WildCARDs: Inflammatory caspases directly detect LPS

Inflammasomes are sensors that serve as activation platforms for caspase-1 — a mechanism that set the prevailing paradigm for inflammatory caspase activation. A recent Nature paper by Shi et al. upends this paradigm by describing an unprecedented model for caspase activation whereby caspase-4, -5, and -11 directly bind their agonist, cytosolic LPS, triggering auto-activation and subsequent pyroptotic cell death.The inflammatory caspases — among them caspase-1, murine caspase-11, and human caspase-4 and -5 (homologs of murine caspase-11) — are central to depriving infectious agents of intracellular replication niches. Upon responding to a given stimulus, they become catalytically active and initiate a form of programmed inflammatory cell death termed pyroptosis. By examining their homology and adjacent chromosomal arrangement in humans and other mammals, it is apparent that the inflammatory caspases originate from a series of gene duplications and subsequent divergences.A balanced caspase-1 response is critical to defense against a variety of infectious agents, whereas its aberrant activation underlies a number of immune pathologies. Less is known about caspase-11, -4, and -5 in infection; however, we have shown that one physiological role of caspase-11 is to detect and help clear cytosol invasive infections, such as those caused by Burkholderia thailandensis¹. More recent work has shown that caspase-11 mediates resistance to DSS-induced colitis² and clearance of Salmonella enterica serovar Typhimurium-infected cells in the intestinal epithelium³, perhaps limited to the times when these bacteria enter the cytosol. Likewise, caspase-4 responds to S. Typhimurium, enteropathogenic E. coli³, and Shigella flexneri⁴ infections in human intestinal epithelial cells. As with caspase-1, moderation of caspase-11 activity is key to limiting immune pathology: much of the lethality of bolus lipopolysaccharide (LPS) injection is mediated by caspase-11^5,6,7,8. Shedding light on the mechanisms underlying these observations, our lab and that of Dr Vishva Dixit independently determined that caspase-11 is activated in response to cytosolic LPS^7,8; however, whether caspase-4 (and/or -5) functions similarly was not determined.Of the inflammatory caspases, the activation mechanism of caspase-1 is the best described. Via its N-terminal CARD domain and an adaptor protein called ASC, caspase-1 interacts with a family of cytosolic proteins, the inflammasomes, that detect signatures of infection (Figure 1). It then initiates pyroptosis and directs proinflammatory cytokine secretion. Inflammasomes thus follow the paradigm of apoptotic caspase activation, where apoptosis initiators caspase-2, -8 and -9 are recruited and activated by death domain family-containing upstream sensors: the piddosome, DISC, and apoptosome, respectively. Therefore, we and others assumed that the model of upstream sensor activating downstream caspase would hold for the other inflammatory caspases as well. For example, Kayagaki and colleagues coined the term ''noncanonical inflammasome pathway'' to describe activation of caspase-11 by a putative LPS sensor^5,6. However, a recent elegant paper by Shi et al.⁹ proves this hypothesis wrong and describes an entirely novel paradigm of caspase activation. Moreover, the authors address many of the gaps in our understanding of caspase-11, -4, and -5 biology.Open in a separate windowFigure 1Schematic of canonical and noncanonical inflammasome pathways for inflammatory caspase activation. Left: Inflammasomes such as AIM2, NLRP3, and NLRC4 detect contamination of the cytosol with microbial ligands (e.g., DNA, flagellin, bacterial type 3 secretion system components) or certain cellular perturbations. Via the adaptor protein ASC, they subsequently activate caspase-1 (in the case of certain CARD-containing inflammasomes, such as NLRC4, direct interaction with caspase-1 can also occur), which initiates pyroptosis and secretion of the proinflammatory cytokines IL-1β and IL-18. Right: Caspase-4, -5, and -11 directly bind cytosolic LPS from Gram-negative