Assemblages and isotopic signals of modern ostracodes and host waters from Patagonia, Argentine

Ostracode species assemblages and stable oxygen and carbon isotope ratios of living and recent ostracodes, together with delta18O and delta13C_DIC values of host water samples, provide a first data set that characterizes a wide range of modern aquatic environments in the Laguna Cari-Laufquen (41°S, 68 - 69°W) and the Lago Cardiel area (48 - 49°S, 70 - 71°W) in Patagonia, Argentina. This data set will ultimately be used to interpret and calibrate data acquired from lake sediment cores with the goal of reconstructing past climate. Species assemblages and isotope values can be assigned to three groups; (1) springs, seeps and streams, (2) permanent ponds and lakes, and (3) ephemeral ponds and lakes. Springs, seeps and streams are characterized by Darwinula sp., Heterocypris incongruens, Eucypris fontana, Amphicypris nobilis and Ilyocypris ramirezi. Ostracode and water isotope values range between -13 and -5 per mil for oxygen, and between -15 and -3 per mil for carbon. They are the most negative of the entire sample set, reflecting ground water input with little or no evaporative enrichment. Limnocythere patagonica, Eucypris labyrinthica, Limnocythere sp. and Eucypris aff. fontana are typical species of permanent ponds and lakes. Isotope values indicate high degree of evaporation of lake waters relative to feeder springs and streams and range between -7 and +5 per mil for oxygen, and -5 and +4 per mil for carbon. Limnocythere rionegroensis is the dominant species in ephemeral ponds and lakes. These systems display the most enriched isotope values in both ostracodes and host waters, extending from -5 to +7 per mil for oxygen, and from -5 to +6 per mil for carbon. Living ostracodes show a positive offset from equilibrium values of up to 2 per mil for oxygen. Carbon-isotope values are up to 6? more negative than equilibrium values in highly productive pools. Comparison of ostracode and host water isotope signals permits assessment of the life span of the aquatic environments. Valves from dead ostracodes collected from ephemeral ponds and lakes show a wide scatter with each sample providing a snapshot of the seasonal history of the host water. The presence of the stream species Ilyocypris ramirezi and a wide range of ostracode isotope values suggest that ephemeral ponds and lakes are fed by streams during spring run-off and seasonally dry. A temporary character is also indicated by Heterocypris incongruens, a drought-resistant species that occupies most springs and seeps. In addition, Limnocythere rionegroensis has adjusted its reproduction strategies to its environment. Whereas only females were collected in fresh host waters, males were found in ephemeral ponds and lakes with higher solute content. Sexual reproduction seems to be the more successful reproduction strategy in high and variable salinities and seasonal droughts. The temporary character of the aquatic environments shows that the availability of meteoric water controls the life span of host waters and underlines the sensitivity of the area to changes in precipitation.

Characteristics of introduced Aphididae species, an endemic fly (Anatalanta aptera) and an introduced ground beetle (Merizodus soledadinus) on the Kerguelen Islands

The suite of environments and anthropogenic modifications of sub-Antarctic islands provide key opportunities to improve our understanding of the potential consequences of climate change and biological species invasions on terrestrial ecosystems. The profound impact of human introduced invasive species on indigenous biota, and the facilitation of establishment as a result of changing thermal conditions, has been well documented on the French sub-Antarctic Kerguelen Islands (South Indian Ocean). The present study provides an overview of the vulnerability of sub-Antarctic terrestrial communities with respect to two interacting factors, namely climate change and alien insects. We present datasets assimilated by our teams on the Kerguelen Islands since 1974, coupled with a review of the literature, to evaluate the mechanism and impact of biological invasions in this region. First, we consider recent climatic trends of the Antarctic region, and its potential influence on the establishment, distribution and abundance of alien insects, using as examples one fly and one beetle species. Second, we consider to what extent limited gene pools may restrict alien species' colonisations. Finally, we consider the vulnerability of native communities to aliens using the examples of one beetle, one fly, and five aphid species taking into consideration their additional impact as plant virus vectors. We conclude that the evidence assimilated from the sub-Antarctic islands can be applied to more complex temperate continental systems as well as further developing international guidelines to minimise the impact of alien species.

Chemical composition of phosphorites and host sediments

Paleotemperature curves were drawn from oxygen-isotope ratios in CaCO3 of planktonic foraminiferal tests and by the micropaleontological method using quantitative relationships of their species. Two series of curves yield similar results. These data confirm that isotope composition of oxygen reflects primarily temperature, and not isotope composition in ocean water. Temperature of the upper layer of ocean water increased from north to south both during the last two glaciations and in the interglacials. All three sediment cores collected from different latitudes show approximately the same amplitudes of fluctuation of mean annual temperature during times of their accumulation, as determined independently by different methods; these amplitudes are estimated as 5-7°C.

