Impact of acidification and eutrophication on macrophyte communities in soft waters. II. Experimental studies

In The Netherlands, there has been a dramatic decline during the last 30 years in the number of stands belonging to the phytosociological alliance Littorellion. Generally, the communities classified within this alliance occur in poorly buffered, oligotrophic waters, with very low phosphate, nitrogen and carbon dioxide levels in the water layer and considerably higher nutrient levels in the sediment. The plant species dominating these communities are isoetids such as Litoorella uniflora (L.) Aschers., Lobelia dortmanna L. and Isoetes lacustris L., which show various adaptations to make successful growth possible under these conditions.Field observations showed that the water where Littorella uniflora had disappeared or strongly decreased could be divided into two groups. A major group (77%) was characterized by the presence of submerged Juncus bulbosus L. and/or Sphagnum species. These water appeared to be strongly acidified (pH < 4.5) and had increased nitrogen levels with ammonium as the dominant N-source. Within this group, the waters with luxuriant growth of Juncus bulbosus and/or Sphagnum spp. had strongly increased carbon dioxide levels in both sediment and water.Different types of experiments proved causal relationships between the observed changes in macrophytes and the changed physico-chemical parameters. Ecophysiological experiments showed that Juncus bulbosus lacks the typical adaptations of the isoetid plant species, i.e. it uses very low amounts of sediment-CO₂ and releases only a little oxygen from the roots. However, Juncus bulbosus is more able than Littorella uniflora to use CO₂ from the water layer. From the nutrient-uptake experiments, the decreased nitrate and increased ammonium levels seem to be favourable to Juncus bulbosus. The culture experiments clearly demonstrated that the biomass of Juncus bulbosus only increased strongly when the sediment was poorly buffered and the pH of water was low. When combining factors like CO₂ enrichment of the sediment, with and without phosphate, and/or ammonium enrichment of the water in the culture experiments, it is clearly shown that phosphate and/or ammonium enrichment without CO₂ enrichment do not lead to an increase in biomass of Juncus bulbosus. Therefore, it is obvious that the changes in the macrophyte community can be ascribed primarily to changes in the carbon budget as a result of acidification.A minor group of waters (23%) was characterized by the absence of submerged Juncus bulbosus and/or Sphagnum spp. In most of these waters, submerged plant species occurred, such as Myriophyllum alterniflorum DC or non-rooted species such as Riccia fluitans L. These waters were not acidified, and generally had an increased alkalinity and higher nitrogen and phosphate levels of sediment and/or water. Culture experiments showed that phosphate enrichment of the sediment alone leads to luxuriant growth of submerged macrophyte species such as Myriophyllum alterniflorum, whereas phosphate enrichment of both sediment and water leads to mass development of non-rooted plant species such as Riccia fluitans.     

Expression of bacteriophage M13 dna in vivo. I. Synthesis of phage-specific RNA and protein in minicells.

It is demonstrated that after infection of the appropriate minicell-producing strain of Escherichia coli with the filamentous bacteriophage M13, its replicative form DNA is segregated into minicells. Consequently these minicells have acquired the capability to direct the synthesis of phage-specific RNA and protein. Comparision of the electrophoretic mobilities of phage-specific RNA species made in vitro with those made in M13 replicative form DNA harbouring minicells, have indicated that almost all in vitro synthesized G-start RNAs have an equivalent among the in vivo synthesized RNA products. Furthermore it could be demonstrated that in M13 replicative form DNA harbouring minicells the phage-specific proteins encoded by genes III, IV, V and VIII are made. In addition the synthesis of a phage-specific polypeptide (molecular weight approx. 3000) co-migrating with the recently discovered capsid protein (designated C-protein) could be demonstrated. The meaning of these results for the resolution of the regulatory mechanisms operative during the life cycle of this phage will be discussed.     

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV‐positive tumour cells

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.     

Mitochondrial enzymes discriminate between mitochondrial disorders and chronic fatigue syndrome

We studied the extent of mitochondrial involvement in chronic fatigue syndrome (CFS) and investigated whether measurement of mitochondrial respiratory chain complex (RCC) activities discriminates between CFS and mitochondrial disorders. Mitochondrial content was decreased in CFS compared to healthy controls, whereas RCC activities corrected for mitochondrial content were not. Conversely, mitochondrial content did not discriminate between CFS and two groups of mitochondrial disorders, whereas ATP production rate and complex I, III and IV activity did, all with higher activities in CFS. We conclude that the ATP production rate and RCC activities can reliably discriminate between mitochondrial disorders and CFS.     

