Evaluation of tadpole mouthpart depigmentation as a diagnostic test for infection by Batrachochytrium dendrobatidis for four California anurans

The objective of this study was to evaluate the utility of gross morphologic examination of larval mouthpart defects as a diagnostic screening test to detect Batrachochytrium dendrobatidis infection in four California, USA, anuran species. We examined mouthparts of 2,034 tadpoles of Bufo boreas, Pseudacris regilla, and Rana catesbeiana collected in 2003 and 2004 and Bufo canorus collected in 2004. Data were recorded for three morphologic features: upper toothrows, lower toothrows, and combined jaw sheaths. Mouthpart defects were observed in all four species (n=757), but only two species were infected with B. dendrobatidis (n=84). Sensitivity and specificity of the mouthparts test were 76% and 58%, respectively. Forty-two percent of B. dendrobatidis-negative animals would have been designated positive based on mouthpart defects. Observed prevalence was 43%, and true prevalence was 3.0%. Tests of the null hypothesis using logistic regression analysis showed that anuran larval mouthpart defects were not associated with B. dendrobatidis infection whether mouthparts scores were tested by individual morphologic feature or in combination (P=0.37). We conclude that B. dendrobatidis infection and anuran larval mouthpart defects are two separate processes that may occur concurrently and that evaluation of tadpole oral morphology is neither an accurate nor a reliable diagnostic test for B. dendrobatidis infection for the four species tested.     

Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.     

Adhesion and degranulation-promoting adapter protein (ADAP) positively regulates T cell sensitivity to antigen and T cell survival

The hemopoietic specific adapter protein ADAP (adhesion and degranulation-promoting adapter protein) positively regulates TCR-dependent, integrin-mediated adhesion and participates in signaling pathways downstream of the TCR that result in T cell activation. The specific role of ADAP in regulating Ag-dependent T cell interactions with APCs and T cell activation following Ag stimulation is not known. We used ADAP-/- DO11.10 T cells to demonstrate that ADAP promotes T cell conjugation to Ag-laden APCs. Complementary in vitro and in vivo approaches reveal that ADAP controls optimal T cell proliferation, cytokine production, and expression of the prosurvival protein Bcl-xL in response to limiting Ag doses. Furthermore, ADAP is critical for clonal expansion in vivo independent of Ag concentration under conditions of low clonal abundance. These results suggest that ADAP regulates T cell activation by promoting Ag-dependent T cell-APC interactions, resulting in enhanced T cell sensitivity to Ag, and by participating in prosurvival signaling pathways initiated by Ag stimulation.     

Role of Microbial Immigration in the Colonization of Apple Leaves by Aureobasidium pullulans

The role of microbial immigration in the veinal colonization pattern of Aureobasidium pullulans on the adaxial surface of apple leaves was investigated in two experiments at two periods (early and late seasons) in 2004 by applying green fluorescent protein (GFP)-tagged blastospores to the foliage of orchard trees. Individual leaves were resampled by a semidestructive method immediately after inoculation (t₀) and about 1 (t₁), 2 (t₂), and 3 (t₃) weeks later. At t₀, there were no significant (P ≤ 0.05) differences in densities (cells/mm²) on veinal (excluding midvein) sites and those on interveinal sites, but at all points thereafter, densities were significantly higher on veins. GFP-tagged A. pullulans cells remained primarily as singletons on interveinal regions (≥90% at all points), while ≥20% of cells over veins at t₃ were in colonies of ≥4 cells. The colonies that developed from single cells placed on midveins and other veins were significantly larger than those that developed on interveinal regions of detached field and seedling leaves incubated under controlled conditions. Colonies primarily developed linearly along veins, reaching average colony sizes (72 h) of 24.4 ± 12.7 (mean ± standard deviation) cells. In contrast, colonies on interveinal regions tended to average only 2.9 ± 1.3 cells, with less linearity. To examine the potential role of A. pullulans growth-inhibiting factors associated with interveinal features, single GFP-tagged A. pullulans cells in droplets previously incubated on interveinal sites were placed on midveins and compared to midvein colonies derived from cells in a water-only suspension. No differences in colony size resulted. Our results indicate that immigration limitation and growth-inhibiting factors are not the primary factors responsible for A. pullulans veinal colonization patterns in the field. Rather, indirect evidence suggests that growth-promoting substances occur locally in the veinal areas.     

An evolutionary view of human recombination

Recombination has essential functions in mammalian meiosis, which impose several constraints on the recombination process. However, recent studies have shown that, in spite of these roles, recombination rates vary tremendously among humans, and show marked differences between humans and closely related species. These findings provide important insights into the determinants of recombination rates and raise new questions about the selective pressures that affect recombination over different genomic scales, with implications for human genetics and evolutionary biology.     

Delayed onset of midline netrin expression in Artemia franciscana coincides with commissural axon growth and provides evidence for homology of midline cells in distantly related arthropods

Although many similarities in arthropod central nervous systems (CNS) development exist, differences in midline cell formation and ventral nerve cord axonogenesis have been noted in arthropods. It is possible that changes in the expression of axon guidance molecules such as Netrin, which functions during commissural axon guidance in Drosophila and many other organisms, may parallel these differences. In this investigation, we analyze this hypothesis by examining Netrin accumulation during development of the brine shrimp Artemia franciscana, a branchiopod crustacean. An Artemia franciscana netrin (afrnet) orthologue was cloned. An antibody to the afrNet protein was generated and used to examine the pattern of afrNet accumulation during Artemia development. Despite differences between Drosophila and Artemia nerve cord development, examination of afrNet accumulation suggests that this protein functions to regulate commissure formation during Artemia CNS development. However, detection of afrNet at the midline and on commissural axons occurs at a relatively later time point in Artemia as compared with Drosophila. Detection of afrNet in a subset of midline cells that closely resemble Netrin-expressing cells at the Drosophila midline provides evidence for homology of midline cells in arthropods. Expression of Netrins in many other tissues is comparable, suggesting that Netrin proteins may play many conserved roles during arthropod development.     

Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity

In Alzheimer's disease and tauopathies, tau protein aggregates into neurofibrillary tangles that progressively spread to synaptically connected brain regions. A prion‐like mechanism has been suggested: misfolded tau propagating through the brain seeds neurotoxic aggregation of soluble tau in recipient neurons. We use transgenic mice and viral tau expression to test the hypotheses that trans‐synaptic tau propagation, aggregation, and toxicity rely on the presence of endogenous soluble tau. Surprisingly, mice expressing human P301Ltau in the entorhinal cortex showed equivalent tau propagation and accumulation in recipient neurons even in the absence of endogenous tau. We then tested whether the lack of endogenous tau protects against misfolded tau aggregation and toxicity, a second prion model paradigm for tau, using P301Ltau‐overexpressing mice with severe tangle pathology and neurodegeneration. Crossed onto tau‐null background, these mice had similar tangle numbers but were protected against neurotoxicity. Therefore, misfolded tau can propagate across neural systems without requisite templated misfolding, but the absence of endogenous tau markedly blunts toxicity. These results show that tau does not strictly classify as a prion protein.     

Identification of Cdk targets that control cytokinesis

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.