The NOD2 p.Leu1007fsX1008 Mutation (rs2066847) Is a Stronger Predictor of the Clinical Course of Crohn's Disease than the FOXO3A Intron Variant rs12212067

Background

Very recently, a sub-analysis of genome-wide association scans revealed that the non-coding single nucleotide polymorphism (SNP) rs12212067 in the FOXO3A gene is associated with a milder course of Crohn''s disease (CD) (Cell 2013;155:57–69). The aim of our study was to evaluate the clinical value of the SNP rs12212067 in predicting the severity of CD by correlating CD patient genotype status with the most relevant complications of CD such as stenoses, fistulas, and CD-related surgery.

Methodology/Principal Findings

We genotyped 550 CD patients for rs12212067 (FOXO3A) and the three common CD-associated NOD2 mutations rs2066844, rs2066847, and rs2066847 and performed genotype-phenotype analyses.

Results

No significant phenotypic differences were found between the wild-type genotype TT of the FOXO3A SNP rs12212067 and the minor genotypes TG and GG independently from NOD2 variants. The allele frequency of the minor G allele was 12.7%. Age at diagnosis, disease duration, body mass index, surgery rate, stenoses, fistula, need for immunosuppressive therapy, and disease course were not significantly different. In contrast, the NOD2 mutant p.Leu1007fsX1008 (rs2066847) was highly associated with penetrating CD (p = 0.01), the development of fistulas (p = 0.01) and stenoses (p = 0.01), and ileal disease localization (p = 0.03). Importantly, the NOD2 SNP rs2066847 was a strong separator between an aggressive and a mild course of CD (p = 2.99×10⁻⁵), while the FOXO3A SNP rs12212067 did not separate between mild and aggressive CD behavior in our cohort (p = 0.35). 96.2% of the homozygous NOD2 p.Leu1007fsX1008 carriers had an aggressive disease behavior compared to 69.3% of the patients with the NOD2 wild-type genotype (p = 0.007).

Conclusion/Significance

In clinical practice, the NOD2 variant p.Leu1007fsX1008 (rs2066847), in particular in homozygous form, is a much stronger marker for a severe clinical phenotype than the FOXO3A rs12212067 SNP for a mild disease course on an individual patient level despite its important impact on the inflammatory response of monocytes.     

High Prevalence and Spatial Distribution of Strongyloides stercoralis in Rural Cambodia

Background

The threadworm, Strongyloides stercoralis, endemic in tropical and temperate climates, is a neglected tropical disease. Its diagnosis requires specific methods, and accurate information on its geographic distribution and global burden are lacking. We predicted prevalence, using Bayesian geostatistical modeling, and determined risk factors in northern Cambodia.

Methods

From February to June 2010, we performed a cross-sectional study among 2,396 participants from 60 villages in Preah Vihear Province, northern Cambodia. Two stool specimens per participant were examined using Koga agar plate culture and the Baermann method for detecting S. stercoralis infection. Environmental data was linked to parasitological and questionnaire data by location. Bayesian mixed logistic models were used to explore the spatial correlation of S. stercoralis infection risk. Bayesian Kriging was employed to predict risk at non-surveyed locations.

Principal Findings

Of the 2,396 participants, 44.7% were infected with S. stercoralis. Of 1,071 strongyloidiasis cases, 339 (31.6%) were among schoolchildren and 425 (39.7%) were found in individuals under 16 years. The incidence of S. stercoralis infection statistically increased with age. Infection among male participants was significantly higher than among females (OR: 1.7; 95% CI: 1.4–2.0; P<0.001). Participants who defecated in latrines were infected significantly less than those who did not (OR: 0.6; 95% CI: 0.4–0.8; P = 0.001). Strongyloidiasis cases would be reduced by 39% if all participants defecated in latrines. Incidence of S. stercoralis infections did not show a strong tendency toward spatial clustering in this province. The risk of infection significantly decreased with increasing rainfall and soil organic carbon content, and increased in areas with rice fields.

Conclusions/Significance

Prevalence of S. stercoralis in rural Cambodia is very high and school-aged children and adults over 45 years were the most at risk for infection. Lack of access to adequate treatment for chronic uncomplicated strongyloidiasis is an urgent issue in Cambodia. We would expect to see similar prevalence rates elsewhere in Southeast Asia and other tropical resource poor countries.     

Simple Fecal Flotation Is a Superior Alternative to Guadruple Kato Katz Smear Examination for the Detection of Hookworm Eggs in Human Stool

Background

Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia.

Methods/Principle Findings

Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification) of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard) was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0%) followed by SNF (44.0%) and quadruple KK smears (36.0%) compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens.

Conclusion/Significance

As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.     

