An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors

Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.This work was supported by grants from the DFG to I.I.-T. and G.R.L. within the collaborative research centre (SFB 665) and from the Alexander von Humboldt Foundation to E.St.J.S

Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the {alpha}5, {alpha}3, and {beta}4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in {alpha}3{beta}4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that {beta}4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of {beta}4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with {alpha}3. We found that {beta}4A90I and {beta}4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant {beta}4D447Y, significantly increased nicotine-evoked current amplitudes, while {beta}4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express {beta}4 or {beta}4 variants in the MHb. Immunoprecipitation studies confirmed that {beta}4 lentiviral-mediated expression leads to specific upregulation of {alpha}3{beta}4 but not {beta}2 nAChRs in the Mhb. Mice injected with the {beta}4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the {beta}4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the {beta}4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular {beta}4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice

Solution conformation of the major adduct between the carcinogen (+)-anti-benzo[ɑ]pyrene diol epoxide and DNA

We have synthesized, separated, and purified ≈10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-anti-benzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N^2-dG adduct positioned opposite dC and flanked by G.C pairs in the d[C1-C2-A3-T4-C5-(BP)G6-C7-T8-A9-C10-C11].d[12- G13-T14-A15-G16-C17-G18-A19-T20-G 21-G22] duplex +ADdesignated (BP)G.C 11-mer+BD. We have determined the solution structure centered about the BP covalent adduct site in the (BP)G.C 11-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugar-phosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8].d[A15-G16-C17-G18-A19] segment centered about the adduct site with Watson-Crick alignment for both the (BP)G6.C17 pair and flanking G.C pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of ≈45° with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other steroisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomer-dependent spectrum of mutational and carcinogenic activities

Association of size at birth with adolescent hormone levels, body size and age at menarche: relevance for breast cancer risk

Birth size has been positively associated with age at menarche and height in adolescence and adulthood, but the relevant biological mechanisms remain unclear. Among 262 Norwegian term-born singleton girls, birth size measures (weight, length, ponderal index, head circumference and subscapular skin-fold thickness) were analysed in relation to adolescent hormone levels (oestradiol, prolactin, dehydroepiandrosterone sulphate, androstenedione and free testosterone index), age at menarche and adolescent (ages 12.7–15.5 years) and body size (height, weight, body mass index and waist-to-hip ratio) using survival analysis and general linear modelling. The results were adjusted for gestational age at birth, age and menarcheal status at measurement in adolescence and maternal age at menarche. Birth weight, birth length and head circumference were positively associated with adolescent weight and height, and small birth size was associated with earlier age at menarche. Subscapular skin-fold thickness at birth was not associated with adolescent body size, but low fold-thickness was associated with earlier age at menarche. Measures of birth size were inversely related to circulating levels of dehydroepiandrosterone sulphate in adolescence, but there was no clear association with other hormones. These results suggest that physical and sexual development in puberty and adolescence is influenced by prenatal factors, and in combination, these factors may influence health and disease later in life

Minhocas em sistemas de uso do solo: estudo de caso em Assistência, SP.

Resumo. 5° ELAETAO

Indicadores de qualidade do solo em sistemas de uso do solo: estudo de caso em Assistência, SP.

Resumo. 5° ELAETAO

Evolutionary winners are ecological losers among oceanic island plants

Aim Adaptive radiation, in which successful lineages proliferate by exploiting untapped niche space, provides a popular but potentially misleading characterization of evolution on oceanic islands. Here we analyse the respective roles of members of in situ diversified vs. non-diversified lineages in shaping the main ecosystems of an archipelago to explore the relationship between evolutionary and ecological ‘success’. Location Canary Islands. Taxon Vascular plants. Methods We quantified the abundance/rarity of the native flora according to the geographical range (number of islands where present and geographical extent of the range), habitat breadth (climatic niche) and local abundance (cover) using species distribution data based on 500 × 500 m grid cells and 2000 vegetation inventories located all over the archipelago. Results Species of diversified lineages have significantly smaller geographic ranges, narrower climatic niches and lower local abundances than those of non-diversified lineages. Species rarity increased with the degree of diversification. The diversified Canarian flora is mainly comprised by shrubs. At both archipelagic and island level, the four core ecosystems (Euphorbia scrub, thermophilous woodlands, laurel forest and pine forest) were dominated by non-diversified lineages species, with diversified lineages species providing <25% cover. Species of diversified lineages, although constituting 54% of the archipelagic native flora, were only abundant in two rare ecosystems: high mountain scrub and rock communities. Main conclusions Radiated species, endemic products of in situ speciation, are mostly rare in all three rarity axes and typically do not play an important role in structuring plant communities on the Canaries. The vegetation of the major ecosystem types is dominated by plants representing non-diversified lineages (species that derive from immigration and accumulation), while species of evolutionarily successful lineages are abundant only in marginal habitats and could, therefore, be considered ecological losers. Within this particular oceanic archipelago, and we posit within at least some others, evolutionary success in plants is accomplished predominantly at the margins.publishedVersio

