Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure

In zoonotic infections, the relationships between animals and humans lead to parasitic disease with severity that ranges from mild symptoms to life-threatening conditions. In cities and their surrounding areas, this statement is truer with the overcrowding of the protagonists of the parasites’ life cycle. The present study aims to investigate the distribution of a parasite, Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis, using copro-sampling in historically endemic rural settlements of the eastern part of France and in newly endemic areas including urban parks and settlements surrounding Paris. Based on 2741 morphologically identified and geolocalized copro-samples, the density of fox faeces was generally higher in the surrounding settlements, except for one rural area where the faeces were at larger density downtown in the winter. Fox faeces are rare but present in urban parks. Dog faeces are concentrated in the park entrances and in the centre of the settlements. DNA was extracted for 1530 samples that were collected and identified from fox, dog, cat, stone marten and badger carnivore hosts. Echinococcus multilocularis diagnosis and host faecal tests were performed using real-time PCR. We failed to detect the parasite in the surroundings of Paris, but the parasite was found in the foxes, dogs and cats in the rural settlements and their surroundings in the historically endemic area. A spatial structuring of the carnivore stool distribution was highlighted in the present study with high densities of carnivore stools among human occupied areas within some potentially high-risk locations.     

A global study of relationships between leaf traits, climate and soil measures of nutrient fertility

Aim This first global quantification of the relationship between leaf traits and soil nutrient fertility reflects the trade‐off between growth and nutrient conservation. The power of soils versus climate in predicting leaf trait values is assessed in bivariate and multivariate analyses and is compared with the distribution of growth forms (as a discrete classification of vegetation) across gradients of soil fertility and climate. Location All continents except for Antarctica. Methods Data on specific leaf area (SLA), leaf N concentration (LNC), leaf P concentration (LPC) and leaf N:P were collected for 474 species distributed across 99 sites (809 records), together with abiotic information from each study site. Individual and combined effects of soils and climate on leaf traits were quantified using maximum likelihood methods. Differences in occurrence of growth form across soil fertility and climate were determined by one‐way ANOVA. Results There was a consistent increase in SLA, LNC and LPC with increasing soil fertility. SLA was related to proxies of N supply, LNC to both soil total N and P and LPC was only related to proxies of P supply. Soil nutrient measures explained more variance in leaf traits among sites than climate in bivariate analysis. Multivariate analysis showed that climate interacted with soil nutrients for SLA and area‐based LNC. Mass‐based LNC and LPC were determined mostly by soil fertility, but soil P was highly correlated to precipitation. Relationships of leaf traits to soil nutrients were stronger than those of growth form versus soil nutrients. In contrast, climate determined distribution of growth form more strongly than it did leaf traits. Main conclusions We provide the first global quantification of the trade‐off between traits associated with growth and resource conservation ‘strategies’ in relation to soil fertility. Precipitation but not temperature affected this trade‐off. Continuous leaf traits might be better predictors of plant responses to nutrient supply than growth form, but growth forms reflect important aspects of plant species distribution with climate.     

Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis.

The active site serine residue of penicillin-binding protein 3 of Escherichia coli that is acylated by penicillin (Ser-307) has been converted to a cysteine residue using a simple and efficient two primer method of site-directed mutagenesis. The resulting thiol-penicillin-binding protein 3 was expressed under the control of the lacUV5 promoter in a high copy number plasmid. Constitutive expression of the thiol-enzyme (but not of the wild-type enzyme) was lethal, and the plasmid could only be maintained in E. coli strains that carried the lacIq mutation. Induction of the expression of the thiol-enzyme resulted in inhibition of cell division and the growth of the bacteria into very long filamentous cells. The inhibition of septation was probably due to interference of the function of the wild-type penicillin-binding protein 3 in cell division by the enzymatically inactive thiol-enzyme, and this implies that penicillin-binding protein 3 acts as part of a complex in vivo. We were unable to detect any acylation of the thiol-enzyme by penicillin, but it is not yet clear if this was because the thioester was not formed at an appreciable rate, or if it was formed but was too unstable to be detected by a modified penicillin-binding protein assay.     

Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.