Selecting cases from nuclear families for case-control association analysis

We examine the efficiency of a number of schemes to select cases from nuclear families for case-control association analysis using the Genetic Analysis Workshop 14 simulated dataset. We show that with this simulated dataset comparing all affected siblings with unrelated controls is considerably more powerful than all of the other approaches considered. We find that the test statistic is increased by almost 3-fold compared to the next best sampling schemes of selecting all affected sibs only from families with affected parents (AF aff), one affected sib with most evidence of allele-sharing from each family (SF), and all affected sibs from families with evidence for linkage (AF L). We consider accounting for biological relatedness of samples in the association analysis to maintain the correct type I error. We also discuss the relative efficiencies of increasing the ratio of unrelated cases to controls, methods to confirm associations and issues to consider when applying our conclusions to other complex disease datasets.     

Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis Factor-alpha

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFalpha; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFalpha vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFalpha trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.     

Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus

It is well documented that long term potentiation (LTP) is impaired in the hippocampus of the aged animal. Among the changes that contribute to this impairment is an increase in hippocampal concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and increased IL-1beta-induced signaling. In this study we investigated the possibility that these changes were a consequence of decreased concentration of the anti-inflammatory cytokine, IL-4, and decreased IL-4-stimulated signaling. We report that functional IL-4 receptors are expressed on granule cells of the dentate gyrus and that receptor activation results in phosphorylation of JAK1 and STAT6. Hippocampal IL-4 concentration was decreased with age, and this was accompanied by a decrease in phosphorylation of JAK1 and STAT6. The evidence indicates that IL-4 modulates expression of IL-1beta mRNA and protein and that it attenuates IL-1beta-induced impairment of LTP and phosphorylation of JNK and c-Jun. We argued that, if a decrease in hippocampal IL-4 concentration significantly contributed to the age-related impairment in LTP, then restoration of IL-4 should restore LTP. To test this, we treated rats with VP015 (phospholipid microparticles-incorporating phosphatidylserine), which increases IL-4 concentration in hippocampus. The data indicate that the VP015-induced increase in IL-4 concentration in hippocampus of aged rats and lipopolysaccharide (LPS)-treated rats was accompanied by a reversal of the age-related and LPS-induced impairment in LTP in perforant path granule cell synapses. We propose that interplay between pro-inflammatory and anti-inflammatory responses impact significantly on synaptic function in the hippocampus of the aged rat.     

Genetic evidence for a distinct subtype of schizophrenia characterized by pervasive cognitive deficit

A novel phenotyping strategy in schizophrenia, targeting different neurocognitive domains, neurobehavioral features, and selected personality traits, has allowed us to identify a homogeneous familial subtype of the disease, characterized by pervasive neurocognitive deficit. Our genome scan data indicate that this subtype, which accounts for up to 50% of our sample, has a distinct genetic basis and explains linkage to chromosome 6p24 reported previously. If representative of other populations, the ratio of schizophrenia subtypes observed in our families could have a profound impact on sample heterogeneity and on the power of genetic studies to detect linkage and association. Our proposed abbreviated battery of tests should facilitate phenotype characterization for future genetic analyses and allow a focus on a crisply defined schizophrenia subtype, thus promoting a more informed search for susceptibility genes.     

The Escherichia coli fadK (ydiD) gene encodes an anerobically regulated short chain acyl-CoA synthetase

We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources. This beta-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD. The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain. The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components. We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms). The enzymatic mechanism of FadK is similar to other acyl-CoA synthetases in that it forms an acyl-AMP intermediate prior to the formation of the final acyl-CoA product. Expression of FadK is repressed during aerobic growth and is maximally expressed under anaerobic conditions in the presence of the terminal electron acceptor, fumarate.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

Killer whales are capable of vocal learning

The production learning of vocalizations by manipulation of the sound production organs to alter the physical structure of sound has been demonstrated in only a few mammals. In this natural experiment, we document the vocal behaviour of two juvenile killer whales, Orcinus orca, separated from their natal pods, which are the only cases of dispersal seen during the three decades of observation of their populations. We find mimicry of California sea lion (Zalophus californianus) barks, demonstrating the vocal production learning ability for one of the calves. We also find differences in call usage (compared to the natal pod) that may reflect the absence of a repertoire model from tutors or some unknown effect related to isolation or context.     

Division of labor: Subsets of dorsal-appendage-forming cells control the shape of the entire tube

The function of an organ relies on its form, which in turn depends on the individual shapes of the cells that create it and the interactions between them. Despite remarkable progress in the field of developmental biology, how cells collaborate to make a tissue remains an unsolved mystery. To investigate the mechanisms that determine organ structure, we are studying the cells that form the dorsal appendages (DAs) of the Drosophila melanogaster eggshell. These cells consist of two differentially patterned subtypes: roof cells, which form the outward-facing roof of the lumen, and floor cells, which dive underneath the roof cells to seal off the floor of the tube. In this paper, we present three lines of evidence that reveal a further stratification of the DA-forming epithelium. Laser ablation of only a few cells in the anterior of the region causes a disproportionately severe shortening of the appendage. Genetic alteration through the twin peaks allele of tramtrack69 (ttk^twk), a female-sterile mutation that leads to severely shortened DAs, causes no such shortening when removed from a majority of the DA-forming cells, but rather, produces short appendages only when removed from cells in the very anterior of the tube-forming tissue. Additionally we show that heterotrimeric G-protein function is required for DA morphogenesis. Like TTK69, Gβ13F is not required in all DA-forming follicle cells but only in the floor and leading roof cells. The different phenotypes that result from removal of Gβ13F from each region demonstrate a striking division of function between different DA-forming cells. Gβ mutant floor cells are unable to control the width of the appendage while Gβ mutant leading roof cells fail to direct the elongation of the appendage and the convergent-extension of the roof-cell population.     

AqpZ-mediated water permeability in Escherichia coli measured by stopped-flow spectroscopy

We report that the water permeability of wild-type Escherichia coli during exponential growth is comparable to that of an aqpZ disruption mutant. In contrast, an increase in permeability is observed for the wild type at the onset of the stationary stage with no significant corresponding change in the permeability of the mutant.