bacteria. They subsequently oligomerize, activate, and initiate pyroptosis.Using electroporation to deliver bacterial components into the cytosol of cells, the authors first determined that caspase-4 responds to LPS in human monocytes by triggering pyroptosis. These findings extended to non-myeloid cells, where caspase-4 is constitutively expressed. The authors then demonstrated that caspase-4 and caspase-11 are functionally interchangable, supporting that they are homologs.Shi and colleagues next began identifying the molecule that actually binds LPS in the cytosol. They screened a number of NLRs and CARD domain-containing proteins, but no candidates emerged. In agreement with this, unpublished work from our lab also ruled out virtually all known CARD-containing proteins as the LPS sensor. Clues to the identity of the sensor arose from the following astute observations: First, Shi et al. noticed that both caspase-4 and caspase-11, when purified from E. coli, eluted from columns as large oligomers, suggesting activation, whereas they eluted as monomers when expressed in and purified from insect cells. Second, they found that the LPS contents of caspase-4 and -11 purified from E. coli were three orders of magnitude higher than what they typically observed when purifying bacterial proteins. Together, these results suggested that caspase-4 and -11 directly bind LPS. A series of pull-downs and surface plasmon resonance experiments confirmed this notion, revealing stable interaction of LPS with caspase-4 and -11 in cells transfected with LPS. Furthermore, the authors showed that caspase-5 similarly binds LPS. In all cases, LPS binding and caspase oligomerization was CARD domain dependent; indeed, purified caspase-4 and -11 CARD domains were sufficient to bind LPS and oligomerize. Three regions of basic residues in the caspase-11 CARD domain — mostly conserved in caspase-4 and -5, but not caspase-1 — were critical for LPS binding. Last, the authors determined that caspase-11 and -4 oligomerization stimulates activation, as measured by cleavage of a fluorogenic substrate. Interestingly, the known antagonists of caspase-11 activation Lipid IVa and atypical LPS from Rhodobacter sphaeroides bound caspase-4 and -11, but failed to induce oligomerization and activation.The Shi et al. paper brings to light a number of fascinating perspectives. First, binding of LPS by caspase-4, -5, and -11 establishes a new paradigm for caspase activation: Direct detection of a cell death-inducing ligand by a caspase. As the authors noted, this is analogous to horseshoe crab factors C and G, which bind LPS and β-(1,3)-D-glucan, respectively, and initiate coagulation cascades in haemolymphs.Second, the cell expression patterns of caspase-11, -4, and -5 may have important implications in future strategies for treating endotoxemia and Gram-negative sepsis. Caspase-11 expression is inducible in myeloid cells, where its basal expression is low; in contrast, caspase-4 appears to be constitutively expressed in human myeloid cells. Therefore, aberrant translocation of LPS into the cytosol of human myeoloid cells may not require priming to activate caspase-4 and initiate pyroptosis, perhaps sensitizing humans to the deleterious effects of LPS compared to mice. Investigating other cell type expression differences in this context will be informative.In answering so many questions about the biology of the inflammatory caspases, the work of Shi and colleagues raises many more. Among them: Why do antagonists of caspase-11 fail to induce oligomerization? How do the CARD domains of these caspases “see” LPS? During binding of LPS by MD2, the acyl chains of lipid A extend into the binding cleft of MD2¹⁰ in a manner sensitive to acyl chain length; in contrast, caspase-11 detects very diverse lipid A acyl chain lengths and structures, such as those of Salmonella and Legionella species^7,8,11, suggesting that the CARD domain may wrap around the lipid groups of LPS near the phosphate head groups of lipid A. Insights into these questions will surely come from crystal structures of caspase-11, -4, and -5 bound to various LPS structures.     

