Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide

Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months.     

Biological diversification in a complex region: a spatial analysis of faunistic diversity and biogeography of the Andes of Colombia

Aim Understanding large‐scale patterns of beta diversity and endemism is essential for ecoregional conservation planning. We present a study of spatial patterns of faunal diversification and biogeographical relationships in the Andean region of Colombia. This region has a great geomorphological complexity, as it is formed by several mountain ranges with different geologic origins. We hypothesize that this complexity results in a high turnover in species composition among subregions. Location The Andean region of Colombia, including the Santa Marta and Macarena mountain ranges. Methods The region was divided into subregions, represented by the eastern and western slopes of each of the three Andean cordilleras, the Cauca and Magdalena valley bottoms, and the peripheral mountain ranges of Perijá, Macarena and Sierra Nevada de Santa Marta. Species lists for five animal taxa (rodents, bats, birds, frogs and butterflies) were compiled for each subregion and similarities in species composition were determined by cluster analysis. To explore biogeographical relationships, species were classified into one of four distributional categories: endemic, tropical Andean, Andean‐Central American and wide continental distribution. Results The highest species richness in the region was found in the Pacific and eastern versants of the Andes, and the lowest in the Cauca and Magdalena valley bottoms. Inter‐Andean slopes were intermediate in species richness. However, when species richness was calculated per unit area, the most diverse regions were the Santa Marta and Macarena ranges, the Cauca Valley watershed and the Pacific slope. Although each taxonomic group had a different branching pattern, dendrograms indicated five common subregional clusterings: (1) Perijá‐Sierra Nevada, (2) the Pacific slope, (3) the eastern Andean slope, (4) the Cauca and Magdalena valley bottoms, and (5) the inter‐Andean slopes. Clustering patterns of inter‐Andean slopes varied among taxa. In birds, bats and rodents, grouping was by opposite slopes of the same valley, whereas frogs were grouped by mountain ranges and butterflies by valleys and their respective slopes. Seventy‐five per cent of species in all taxa were found in less than five subregions. The fauna of the Magdalena and Cauca valley bottoms was composed mostly of lowland species with wide geographical distributions, whereas the cordilleran fauna was mostly restricted to the tropical Andes. Main conclusions The western and eastern versants of the Andes have the highest species richness, but are also the largest subregions. On a per unit area basis, the peripheral ranges (Santa Marta and Macarena) are the richest, followed by the western portion of the Andes (the Cauca Valley watershed and the Pacific versant). Clustering patterns in dendrograms suggest two major patterns of differentiation of the Andean fauna: one elevational (lowlands vs. highlands) and one horizontal (among ranges and/or slopes). Biogeographical affinities of the inter‐Andean valley bottoms are with the lowland faunas of tropical America. In contrast, Andean faunas diversified locally, resulting in the evolution of a large number of endemic species, particularly among the less vagile taxa. Three different main branches of Andean fauna can be recognized, one confined to the Pacific, another to the eastern (Amazonian‐Llanos) versant of the Andes, and the third one composed by the inter‐Andean slopes of the Cauca and Magdalena valleys. The identification of five main biogeographical units in the Andean region of Colombia has important implications for the conservation of the regional biota. Conservation initiatives that seek to preserve representative samples of the regional biodiversity should take into account the patterns of diversification described here, and the evolutionary processes that gave rise to these patterns.     

Imaging Mass Spectrometry Reveals Alterations in N-Linked Glycosylation That Are Associated With Histopathological Changes in Nonalcoholic Steatohepatitis in Mouse and Human

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation, hepatocyte injury, and fibrosis. Further, NASH is a risk factor for cirrhosis and hepatocellular carcinoma. Previous research demonstrated that serum N-glycan profiles can be altered in NASH patients. Here, we hypothesized that these N-glycan modifications may be associated with specific liver damage in NAFLD and NASH. To investigate the N-glycome profile in tissue, imaging mass spectrometry was used for a qualitative and quantitative in situ N-linked glycan analysis of mouse and human NAFLD/NASH tissue. A murine model was used to induce NAFLD and NASH through ad libitum feeding with either a high-fat diet or a Western diet, respectively. Mice fed a high-fat diet or Western diet developed inflammation, steatosis, and fibrosis, consistent with NAFLD/NASH phenotypes. Induction of NAFLD/NASH for 18 months using high caloric diets resulted in increased expression of mannose, complex/fucosylated, and hybrid N-glycan structures compared to control mouse livers. To validate the animal results, liver biopsy specimens from 51 human NAFLD/NASH patients representing the full range of NASH Clinical Research Network fibrosis stages were analyzed. Importantly, the same glycan alterations observed in mouse models were observed in human NASH biopsies and correlated with the degree of fibrosis. In addition, spatial glycan alterations were localized specifically to histopathological changes in tissue like fibrotic and fatty areas. We demonstrate that the use of standard staining’s combined with imaging mass spectrometry provide a full profile of the origin of N-glycan modifications within the tissue. These results indicate that the spatial distribution of abundances of released N-glycans correlate with regions of tissue steatosis associated with NAFLD/NASH.