM, 50 mM for butane 2,3 diol). Ca²⁺ transients depended on external Ca²⁺ (0.1-10 mM), and were blocked by La³⁺ (5 mM). The metabolites affected growth, methylglyoxal being most potent, blocking growth completely up to 5 h without killing the cells. But there was no affect on the number of viable cells after 24 h. These results were consistent with carbohydrate products activating a La³⁺-sensitive Ca²⁺ channel, rises in cytosolic Ca²⁺ possibly protecting against certain toxins. They have important implications in bacterial-host cell signalling, and where numbers of different bacteria compete for the same substrates, e.g., the gut in lactose and food intolerance.     

Treeness triangles: visualizing the loss of phylogenetic signal

It is well known that molecular data "saturates" with increasing sequence divergence (thereby losing phylogenetic information) and that in addition the accumulation of misleading information due to chance similarities or to systematic bias may accompany saturation as well. Exploratory data analysis methods that can quantify the extent of signal loss or convergence for a given data set are scarce. Such methods are needed because genomics delivers very long sequence alignments spanning substantial phylogenetic depth, where site saturation may be compounded by systematic biases or other alternative signals. Here we introduce the Treeness Triangle (TT) graph, in which signals detectable by Hadamard (spectral) analysis are summed into 3 categories--those supporting 1) external and 2) internal branches in the optimal tree, in addition to 3) the residuals (potential internal branches not present in the optimal tree). These 3 values are plotted in a standard ternary coordinate system. The approach is illustrated with simulated and real data sets, the latter from complete chloroplast genomes, where potential problems of paralogy or lateral gene acquisition can be excluded. The TT uncovers the divergence-dependent loss of phylogenetic signal as subsets of chloroplast genomes are investigated that span increasingly deeper evolutionary timescales. The rate of signal loss (or signal retention) varies with the gene and/or the method of analysis.     

Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.     

Utility of multispectral imaging for nuclear classification of routine clinical histopathology imagery

Background

We present an analysis of the utility of multispectral versus standard RGB imagery for routine H&;E stained histopathology images, in particular for pixel-level classification of nuclei. Our multispectral imagery has 29 spectral bands, spaced 10 nm within the visual range of 420–700 nm. It has been hypothesized that the additional spectral bands contain further information useful for classification as compared to the 3 standard bands of RGB imagery. We present analyses of our data designed to test this hypothesis.

Results

For classification using all available image bands, we find the best performance (equal tradeoff between detection rate and false alarm rate) is obtained from either the multispectral or our "ccd" RGB imagery, with an overall increase in performance of 0.79% compared to the next best performing image type. For classification using single image bands, the single best multispectral band (in the red portion of the spectrum) gave a performance increase of 0.57%, compared to performance of the single best RGB band (red). Additionally, red bands had the highest coefficients/preference in our classifiers. Principal components analysis of the multispectral imagery indicates only two significant image bands, which is not surprising given the presence of two stains.

Conclusion

Our results indicate that multispectral imagery for routine H&;E stained histopathology provides minimal additional spectral information for a pixel-level nuclear classification task than would standard RGB imagery.

     

Amphioxus AmphiDelta: evolution of Delta protein structure, segmentation, and neurogenesis

The amphioxus genome has a single Delta gene (AmphiDelta) encoding a protein 766 amino acids long. Comparison of Delta proteins of amphioxus and other animals indicates that AmphiDelta retains features of a basal bilaterian Delta protein--in having nine epidermal growth factor (EGF) repeats and also in having characteristic numbers of amino acids separating successive cysteines between and within EGF repeats. During development, AmphiDelta is expressed in the forming somites, in some regions of pharyngeal endoderm, and in cells (presumably differentiating neurons) scattered in both the neural plate and ectoderm. Expression is strongly associated with cells initiating movements to separate themselves from parent epithelia, either en masse by evagination (endoderm and mesoderm) or by delamination as isolated cells (ectoderm). The AmphiDelta-expressing cells delaminating from the ectoderm apparently migrate beneath it as they begin differentiating into probable sensory neurons, suggesting a scenario for the evolutionary origin of the placode-derived neurons of vertebrate cranial ganglia.     