Temperature regimes and aphid density interactions differentially influence VOC emissions in Arabidopsis

The effects of volatile emissions from plants exposed to individual abiotic and biotic stresses are well documented. However, the influence of multiple stresses on plant photosynthesis and defense responses, resulting in a variety of volatile profiles has received little attention. In this study, we investigated how temperature regimes in the presence and absence of the sucking insect Myzus persicae affected volatile organic compound (VOC) emissions in Arabidopsis over three time periods (0–24, 24–48, and 48–72 h). Headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry was used to evaluate Arabidopsis VOCs. The results showed that under laboratory conditions, eight volatile classes [alcohols (mainly 2-ethyl-hexan-1-ol), ketone (6-methyl hept-5-en-2-one), esters (mainly (Z)-3-hexenyl acetate), aldehydes (mainly phenylacetaldehyde), isothiocyanates (mainly 4-methylpentyl isothiocyanate), terpenes (mainly (E,E)-α-farnesene), nitrile (5-(methylthio) pentanenitrile), and sulfide (dimethyl trisulfide)] were observed on plants exposed to stress combinations, whereas emissions of six volatile classes were observed during temperature stress treatments alone (with the exception of nitriles and sulfides). Aphid density at high temperature combinations resulted in significantly higher isothiocyanate, ester, nitrile, and sulfide proportions. The results of the present study provide an insight into the effects of temperature–aphid interactions on plant volatile emissions.     

Genomic regions underlying agronomic traits in linseed (Linum usitatissimum L.) as revealed by association mapping OA简

The extreme climate of the Canadian Prairies poses a major challenge to improve yield. Although it is possible to breed for yield per se, focusing on yield‐related traits could be advantageous because of their simpler genetic architecture. The Canadian flax core collection of 390 accessions was genotyped with 464 simple sequence repeat markers, and phenotypic data for nine agronomic traits including yield, bolls per area, 1,000 seed weight, seeds per boll, start of flowering, end of flowering, plant height, plant branching, and lodging collected from up to eight environments was used for association mapping. Based on a mixed model (principal component analysis (PCA) + kinship matrix (K)), 12 significant marker‐trait associations for six agronomic traits were identified. Most of the associations were stable across environments as revealed by multivariate analyses. Statistical simulation for five markers associated with 1000 seed weight indicated that the favorable alleles have additive effects. None of the modern cultivars carried the five favorable alleles and the maximum number of four observed in any accessions was mostly in breeding lines. Our results confirmed the complex genetic architecture of yield‐related traits and the inherent difficulties associated with their identification while illustrating the potential for improvement through marker‐assisted selection.     

Salmonella enterica Flagellin Is Recognized via FLS2 and Activates PAMP-Triggered Immunity in Arabidopsis thaliana

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogenassociated molecular pattern （PAMP） in Arabidopsis thaliana, which induces PAMP-triggered immunity （PTI） via the recognition of the fig22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system （T3SS） mutant prgH- induced stronger defense gene expression than wild-type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the fig22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether, these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.     

Assessment of Coupling between Trans-Abdominally Acquired Fetal ECG and Uterine Activity by Bivariate Phase-Rectified Signal Averaging Analysis

Couplings between uterine contractions (UC) and fetal heart rate (fHR) provide important information on fetal condition during labor. At present, couplings between UC and fHR are assessed by visual analysis and interpretation of cardiotocography. The application of computerized approaches is restricted due to the non-stationarity of the signal, missing data and noise, typical for fHR. Herein, we propose a novel approach to assess couplings between UC and fHR, based on a signal-processing algorithm termed bivariate phase-rectified signal averaging (BPRSA).

Methods

Electrohysterogram (EHG) and fetal electrocardiogram (fECG) were recorded non-invasively by a trans-abdominal device in 73 women at term with uneventful singleton pregnancy during the first stage of labor. Coupling between UC and fHR was analyzed by BPRSA and by conventional cross power spectral density analysis (CPSD). For both methods, degree of coupling was assessed by the maximum coefficient of coherence (C_PRSA and C_RAW, respectively) in the UC frequency domain. Coherence values greater than 0.50 were consider significant. C_PRSA and C_RAW were compared by Wilcoxon test.

Results

At visual inspection BPRSA analysis identified coupled periodicities in 86.3% (63/73) of the cases. 11/73 (15%) cases were excluded from further analysis because no 30 minutes of fECG recording without signal loss was available for spectral analysis. Significant coupling was found in 90.3% (56/62) of the cases analyzed by BPRSA, and in 24.2% (15/62) of the cases analyzed by CPSD, respectively. The difference between median value of C_PRSA and C_RAW was highly significant (0.79 [IQR 0.69–0.90] and 0.29 [IQR 0.17–0.47], respectively; p<0.0001).