Impacts of ocean acidification on multiplication and caste organisation of parasitic trematodes in their gastropod host

Ocean acidification is predicted to impact the structure and function of all marine ecosystems in this century. As focus turns towards possible impacts on interactions among marine organisms, its effects on the biology and transmission potential of marine parasites must be evaluated. In the present study, we investigate two marine trematode species (Philophthalmus sp. and Parorchis sp., both in the family Philophthalmidae) infecting two marine gastropods. These trematodes are unusual in that their asexually multiplying stages within snails display a division of labour, with two distinct castes, a large-bodied morph producing infective stages and a smaller morph playing a defensive role against other competing parasites. Using a potentiometric ocean acidification simulation system, we test the impacts of acidified seawater (7.8 and 7.6 pH) on the production of free-living infective stages (cercariae), the size and survival of encysted resting stages (metacercariae), and the within-host division of labour measured as the ratio between numbers of the two morphs. In general, low pH conditions caused an increase in cercarial production and a reduction in metacercarial survival. The ratio of the two castes within snail hosts tended to shift towards more of the smaller defensive morphs under low pH. However, the observed effects of reduced pH were species specific and not always unimodal. These results suggest that ocean acidification can affect the biology of marine parasites and may also impact transmission success and parasite abundance of some trematodes, with possible consequences for marine communities and ecosystems.

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Effect of the Legume Plant Host on Rhizobial Soil Population Dynamics

Rhizobium leguminosarum bv viciae (Rlv) is a soil bacterium able to establish specific root-nodule symbioses with legumes of four different genera: Pisum, Vicia, Lens and Lathyrus. Rlv isolates from nodules of any of these legumes can nodulate any of them; however, it has been shown that plants select specific rhizobial genotypes from those present in the soil (1,2). We have previously shown this at the genomic level by following a population genomics approach. Pool genomic sequences from 100 isolates from each of four plant species: P. sativum, L. culinaris, V. faba and V. sativa, show different, specific profiles at the single nucleotide polymorphism (SNP) level for relevant genes. In this work, the extent of Rlv selection from a well-characterized soil population by different legume plant hosts: P. sativum, L. culinaris, V. faba and V. sativa, after a medium-term mesocosm study is described. Direct soil isolates from each of these mesocosm studies have been tested for specific rhizobial genes (glnII and fnrN) and symbiotic genes (nodC and nifH). Different populations were characterized further by Sanger sequencing of both the rpoB phylogenetic marker gene and the symbiotic genes nodC and nifH. The distribution and size of the rhizobial population for each legume host showed changes during the medium-term mesocosm study. Particularly, a non-symbiotic group of rhizobia was enriched by all four hosts, in contrast to the symbiotic rhizobia profile, which was specific for each legume plant host.

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Macroevolution of insect–plant associations: The relevance of host biogeography to host affiliation

Identifying the factors that have promoted host shifts by phytophagous insects at a macroevolutionary scale is critical to understanding the associations between plants and insects. We used molecular phylogenies of the beetle genus Blepharida and its host genus Bursera to test whether these insects have been using hosts with widely overlapping ranges over evolutionary time. We also quantified the importance of host range coincidence relative to host chemistry and host phylogenetic relatedness. Overall, the evolution of host use of these insects has not been among hosts that are geographically similar. Host chemistry is the factor that best explains their macroevolutionary patterns of host use. Interestingly, one exceptional polyphagous species has shifted among geographically close chemically dissimilar plants.

Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification.

The common nodABC genes of Rhizobium meliloti are host-range determinants

Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti. In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R. meliloti, in contrast to NodA of R. tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.

A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum

Converging TGF-β and insulin-like neuroendocrine signaling pathways regulate whether Caenorhabditis elegans develops reproductively or arrests at the dauer larval stage. We examined whether neurotransmitters act in the dauer entry or recovery pathways. Muscarinic agonists promote recovery from dauer arrest induced by pheromone as well as by mutations in the TGF-β pathway. Dauer recovery in these animals is inhibited by the muscarinic antagonist atropine. Muscarinic agonists do not induce dauer recovery of either daf-2 or age-1 mutant animals, which have defects in the insulin-like signaling pathway. These data suggest that a metabotropic acetylcholine signaling pathway activates an insulin-like signal during C. elegans dauer recovery. Analogous and perhaps homologous cholinergic regulation of mammalian insulin release by the autonomic nervous system has been noted. In the parasitic nematode Ancylostoma caninum, the dauer larval stage is the infective stage, and recovery to the reproductive stage normally is induced by host factors. Muscarinic agonists also induce and atropine potently inhibits in vitro recovery of A. caninum dauer arrest. We suggest that host or parasite insulin-like signals may regulate recovery of A. caninum and could be potential targets for antihelminthic drugs.

Species-barrier-independent prion replication in apparently resistant species

Transmission of prions between mammalian species is thought to be limited by a “species barrier,” which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.