1H, 15N and 13C resonance assignment of the N-terminal C39 peptidase-like domain of the ABC transporter Haemolysin B (HlyB)

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins, which catalyze the translocation of molecules across biological membranes in an ATP-dependent manner. Despite the diversity in the transported substrates, they all share the same architecture, comprised of two transmembrane (TMD) and two nucleotide-binding domains (NBD). Members of the bacteriocin ABC transporter subfamily feature a special domain, belonging to the C39 (cystein protease family 39) peptidase protein family. These domains are assumed to cleave a C-terminal signal sequence from the protein or peptide substrate before or during the transport process. Although the C39 peptidase-like domain of the ABC transporter haemolysin B from E. coli shows no proteolytic activity, it is essential for the function of this transporter. In order to elucidate the contribution of the isolated C39 peptidase-like domain in the whole transport process, the backbone and side chain ¹H, ¹⁵N and ¹³C-NMR chemical shifts have been assigned.     

Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.     

High-throughput evaluation of the critical micelle concentration of detergents

Determination of the critical micelle concentration (CMC) value of detergents routinely used in biological applications is necessary to follow possible changes due to different buffer compositions (e.g., temperature, pH) such as those in solutions that are used for protein activity assays or crystallization. Here we report a method to determine the CMC values of detergents through a fast and robust assay that relies on the fluorescence of Hoechst 33342 using a 96-well plate reader. Furthermore, this assay provides the possibility and sensitivity to measure the CMC of detergent mixtures. The examples described here emphasize the potential and applicability of this assay and demonstrate that analysis of the physicochemical parameters of detergents can now be investigated in virtually every laboratory.     

Depuration processes affect the Vibrio community in the microbiota of the Manila clam,Ruditapes philippinarum

As filter-feeders, bivalve molluscs accumulate Vibrio into edible tissues. Consequently, an accurate assessment of depuration procedures and the characterization of the persistent Vibrio community in depurated shellfish represent a key issue to guarantee food safety in shellfish products. The present study investigated changes in the natural Vibrio community composition of the Ruditapes philippinarum microbiota with specific focus on human pathogenic species. For this purpose, the study proposed a MLSA-NGS approach (rRNA 16S, recA and pyrH) for the detection and identification of Vibrio species. Clam microbiota were analysed before and after depuration procedures performed in four depuration plants, using culture-dependent and independent approaches. Microbiological counts and NGS data revealed differences in terms of both contamination load and Vibrio community between depuration plants. The novel MLSA-NGS approach allowed for a clear definition of the Vibrio species specific to each depuration plant. Specifically, depurated clam microbiota showed presence of human pathogenic species. Ozone treatments and the density of clams in the depuration tank probably influenced the level of contamination and the Vibrio community composition. The composition of Vibrio community specific to each plant should be carefully evaluated during the risk assessment to guarantee a food-safe shellfish-product for the consumer.     

Living Rivers: Trends and Challenges in Science and Management

This paper discusses and summarizes the major conclusions of the special issue entitled ‘Living rivers: trends and challenges in science and management’ of Hydrobiologia/Developments in Hydrobiology. It is structured along three lines: (1) Functioning of river systems; (2) Ecological rehabilitation of river systems; (3) Challenges and obstacles to sustainable management. Sustainable river basin management requires a thorough understanding of natural, physical, chemical and ecological processes as well as socio-economic functioning. Environmental rehabilitation of river systems inevitably requires an ecosystem-based approach involving solutions supported by the public. The challenge is to combine ecological and socio-economic sustainability. Rethinking our efforts in both directions must lead to more innovative solutions than those available at present.     

In search of an uncultured human-associated TM7 bacterium in the environment

We have identified an environmental bacterium in the Candidate Division TM7 with ≥98.5% 16S rDNA gene homology to a group of TM7 bacteria associated with the human oral cavity and skin. The environmental TM7 bacterium (referred to as TM7a-like) was readily detectable in wastewater with molecular techniques over two years of sampling. We present the first images of TM7a-like cells through FISH technique and the first images of any TM7 as viable cells through the STARFISH technique. In situ quantification showed TM7 concentration in wastewater up to five times greater than in human oral sites. We speculate that upon further characterization of the physiology and genetics of the TM7a-like bacterium from environmental sources and confirmation of its genomic identity to human-associated counterparts it will serve as model organisms to better understand its role in human health. The approach proposed circumvents difficulties imposed by sampling humans, provides an alternative strategy to characterizing some diseases of unknown etiology, and renders a much needed understanding of the ecophysiological role hundreds of unique Bacteria and Archaea strains play in mixed microbial communities.