Increasing membrane cholesterol of neurons in culture recapitulates Alzheimer’s disease early phenotypes

Background

It is suspected that excess of brain cholesterol plays a role in Alzheimer’s disease (AD). Membrane-associated cholesterol was shown to be increased in the brain of individuals with sporadic AD and to correlate with the severity of the disease. We hypothesized that an increase of membrane cholesterol could trigger sporadic AD early phenotypes.

Results

We thus acutely loaded the plasma membrane of cultured neurons with cholesterol to reach the 30% increase observed in AD brains. We found changes in gene expression profiles that are reminiscent of early AD stages. We also observed early AD cellular phenotypes. Indeed we found enlarged and aggregated early endosomes using confocal and electron microscopy after immunocytochemistry. In addition amyloid precursor protein vesicular transport was inhibited in neuronal processes, as seen by live-imaging. Finally transient membrane cholesterol loading lead to significantly increased amyloid-β42 secretion.

Conclusions

Membrane cholesterol increase in cultured neurons reproduces most early AD changes and could thus be a relevant model for deciphering AD mechanisms and identifying new therapeutic targets.

     

Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae,Creates a Binding Site for Host 14-3-3 Proteins

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.A multicomponent defense response is initiated when plant pattern recognition receptors perceive microbially derived structural components (Nürnberger and Brunner, 2002), which are referred to as pathogen-associated molecular patterns. Many bacterial pathogens have developed type III secretion system (TTSS) effectors that can suppress or modulate pathogen-associated molecular pattern-triggered immunity (Jones and Dangl, 2006). Effector-triggered immunity represents a second layer of defense, whereby plants have evolved mechanisms that rely upon Resistance (R) proteins to sense and respond to cognate TTSS effectors. Thus, the expression of a specific bacterial effector can either sustain disease in susceptible plants or render the pathogen avirulent in resistant plants that express the corresponding R protein. Several lines of evidence suggest an involvement of scaffold proteins from the 14-3-3 family in mediating these defense responses at various levels (Yang et al., 2009; Oh et al., 2010). Some R proteins have been shown to bind 14-3-3 proteins directly. RPW2.8, which confers resistance to fungal pathogens of Golovinomyces spp., associates specifically with the 14-3-3 isoform λ (designated GF14λ) from Arabidopsis (Arabidopsis thaliana; Yang et al., 2009). Moreover, both types of resistance were compromised in Arabidopsis lacking the λ isoform. Consistently, ectopic overexpression of GF14λ in transgenic Arabidopsis results in enhanced resistance to powdery mildew (Golovinomyces cichoracearum; Yang et al., 2009). Tobacco (Nicotiana tabacum) N protein, which mediates resistance to Tobacco mosaic virus, also binds 14-3-3 protein (Ueda et al., 2006). The viral p50 replicase helicase domain is the cognate ligand for N protein. Since this domain also interacts with 14-3-3s, it is possible that 14-3-3s might function in the formation of the receptor-ligand recognition complex (Ueda et al., 2006). In addition, the tomato (Solanum lycopersicum) 14-3-3 protein TF7 has been shown to exhibit positive regulation on the mitogen-activated protein kinase cascade, which is activated rapidly by pathogen recognition (Oh et al., 2010; Oh and Martin, 2011).There is increasing evidence that many intracellular pathways are regulated by the modulation of scaffold protein properties rather than the activities of integral components in the signaling cascades (Good et al., 2011). This strategy enables signal transduction to be turned on or off rapidly via the assembly or disassembly of complexes. This same mechanism also allows the intensity and kinetics of a response to be fine-tuned to the stimulus (Good et al., 2011). It was recently suggested that the manipulation of scaffolding may be one strategy employed by pathogens to interfere with the host defense response. The best-characterized example of scaffolding manipulation is the phytotoxin fusicoccin, which is secreted by the fungus Fusicoccum amygdali. Fusicoccin targets a 14-3-3 protein that regulates guard cell H⁺-ATPases, and its activity results in stomatal opening, facilitating pathogen entry (Oecking et al., 1994). Some bacterial virulence factors simply require scaffold proteins to reach their destination within host cells or to become enzymatically active, while others target the host scaffold proteins to suppress defenses. Yersinia species secrete the TTSS effector YopK (for Yersinia outer protein K), which binds to the Receptor for Activated C Kinase1 in mammals (Thorslund et al., 2011). It is hypothesized that this interaction blocks phagocytosis, allowing efficient extracellular proliferation of the bacteria. Yersinia spp. has also acquired the virulence factor YopM, which mimics eukaryotic scaffolds and forces bridging of host kinases (McDonald et al., 2003). Similarly, enterohemorrhagic Escherichia coli strains use EspG to form an artificial complex that effectively reprograms host signaling (Selyunin et al., 2011).HopQ1 (for Hrp outer protein Q [also known as HopQ1-1]; {"type":"entrez-protein","attrs":{"text":"AAZ37975.1","term_id":"71558765","term_text":"AAZ37975.1"}}AAZ37975.1) is a type III effector that has been acquired recently by Pseudomonas syringae strains (Rohmer et al., 2004), whereas its xenologs from Ralstonia solanacearum and Xanthomonas spp. appear to be ancient. HopQ1 contributes to host specificity, but its exact role in pathogenesis remains undefined. This study shows that HopQ1 must undergo a specific phosphorylation event in planta as a prerequisite for its binding to host 14-3-3 and that its properties depend upon the formation of the effector-host protein complex.     

Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide–sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.     

Competition components along productivity gradients – revisiting a classic dispute in ecology

Competition is ubiquitous in plant communities with various effects on plant fitness and community structure. A long-standing debate about different approaches to explain competition is the controversy between David Tilman and Philip Grime. Grime stated that the importance of competition relative to the impact of the environment increases along a productivity gradient, while Tilman argued that the intensity of competition is independent of productivity. To revisit this controversy, we assumed that the effects of plant–plant interactions are additive and applied the new competition indices by Díaz-Sierra et al. (2017) in a field experiment along a productivity gradient in S-Germany, using the rare arable plant Arnoseris minima as a study species. The ‘target technique' was applied, to separate the effects of root and shoot competition. The study plants were exposed to five competition treatments with three replicates in 18 sites, respectively. We investigated the expectation that root competition is more intense in unproductive sites than shoot competition. Additionally, we predicted survival to be less affected by competition than growth-related plant parameters. Using the biomass of individuals without competition as a proxy for site productivity there was a positive relationship with competition importance but no relationship with competition intensity when plants experienced full competition. Survival of the target plants was unaffected by competition. Root competition was the main mechanism determining the performance of the target plants, whereas the effect of shoot competition was relatively low albeit increasing with productivity. We conclude that when considering plant–plant interactions additive both Grime's and Tilman's theories can be supported.     

Signaling pathways of heat- and hypersalinity-induced polyp bailout in Pocillopora acuta

Polyp bailout is a drastic response to acute stress where coral coloniality breaks down and polyps detach. We induced polyp bailout in Pocillopora acuta with heat stress and tested for differential gene expression using RNAseq and a qPCR assay. Furthermore, we induced polyp bailout with hypersalinity and compared the results to identify stressor-independent signals and pathways active during polyp bailout. Both stressors led to the onset of polyp bailout and the detachment of vital polyps. We observed activation of microbe-associated molecular pattern receptors and downstream signaling pathways of the innate immune system. Further, we detected growth factors and genes active during Wnt-signaling potentially contributing to wound healing, regeneration, and proliferation. Upregulation of several genes encoding for matrix metalloproteinases and the fibroblast growth factor signaling pathway are the most likely involved in the remodeling of the extracellular matrix, as well as in the detachment of polyps from the calcareous skeleton during polyp bailout. Expression of genes of interest in our qPCR assay of vital polyps from our heat-stress experiment, showed a trend for a normalization of gene expression after polyp bailout. Our results provide new insights into the signaling cascades leading to the observed physiological responses during polyp bailout. Comparison between the two stressors showed that certain signaling pathways are independent of the stressor and suggested that polyp bailout is a general response of corals to acute stress. Furthermore, immune system responses during polyp bailout indicate that microbe-associated partners of corals may lead to the polyp bailout response.

     

Dragondrop: a novel passive mechanism for aerial righting in the dragonfly

Dragonflies perform dramatic aerial manoeuvres when chasing targets but glide for periods during cruising flights. This makes dragonflies a great system to explore the role of passive stabilizing mechanisms that do not compromise manoeuvrability. We challenged dragonflies by dropping them from selected inverted attitudes and collected 6-degrees-of-freedom aerial recovery kinematics via custom motion capture techniques. From these kinematic data, we performed rigid-body inverse dynamics to reconstruct the forces and torques involved in righting behaviour. We found that inverted dragonflies typically recover themselves with the shortest rotation from the initial body inclination. Additionally, they exhibited a strong tendency to pitch-up with their head leading out of the manoeuvre, despite the lower moment of inertia in the roll axis. Surprisingly, anaesthetized dragonflies could also complete aerial righting reliably. Such passive righting disappeared in recently dead dragonflies but could be partially recovered by waxing their wings to the anaesthetised posture. Our kinematics data, inverse dynamics model and wind-tunnel experiments suggest that the dragonfly''s long abdomen and wing posture generate a rotational tendency and passive attitude recovery mechanism during falling. This work demonstrates an aerodynamically stable body configuration in a flying insect and raises new questions in sensorimotor control for small flying systems.