Mapping resistance to Southern rust in a tropical by temperate maize recombinant inbred topcross population

Southern rust, caused by Puccinia polysora Underw, is a foliar disease that can severely reduce grain yield in maize (Zea mays L.). Major resistance genes exist, but their effectiveness can be limited in areas where P. polysora is multi-racial. General resistance could be achieved by combining quantitative and race-specific resistances. This would be desirable if the resistance alleles maintained resistance across environments while not increasing plant maturity. Recombinant inbred (RI) lines were derived from a cross between NC300, a temperate-adapted all-tropical line, and B104, an Iowa Stiff Stalk Synthetic line. The RI lines were topcrossed to the tester FR615 × FR697. The 143 topcrosses were scored for Southern rust in four environments. Time to flowering was measured in two environments. The RI lines were genotyped at 113 simple sequence repeat markers and quantitative trait loci (QTL) were mapped for both traits. The entry mean heritability estimate for Southern rust resistance was 0.93. A multiple interval mapping model, including four QTL, accounted for 88% of the variation among average disease ratings. A major QTL located on the short arm of chromosome 10, explained 83% of the phenotypic variation, with the NC300 allele carrying the resistance. Significant (P < 0.001), but relatively minor, topcross-by-environment interaction occurred for Southern rust, and resulted from the interaction of the major QTL with the environment. Maturity and Southern rust rating were slightly correlated, but QTL for the two traits did not co-localize. Resistance was simply inherited in this population and the major QTL is likely a dominant resistant gene that is independent of plant maturity.     

QTL mapping with near-isogenic lines in maize

A set of 89 near-isogenic lines (NILs) of maize was created using marker-assisted selection. Nineteen genomic regions, identified by restriction fragment length polymorphism loci and chosen to represent portions of all ten maize chromosomes, were introgressed by backcrossing three generations from donor line Tx303 into the B73 genetic background. NILs were genotyped at an additional 128 simple sequence repeat loci to estimate the size of introgressions and the amount of background introgression. Tx303 introgressions ranged in size from 10 to 150 cM, with an average of 60 cM. Across all NILs, 89% of the Tx303 genome is represented in targeted and background introgressions. The average proportion of background introgression was 2.5% (range 0–15%), significantly lower than the expected value of 9.4% for third backcross generation lines developed without marker-assisted selection. The NILs were grown in replicated field evaluations in two years to map QTLs for flowering time traits. A parallel experiment of testcrosses of each NIL to the unrelated inbred, Mo17, was conducted in the same environments to map QTLs in NIL testcross hybrids. QTLs affecting days to anthesis, days to silking, and anthesis-silk interval were detected in both inbreds and hybrids in both environments. The testing environments differed dramatically for drought stress, and different sets of QTLs were detected across environments. Furthermore, QTLs detected in inbreds were typically different from QTLs detected in hybrids, demonstrating the genetic complexity of flowering time. NILs can serve as a valuable genetic mapping resource for maize breeders and geneticists. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Naegeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosa reticularis: two allelic ectodermal dysplasias caused by dominant mutations in KRT14

Naegeli-Franceschetti-Jadassohn syndrome (NFJS) and dermatopathia pigmentosa reticularis (DPR) are two closely related autosomal dominant ectodermal dysplasia syndromes that clinically share complete absence of dermatoglyphics (fingerprint lines), a reticulate pattern of skin hyperpigmentation, thickening of the palms and soles (palmoplantar keratoderma), abnormal sweating, and other subtle developmental anomalies of the teeth, hair, and skin. To decipher the molecular basis of these disorders, we studied one family with DPR and four families with NFJS. We initially reassessed linkage of NFJS/DPR to a previously established locus on 17q11.2-q21. Combined multipoint analysis generated a maximal LOD score of 8.3 at marker D17S800 at a recombination fraction of 0. The disease interval was found to harbor 230 genes, including a large cluster of keratin genes. Heterozygous nonsense or frameshift mutations in KRT14 were found to segregate with the disease trait in all five families. In contrast with KRT14 mutations affecting the central alpha -helical rod domain of keratin 14, which are known to cause epidermolysis bullosa simplex, NFJS/DPR-associated mutations were found in a region of the gene encoding the nonhelical head (E1/V1) domain and are predicted to result in very early termination of translation. These data suggest that KRT14 plays an important role during ontogenesis of dermatoglyphics and sweat glands. Among other functions, the N-terminal part of keratin molecules has been shown to confer protection against proapoptotic signals. Ultrastructural examination of patient skin biopsy specimens provided evidence for increased apoptotic activity in the basal cell layer where KRT14 is expressed, suggesting that apoptosis is an important mechanism in the pathogenesis of NFJS/DPR.