Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms

A new approach for quantification of extracellular DNA (eDNA) in mixed biofilms at microscale resolution was developed and combined with other staining techniques to assess the origin, abundance and role of eDNA in activated sludge biofilms. Most eDNA was found in close proximity to living cells in microcolonies, suggesting that most of it originated from an active secretion or alternatively, by lysis of a sub-population of cells. When the staining was combined with fluorescence in situ hybridization for identification of the microorganisms, it was found that the eDNA content varied among the different probe-defined species. The highest amount of eDNA was found in and around the microcolonies of denitrifiers belonging to the genera Curvibacter and Thauera, the ammonium-oxidizing Nitrosomonas and the nitrite-oxidizing Nitrospira. Other floc-formers also produced eDNA, although in lower amounts. The total eDNA content in activated sludge varied from 4 to 52 mg per gram volatile suspended solids in different wastewater treatment plants. Very high local concentrations within some microcolonies were found with up to approximately 300 mg of eDNA per g of organic matter. DNase digestion of activated sludge led to general floc disintegration and disruption of the microcolonies with high eDNA content, implying that eDNA was an important structural component in activated sludge biofilms.     

Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes

Background

SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1.

Methodology/Principal Finding

We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-β-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C.

Conclusion/Significance

The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.     

In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm

Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e- equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers.     

Defined inflammatory states in astrocyte cultures: correlation with susceptibility towards CD95-driven apoptosis

A complete cytokine mix (CCM) or its individual components tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) were used to switch resting murine astrocytes to reactive states. The transformation process was characterized by differential up-regulation of interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) mRNA and protein and a subsequent release of prostaglandin E2, nitric oxide (NO) and IL-6. Both CD95L and anti-CD95 antibodies triggered caspase activation followed by apoptotic death in fully pro-inflammatory astrocytes, whereas resting cells were totally resistant. Two other death-inducing ligands, TNF and TNF-related apoptosis-inducing ligand (TRAIL) did not induce apoptosis in reactive astrocytes. The switch in astrocyte sensitivity was accompanied by up-regulation of caspase-8 and CD95 as well as the capacity to recruit Fas-associated death domain (FADD) to the activated death receptor complex. Neither CD95-mediated death, nor other inflammatory parameters were affected by inhibition of iNOS or COX, respectively. Accordingly, IFN-gamma was absolutely essential for up-regulation of iNOS, but not for the switch in apoptosis sensitivity. In contrast, p38 kinase activity was identified as an important controller of both the inflammatory reaction and apoptosis both in astrocytes stimulated with CCM and in glia exposed to TNF and IL-1 only.     

A new cercaria and metacercaria of Acanthoparyphium (Echinostomatidae) found in an intertidal snail Zeacumantus subcarinatus (Batillaridae) from New Zealand

A new 23-collar-spined cercaria and metacercaria are described from intertidal molluscs of the coast of New Zealand. The new cercaria found emerging from the mud snails Zeacumantus subcarinatus (Sowerby) (Prosobranchia: Batillariidae) is characterized mainly by the number and arrangement of the cephalic glands, the size of the suckers, and the size and number of the collar spines. The cercaria encysts in the cockle Austrovenus stutchburyi (Wood) (Bivalvia: Veneridae) which lives in sympatry with the first intermediate host throughout New Zealand. Laboratory infections of cockles by cercariae from naturally infected snails resulted in metacercariae identical to those found in naturally infected cockles. The main features of the cercaria and metacercaria are the presence of a reniform collar with 23 spines, two pairs of small cephalic glands at the oral sucker level, another two pairs of much longer ones posterior to the pharynx, and the excretory vesicle Y-shaped with the main collecting canals extending to the anterior level of the ventral sucker. Each of the main collecting canals had 10-11 pairs of bilateral diverticula between the anterior edge of the ventral sucker and anterior body end. A brief discussion of its possible life cycle and ecology is also provided.     

