Lack of association of Mycobacterium avium subsp. paratuberculosis with oocytes and embryos from moderate shedders of the pathogen

Paratuberculosis is a chronic and progressive disease of the intestine in ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). The bacterium is transmitted to young animals, becomes manifest in adulthood and leads to economic losses. The aim of this study is to investigate if cows shedding Map possess oocytes and embryos that are carriers of the bacterium. New genetical material can enter the dairy farm using embryo transfer but the question as to whether this technique is safe with respect to transmission of paratuberculosis has yet to be addressed. We selected and bought 16 cows, all proven to be moderate shedders of the bacterium in the faeces immediately prior to the experiment but none were clinically sick. One sample of uterine content was collected from each animal by flushing the uterus on the day of heat and five samples of homogenised uterine tissue were collected on the eighth day of the same cycle by biopsy. In addition, 217 cumulus-oocyte complexes (COCs), ranging from 3 to 35 COCs per animal, were collected using ultrasound guided transvaginal puncture of the ovarian follicles (OPU). On the seventh day of the subsequent cycle 31 embryos were obtained using the classic technique of super ovulation induction, artificial insemination (AI), followed by flushing of the uterus. These embryos have been washed and trypsinised. Fourteen of the 16 cows were treated again for super ovulation in the subsequent cycle and 19 foetuses were collected by opening of the uterus after euthanasia on Days 35-49 of the cycle. All samples were cultured for presence of Map and checked every 2 months during 1 year for bacterial growth. None of the samples showed growth of Map after 12 months of culture. Pathological examination of the cows revealed different degrees of severity of pathological alterations of the intestinal tract and mesenteric lymph nodes. However, the results suggest that neither in vivo embryo's nor oocytes are carriers of the bacteria and do not form an extra risk at transfer. However, due to the limited size of the experiment (sample size of 16 cows), a certain margin for error remains.     

Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces

Flow chambers are commonly used to study microbial adhesion to surfaces under environmentally relevant hydrodynamic conditions. The parallel plate flow chamber (PPFC) is the most common design, and mass transport occurs through slow convective diffusion. In this study, we analyzed four different PPFCs to determine whether the expected hydrodynamic conditions, which control both mass transport and detachment forces, are actually achieved. Furthermore, the different PPFCs were critically evaluated based on the size of the area where the velocity profile was established and constant with a range of flow rates, indicating that valid observations could be made. Velocity profiles in the different chambers were calculated by using a numerical simulation model based on the finite element method and were found to coincide with the profiles measured by particle image velocimetry. Environmentally relevant shear rates between 0 and 10,000 s(-1) could be measured over a sizeable proportion of the substratum surface for only two of the four PPFCs. Two models appeared to be flawed in the design of their inlets and outlets and allowed development of a stable velocity profile only for shear rates up to 0.5 and 500 s(-1). For these PPFCs the inlet and outlet were curved, and the modeled shear rates deviated from the calculated shear rates by up to 75%. We concluded that PPFCs used for studies of microbial adhesion to surfaces should be designed so that their inlets and outlets are in line with the flow channel. Alternatively, the channel length should be increased to allow a greater length for the establishment of the desired hydrodynamic conditions.     

Direct random insertion mutagenesis of Helicobacter pylori

Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or instability of the cloned DNA, or toxicity of the encoded products. We therefore created two mutant libraries in the human pathogen Helicobacter pylori using a simple, direct mutagenesis technique, which does not require E. coli as intermediate. H. pylori total DNA was digested, circularized and digested again with a frequently cutting restriction enzyme, and the resulting fragments were ligated to a kanamycin antibiotic resistance cassette. Subsequently, the ligation mixture was transformed into the parental H. pylori strain 1061. Insertion of the kanamycin cassette by double homologous recombination into the genome of H. pylori 1061 resulted in approximately 2500 kanamycin resistant colonies. Heterogeneity of kanamycin cassette insertion was confirmed by Southern blotting. The isolation of two independent H. pylori mutants defective in production of urease from this library underlines the potential of this mutagenesis strategy.     

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.     

MODBASE, a database of annotated comparative protein structure models

MODBASE is a queryable database of annotated comparative protein structure models. The models are derived by MODPIPE, an automated modeling pipeline relying on the programs PSI-BLAST and MODELLER. The database currently contains 3D models for substantial portions of approximately 17 000 proteins from 10 complete genomes, including those of Caenorhabditis elegans, Saccharomyces cerevisiae and Escherichia coli, as well as all the available sequences from Arabidopsis thaliana and Homo sapiens. The database also includes fold assignments and alignments on which the models were based. In addition, special care is taken to assess the quality of the models. ModBase is accessible through a web interface at http://guitar.rockefeller.edu/modbase/     

Highly efficient immobilization of glycosylated enzymes into polyurethane foams

Glycosylated enzymes, including aminoacylase from Aspergillus melleus, chloroperoxidase from Caldariomyces fumago, and phytase from Aspergillus ficuum, were covalently immobilized into polyurethane foams with very high enzyme loadings of up to 0.2 g protein per gram dry foam. The immobilization efficiency (retained activity) ranged from 100% at a low loading to 60% at high loadings. In contrast to many other immobilization methods no leaching of the enzyme from the support took place under the reaction conditions. In short, a universal method for the immobilization of enzymes from fungal sources was developed, affording a highly active, stable, and reusable biocatalyst.     

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.     

Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coii

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999 bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33,905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid Cro-LacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to 'normal' K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.     

The effect of fluctuations in tidal inundation frequency on a salt-marsh vegetation

Over a period of 15 years recordings were made of the species cover in permanent plots on the salt marsh of one of the West Frisian Islands, Schiermonnikoog (The Netherlands). Correlations between annual changes in the cover of the major species, and fluctuations in the monthly frequency of inundation by seawater were studied. First, a spectral analysis was carried out on the inundation frequency data to look for predictable patterns. Subsequently, fluctuations were defined as deviations from these predictable patterns. In a repeated multiple regression model, the effects of the season in which the fluctuations occurred, and the elevational position of the plots on the salt marsh were studied as factors influencing the correlation patterns. The behaviour of various species is discussed in relation to their seed bank characteristics and their salt tolerance.     

Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.