Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.     

Tumour necrosis factor alpha inhibits purinergic calcium signalling in blood-brain barrier endothelial cells

The breaching of the blood-brain barrier is an essential aspect in the pathogenesis of neuroinflammatory diseases, in which tumour necrosis factor alpha (TNF-alpha) as well as endothelial calcium ions play a key role. We investigated whether TNF-alpha could influence the communication of calcium signals between brain endothelial cells (GP8 and RBE4). Intercellular calcium waves triggered by mechanical stimulation or photoliberation of InsP3 in single cells were significantly reduced in size after TNF-alpha exposure (1000 U/mL, 2 and 24 h). Calcium signals are communicated between cells by means of gap junctional and paracrine purinergic signalling. TNF-alpha significantly inhibited gap junctional coupling, stimulated the basal release of ATP, and dose-dependently blocked the triggered component of ATP release. The cytokine displayed similar effects on the uptake of a fluorescent reporter dye into the cells. Previous work with connexin mimetic peptides demonstrated that the triggered ATP release in these cells is connexin-related; these peptides did, however, not influence the elevated basal ATP release caused by TNF-alpha. We conclude that TNF-alpha depresses calcium signal communication in blood-brain barrier endothelial cells, by reducing gap junctional coupling and by inhibiting triggered ATP release. The cytokine thus inhibits connexin-related communication pathways like gap junctions and connexin hemichannels.     

Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26

Calcium signals can be communicated between cells by the diffusion of a second messenger through gap junction channels or by the release of an extracellular purinergic messenger. We investigated the contribution of these two pathways in endothelial cell lines by photoliberating InsP(3) or calcium from intracellular caged precursors, and recording either the resulting intercellular calcium wave or else the released ATP with a luciferin/luciferase assay. Photoliberating InsP(3) in a single cell within a confluent culture triggered an intercellular calcium wave, which was inhibited by the gap junction blocker alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptide gap 26, the purinergic inhibitors suramin, PPADS and apyrase and by purinergic receptor desensitisation. InsP(3)-triggered calcium waves were able to cross 20 microm wide cell-free zones. Photoliberating InsP(3) triggered ATP release that was blocked by buffering intracellular calcium with BAPTA and by applying gap 26. Gap 26, however, did not inhibit the gap junctional coupling between the cells as measured by fluorescence recovery after photobleaching. Photoliberating calcium did not trigger intercellular calcium waves or ATP release. We conclude that InsP(3)-triggered ATP release through connexin hemichannels contributes to the intercellular propagation of calcium signals.     

Characterization of bacterial communities in four freshwater lakes differing in nutrient load and food web structure

The phylogenetic composition of bacterioplankton communities in the water column of four shallow eutrophic lakes was analyzed by partially sequencing cloned 16S rRNA genes and by PCR-DGGE analysis. The four lakes differed in nutrient load and food web structure: two were in a clearwater state and had dense stands of submerged macrophytes, while two others were in a turbid state characterized by the occurrence of phytoplankton blooms. One turbid and one clearwater lake had very high nutrient levels (total phosphorus > 100 microg/l), while the other lakes were less nutrient rich (total phosphorus < 100 microg/l). Cluster analysis, multidimensional scaling and ANOSIM (analysis of similarity) were used to investigate differences among the bacterial community composition in the four lakes. Our results show that each lake has its own distinct bacterioplankton community. The samples of lake Blankaart differed substantially from those of the other lakes; this pattern was consistent throughout the year of study. The bacterioplankton community composition in lake Blankaart seems to be less diverse and less stable than in the other three lakes. Clone library results reveal that Actinobacteria strongly dominated the bacterial community in lake Blankaart. The relative abundance of Betaproteobacteria was low, whereas this group was dominant in the other three lakes. Turbid lakes had a higher representation of Cyanobacteria, while clearwater lakes were characterized by more representatives of the Bacteroidetes. Correlating our DGGE data with environmental parameters, using the BIOENV procedure, suggests that differences are partly related to the equilibrium state of the lake.     