Detection of activity among uncultured Actinobacteria in a drinking water reservoir

The abundance, identity and activity of uncultured Bacteria and Actinobacteria present in a drinking water reservoir (North Pine Dam, Brisbane, Australia) were determined using a combination of fluorescence in situ hybridization (FISH) alone or with catalysed reporter deposition (CARD-FISH) with microautoradiography. The CARD-FISH technique was modified relative to previous described procedures and performed directly on gelatine cover slips in order to allow simultaneous combination with microautoradiography. Almost twofold higher numbers of microorganisms could be identified as either Bacteria or Actinobacteria using the CARD-FISH technique as compared with the traditional FISH technique. A combination of FISH or CARD-FISH with microautoradiography showed generally higher activity among the Actinobacteria than among all Bacteria. Another important observation was that many cells within the FISH-negative populations of both Actinobacteria and Bacteria were actively assimilating thymidine. Thus, great care should be taken when extrapolating the active fraction of a prokaryotic community to be equivalent to the FISH-detectable population in such environments. Bacterial groups within Actinobacteria produce the odours geosmin and 2-methylisoborneol, which lower the quality of surface water when used for drinking. The results indicate that combined microautoradiography and CARD-FISH may serve as an effective tool when studying identity and activity of microorganisms within freshwater environments.     

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.     

Pathological and incidental findings on brain MRI in a single-center study of 229 consecutive girls with early or precocious puberty

Central precocious puberty may result from organic brain lesions, but is most frequently of idiopathic origin. Clinical or biochemical factors which could predict a pathological brain MRI in girls with CPP have been searched for. With the recent decline in age at pubertal onset among US and European girls, it has been suggested that only girls with CPP below 6 years of age should have brain MRI performed.

Objective

To evaluate the outcome of brain MRI in girls referred with early signs of puberty in relation to age at presentation as well as clinical and biochemical parameters.

Method

A single-center study of 229 consecutive girls with early or precocious puberty who had brain imaging performed. We evaluated medical history, clinical and biochemical factors, and four groups were defined based on the outcome of their MRI.

Results

Thirteen out of 208 (6.3%) girls with precocious puberty, but no other sign of CNS symptoms, had a pathological brain MRI. Importantly, all 13 girls were above 6 years of age, and 6 girls were even 8–9 years old. Twenty girls (9.6%) had incidental findings on brain MRI. Furthermore, 21 girls had known CNS pathology at time of evaluation. Basal LH was significantly higher in girls with newly diagnosed CNS pathology compared to girls with a non-pathological MRI (p = 0.025); no cut of value was found as values overlapped.

Conclusion

A high frequency of 6–8 year old girls with precocious puberty in our study had a pathological brain MRI, which could not be predicted from any clinical nor biochemical parameters. Thus, we believe that girls with precocious pubertal development of central origin before 8 years of age should continue to be examined by a brain MRI.     

The Effect of Elevated Body Mass Index on Ischemic Heart Disease Risk: Causal Estimates from a Mendelian Randomisation Approach

Background

Adiposity, assessed as elevated body mass index (BMI), is associated with increased risk of ischemic heart disease (IHD); however, whether this is causal is unknown. We tested the hypothesis that positive observational associations between BMI and IHD are causal.

Methods and Findings

In 75,627 individuals taken from two population-based and one case-control study in Copenhagen, we measured BMI, ascertained 11,056 IHD events, and genotyped FTO(rs9939609), MC4R(rs17782313), and TMEM18(rs6548238). Using genotypes as a combined allele score in instrumental variable analyses, the causal odds ratio (OR) between BMI and IHD was estimated and compared with observational estimates. The allele score-BMI and the allele score-IHD associations used to estimate the causal OR were also calculated individually. In observational analyses the OR for IHD was 1.26 (95% CI 1.19–1.34) for every 4 kg/m² increase in BMI. A one-unit allele score increase associated with a 0.28 kg/m² (95 CI% 0.20–0.36) increase in BMI and an OR for IHD of 1.03 (95% CI 1.01–1.05) (corresponding to an average 1.68 kg/m² BMI increase and 18% increase in the odds of IHD for those carrying all six BMI increasing alleles). In instrumental variable analysis using the same allele score the causal IHD OR for a 4 kg/m² increase in BMI was 1.52 (95% CI 1.12–2.05).

Conclusions

For every 4 kg/m² increase in BMI, observational estimates suggested a 26% increase in odds for IHD while causal estimates suggested a 52% increase. These data add evidence to support a causal link between increased BMI and IHD risk, though the mechanism may ultimately be through intermediate factors like hypertension, dyslipidemia, and type 2 diabetes. This work has important policy implications for public health, given the continuous nature of the BMI-IHD association and the modifiable nature of BMI. This analysis demonstrates the value of observational studies and their ability to provide unbiased results through inclusion of genetic data avoiding confounding, reverse causation, and bias. Please see later in the article for the Editors'' Summary