Structural Basis for dsRNA Recognition,Filament Formation,and Antiviral Signal Activation by MDA5

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Highlights? MDA5 forms an open, C-shaped ring around the viral dsRNA stem ? The CTD of MDA5 has a different orientation and flexibility compared to RIG-I ? MDA5 forms filaments by stacking monomers head-to-tail with a 70° turn per monomer ? The 2CARD domain assembles into oligomers that activate interferon signaling via MAVS     

Models for assessing local‐scale co‐abundance of animal species while accounting for differential detectability and varied responses to the environment

We developed a new modeling framework to assess how the local abundance of one species influences the local abundance of a potential competitor while explicitly accounting for differential responses to environmental conditions. Our models also incorporate imperfect detection as well as abundance estimation error for both species. As a case study, we applied the model to four pairs of mammal species in Borneo, surveyed by extensive and spatially widespread camera trapping. We detected different responses to elevation gradients within civet, macaque, and muntjac deer species pairs. Muntjac and porcupine species varied in their response to terrain ruggedness, and the two muntjac responded different to river proximity. Bornean endemic species of civet and muntjac were more sensitive than their widespread counterparts to habitat disturbance (selective logging). Local abundance within several species pairs was positively correlated, but this is likely due to the species having similar responses to (unmodeled) environmental conditions or resources rather than representing facilitation. After accounting for environment and correcting for false absences in detection, negative correlations in local abundance appear rare in tropical mammals. Direct competition may be weak in these species, possibly because the ‘ghost of competition past’ or habitat filtering have already driven separation of the species in niche space. The analytical framework presented here could increase basic understanding of how ecological interactions shape patterns of abundance across the landscape for a range of taxa, and also provide a powerful tool for forecasting the impacts of global change.     

The Effectiveness of Community Action in Reducing Risky Alcohol Consumption and Harm: A Cluster Randomised Controlled Trial

Background

The World Health Organization, governments, and communities agree that community action is likely to reduce risky alcohol consumption and harm. Despite this agreement, there is little rigorous evidence that community action is effective: of the six randomised trials of community action published to date, all were US-based and focused on young people (rather than the whole community), and their outcomes were limited to self-report or alcohol purchase attempts. The objective of this study was to conduct the first non-US randomised controlled trial (RCT) of community action to quantify the effectiveness of this approach in reducing risky alcohol consumption and harms measured using both self-report and routinely collected data.

Methods and Findings

We conducted a cluster RCT comprising 20 communities in Australia that had populations of 5,000–20,000, were at least 100 km from an urban centre (population ≥ 100,000), and were not involved in another community alcohol project. Communities were pair-matched, and one member of each pair was randomly allocated to the experimental group. Thirteen interventions were implemented in the experimental communities from 2005 to 2009: community engagement; general practitioner training in alcohol screening and brief intervention (SBI); feedback to key stakeholders; media campaign; workplace policies/practices training; school-based intervention; general practitioner feedback on their prescribing of alcohol medications; community pharmacy-based SBI; web-based SBI; Aboriginal Community Controlled Health Services support for SBI; Good Sports program for sports clubs; identifying and targeting high-risk weekends; and hospital emergency department–based SBI. Primary outcomes based on routinely collected data were alcohol-related crime, traffic crashes, and hospital inpatient admissions. Routinely collected data for the entire study period (2001–2009) were obtained in 2010. Secondary outcomes based on pre- and post-intervention surveys (n = 2,977 and 2,255, respectively) were the following: long-term risky drinking, short-term high-risk drinking, short-term risky drinking, weekly consumption, hazardous/harmful alcohol use, and experience of alcohol harm. At the 5% level of statistical significance, there was insufficient evidence to conclude that the interventions were effective in the experimental, relative to control, communities for alcohol-related crime, traffic crashes, and hospital inpatient admissions, and for rates of risky alcohol consumption and hazardous/harmful alcohol use. Although respondents in the experimental communities reported statistically significantly lower average weekly consumption (1.90 fewer standard drinks per week, 95% CI = −3.37 to −0.43, p = 0.01) and less alcohol-related verbal abuse (odds ratio = 0.58, 95% CI = 0.35 to 0.96, p = 0.04) post-intervention, the low survey response rates (40% and 24% for the pre- and post-intervention surveys, respectively) require conservative interpretation. The main limitations of this study are as follows: (1) that the study may have been under-powered to detect differences in routinely collected data outcomes as statistically significant, and (2) the low survey response rates.

Conclusions

This RCT provides little evidence that community action significantly reduces risky alcohol consumption and alcohol-related harms, other than potential reductions in self-reported average weekly consumption and experience of alcohol-related verbal abuse. Complementary legislative action may be required to more effectively reduce alcohol harms.

