Dietary protein content affects the response of meadow voles, <Emphasis Type="Italic">Microtus pennsylvanicus</Emphasis>, to over-marks

The response to signals, including scent marks, from opposite-sex conspecifics can be affected by the nutritional state of both the sender and receiver of these signals. Protein content of the diet affects how meadow voles (Microtus pennsylvanicus) respond to single scent marks, but it is unknown how it affects an individual’s response to the overlapping scent marks of two donors (an over-mark). In experiment 1, we tested the hypothesis that protein content of the diet affects the amount of time voles spend investigating the marks of the top- and bottom-scent donors of an over-mark. Males and females fed a 22% protein diet spent more time investigating the scent mark of the top-scent donor than that of the bottom-scent donor; voles fed 9% and 13% protein diets spent similar amounts of time investigating the top- and bottom-scent donors. In experiment 2, we tested the hypothesis that protein content of the diet of the top- and bottom-scent donors affects the amount of time conspecifics spend investigating their scent marks. Female voles spent more time investigating the mark of the top-scent male than that of the bottom-scent male, independent of the differences in protein content of the diets of the top- and bottom-scent donors. Male voles, however, spent more time investigating the top-scent female when she was fed a diet higher in protein content than that of the bottom-scent female. Our results are discussed within the context of the natural history of voles.     

Retrograde transport of pertussis toxin in the mammalian cell

Pertussis toxin (PT), an AB5 exotoxin and important virulence factor of Bordetella pertussis , is hypothesized to traffic along a retrograde transport pathway in mammalian cells. This pathway includes endosomal uptake, transport to the Golgi complex and endoplasmic reticulum (ER), dissociation of the holotoxin in the ER and translocation of the A subunit (S1) to the cytosol, where it ADP-ribosylates its G protein targets. In this study, PT was visualized in the Golgi complex by immunofluorescence microscopy, but transport beyond the Golgi could not be detected by this method. To gain evidence for the retrograde pathway, peptide tags with target sites for tyrosine sulfation (a trans -Golgi network-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained in vitro enzymatic and cellular activity as assessed by ADP-ribosylation assays. Peptide-tagged PT subunits were found to be modified by tyrosine sulfation, and, at later time points, by N-glycosylation. Appearance of sulfated PT subunits was inhibited by pretreatment of cells with brefeldin A. In some cell types, much of the S4 glycosylation, but not that of S1, was resistant to endoglycosidase H, suggesting that, subsequent to core N-glycosylation in the ER, S4 was transported anterograde to the Golgi, where further glycosylation occurred. When cells were pretreated with methyl-β-cyclodextrin, sulfation of PT subunits and PT cytotoxicity were reduced, suggesting that PT transport is dependent on cellular cholesterol content. These data support a retrograde pathway for PT intracellular transport.     

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

Electrodeless dielectrophoresis of single- and double-stranded DNA

Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force.     

Functional Cooperation of the Interleukin-2 Receptor β Chain and Jak1 in Phosphatidylinositol 3-Kinase Recruitment and Phosphorylation

Phosphatidylinositol 3-kinase (PI 3-K) plays an important role in signaling via a wide range of receptors such as those for antigen, growth factors, and a number of cytokines, including interleukin-2 (IL-2). PI 3-K has been implicated in both IL-2-induced proliferation and prevention of apoptosis. A number of potential mechanisms for the recruitment of PI 3-K to the IL-2 receptor have been proposed. We now have found that tyrosine residues in the IL-2 receptor β chain (IL-2Rβ) are unexpectedly not required for the recruitment of the p85 component of PI 3-K. Instead, we find that Jak1, which associates with membrane-proximal regions of the IL-2Rβ cytoplasmic domain, is essential for efficient IL-2Rβ–p85 interaction, although some IL-2Rβ–p85 association can be seen in the absence of Jak1. We also found that Jak1 interacts with p85 in the absence of IL-2Rβ and that IL-2Rβ and Jak1 cooperate for the efficient recruitment and tyrosine phosphorylation of p85. This is the first report of a PI 3-K–Jak1 interaction, and it implicates Jak1 in an essential IL-2 signaling pathway distinct from the activation of STAT proteins.     

A total crapshoot? Evaluating bioinformatic decisions in animal diet metabarcoding analyses

Metabarcoding studies provide a powerful approach to estimate the diversity and abundance of organisms in mixed communities in nature. While strategies exist for optimizing sample and sequence library preparation, best practices for bioinformatic processing of amplicon sequence data are lacking in animal diet studies. Here we evaluate how decisions made in core bioinformatic processes, including sequence filtering, database design, and classification, can influence animal metabarcoding results. We show that denoising methods have lower error rates compared to traditional clustering methods, although these differences are largely mitigated by removing low‐abundance sequence variants. We also found that available reference datasets from GenBank and BOLD for the animal marker gene cytochrome oxidase I (COI) can be complementary, and we discuss methods to improve existing databases to include versioned releases. Taxonomic classification methods can dramatically affect results. For example, the commonly used Barcode of Life Database (BOLD) Classification API assigned fewer names to samples from order through species levels using both a mock community and bat guano samples compared to all other classifiers (vsearch‐SINTAX and q2‐feature‐classifier's BLAST + LCA, VSEARCH + LCA, and Naive Bayes classifiers). The lack of consensus on bioinformatics best practices limits comparisons among studies and may introduce biases. Our work suggests that biological mock communities offer a useful standard to evaluate the myriad computational decisions impacting animal metabarcoding accuracy. Further, these comparisons highlight the need for continual evaluations as new tools are adopted to ensure that the inferences drawn reflect meaningful biology instead of digital artifacts.     

Evidence for an increased rate of choline efflux across erythrocyte membranes in Alzheimer's disease

Alzheimer's disease (AD), the major dementing disorder of the elderly, is associated with cholinergic neuronal loss and decreased activity of choline acetyl-transferase (CAT). Previous biophysical studies had suggested an altered conformation of membrane proteins in AD erythrocyte ghosts. Since erythrocytes have a choline transport system and cholinergic neurons are implicated in AD, the present experiments were undertaken to determine if the efflux rate of [¹⁴C]choline was altered in AD erythrocytes. The mean efflux rate constant was highly significantly increased (P<0.01) by greater than 25% in 9 drug-free AD patients compared to 9 sex-matched, drug-free controls of similar age. These results are discussed in terms of potential molecular mechanisms to account for cholinergic neuronal loss in AD.