Rapid species and strain differentiation of non-tubercoulous mycobacteria by Fourier-Transform Infrared microspectroscopy

Rapid identification of non-tuberculous mycobacteria (NTM) species is important in clinical laboratories to stipulate the appropriate therapy and to offer a comprehensive infection control. We applied Fourier-Transform Infrared microspectroscopy to evaluate, whether the most frequent species of NTM can be rapidly and uniformly identified by this method using microcolonies of NTM growing on solid nutrient agar plates. To establish a standardized protocol, the heterogeneity of cell growth within the microcolonies and the reproducibility of measuring the IR spectra from whole mycobacterial microcolonies were first studied. Hierarchical cluster analysis applied to spectra obtained by linear mapping across microcolony imprints from fast- and slow-growing NTM revealed only little spectral variance between the various microcolony zones. In parallel, when repetitive measurements were performed on independently grown whole single microcolonies with diameters of 80 and 140 mum, excellent reproducibility could be achieved, verifying that mycobacterial microcolonies are well suited for FT-IR-based identification. Twenty-eight different and well-defined strains, comprising the most frequent species of NTM isolated in clinical laboratories, were used to create a classification system based on FT-IR spectra from single microcolonies. Hierarchical cluster analysis allowed the assignment of all isolates measured in replicates to their correct species-specific clusters. Additionally, a clear separation of all strains into strain-specific sub-clusters was observed. These results demonstrate the potential of FT-IR microspectroscopy to rapidly differentiate NTM at the species and strain level. The data so far obtained suggest that an extended spectral database, containing more NTM strains and covering a broader biological variance, may provide a practical solution to rapidly identify unknown NTM isolates in routine clinical-microbiological laboratories with the additional possibility to type these microorganisms at the sub-species level.     

Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.     

Spontaneous cross-link of mutated alpha1 subunits during GABA(A) receptor assembly

gamma-Aminobutyric acid, type A (GABA(A)) receptor alpha1 subunits containing a cysteine mutation at a position in the channel mouth (H109C) surprisingly formed a spontaneous cross-link with each other in receptors composed of alpha1H109C, beta3, and gamma2 subunits. Cross-linking of two alpha1H109C subunits did not significantly change the affinity of [(3)H]muscimol or [(3)H]Ro15-1788 binding in alpha1H109Cbeta3gamma2 receptors, but GABA displayed a reduced potency for activating chloride currents. On reduction of the disulfide bond, however, GABA activation as well as diazepam modulation was similar in mutated and wild-type receptors, suggesting that these receptors exhibited the same subunit stoichiometry and arrangement. Disulfide bonds could not be reoxidized by copper phenanthroline after having been reduced in completely assembled receptors, suggesting that cross-linking can only occur at an early stage of assembly. The cross-link of alpha1H109C subunits and the subsequent transport of the resulting homodimers to the cell surface caused a reduction of the intracellular pool of alpha1H109C subunits and a reduced formation of completely assembled receptors. The formation of alpha1H109C homodimers as well as of correctly assembled GABA(A) receptors containing cross-linked alpha1H109C subunits could indicate that homodimerization of alpha1 subunits via contacts located in the channel mouth might be one starting point of GABA(A) receptor assembly. Alternatively the assembly mechanism might have started with the formation of heterodimers followed by a cross-link of mutated alpha1 subunits at the heterotrimeric stage. The formation of cross-linked alpha1H109C homodimers would then have occurred independently in a separate pathway.     

Molecular phylogeny, biogeography, and systematics of Dicerandra (Lamiaceae), a genus endemic to the southeastern United States

Dicerandra, an endemic mint of the southeastern United States, comprises nine species, all of which are threatened or endangered and restricted to sandhill vegetation and a mosaic of scrub habitats. Molecular phylogenetic analyses of Dicerandra based on data from the nuclear and plastid genomes for all 13 taxa of the genus, identified two strongly supported clades, corresponding to the four annual and to the five perennial species of Dicerandra. However, the nuclear and plastid trees were incongruent in their placement of two perennial taxa, D. cornutissima and D. immaculata var. savannarum, perhaps due to ancient hybridization or to lineage sorting. Based on these analyses, the widespread D. linearifolia is not monophyletic, with populations of D. linearifolia var. linearifolia falling into either western or eastern clades. The western clade, comprising populations of D. linearifolia var. linearifolia and var. robustior, occurs in an area drained by rivers flowing toward the Gulf of Mexico, whereas the eastern clade, comprising populations of D. linearifolia var. linearifolia, D. densiflora, D. odoratissima, and D. radfordiana (i.e., all the annual species), occupies a region drained by rivers flowing to the Atlantic Ocean. Although this pattern of genetic differentiation between populations from these two river drainages has been documented in several animal species, it has not previously been reported for plants. A revised subgeneric classification is presented to reflect the annual and perennial clades.     

