The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.     

Ten years of progress in vaccination against cancer: the need to counteract cancer evasion by dual targeting in future therapies

Although cancer immunology has made vigorous progress over the last decade, its future remains uncertain. Tumors have clearly proved subject to immune surveillance, leading to antigenic editing, and means of activating both T and B arms of the immune system have been devised. Therapeutic vaccination and monoclonal antibody therapy have so far proved disappointing, because tumors prove adept at evasion from immune control. Dual targeting could well counteract evasion, provided that the two targets are independent and are attacked simultaneously. This stage has nearly but not quite been reached in several forms of immunotherapy, particularly of B-cell cancers, although such treatment also carries hazards.     

Estimation of capture probabilities using generalized estimating equations and mixed effects approaches

Modeling individual heterogeneity in capture probabilities has been one of the most challenging tasks in capture–recapture studies. Heterogeneity in capture probabilities can be modeled as a function of individual covariates, but correlation structure among capture occasions should be taking into account. A proposed generalized estimating equations (GEE) and generalized linear mixed modeling (GLMM) approaches can be used to estimate capture probabilities and population size for capture–recapture closed population models. An example is used for an illustrative application and for comparison with currently used methodology. A simulation study is also conducted to show the performance of the estimation procedures. Our simulation results show that the proposed quasi‐likelihood based on GEE approach provides lower SE than partial likelihood based on either generalized linear models (GLM) or GLMM approaches for estimating population size in a closed capture–recapture experiment. Estimator performance is good if a large proportion of individuals are captured. For cases where only a small proportion of individuals are captured, the estimates become unstable, but the GEE approach outperforms the other methods.     

Formation of L-canavanine in in vitro cultures of Canavalia ensiformis (L.) DC.

In vitro tissue cultures of Canavalia ensiformis (L.) D.C. derived from hypocotyl have been obtained. They were found to accumulate L-canavanine depending on the medium where they were grown. Addition of polyethylenglycol (4%) to the culture medium led to a reduced accumulation of l-canavanine and an increase in the amino acids and the quaternary ammonium compounds contents.     

Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization

Background and Aims: The aims of this study were to set up proliferation conditionsfor hairy roots of Coffea arabica regenerated after transformationby Agrobacterium rhizogenes strain A4-RS, and to carry out themorphological and molecular characterization of hairy root clonesmaintained over the long term. Methods: Auxin supply, light conditions and sucrose concentration weremodified with the aim of establishing efficient root proliferationconditions. The morphological variability among 62 establishedhairy root clones was phenotyped by scanning the roots and analysingthe images using ‘whinRHIZO’ software procedures.PCR analysis of integration in transformed root cells of roland aux oncogenes from the T-DNA of the Ri plasmid was usedto study the molecular variability among clones. Key Results: Auxin supply was necessary to obtain and stimulate growth andbranching, and IBA applied at 0·5 µM was the mostefficient auxin. Significant differences were shown among the62 clones for total root length and for the percentage of fineroots. These variables were stable across subcultures and couldhence be used for efficient characterization of hairy root clones.The majority of hairy root clones (86 %) exhibited non-significantphenotype differences with non-transformed roots. Eight cloneswere significantly different from the non-transformed controlsin that they possessed a low proportion of fine roots. Two otherhairy root clones grew significantly faster than the other clones.The PCR analysis revealed a low variability in the integrationof rol and aux oncogenes in transformed root cells. The T_R-DNAwas never integrated as aux1 and aux2 genes were not found,although rolB and rolC genes from the T_L-DNA were always present. Conclusions: The discovery of low morphological variability among coffeehairy roots together with the identification of morphologicalvariables allowing easy identification of phenotypically alteredclones represent two important results. They make hairy rootsa possible, and efficient, tool for functional-genomic studiesof coffee root genes.     

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.     

Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots

The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The A. rhizogenes strain A4RS proved to be the most virulent. High transformation efficiencies (70%) were obtained using a 2-week co-cultivation period at a temperature of 15–18°C. Using a p35S-gusA-int construct inserted in the pBIN19 binary plasmid, we could estimate that 35% of transformed roots were GUS positive (co-transformed). Using the GUS assay as visual marker, 40% composite plants bearing a branched co-transformed rootstock could be obtained after only 12 weeks without selection with herbicides or antibiotics. Transgenic coffee roots obtained with A. rhizogenes did not exhibit the ‘hairy’ disturbed phenotype and were morphologically similar to normal roots. PCR analyses demonstrated that all co-transformed roots were positive for the expected rolB and gusA genes. Transformed and non-transformed root systems from both susceptible and resistant varieties were inoculated with Meloidogyne exigua nematode individuals. Inoculation of composite plants from the Caturra susceptible variety resulted in the normal development of nematode larvae. Numbers of extracted nematodes demonstrated that transformed roots retain the resistance/sensibility phenotype of varieties from which they are derived. These results suggest that composite plants constitute a powerful tool for studying nematode resistance genes.     

Looking for a needle in a haystack: inference about individual fitness components in a heterogeneous population

Studies of wild vertebrates have provided evidence of substantial differences in lifetime reproduction among individuals and the sequences of life history ‘states’ during life (breeding, nonbreeding, etc.). Such differences may reflect ‘fixed’ differences in fitness components among individuals determined before, or at the onset of reproductive life. Many retrospective life history studies have translated this idea by assuming a ‘latent’ unobserved heterogeneity resulting in a fixed hierarchy among individuals in fitness components. Alternatively, fixed differences among individuals are not necessarily needed to account for observed levels of individual heterogeneity in life histories. Individuals with identical fitness traits may stochastically experience different outcomes for breeding and survival through life that lead to a diversity of ‘state’ sequences with some individuals living longer and being more productive than others, by chance alone. The question is whether individuals differ in their underlying fitness components in ways that cannot be explained by observable ‘states’ such as age, previous breeding success, etc. Here, we compare statistical models that represent these opposing hypotheses, and mixtures of them, using data from kittiwakes. We constructed models that accounted for observed covariates, individual random effects (unobserved heterogeneity), first‐order Markovian transitions between observed states, or combinations of these features. We show that individual sequences of states are better accounted for by models incorporating unobserved heterogeneity than by models including first‐order Markov processes alone, or a combination of both. If we had not considered individual heterogeneity, models including Markovian transitions would have been the best performing ones. We also show that inference about age‐related changes in fitness components is sensitive to incorporation of underlying individual heterogeneity in models. Our approach provides insight into the sources of individual heterogeneity in life histories, and can be applied to other data sets to examine the ubiquity of our results across the tree of life.     

Lack of correlation between the amplitudes of TRP channel-mediated responses to weak and strong stimuli in intracellular Ca imaging experiments

It is often observed in intracellular Ca²⁺ imaging experiments that the amplitudes of the Ca²⁺ signals elicited by newly characterized TRP agonists do not correlate with the amplitudes of the responses evoked subsequently by a specific potent agonist. We investigated this rather controversial phenomenon by first testing whether it is inherent to the comparison of the effects of weak and strong stimuli. Using five well-characterized TRP channel agonists in commonly used heterologous expression systems we found that the correlation between the amplitudes of the Ca²⁺ signals triggered by two sequentially applied stimuli is only high when both stimuli are strong. Using mathematical simulations of intracellular Ca²⁺ dynamics we illustrate that the innate heterogeneity in expression and functional properties of Ca²⁺ extrusion (e.g. plasma membrane Ca²⁺ ATPase) and influx (TRP channels) pathways across a cellular population is a sufficient condition for low correlation between the amplitude of Ca²⁺ signals elicited by weak and strong stimuli. Taken together, our data demonstrate that this phenomenon is an expected outcome of intracellular Ca²⁺ imaging experiments that cannot be taken as evidence for lack of specificity of low-efficacy stimuli, or as an indicator of the need of other cellular components for channel stimulation.