Training Programmes Can Change Behaviour and Encourage the Cultivation of Over-Harvested Plant Species

Cultivation of wild-harvested plant species has been proposed as a way of reducing over-exploitation of wild populations but lack of technical knowledge is thought to be a barrier preventing people from cultivating a new species. Training programmes are therefore used to increase technical knowledge to encourage people to adopt cultivation. We assessed the impact of a training programme aiming to encourage cultivation of xaté (Chamaedorea ernesti-augusti), an over-harvested palm from Central America. Five years after the training programme ended, we surveyed untrained and trained individuals focusing on four potential predictors of behaviour: technical knowledge, attitudes (what individuals think about a behaviour), subjective norms (what individuals perceive others to think of a behaviour) and perceived behavioural control (self assessment of whether individuals can enact the behaviour successfully). Whilst accounting for socioeconomic variables, we investigate the influence of training upon these behavioural predictors and examine the factors that determine whether people adopt cultivation of a novel species. Those who had been trained had higher levels of technical knowledge about xaté cultivation and higher belief in their ability to cultivate it while training was not associated with differences in attitudes or subjective norms. Technical knowledge and perceived behavioural control (along with socio-economic variables such as forest ownership and age) were predictors of whether individuals cultivate xaté. We suggest that training programmes can have a long lasting effect on individuals and can change behaviour. However, in many situations other barriers to cultivation, such as access to seeds or appropriate markets, will need to be addressed

Interleukin 15 Levels in Serum May Predict a Severe Disease Course in Patients with Early Arthritis

Background: Interleukin-15 (IL-15) is thought to be involved in the physiopathological mechanisms of RA and it can be detected in the serum and the synovial fluid of inflamed joints in patients with RA but not in patients with osteoarthritis or other inflammatory joint diseases. Therefore, the objective of this work is to analyse whether serum IL-15 (sIL-15) levels serve as a biomarker of disease severity in patients with early arthritis (EA). Methodology and Results: Data from 190 patients in an EA register were analysed (77.2% female; median age 53 years; 6-month median disease duration at entry). Clinical and treatment information was recorded systematically, especially the prescription of disease modifying anti-rheumatic drugs. Two multivariate longitudinal analyses were performed with different dependent variables: 1) DAS28 and 2) a variable reflecting intensive treatment. Both included sIL-15 as predictive variable and other variables associated with disease severity, including rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA). Of the 171 patients (638 visits analysed) completing the follow-up, 71% suffered rheumatoid arthritis and 29% were considered as undifferentiated arthritis. Elevated sIL-15 was detected in 29% of this population and this biomarker did not overlap extensively with RF or ACPA. High sIL-15 levels (β Coefficient [95% confidence interval]: 0.12 [0.06-0.18]; p&0.001) or ACPA (0.34 [0.01-0.67]; p = 0.044) were significantly and independently associated with a higher DAS28 during follow-up, after adjusting for confounding variables such as gender, age and treatment. In addition, those patients with elevated sIL-15 had a significantly higher risk of receiving intensive treatment (RR 1.78, 95% confidence interval 1.18-2.7; p = 0.007). Conclusions: Patients with EA displaying high baseline sIL-15 suffered a more severe disease and received more intensive treatment. Thus, sIL-15 may be a biomarker for patients that are candidates for early and more intensive treatmentThe work of Belen Díaz-Sánchez was supported by the RETICS Programme (Programa de Redes Temáticas de Investigación Colaborativa [Colaborative Research Thematic Network Programme]; RD08/0075 - RIER [Red de Inflamación y Enfermedades Reumáticas; Inflammation and Rheumatic Diseases Network]) from the Instituto de Salud Carlos III, Spain (URL: www.isciii.es) within the VI National Plan for I+D+I 2008–2011 (FEDER). The work of Isidoro González-Álvaro was in part supported by a grant for the Intensification of the Research Tasks in the National Health Care System from Instituto de Salud Carlos III, Spain. The consumables for measurements and data analysis were supported by a Fondo de Investigación Sanitaria grant (08/0754) from the Instituto de Salud Carlos II