Genetic differentiation among populations of Cicerbita alpina (L.) Wallroth (asteraceae) in the western Alps

In this study we analyzed the genetic population structure of the hygrophilous tall-herb Cicerbita alpina in the western Alps because this group of mountain plants is underrepresented in the biogeographical literature. AFLP (amplified fragment length polymorphism) fingerprints of 40 samples were analyzed from four populations situated in a transect from the southwestern Alps to the eastern part of the western Alps and one population from the Black Forest outside the Alps. Two genetic groups can be distinguished. The first group (A) comprises the populations from the northern and eastern parts of the western Alps, and the second group (B) comprises the populations from the southwestern Alps and the Black Forest. Group A originates most likely from at least one refugium in the southern piedmont regions of the Alps. This result provides molecular evidence for a humid climate at the southern margin of the Alps during the Würm glaciation. Group B originates presumably from western or northern direction and we discuss two possible scenarios for the colonization of the Alps, i.e. (1) long-distance dispersal from southwestern refugia and (2) colonization from nearby refugia in the western and/or northern Alpine forelands. The study demonstrates that the target species harbours considerable genetic diversity, even on a regional scale, and therefore is a suitable model for phylogeographic research.     

Alternative profiling platform based on MELDI and its applicability in clinical proteomics

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.     

A new analytical material-enhanced laser desorption ionization (MELDI) based approach for the determination of low-mass serum constituents using fullerene derivatives for selective enrichment

60]fullerene derivatives (dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA-[60]fullerene) were prepared and subjected to a comprehensive characterization study including protein binding properties and capacity. These fullerene derivatives were successfully applied as material-enhanced laser desorption/ionization (MELDI) carrier materials. It is shown that diverse functionalities result in characteristic human serum peak patterns (m/z 2000-20 000) in terms of signal intensity as well as the number of detectable masses. In addition, the fullerene derivatives clearly provided differences in the low molecular weight mass region (m/z 1000-4000) after elution of the adsorbed serum constituents, and [60]fullerenoacetic acid was the most effective carrier material. Novel high-speed, monolithic, high-resolution capillary columns, prepared by thermally initiated copolymerization of methylstyrene (MSt) and 1,2-bis(p-vinylphenyl)ethane (BVPE) were employed for eluate separation and target spotting. Thus, serum compounds in the low-mass range were successfully fractionated and subjected to MALDI-MS/MS analysis. This contribution, hence, proposes a new "top-down" strategy for proteome research enabling protein profiling as well as biomarker identification in the low-mass range using selective enrichment, high-resolution separation, and offline MALDI-MS/MS evaluation.     

Assessment of angiogenesis-related parameters in juvenile idiopathic arthritis-associated uveitis

Molecular Biology Reports - Juvenile idiopathic arthritis-associated uveitis (JIAU) may run a chronic and treatment-resistant course, and occasionally, alterations of the iris vasculature may be...     

Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences

The significance of bacteria for eukaryotic functioning is increasingly recognized. Coral reef ecosystems critically rely on the relationship between coral hosts and their intracellular photosynthetic dinoflagellates, but the role of the associated bacteria remains largely theoretical. Here, we set out to relate coral‐associated bacterial communities of the fungid host species Ctenactis echinata to environmental settings (geographic location, substrate cover, summer/winter, nutrient and suspended matter concentrations) and coral host abundance. We show that bacterial diversity of C. echinata aligns with ecological differences between sites and that coral colonies sampled at the species’ preferred habitats are primarily structured by one bacterial taxon (genus Endozoicomonas) representing more than 60% of all bacteria. In contrast, host microbiomes from lower populated coral habitats are less structured and more diverse. Our study demonstrates that the content and structure of the coral microbiome aligns with environmental differences and denotes habitat adequacy. Availability of a range of coral host habitats might be important for the conservation of distinct microbiome structures and diversity.