New Spatially Explicit Method for Detecting Extracellular Protease Activity in Biofilms

A novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.     

Der faulschlamm und seine ziliaten leitformen

Ohne Zusammenfassung     

The Parvidrilidae – a diversified groundwater family: description of six new species from southern Europe,and clues for its phylogenetic position within Clitellata (Annelida)

The Parvidrilidae Erséus, 1999 constitute the most recently described family of oligochaete microdriles. Prior to this study, Parvidrilus strayeri Erséus, 1999, and Parvidrilus spelaeus Martínez‐Ansemil, Sambugar & Giani, 2002, found in groundwaters of the USA (Alabama) and Europe (Slovenia and Italy), respectively, were the only two species in this family. In this paper, six new species – Parvidrilus camachoi , Parvidrilus gianii , Parvidrilus jugeti , Parvidrilus meyssonnieri , Parvidrilus stochi , and Parvidrilus tomasini – and Parvidrilus gineti (Juget, 1959) comb. nov. are added to the family. With all species being stygobiont, the Parvidrilidae is unique in being the only family of oligochaetes worldwide comprising taxa that are restricted to groundwater habitats. Parvidrilids are exceedingly small worms whose principal morphological characteristics are the presence of hair setae in ventral bundles, the markedly posterior position of setae within the segments, the presence of mid‐dorsal glandular pouches in mesosomial segments, the lateral development of the clitellum, the presence of a single male pore in segment XII, and the presence (or absence) of a single spermatheca. The phylogenetic relationships of the Parvidrilidae within the Clitellata were investigated using the nuclear 18S rRNA gene, and the most representative and taxonomically balanced data set of clitellate families available to date. The data were analysed by parsimony, maximum likelihood, and Bayesian inference. Irrespective of the method used, Parvidrilidae were placed far from Capilloventridae, one family once suggested to be closely related to parvidrilids. Although closer to Enchytraeidae than Phreodrilidae, two other suggested putative sister families, the exact position of Parvidrilidae within Clitellata still remained uncertain in the absence of branch support. The examination of reproductive structures, together with the similarity of other important anatomical traits of the new species herein described, reinforced the idea that phreodrilids were the best candidate to be the sister group to parvidrilids on morphological grounds. A fragment of the mitochondrial cytochrome oxidase I gene, used as a barcode, also genetically characterized a few Parvidrilus species. The observation that two species diverge from each other by high genetic distances, even though their type localities are more or less only 100 km apart, is interpreted in the context of low dispersal abilities of inhabitants of the subterranean aquatic ecosystem, and habitat heterogeneity. The Parvidrilidae appear to be a diversified, Holarctic, and probably widely distributed family in groundwater, but very often overlooked because of the small size and external similarity with the polychaete family Aeolosomatidae of its members. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 530–558.     

Stepwise unfolding of ankyrin repeats in a single protein revealed by atomic force microscopy

Using single-molecule atomic force microscopy, we find that a protein consisting of six identical ankyrin repeat units flanked by N- and C-terminal modules (N6C) unfolds in a stepwise, unit-by-unit fashion under a mechanical force. Stretching a N6C molecule results in a sawtooth pattern fingerprint, with as many as six peaks separated by approximately 10 nm and an average unfolding force of 50 +/- 20 pN. Our results demonstrate that a stretching force can unfold multiple repeat units individually in a single protein molecule, despite extensive hydrophobic interactions between adjacent units.     

Clonal Dissemination of Escherichia coli O157:H7 Subtypes among Dairy Farms in Northeast Ohio

To ascertain the extent to which indistinguishable strains of Escherichia coli O157:H7 are shared between farms, molecular characterization was performed on E. coli O157:H7 isolates recovered during a longitudinal study of 20 dairy farms in northeast Ohio. Of the 20 dairy farms sampled, 16 were located in a primary area and 4 were located in two other distant geographical areas. A total of 92 E. coli O157:H7 isolates obtained from bovine fecal samples, water trough sediment samples, free-stall bedding, and wild-bird excreta samples were characterized. Fifty genetic subtypes were observed among the isolates using XbaI and BlnI restriction endonucleases. Most restriction endonuclease digestion profiles (REDPs) were spatially and temporally clustered. However, four REDPs from multiple sources were found to be indistinguishable by pulsed-field gel electrophoresis between four pairs of farms. The geographical distance between farms which shared an indistinguishable E. coli O157:H7 REDP ranged from 9 to 50 km, and the on-farm sources sharing indistinguishable REDPs included cattle and wild bird feces and free-stall bedding. Within the study population, E. coli O157:H7 REDP subtypes were disseminated with considerable frequency among farms in close geographic proximity, and nonbovine sources may contribute to the transmission of this organism between farms.     

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.     

Comparison of microbial diversity of edible flowers and basil grown with organic versus conventional methods

The consumption and use of edible flowers as food is growing; however, no study has been conducted to evaluate their role in the cause of food-borne illness or in food safety. Recent food-borne outbreaks traced to fresh herbs have raised concern about their processing and handling. Basil, one of the most commonly used fresh herbs, has been identified as a source of food-borne illness. Baseline assessments of microflora were performed, and the microbial diversity between growing methods (organic vs. conventional) was compared. DNA sequencing was used to identify the microbial flora present on fresh edible flower and basil samples. The most predominant species identified were Enterobacter hormaechei (10%), Acinetobacter calcoaceticus (10%), Enterobacter ludwigii (10%), Enterobacter asburiae (6%), and Enterobacter cowanii (6%). Pseudomonas aeruginosa (6%), Salmonella enterica (6%), and Bacillus amyloliquefaciens (2%) were also isolated. Phylogenetic analysis showed that most species of isolated bacteria belonged to the phyla Gammaproteobacteria (81.2%) and Firmicutes (18.8%). Statistical analysis, diversity index for species richness, and lineage-per-time plots showed that for basil, organically grown samples had a higher microbial diversity than conventionally grown samples. Edible flowers and basil are often grown using organic methods and are commonly consumed raw without any washing or cooking, to preserve aesthetic value, but these practices may pose a potential risk for food-borne illness. The baseline assessment, together with phylogenetic and statistical analyses, indicated possible microbial contamination in edible flowers and basil. The use of statistical estimation of molecular diversity based on the 16S rRNA sequences and lineage-per-time plots with phylogenetic analysis well served as a means for comparing microbial diversity in food samples between the growing methods (organic vs. conventional).     

Exercise increases interleukin-10 levels both intraarticularly and peri-synovially in patients with knee osteoarthritis: a randomized controlled trial

Introduction  

The microdialysis method was applied to the human knee joint with osteoarthritis (OA) in order to reveal changes in biochemical markers of cartilage and inflammation, intraarticularly and in the synovium, in response to a single bout of mechanical joint loading.