Nocardia vulneris sp. nov., isolated from wounds of human patients in North America

Nocardia species are ubiquitous in the environment with an increasing number of species isolated from clinical sources. From 2005 to 2009, eight isolates (W9042, W9247, W9290, W9319, W9846, W9851^T, W9865, and W9908) were obtained from eight patients from three states in the United States and Canada; all were from males ranging in age from 47 to 81 years old; and all were obtained from finger (n = 5) or leg (n = 3) wounds. Isolates were characterized by polyphasic analysis using molecular, phenotypic, morphologic and chemotaxonomic methods. Sequence analysis of 16S rRNA gene sequences showed the eight isolates are 100 % identical to each other and belong in the genus Nocardia. The nearest phylogenetically related neighbours were found to be the type strains for Nocardia altamirensis (99.33 % sequence similarity), Nocardia brasiliensis (99.37 %), Nocardia iowensis (98.95 %) and Nocardia tenerifensis (98.44 %). The G+C content of isolate W9851^T was determined to be 68.4 mol %. The DNA–DNA relatedness between strain W9851^T and the N. brasiliensis type strain was 72.8 % and 65.8 % when measured in the laboratory and in silico from genome sequences, respectively, and 95.6 % ANI. Whole-cell peptidoglycan was found to contain meso-diaminopimelic acid; MK-8-(H₄)_ω-cyc was identified as the major menaquinone; the major fatty acids were identified as C_16:0, 10 Me C_18:0, and C_{18:1 w9c}, the predominant phospholipids were found to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides; whole-cell sugars detected were arabinose and galactose; and mycolic acids ranging from 38 to 60 carbon atoms were found to be present. These chemotaxonomic analyses are consistent with assignment of the isolates to the genus Nocardia. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of the clinical isolates showed genus and species level profiles that were different from other Nocardia species. All isolates were resistant to ciprofloxacin, clarithromycin and imipenem but were susceptible to amikacin, amoxicillin/clavulanate, linezolid and trimethoprim/sulfamethoxazole. The results of our polyphasic analysis suggest the new isolates obtained from wound infections represent a novel species within the genus Nocardia, for which the name Nocardia vulneris sp. nov. is proposed, with strain W9851^T (= DSM 45737^T = CCUG 62683^T = NBRC 108936^T) as the type strain.     

Estimates of dietary threshold levels for crude protein and amino acids to obtain plateau values of apparent ileal crude protein and amino acid digestibilities in newly weaned pigs

This study was conducted to estimate dietary threshold levels for crude protein (CP) and amino acids (AA) at which apparent ileal digestibilities (AID) of CP and AA in assay diets for newly weaned pigs reach plateaus. A total of 14 (12 + 2 for replacement) three-week old barrows were fitted with simple T cannulas at the distal ileum. Corn starch-based diets containing six graded levels of CP from casein, 90, 155, 220, 285, 350, or 415 g CP/kg assay diet (as-fed), were formulated. At 28 days of age, the pigs were randomly allocated to the six dietary treatments with two pigs per CP level in four weekly repeated measurement periods. They were fed twice daily a total of 30 g (as-fed) per kg of individual body weight at 8:00 and 20:00 h. The dietary CP and AA levels affected AID of CP and most AA (p = 0.005 to p = 0.040) in the assay diets. The AID of CP and AA were higher at 155 and 220 compared to 90 g CP/kg assay diet (p < 0.001 to p = 0.047). Initially, the AID of CP and AA increased sharply then gradually reached, at individual break points (p < 0.001 to p = 0.047), their plateaus (p < 0.001), which did not change up to dietary CP levels of 415 g/kg assay diet and the AID values became independent of the dietary AA levels. The piglets' capacity to digest CP and absorb AA was not limiting under these experimental conditions. There was no effect of age on AID of CP and AA (p = 0.056 to p = 0.899) except for a linear increase (p = 0.045) in AID of glycine from Period 1 to 4. Segmented quadratic with plateau relationships between the AID of CP and AA and their dietary contents were fitted for CP and each AA. The lower end points of 95% confidence intervals of the plateau AID values were defined to represent the initial plateau AID. The dietary CP and AA contents, corresponding to the initial plateau AID values, represent the dietary threshold levels. For CP and the indispensable amino acids, the plateau AID [%] and the dietary threshold levels [g/kg DM], respectively, in casein were: CP, 94.2 and 176; arginine, 95.1 and 7; histidine, 96.0 and 5; isoleucine, 96.4 and 8; leucine, 96.8 and 16; lysine, 96.8 and 12; methionine, 97.9 and 5; phenylalanine, 96.2 and 10; threonine, 93.4 and 9; tryptophan, 94.3 and 2; valine, 95.9 and 11. For the determination of plateau AID in piglets, the crude protein and amino acid contents in the assay diets should meet or exceed the corresponding dietary threshold levels.     

Background correction of two-colour cDNA microarray data using spatial smoothing methods

The analysis of two-colour cDNA microarray data usually involves subtracting background values from foreground values prior to normalization and further analysis. This approach has the advantage of reducing bias and the disadvantage of blowing up the variance of lower abundant spots. Whenever background subtraction is considered, it implicitly assumes locally constant background values. In practice, this assumption is often not met, which casts doubts on the usefulness of simple background subtraction. In order to improve background correction, we propose local background smoothing within the pre-processing pipeline of cDNA microarray data prior to background correction. For this purpose, we employ a geostatistical framework with ordinary kriging using both isotropic and anisotropic models of spatial correlation and 2-D locally weighted regression. We show that application of local background smoothing prior to background correction is beneficial in comparison to using raw background estimates. This is done using data of a self-versus-self experiment in Arabidopsis where subsets of differentially expressed genes were simulated. Using locally smoothed background values in conjunction with existing background correction methods increases the power, increases the accuracy and decreases the number of false positive results.     

