Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors

To study the role of various caspases during apoptosis, we have designed a series of caspase inhibitors based on the cowpox virus cytokine response modifier A (crmA) protein. Wild-type crmA inhibits caspases 1 and 8 and thereby protects cells from apoptosis triggered by ligation of CD95 or tumour necrosis factor (TNF) receptors, but it does not protect against death mediated by other caspases. By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the different families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspase inhibitors that show altered ability to protect against cell death. Although DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded rapidly and was a weaker inhibitor than crmA DQMD, which was not degraded. Unlike wild-type crmA and crmA DEVD, crmA DQMD was able to inhibit apoptosis caused by direct activation of caspase 3 and protected lymphoid cells from death induced by radiation and dexamethasone. Significantly, the protected cells were capable of sustained growth.     

MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options—especially during pregnancy.     

CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42^Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.     

The French accelerator mass spectrometry facility ASTER: Improved performance and developments

Following the installation and acceptance test of the French 5 MV AMS facility ASTER, the focus has been on improving the capability for routine measurements of ¹⁰Be and ²⁶Al. Quality assurance has been established by the introduction of traceable AMS standards for each nuclide, by self-monitoring through participation in round-robin exercises and proficiency testing, and by surveillance of long- and short-time variability of blank and reference materials. A background level of 3 × 10^?14 makes ASTER well-suited for measuring ⁴¹Ca/⁴⁰Ca in the10^?12 region, which is sufficient for a wide range of applications. Routine AMS measurements of volatile elements like ³⁶Cl and ¹²⁹I will most likely become feasible in the very near future as the result of significant improvements in the ion source design.     

Insectivorous Birds Are Attracted by Plant Traits Induced by Insect Egg Deposition

Insectivorous birds feed upon all developmental stages of herbivorous insects, including insect eggs if larvae and adults are unavailable. Insect egg deposition on plants can induce plant traits that are subsequently exploited by egg parasitoids searching for hosts. However, it is unknown whether avian predators can also use egg-induced plant changes for prey localization. Here, we studied whether great tits (Parus major) and blue tits (Cyanistes caeruleus) are attracted by traits of the Scots pine (Pinus sylvestris) induced by pine sawfly (Diprion pini) egg deposition. We chose this plant – insect system because sawfly egg deposition on pine needles is known to locally and systemically induce a change in pine volatile organic compounds (VOCs), and tits are known to prey upon sawfly eggs. In dual choice laboratory experiments, we simultaneously offered the birds an egg-free control branch and a systemically egg-induced branch. Significantly more birds visited the egg-induced branch first. We confirmed by GC-MS analyses that systemically egg-induced branches released more (E)-β-farnesene compared to control branches. Spectrophotometric analyses showed that control branches reflected more light than egg-induced branches throughout the avian visual range. Although a discrimination threshold model for blue tits suggests that the birds are poor at discriminating this visual difference, the role of visual stimuli in attracting the birds to egg-induced pines cannot be discounted. Our study shows, for the first time, that egg-induced odorous and/or visual plant traits can help birds to locate insect eggs without smelling or seeing those eggs.     

TextWiki: a superlative resource

Although superlatives are commonly used in natural language, so far there has been no large-scale computational investigation of the types of comparisons they express. This article describes a comprehensive annotation scheme for superlatives, which classifies superlatives according to their surface forms and motivates an initial focus on so-called “ISA superlatives”. This type of superlative comparison is especially suitable for a computational approach because both their targets and comparison sets are explicitly realised in the text, and the proposed annotation scheme offers guidelines for annotating the spans of such comparative elements. The annotations are tested and evaluated on 500 tokens of superlatives with good inter-annotator agreement. In addition to providing a platform for investigating superlatives on a larger scale, this research also introduces a new text-based Wikipedia corpus in which all superlative instances have been annotated according to the proposed annotation scheme, and which has been used to develop a tool that can reliably distinguish between different superlative types, and identify the comparative components of ISA superlatives.     

Experiments and modeling of the autoignition of methylcyclohexane at high pressure

New experimental data are collected for methyl-cyclohexane (MCH) autoignition in a heated rapid compression machine (RCM). Three mixtures of MCH/O₂/N₂/Ar at equivalence ratios of ? = 0.5, 1.0, and 1.5 are studied and the ignition delays are measured at compressed pressure of 50 bar and for compressed temperatures in the range of 690–900 K. By keeping the fuel mole fraction in the mixture constant, the order of reactivity, in terms of inverse ignition delay, is measured to be ? = 0.5 > ? = 1.0 > ? = 1.5, demonstrating the dependence of the ignition delay on oxygen concentration. In addition, an existing model for the combustion of MCH is updated with new reaction rates and pathways, including substantial updates to the low-temperature chemistry. The new model shows good agreement with the overall ignition delays measured in this study, as well as the ignition delays measured previously in the literature using RCMs and shock tubes. This model therefore represents a strong improvement compared to the previous version, which uniformly over-predicted the ignition delays. Chemical kinetic analyses of the updated mechanism are also conducted to help understand the fuel decomposition pathways and the reactions controlling the ignition. Combined, these results and analyses suggest that further investigation of several of the low-temperature fuel decomposition pathways is required.     

Quality control of beer using high-resolution nuclear magnetic resonance spectroscopy and multivariate analysis

High-resolution nuclear magnetic resonance (NMR) spectroscopy is introduced for the quality control and authenticity assessment of beer in official food control. Measurements were performed using a 400-MHz NMR spectrometer using flow injection technology for automatic sample changing. Only degassing and addition of buffer (pH 5.6 in D₂O for locking and 0.1% TSP for referencing) is required to prepare the beer samples. Differences in the spectral profiles of beers varying in type and origin were studied by principal component analysis (PCA), considering the spectrum to be a characteristic fingerprint. For the first time, the high throughput of a Flow-Injection NMR system allowed a comprehensive database of beer spectra for PCA classification to be established efficiently. Beers made with barley malt could be distinguished from those made with wheat malt. Clustering of beers from the same brewing sites was observed, as well as significant discrimination of beers with deteriorated quality. Using the partial least squares (PLS) method to correlate NMR spectra with results from reference methods, models for calculating the original gravity, ethanol and lactic acid were established. The results obtained suggest that NMR is a useful tool in the quality control of beer samples, since quantitative determination of essential compounds as well as chemometric classification are simultaneously possible. Compared to conventional methods, ¹H NMR spectroscopy is faster and requires simpler sample preparation.