Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis

Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-Å-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.     

Effects of the Hemlock Woolly Adelgid on Nitrogen Losses from Urban and Rural Northern Forest Ecosystems

The objectives of this study were to quantify rates of nitrogen inputs to the forest floor, determine rates of nitrogen losses via leaching and to partition the sources of NO₃ ⁻ from healthy, declining, and salvage or preemptively cut eastern hemlock (Tsuga canadensis) stands in both an urban forest at the Arnold Arboretum in Boston, MA and a rural forest at Harvard Forest in Petersham, MA. Rates of nitrogen inputs (NH₄ ⁺ and NO₃ ⁻) to the forest floor were 4–5 times greater, and rates of nitrogen losses via leachate were more than ten times greater, at the Arnold Arboretum compared to Harvard Forest. Nitrate that was lost via leachate at Harvard Forest came predominantly from atmospheric deposition inputs, whereas NO₃ ⁻ losses at the Arnold Arboretum came predominantly from nitrification. Although our study was limited to one urban and one rural site, our results suggest that current management regimes used to control the hemlock woolly adelgid (Adelges tsugae), such as salvage cutting, may be reducing nitrogen losses in urban areas due to rapid regrowth of vegetation and uptake of nitrogen by those plants. In contrast, preemptive cutting of trees in rural areas may be leading to proportionately greater losses of nitrogen in those sites, though the total magnitude of nitrogen lost is still smaller than in urban sites. Results of our study suggest that the combination of the hemlock woolly adelgid, nitrogen inputs, and management practices lead to changes in the movement and source of NO₃ ⁻ losses from eastern hemlock forest ecosystems.     

Fat cadherin modulates organ size in Drosophila via the Salvador/Warts/Hippo signaling pathway

BACKGROUND: The atypical Fat cadherin has long been known to control cell proliferation and organ size in Drosophila, but the mechanism by which Fat controls these processes has remained elusive. A newly emerging signaling pathway that controls organ size during development is the Salvador/Warts/Hippo pathway. RESULTS: Here we demonstrate that Fat limits organ size by modulating activity of the Salvador/Warts/Hippo pathway. ft interacts genetically with positive and negative regulators of this pathway, and tissue lacking fat closely phenocopies tissue deficient for genes that normally promote Salvador/Warts/Hippo pathway activity. Cells lacking fat grow and proliferate more quickly than their wild-type counterparts and exhibit delayed cell-cycle exit as a result of elevated expression of Cyclin E. fat mutant cells display partial insensitivity to normal developmental apoptosis cues and express increased levels of the anti-apoptotic DIAP1 protein. Collectively, these defects lead to increased organ size and organism lethality in fat mutant animals. Fat modulates Salvador/Warts/Hippo pathway activity by promoting abundance and localization of Expanded protein at the apical membrane of epithelial tissues. CONCLUSIONS: Fat restricts organ size during Drosophila development via the Salvador/Warts/Hippo pathway. These studies aid our understanding of developmental organ size control and have implications for human hyperproliferative disorders, such as cancers.     

Marfan syndrome-causing mutations in fibrillin-1 result in gross morphological alterations and highlight the structural importance of the second hybrid domain

Mutations in fibrillin-1 result in Marfan syndrome, which affects the cardiovascular, skeletal and ocular systems. The multiorgan involvement and wide spectrum of associated phenotypes highlights the complex pathogenesis underlying Marfan syndrome. To elucidate the genotype to phenotype correlations, we engineered four Marfan syndrome causing mutations into a fibrillin-1 fragment encoded by exons 18-25, a region known to interact with tropoelastin. Biophysical and biochemical approaches, including small angle x-ray scattering, analytical ultracentrifugation, and circular dichroism, were used to study the impact of these mutations upon the structure and function of the protein. Mutations G880S, C862R, and C908R, situated within the second hybrid domain, disrupted the ratio of alpha-helix to beta-sheet leading to a more compact conformation. These data clearly demonstrate the importance of the previously uncharacterized hybrid domain in fibrillin-1 structure. In contrast, mutation K1023N situated within the linker region between the third eight cysteine motif and cbEGF 11 markedly extended the length of the fragment. However, none of the mutations affected tropoelastin binding. The profound effects of all four mutations on fragment conformation suggest that they contribute to the pathogenesis of Marfan syndrome by disrupting protein folding and its assembly into fibrillin-rich microfibrils.     

Efficacy and Safety of Inhaled Carbon Monoxide during Pulmonary Inflammation in Mice

Background

Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.

Methodology

Mice were exposed to 0–500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow.

Principal Findings

When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40–50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.

Conclusions

Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.     

Deterministic epidemic models on contact networks: correlations and unbiological terms

The relationship between system-level and subsystem-level master equations is investigated and then utilised for a systematic and potentially automated derivation of the hierarchy of moment equations in a susceptible-infectious-removed (SIR) epidemic model. In the context of epidemics on contact networks we use this to show that the approximate nature of some deterministic models such as mean-field and pair-approximation models can be partly understood by the identification of implicit anomalous terms. These terms describe unbiological processes which can be systematically removed up to and including the nth order by nth order moment closure approximations. These terms lead to a detailed understanding of the correlations in network-based epidemic models and contribute to understanding the connection between individual-level epidemic processes and population-level models. The connection with metapopulation models is also discussed. Our analysis is predominantly made at the individual level where the first and second order moment closure models correspond to what we term the individual-based and pair-based deterministic models, respectively. Matlab code is included as supplementary material for solving these models on transmission networks of arbitrary complexity.     

Trafficking kinesin protein (TRAK)-mediated transport of mitochondria in axons of hippocampal neurons

In neurons, the proper distribution of mitochondria is essential because of a requirement for high energy and calcium buffering during synaptic neurotransmission. The efficient, regulated transport of mitochondria along axons to synapses is therefore crucial for maintaining function. The trafficking kinesin protein (TRAK)/Milton family of proteins comprises kinesin adaptors that have been implicated in the neuronal trafficking of mitochondria via their association with the mitochondrial protein Miro and kinesin motors. In this study, we used gene silencing by targeted shRNAi and dominant negative approaches in conjunction with live imaging to investigate the contribution of endogenous TRAKs, TRAK1 and TRAK2, to the transport of mitochondria in axons of hippocampal pyramidal neurons. We report that both strategies resulted in impairing mitochondrial mobility in axonal processes. Differences were apparent in terms of the contribution of TRAK1 and TRAK2 to this transport because knockdown of TRAK1 but not TRAK2 impaired mitochondrial mobility, yet both TRAK1 and TRAK2 were shown to rescue transport impaired by TRAK1 gene knock-out. Thus, we demonstrate for the first time the pivotal contribution of the endogenous TRAK family of kinesin adaptors to the regulation of mitochondrial mobility.