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development

Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.     

Elucidation of the ipso-substitution mechanism for side-chain cleavage of alpha-quaternary 4-nonylphenols and 4-t-butoxyphenol in Sphingobium xenophagum Bayram

Recently we showed that degradation of several nonylphenol isomers with alpha-quaternary carbon atoms is initiated by ipso-hydroxylation in Sphingobium xenophagum Bayram (F. L. P. Gabriel, A. Heidlberger, D. Rentsch, W. Giger, K. Guenther, and H.-P. E. Kohler, J. Biol. Chem. 280:15526-15533, 2005). Here, we demonstrate with 18O-labeling experiments that the ipso-hydroxy group was derived from molecular oxygen and that, in the major pathway for cleavage of the alkyl moiety, the resulting nonanol metabolite contained an oxygen atom originating from water and not from the ipso-hydroxy group, as was previously assumed. Our results clearly show that the alkyl cation derived from the alpha-quaternary nonylphenol 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol through ipso-hydroxylation and subsequent dissociation of the 4-alkyl-4-hydroxy-cyclohexadienone intermediate preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the metabolism of certain alpha,alpha-dimethyl-substituted nonylphenols appears to also involve a reaction of the cation with the ipso-hydroxy group to form the corresponding 4-alkoxyphenols. Growth, oxygen uptake, and 18O-labeling experiments clearly indicate that strain Bayram metabolized 4-t-butoxyphenol by ipso-hydroxylation to a hemiketal followed by spontaneous dissociation to the corresponding alcohol and p-quinone. Hydroquinone effected high oxygen uptake in assays with induced resting cells as well as in assays with cell extracts. This further corroborates the role of hydroquinone as the ring cleavage intermediate during degradation of 4-nonylphenols and 4-alkoxyphenols.     

Potential causes of linkage disequilibrium in a European maize breeding program investigated with computer simulations

Knowledge about the forces generating and conserving linkage disequilibrium (LD) is important for drawing conclusions about the prospects and limitations of association mapping. The objectives of our research were to examine the importance of (1) selection, (2) mutation, and (3) genetic drift for generating LD in a typical maize breeding program. We conducted computer simulations based on genotypic data of Central European maize open-pollinated varieties which have played an important role as founders of the European flint heterotic group. The breeding scheme and the dimensioning underlying our simulations reflect essentially the maize breeding program of the University of Hohenheim. Results suggested that in a plant breeding program of the examined dimension and breeding scheme, genetic drift and selection are major forces generating LD. The currently used population-based association mapping tests do not explicitly correct for LD caused by these two forces. Therefore, increased type I error rates are expected if these tests are applied to plant breeding populations. As a consequence, we recommend to use family-based association tests for association mapping approaches in plant breeding populations.     

Molecular identification of two strains of third-stage larvae of Contracaecum rudolphii sensu lato (Nematoda: Anisakidae) from fish in Poland

Contracaecum sp. larvae (L3) from fish were identified using nucleotide sequences of the internal transcribed spacers ITS-1 and ITS-2 of the ribosomal DNA. The nematode larvae originated from fish in a freshwater situation (crucian carp Carassius carassius, from Selment Wielki Lake in Mazury, northeastern Poland) and a brackish-water region (Caspian round goby Neogobius melanostomus from the Baltic Sea, Gdafisk Bay at the Polish coast). Two strains (Contracaecum rudolphii A and B) of Contracaecum rudolphii senso lato, a parasite common at the adult stage in fish-eating birds, were identified. In fish from the freshwater site, only the strain temporarily designated C. rudolphii B was identified; in the brackish-water region, both strains were found, suggesting that fish serve as paratenic host for both genotypes. Contracaecum rudolphii sensu lato has been recorded in several species of fish-eating birds in Poland, particularly in the great cormorant, Phalacrocorax carbo, in which the abundance is highest. The results, although based on a restricted number of larvae, suggest that the life cycles of both genotypes can be completed in the Polish region and that at least one of them, C. rudolphii B, can develop both in fresh and brackish water.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

Integral and associated lysosomal membrane proteins

We searched for novel proteins in lysosomal membranes, tentatively participating in molecular transport across the membrane and/or in interactions with other compartments. In membranes purified from placental lysosomes, we identified 58 proteins, known to reside at least partially in the lysosomal membrane. These included 17 polypeptides comprising or associated with the vacuolar adenosine triphosphatase. We report on additional 86 proteins that were significantly enriched in the lysosomal membrane fraction. Among these, 12 novel proteins of unknown functions were found. Three were orthologues of rat proteins that have been identified in tritosomes by Bagshaw RD et al. (A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle. Mol Cell Proteomics 2005;4:133-143). Here, the proteins encoded by LOC201931 (FLJ38482) and LOC51622 (C7orf28A) were expressed with an appended fluorescent tag in HeLa cells and found to be present in lysosomal organelles. Among the lysosomally enriched proteins, also 16 enzymes and transporters were detected that had not been assigned to lysosomal membranes previously. Finally, our results identified a particular set of proteins with known functions in signaling and targeting to be at least partially associated with lysosomes.     

Diffusion tensor imaging and tractwise fractional anisotropy statistics: quantitative analysis in white matter pathology

Background  

Information on anatomical connectivity in the brain by measurements of the diffusion of water in white matter tracts lead to quantification of local tract directionality and integrity.