Trial registration

Australian New Zealand Clinical Trials Registry ACTRN12607000123448 Please see later in the article for the Editors'' Summary     

A brachylaimid trematode from marsupial mice with observations on its life cycle

A species of digenetic trematode, Brachylaime antechini (Family Brachylaimidae), is described from adult worms recovered from the intestine and rectum of two species of marsupial mice, Antechinus stuartii(10 of 56 infected) and Antechinus swainsonii (2 of 5 infected). On experimental evidence unencysted metacercariae from the kidney of a terrestrial gastropod, Strangesta capillacea, are considered to be larval stages of Brachylaime antechini. Miracidia, sporocysts and cercariae have not been recovered, but circumstantial evidence suggests that Strangesta capillacea fulfils the role of first intermediate host. Brachylaime antechini most closely resembles Brachylaime chiapensis, a parasite of deer mice in Mexico.     

Destruction complex function in the Wnt signaling pathway of Drosophila requires multiple interactions between Adenomatous polyposis coli 2 and Armadillo

The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, β-catenin (βcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to βcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds βcat and the affinity is significantly increased. These distinct APC-βcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for βcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.     

High-level expression of a soluble and functional fibronectin type II domain from MMP-2 in the Escherichia coli cytoplasm for solution NMR studies

We report a method for the expression in Escherichia coli of the isolated second type II fibronectin domain from MMP-2 (FNII-2). FNII-2 was expressed as a His(6)thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation. When cultured at 37 degrees C, the expressed protein is located exclusively in the soluble fraction of the E. coli lysate. The fusion protein from the soluble fraction was purified and the His(6)thioredoxin-tag was cleaved by thrombin, resulting in a yield of approximately 40 mg/L. The recombinant FNII-2 was demonstrated to be functional by its ability to bind to gelatin-Sepharose, correct folding of the purified protein was confirmed by NMR spectroscopy. This approach may generally be applicable to all FNII domains and is a significant simplification relative to existing techniques involving refolding from inclusion bodies or expression in the eukaryotic host, Pichia pastoris.     

Quantifying trophoblast migration: In vitro approaches to address in vivo situations

ABSTRACT

When trophoblasts migrate and invade in vivo, they do so by interacting with a range of other cell types, extracellular matrix proteins, chemotactic factors and physical forces such as fluid shear stress. These factors combine to influence overall trophoblast migration and invasion into the decidua, which in turn determines the success of spiral artery remodelling, and pregnancy itself. Our understanding of these important but complex processes is limited by the simplified conditions in which we often study cell migration in vitro, and many discrepancies are observed between different in vitro models in the literature. On top of these experimental considerations, the migration of individual trophoblasts can vary widely. While time-lapse microscopy provides a wealth of information on trophoblast migration, manual tracking of individual cell migration is a time consuming task that ultimately restricts the numbers of cells quantified, and thus the ability to extract meaningful information from the data. However, the development of automated imaging algorithms is likely to aid our ability to accurately interpret trophoblast migration in vitro, and better allow us to relate these observations to in vivo scenarios. This commentary discusses the advantages and disadvantages of techniques commonly used to quantify trophoblast migration and invasion, both from a cell biology and a mathematical perspective, and examines how such techniques could be improved to help us relate trophoblast migration more accurately to in vivo function in the future.     

Lung clearance index in adults with non-cystic fibrosis bronchiectasis

Background

Lung clearance index (LCI) is a measure of abnormal ventilation distribution derived from the multiple breath inert gas washout (MBW) technique. We aimed to determine the clinical utility of LCI in non-CF bronchiectasis, and to assess two novel MBW parameters that distinguish between increases in LCI due to specific ventilation inequality (LCI_vent) and increased respiratory dead space (LCI_ds).

Methods

Forty-three patients with non-CF bronchiectasis and 18 healthy control subjects underwent MBW using the sulphur hexafluoride wash-in technique, and data from 40 adults with CF were re-analysed. LCI_vent and LCI_ds were calculated using a theoretical two-compartment lung model, and represent the proportional increase in LCI above its ideal value due to specific ventilation inequality and increased respiratory dead space, respectively.

Results

LCI was significantly raised in patients with non-CF bronchiectasis compared to healthy controls (9.99 versus 7.28, p < 0.01), and discriminated well between these two groups (area under receiver operating curve = 0.90, versus 0.83 for forced expiratory volume in one second [% predicted]). LCI, LCI_vent and LCI_ds were repeatable (intraclass correlation coefficient > 0.75), and correlated significantly with measures of spirometric airflow obstruction.

Conclusion

LCI is repeatable, discriminatory, and is associated with spirometric airflow obstruction in patients with non-CF bronchiectasis. LCI_vent and LCI_ds are a practical and repeatable alternative to phase III slope analysis and may allow a further level of mechanistic information to be extracted from the MBW test in patients with severe ventilation heterogeneity.