The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.</jats:p

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity

Rolling friction of a viscous sphere on a hard plane

A first-principle continuum-mechanics expression for the rolling friction coefficient is obtained for the rolling motion of a viscoelastic sphere on a hard plane. It relates the friction coefficient to the viscous and elastic constants of the sphere material. The relation obtained refers to the case when the deformation of the sphere $\xi$ is small, the velocity of the sphere $V$ is much less than the speed of sound in the material and when the characteristic time $\xi/V$ is much larger than the dissipative relaxation times of the viscoelastic material. To our knowledge this is the first ``first-principle'' expression of the rolling friction coefficient which does not contain empirical parameters.Comment: 6 pages, 2 figure

Evolutionary trait‐based approaches for predicting future global impacts of plant pathogens in the genus Phytophthora

Plant pathogens are introduced to new geographical regions ever more frequently as global connectivity increases. Predicting the threat they pose to plant health can be difficult without in‐depth knowledge of behaviour, distribution and spread. Here, we evaluate the potential for using biological traits and phylogeny to predict global threats from emerging pathogens. We use a species‐level trait database and phylogeny for 179 Phytophthora species: oomycete pathogens impacting natural, agricultural, horticultural and forestry settings. We compile host and distribution reports for Phytophthora species across 178 countries and evaluate the power of traits, phylogeny and time since description (reflecting species‐level knowledge) to explain and predict their international transport, maximum latitude and host breadth using Bayesian phylogenetic generalised linear mixed models. In the best‐performing models, traits, phylogeny and time since description together explained up to 90%, 97% and 87% of variance in number of countries reached, latitudinal limits and host range, respectively. Traits and phylogeny together explained up to 26%, 41% and 34% of variance in the number of countries reached, maximum latitude and host plant families affected, respectively, but time since description had the strongest effect. Root‐attacking species were reported in more countries, and on more host plant families than foliar‐attacking species. Host generalist pathogens had thicker‐walled resting structures (stress‐tolerant oospores) and faster growth rates at their optima. Cold‐tolerant species are reported in more countries and at higher latitudes, though more accurate interspecific empirical data are needed to confirm this finding. Policy implications. We evaluate the potential of an evolutionary trait‐based framework to support horizon‐scanning approaches for identifying pathogens with greater potential for global‐scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under‐reported species, with similar traits and/or phylogenetic proximity to other high‐impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait‐based horizon‐scanning approaches would benefit from the development of international and cross‐sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny

Environment and Rural Affairs Monitoring & Modelling Programme - ERAMMP Report-78: Interim Report on the Development of Indicator 44 (Status of Biological Diversity in Wales)

i. We report interim progress on work to develop a new indicator of the status of biological diversity for Wales: indicator 44 for the Well-being of Future Generations (Wales) Act (2015). The focus of this work is on combining data into a single indicator of change in the distribution of section 7 species over time. ii. Ongoing work has sought to quantify the additional contribution that could be made by Welsh LERC records. In doing so new tools have been developed to interrogate the LERC data and to identify extra records (combinations of date, species and 1km grid square) over and above those in existing national surveillance scheme datasets for Wales. iii. The scale and complexity of this task has been such that we cannot currently report the size of the LERC contribution. This is also in part because criteria for selecting additional data are likely to need discussion and agreement with our project partners. iv. By the end of the project we plan to have produced new updated annual trends for section 7 species that include national scheme and LERC data where possible. v. In parallel, a new and updated section 7 species indicator for Wales has been produced based on updated national scheme datasets. This combines annual estimates of change in the proportion of occupied sites in 1x1km squares in Wales for 113 species. vi. In the long-term period (1970-2016), the index of distribution change for section 7 priority species in Wales had declined to 87% of its baseline value in 1970. This is considered a statistically significant decrease and the indicator is therefore assessed as decreasing. Over this long-term period, 16% of species showed a strong or weak increase and 34% showed a strong or weak decline. vii. Over the short-term period (2011-2016), the value of the indicator increased from 85 to 87 and was assessed as stable. Between 2011 and 2016, 35% of species showed a strong or weak increase and 19% showed a strong or weak decline. viii. New results for an experimental ‘all-species’ indicator are also presented. ix. Evidence for changes in abundance of section 7 species are reviewed and the merits of developing a new abundance-based indicator for Wales are highlighted as part of a further program of work. x. Finally, we review evidence and data supporting trends for section 7 marine species finding that information is lacking but, based on the outcomes of recent work for Scotland, we highlight additional sources of data that are worth exploring as a basis for trends modelling. Given the wide variety of potentially contributing schemes and ongoing activities we believe a separate expert workshop on marine biodiversity surveillance in Wales would be an efficient way forwards

Evolutionary trait-based approaches for predicting future global impacts of plant pathogens in the genus Phytophthora

1. Plant pathogens are introduced to new geographical regions ever more frequently as global connectivity increases. Predicting the threat they pose to plant health can be difficult without in‐depth knowledge of behaviour, distribution and spread. Here, we evaluate the potential for using biological traits and phylogeny to predict global threats from emerging pathogens. 2. We use a species‐level trait database and phylogeny for 179 Phytophthora species: oomycete pathogens impacting natural, agricultural, horticultural and forestry settings. We compile host and distribution reports for Phytophthora species across 178 countries and evaluate the power of traits, phylogeny and time since description (reflecting species‐level knowledge) to explain and predict their international transport, maximum latitude and host breadth using Bayesian phylogenetic generalised linear mixed models. 3. In the best‐performing models, traits, phylogeny and time since description together explained up to 90%, 97% and 87% of variance in number of countries reached, latitudinal limits and host range, respectively. Traits and phylogeny together explained up to 26%, 41% and 34% of variance in the number of countries reached, maximum latitude and host plant families affected, respectively, but time since description had the strongest effect. 4. Root‐attacking species were reported in more countries, and on more host plant families than foliar‐attacking species. Host generalist pathogens had thicker‐walled resting structures (stress‐tolerant oospores) and faster growth rates at their optima. Cold‐tolerant species are reported in more countries and at higher latitudes, though more accurate interspecific empirical data are needed to confirm this finding. 5. Policy implications. We evaluate the potential of an evolutionary trait‐based framework to support horizon‐scanning approaches for identifying pathogens with greater potential for global‐scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under‐reported species, with similar traits and/or phylogenetic proximity to other high‐impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait‐based horizon‐scanning approaches would benefit from the development of international and cross‐sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny

Is telomere length in peripheral blood lymphocytes correlated with cancer susceptibility or radiosensitivity?

Mean terminal restriction fragment (TRF) lengths in white blood cells (WBCs) have been previously found to be associated with breast cancer. To assess whether this marker could be used as a test for breast cancer susceptibility in women, TRF length was measured in 72 treated female breast cancer patients and 1696 unaffected female controls between the ages of 45 and 77 from the Twin Research Unit at St Thomas' Hospital, as well as 140 newly diagnosed breast cancer cases and 108 mammographically screened unaffected controls from Guy's Hospital. Mean TRF was also tested for correlation with chromosome radiosensitivity and apoptotic response in the Guy's Hospital patients. After adjusting for age, smoking and body mass index, there was no significant difference in TRF lengths between the treated breast cancer patients and unaffected controls (P=0.71). A positive correlation between age-adjusted apoptotic response and mean TRF in newly diagnosed untreated breast cancer patients (P=0.008) was identified but no significant difference in TRF lengths between breast cancer patients and unaffected controls was detected (P=0.53). This suggests that TRF lengths in WBC, is not a marker of breast cancer susceptibility and does not vary significantly between affected women before and after treatment