No general soil carbon sequestration under Central European short rotation coppices

Wood from short rotation coppices (SRCs) is discussed as bioenergy feedstock with good climate mitigation potential inter alia because soil organic carbon (SOC) might be sequestered by a land-use change (LUC) from cropland to SRC. To test if SOC is generally enhanced by SRC over the long term, we selected the oldest Central European SRC plantations for this study. Following the paired plot approach soils of the 21 SRCs were sampled to 80 cm depth and SOC stocks, C/N ratios, pH and bulk densities were compared to those of adjacent croplands or grasslands. There was no general trend to SOC stock change by SRC establishment on cropland or grassland, but differences were very site specific. The depth distribution of SOC did change. Compared to cropland soils, the SOC density in 0–10 cm was significantly higher under SRC (17 ± 2 in cropland and 21 ± 2 kg C m⁻³ in SRC). Under SRC established on grassland SOC density in 0–10 cm was significantly lower than under grassland. The change rates of total SOC stocks by LUC from cropland to SRC ranged from −1.3 to 1.4 Mg C ha⁻¹ yr⁻¹ and −0.6 Mg C ha⁻¹ yr⁻¹ to +0.1 Mg C ha⁻¹ yr⁻¹ for LUC from grassland to SRC, respectively. The accumulation of organic carbon in the litter layer was low (0.14 ± 0.08 Mg C ha⁻¹ yr⁻¹). SOC stocks of both cropland and SRC soils were correlated with the clay content. No correlation could be detected between SOC stock change and soil texture or other abiotic factors. In summary, we found no evidence of any general SOC stock change when cropland is converted to SRC and the identification of the factors determining whether carbon may be sequestered under SRC remains a major challenge.     

Economic Impact of Gene Expression Profiling in Patients with Early-Stage Breast Cancer in France

Background and Aims

The heterogeneous nature of breast cancer can make decisions on adjuvant chemotherapy following surgical resection challenging. Oncotype DX is a validated gene expression profiling test that predicts the likelihood of adjuvant chemotherapy benefit in early-stage breast cancer. The aim of this study is to determine the costs of chemotherapy in private hospitals in France, and evaluate the cost-effectiveness of Oncotype DX from national insurance and societal perspectives.

Methods

A multicenter study was conducted in seven French private hospitals, capturing retrospective data from 106 patient files. Cost estimates were used in conjunction with a published Markov model to assess the cost-effectiveness of using Oncotype DX to inform chemotherapy decision making versus standard care. Sensitivity analyses were performed.

Results

The cost of adjuvant chemotherapy in private hospitals was estimated at EUR 8,218 per patient from a national insurance perspective and EUR 10,305 from a societal perspective. Cost-effectiveness analysis indicated that introducing Oncotype DX improved life expectancy (+0.18 years) and quality-adjusted life expectancy (+0.17 QALYs) versus standard care. Oncotype DX was found cost-effective from a national insurance perspective (EUR 2,134 per QALY gained) and cost saving from a societal perspective versus standard care. Inclusion of lost productivity costs in the modeling analysis meant that costs for eligible patients undergoing Oncotype DX testing were on average EUR 602 lower than costs for those receiving standard care.

Conclusions

As Oncotype DX was found both cost and life-saving from a societal perspective, the test was considered to be dominant to standard care. However, the delay in coverage has the potential to erode the quality of the French healthcare system, thus depriving patients of technologies that could improve clinical outcomes and allow healthcare professionals to better allocate hospital resources to improve the standard of care for all patients.     

Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants

Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.     

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co‐occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co‐incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

Population genomics of natural and experimental populations of guppies (Poecilia reticulata)

Convergent evolution represents one of the best lines of evidence for adaptation, but few cases of phenotypic convergence are understood at the genetic level. Guppies inhabiting the Northern Mountain Range of Trinidad provide a classic example of phenotypic convergent evolution, where adaptation to low or high predation environments has been found for a variety of traits. A major advantage of this system is the possibility of long‐term experimental studies in nature, including transplantation from high to low predation sites. We used genome scans of guppies from three natural high and low predation populations and from two experimentally established populations and their sources to examine whether phenotypic convergent evolution leaves footprints at the genome level. We used population‐genetic modelling approaches to reconstruct the demographic history and migration among sampled populations. Naturally colonized low predation populations had signatures of increased effective population size since colonization, while introduction populations had signatures of decreased effective population size. Only a small number of regions across the genome had signatures of selection in all natural populations. However, the two experimental populations shared many genomic regions under apparent selection, more than expected by chance. This overlap coupled with a population decrease since introduction provides evidence for convergent selection occurring in the two introduced populations. The lack of genetic convergence in the natural populations suggests that convergent evolution is lacking in these populations or that the effects of selection become difficult to detect after a long‐time period.     

CASP10–BCL::Fold efficiently samples topologies of large proteins

During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template‐based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native‐like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE‐only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native‐like assembly of SSEs for further refinement and submission. It was also observed that for some β‐strand proteins model refinement failed as β‐strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non‐natural topologies that require loop regions to pass through the center of the protein. Proteins 2015; 83:547–563. © 2015 Wiley Periodicals, Inc.     

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.