Energy turnover in European hares is centrally limited during early,but not during peak lactation

We investigated metabolizable energy intake (MEI) and milk energy output in European hares throughout gestation and lactation in females raising three young, i.e., close to maximum litter size in this precocial species. We hypothesized that herbivorous hares may face a central limitation of energy turnover during lactation, imposed by maximum capacity of the gastrointestinal tract. Females were provided with low-energy or high-energy diets, either continually, or during lactation only. Unexpectedly, females on either diet reached identical peak MEIs (>6 times BMR) during late lactation, with females on low-energy diet increasing food intake proportionally. Thus, we reject our hypothesis that in lactating hares, peak MEI is centrally limited. During early lactation, MEI and milk transfer was, however, significantly impaired in females on the low-energy diet, indicating a temporal central limitation due to a time-lag caused by the readjustment of energy intake capacity. Importantly, irrespective of the diet, females significantly increased peak MEI late in the breeding season. Consequently, earlier in the season, when energy reserves are still high, energy throughput was not limited by physiological constraints at all. We conclude that extreme MEI may have fitness costs, and that females maximize lifetime reproductive success by actively down-regulating MEI whenever possible.     

Serological evaluation of Mycobacterium ulcerans antigens identified by comparative genomics

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans.     

Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips

Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS), nanoelectrospray-MS/MS (NanoES-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.     

Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex.

High affinity binding of factor VIIa (VIIa) to its cellular receptor tissue factor (TF), as well as association of factor X with phospholipid are required for optimal assembly of the extrinsic activation complex. In addition to the interactions of substrate with phospholipid and enzyme, we here provide evidence that cofactor residues Lys-165 and Lys-166 specifically contribute to the recognition of macromolecular substrate. Ala for Lys replacement in TFA165A166 was compatible with high affinity binding of VIIa when analyzed on cell surfaces as well as in the absence of phospholipid. Dissociation of TFA165A166.VIIa did not occur with a faster rate compared to TF.VIIa, further supporting unaltered VIIa binding function of TFA165A166. Cleavage of chromogenic peptidyl substrate by TFA165A166.VIIa complexes was not diminished, demonstrating that TFA165A166 supported enhancement of catalytic function of the VIIa protease domain. In contrast, factor X activation was reduced in the presence and absence of phospholipid. Further, TFA165A166 effectively competed with wild-type TF in the cleavage of factor X at limited VIIa concentrations. Selective reduction in macromolecular substrate hydrolysis combined with normal VIIa binding by TFA165A166 indicates that the cofactor TF does contribute, either directly or indirectly via specific interactions with VIIa, to factor X recognition.     

Influence of tree age, tree size and crown structure on lichen communities in mature Alpine spruce forests

Testing the relations between tree parameters and the richness and composition of lichen communities in near-natural stands could be a first step to gather information for forest managers interested in conservation and in biodiversity assessment and monitoring. This work aims at evaluating the influence of tree age and age-related parameters on tree-level richness and community composition of lichens on spruce in an Alpine forest. The lichen survey was carried out in four sites used for long-term monitoring. In each site, tree age, diameter at breast height, tree height, the first branch height, and crown projection area were measured for each tree. Trees were stratified into three age classes: (1) <100 years old, immature trees usually not suitable for felling, (2) 100–200 years old, mature trees suitable for felling, and (3) >200 years old, over-mature trees normally rare or absent in managed stands. In each site, seven trees in each age class were selected randomly. Tree age and related parameters proved to influence both tree-level species richness and composition of lichen communities. Species richness increased with tree age and related parameters indicative of tree size. This relation could be interpreted as the result of different joint effects of age per se and tree size with its area-effect. Species turnover is also suspected to improve species richness on over-mature trees. Similarly to species richness, tree-level species composition can be partially explained by tree-related parameters. Species composition changed from young to old trees, several lichens being associated with over-mature trees. This pool of species, including nationally rare lichens, represents a community which is probably poorly developed in managed forests. In accordance to the general aims of near-to-nature forestry, the presence of over-mature trees should be enhanced in the future forest landscape of the Alps especially in protected areas and Natura 2,000 sites, where conservation purposes are explicitly included in the management guidelines.     

Relevant genetic differentiation among Brazilian populations of Anastrepha fraterculus (Diptera,Tephritidae)

We used a population genetic approach to detect the presence of genetic diversity among six populations of Anastrepha fraterculus across Brazil. To this aim, we used Simple Sequence Repeat (SSR) markers, which may capture the presence of differentiative processes across the genome in distinct populations. Spatial analyses of molecular variance were used to identify groups of populations that are both genetically and geographically homogeneous while also being maximally differentiated from each other. The spatial analysis of genetic diversity indicates that the levels of diversity among the six populations vary significantly on an eco-geographical basis. Particularly, altitude seems to represent a differentiating adaptation, as the main genetic differentiation is detected between the two populations present at higher altitudes and the other four populations at sea level. The data, together with the outcomes from different cluster analyses, identify a genetic diversity pattern that overlaps with the distribution of the known morphotypes in the Brazilian area.     

The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.