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

Enhancement of implant osseointegration by high-frequency low-magnitude loading

Background

Mechanical loading is known to play an important role in bone remodelling. This study aimed to evaluate the effect of high- and low-frequency axial loading, applied directly to the implant, on peri-implant bone healing and implant osseointegration.

Methodology

Titanium implants were bilaterally installed in rat tibiae. For every animal, one implant was loaded (test) while the other one was not (control). The test implants were randomly divided into 8 groups according to 4 loading regimes and 2 experimental periods (1 and 4 weeks). The loaded implants were subject to an axial displacement. Within the high- (HF, 40 Hz) or low-frequency (LF, 8 Hz) loading category, the displacements varied 2-fold and were ranked as low- or high-magnitude (LM, HM), respectively. The strain rate amplitudes were kept constant between the two frequency groups. This resulted in the following 4 loading regimes: 1) HF-LM, 40 Hz-8 µm; 2) HF-HM, 40 Hz-16 µm; 3) LF-LM, 8 Hz-41 µm; 4) LF-HM, 8 Hz-82 µm. The tissue samples were processed for resin embedding and subjected to histological and histomorphometrical analyses. Data were analyzed statistically with the significance set at p<0.05.

Principal Findings

After loading for 4 weeks, HF-LM loading (40 Hz-8 µm) induced more bone-to-implant contact (BIC) at the level of the cortex compared to its unloaded control. No significant effect of the four loading regimes on the peri-implant bone fraction (BF) was found in the 2 experimental periods.

Conclusions

The stimulatory effect of immediate implant loading on bone-to-implant contact was only observed in case of high-frequency (40 Hz) low-magnitude (8 µm) loading. The applied load regimes failed to influence the peri-implant bone mass.     

Dispersal-mediated trophic interactions can generate apparent patterns of dispersal limitation in aquatic metacommunities

Dispersal is a major organising force in metacommunities, which may facilitate compositional responses of local communities to environmental change and affect ecosystem function. Organism groups differ widely in their dispersal abilities and their communities are therefore expected to have different adaptive abilities. In mesocosms, we studied the simultaneous compositional response of three plankton communities (zoo-, phyto- and bacterioplankton) to a primary productivity gradient and evaluated how this response was mediated by dispersal intensity. Dispersal enhanced responses in all three planktonic groups, which also affected ecosystem functioning. Yet, variation partitioning analyses indicated that responses in phytoplankton and bacterial communities were not only controlled by dispersal directly but also indirectly through complex trophic interactions. Our results indicate that metacommunity patterns emerging from dispersal can cascade through the food web and generate patterns of apparent dispersal limitation in organisms at other trophic levels.     

Occurrence and survival ofAzospirillum spp. in temperate regions

In order to evaluate the suitability ofAzospirillum spp. as a crop inoculant in temperate regions, the natural occurrence, distribution and survival ofAzospirillum after seed inoculation in Belgian agricultural soils was studied.Azospirillum was present in most of the fields examined, but concentrations never exceeded 1000 cfu per g soil or per g roots. Under field conditions none of the known species was found to be localized inside the roots of barley, wheat, rye, maize or grasses. Also, the distribution ofA. brasilense SpBr 14 within the root system of hydroponic-grown wheat was studied by immunofluorescence. From the rhizosphere samples of the field crops investigated, a number of microaërophilic, diazotrophic bacteria were isolated and identified asA. lipoferum, found only on maize and grass roots, andA. brasilense, present under all crops. In contrast toA. brasilense, A. lipoferum was able to use different amino-acids and some derivatives as sole carbon and nitrogen sources. Use of a peat-based seed inoculant resulted in the establishment of theAzospirillum spp. in the rhizosphere of field-grown winter barley and winter wheat. The established population survived during winter without appreciable change in numbers, but there was no indication of active growth during spring or summer.