Conclusion

BPRSA is a novel computer-based approach that can be reliably applied to trans-abdominally acquired EHG-fECG. It allows the assessment of correlations between UC and fHR patterns in the majority of labors, overcoming the limitations of non-stationarity and artifacts. Compared to standard techniques of cross-correlations, such as CPSD, BPRSA is significantly superior.     

The Gut Microbial Community of Midas Cichlid Fish in Repeatedly Evolved Limnetic-Benthic Species Pairs

Gut bacterial communities are now known to influence a range of fitness related aspects of organisms. But how different the microbial community is in closely related species, and if these differences can be interpreted as adaptive is still unclear. In this study we compared microbial communities in two sets of closely related sympatric crater lake cichlid fish species pairs that show similar adaptations along the limnetic-benthic axis. The gut microbial community composition differs in the species pair inhabiting the older of two crater lakes. One major difference, relative to other fish, is that in these cichlids that live in hypersaline crater lakes, the microbial community is largely made up of Oceanospirillales (52.28%) which are halotolerant or halophilic bacteria. This analysis opens up further avenues to identify candidate symbiotic or co-evolved bacteria playing a role in adaptation to similar diets and life-styles or even have a role in speciation. Future functional and phylosymbiotic analyses might help to address these issues.     

Application and Evaluation of Interactive 3D PDF for Presenting and Sharing Planning Results for Liver Surgery in Clinical Routine

MethodsWe developed an interactive 3D PDF report document format and implemented a software tool to create these reports automatically. After more than 1000 liver CASP cases that have been reported in clinical routine using our 3D PDF report, an international user survey was carried out online to evaluate the user experience.ResultsOur solution enables the user to interactively explore the anatomical configuration and to have different analyses and various resection proposals displayed within a 3D PDF document covering only a single page that acts more like a software application than like a typical PDF file (“PDF App”). The new 3D PDF report offers many advantages over the previous solutions. According to the results of the online survey, the users have assessed the pragmatic quality (functionality, usability, perspicuity, efficiency) as well as the hedonic quality (attractiveness, novelty) very positively.ConclusionThe usage of 3D PDF for reporting and sharing CASP results is feasible and well accepted by the target audience. Using interactive PDF with embedded 3D models is an enabler for presenting and exchanging complex medical information in an easy and platform-independent way. Medical staff as well as patients can benefit from the possibilities provided by 3D PDF. Our results open the door for a wider use of this new technology, since the basic idea can and should be applied for many medical disciplines and use cases.     

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1–3 mutant

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1–3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

L’Observatoire hommes–milieux international Tessékéré (OHMi) : un outil de recherche pour étudier la complexité des écosystèmes arides du Sahel

In the Sahelian zone, the drought phenomenon, combined with anthropic factors (monoculture, bush fires, defect or deficit of manure, overgrazing, etc.), has seriously affected ecological great balances, involving a degradation of the natural resources as well as a fall in agricultural productions, pointing to a process of desertification. To face these challenges, in the course of the 8th ordinary session of the conference of the Heads of States of the African Union held in January 2007 in Addis Ababa (Ethiopia), 11 countries adopted the Panafrican project called the Green Great Wall (GGW). The total objective of the GGW is to contribute i) to the fight against the desert's advance, ii) to the development of the Saharan-Sahelian zones toward a durable management of the natural resources, and iii) to the fight against poverty. It deals with the construction of a set of zones of afforestation crossing the whole African continent in the long term (7000 km of which are in the west). Even if some decisions in the launching phase the GGW must be taken quickly, one cannot do without investment in interdisciplinary research. In particular, associating fundamental research and applied research will allow us to ensure the success in the medium and long term of such a large-scale reforestation project. Research segmented in compartmentalized knowledge fields needed to get adequate tools, among which OHMi Tessékéré, initiated by INNEE (Centre national de la recherche scientifique [CNRS]), in partnership with UCAD, constitutes an example. This suitable scientific tool, capable of action flexibility, of self-financing capacity, anchored in civil society, ready to implement a pragmatic and local interdisciplinarity founded currently on the concept of socio-ecological system (SES), is the one we chose to conduct our studies on the Ferlo arid ecosystems.