Short-term genetic changes: evaluating effective population size estimates in a comprehensively described brown trout (Salmo trutta) population

The effective population size (N(e)) is notoriously difficult to accurately estimate in wild populations as it is influenced by a number of parameters that are difficult to delineate in natural systems. The different methods that are used to estimate N(e) are affected variously by different processes at the population level, such as the life-history characteristics of the organism, gene flow, and population substructure, as well as by the frequency patterns of genetic markers used and the sampling design. Here, we compare N(e) estimates obtained by different genetic methods and from demographic data and elucidate how the estimates are affected by various factors in an exhaustively sampled and comprehensively described natural brown trout (Salmo trutta) system. In general, the methods yielded rather congruent estimates, and we ascribe that to the adequate genotyping and exhaustive sampling. Effects of violating the assumptions of the different methods were nevertheless apparent. In accordance with theoretical studies, skewed allele frequencies would underestimate temporal allele frequency changes and thereby upwardly bias N(e) if not accounted for. Overlapping generations and iteroparity would also upwardly bias N(e) when applied to temporal samples taken over short time spans. Gene flow from a genetically not very dissimilar source population decreases temporal allele frequency changes and thereby acts to increase estimates of N(e). Our study reiterates the importance of adequate sampling, quantification of life-history parameters and gene flow, and incorporating these data into the N(e) estimation.     

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery

Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high‐resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C‐terminal 320 residues adopt the fold of an eight‐bladed β‐propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N‐terminal domain, which in Chlamydomonas ODA16 is located on top of the β‐propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull‐down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.     

Psychological Stress and Hospitalization for Childhood Asthma-a Nationwide Cohort Study in Two Nordic Countries

Objective

Exposures to psychological stress in early life may contribute to the development or exacerbation of asthma. We undertook a cohort study based on data from several population-based registers in Denmark and Sweden to examine whether bereavement in childhood led to increased asthma hospitalization.

Methods

All singleton children born in Denmark during 1977-2008 and in Sweden during 1973-2006 were included in the study (N=5,202,576). The children were followed from birth to the date of first asthma hospitalization, emigration, death, their 18^th birthday, or the end of study (31 December 2007 in Sweden and 31 December 2008 in Denmark), whichever came first. All the children were assigned to the non-bereaved group until they lost a close relative (mother, father or a sibling), from when they were included in the bereaved group. We evaluated the hazard ratio (HR) of first hospitalization for asthma in bereaved children using Cox proportional hazards regression models, compared to those who were in the non-bereaved group. We also did a sub-analysis on the association between bereavement and first asthma medication.

Results

A total of 147,829 children were hospitalized for asthma. The overall adjusted HR of asthma hospitalization in bereaved children was 1.10 (95% confidence interval (CI): 1.04-1.16), compared to non-bereaved children. The risk of asthma hospitalization was increased in those who lost a close relative at age of 14-17 years (HR=1.54, 95% CI: 1.23-1.92), but not in younger age groups. The association between bereavement and asthma hospitalization did not change over time since bereavement. In the sub-analysis in singleton live births during 1996-2008 recorded in the DMBR, bereavement was associated with a lower use of asthma medication (HR=0.87, 95% CI: 0.80-0.95).

Conclusions

Our data suggests that psychological stress following bereavement in late adolescence is associated with an increased risk of asthma hospitalization or lowers the threshold for asthma hospitalization.     

ADAMDEC1 Is a Metzincin Metalloprotease with Dampened Proteolytic Activity

ADAMDEC1 (Decysin-1) is a putative ADAM (a disintegrin and metalloprotease)-like metalloprotease with an unknown physiological role, selectively expressed in mature dendritic cells and macrophages. When compared with other members of the ADAM family, ADAMDEC1 displays some unusual features. It lacks the auxiliary cysteine-rich, EGF, and transmembrane domains, as well as the cytoplasmic tail. The active site of ADAMDEC1 is unique by being the only mammalian ADAM protease with a non-histidine zinc ligand, having an aspartic acid residue instead. Here we demonstrate that ADAMDEC1, despite these unique features, functions as an active metalloprotease. Thus, ADAMDEC1 is secreted as a mature, glycosylated, and proteolytically active metalloprotease, capable of cleaving macromolecular substrates. In the recombinant form, three of the four potential N-linked glycosylation sites are modified by carbohydrate attachment. Substitution of basic residues at the predicted proprotein convertase cleavage site blocks proprotein processing, revealing both specific ADAMDEC1-dependent and specific ADAMDEC1-independent cleavage of the prodomain. The pro-form of ADAMDEC1 does not have proteolytic activity, demonstrating that the prodomain of ADAMDEC1, like in other members of the ADAM family, confers catalytic latency. Interestingly, the proteolytic activity of mature ADAMDEC1 can be significantly enhanced when a canonical ADAM active site with three zinc-coordinating histidine residues is introduced.     

Glass bead cultivation of fungi: Combining the best of liquid and agar media

Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum and Fusarium solani cultivated on agar plates, in shaking liquid culture or on glass beads was compared. Agar plate culture and glass bead cultivation yielded comparable results while liquid culture had lower production of secondary metabolites. RNA extraction from glass beads and liquid cultures was easier than from agar plates and the quality was superior. The system allows simple control of nutrient availability throughout fungal cultivation. This combined with the ease of extraction of nucleic acids and metabolites makes the system highly suitable for the study of gene regulation in response to specific nutrient factors.     

Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits

Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.     

The phosphorylation pattern of human alphas1-casein is markedly different from the ruminant species.

Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, alphas1-casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human alphas1-casein by a combination of matrix-assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphorylated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphorylations were observed in the conserved region covering residues Ser70-Glu78, which is extensively phosphorylated in the ruminant alphas1-caseins.     

PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation

The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the α-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-β-d-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule.     

Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley α-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.