Phylogenetic analyses of C4 photosynthesis evolution in grasses: genetics of convergence and convergence of genes

Summary : During the evolutionary diversification of organisms, similar ecological constraints led to the recurrent appearances of the same traits (phenotypes) in distant lineages, a phenomenon called convergence. In most cases, the genetic origins of the convergent traits remain unknown, but recent studies traced the convergent phenotypes to recurrent alterations of the same gene or, in a few cases, to identical genetic changes. However, these cases remain anecdotal and there is a need for a study system that evolved several times independently and whose genetic determinism is well resolved and straightforward, such as C4 photosynthesis. This adaptation to warm environments, possibly driven by past atmospheric CO2 decreases, consists in a CO2-concentrating pump, created by numerous morphological and biochemical novelties. All genes encoding C4 enzymes already existed in C3 ancestors, and are supposed to have been recruited through gene duplication followed by neo-functionalization, to acquire the cell specific expression pattern and altered kinetic properties that characterize Ca-specific enzymes. These predictions have so far been tested only in species-poor and ecologically marginal C4 dicots. The monocots, and especially the grass family (Poaceae), the most important C4 family in terms of species number, ecological dominance and economical importance, have been largely under-considered as suitable study systems. This thesis aimed at understanding the evolution of the C4 trait in grasses at a molecular level and to use the genetics of C4 photosynthesis to infer the evolutionary history of the C4 phenotype and its driving selective pressures. A molecular phylogeny of grasses and affiliated monocots identified 17 to 18 independent acquisitions of the C4 pathway in the grass family. A relaxed molecular clock was used to date these events and the first C4 evolution was estimated in the Chloridoideae subfamily, between 32-25 million years ago, at a period when atmospheric CO2 abruptly declined. Likelihood models showed that after the COZ decline the probability of evolving the C4 pathway strongly increased, confirming low CO2 as a likely driver of C4 photosynthesis evolution. In order to depict the genetic changes linked to the numerous C4 origins, genes encoding phopshoenolpyruvate carboxylase (PEPC), the key-enzyme responsible for the initial fixation of atmospheric CO2 in the C4 pathway, were isolated from a large sample of C3 and C4 grasses. Phylogenetic analyses were used to reconstruct the evolutionary history of the PEPC multigene family and showed that the evolution of C4-specific PEPC had been driven by positive selection on 21 codons simultaneously in up to eight C4 lineages. These selective pressures led to numerous convergent genetic changes in many different C4 clades, highlighting the repeatability of some evolutionary processes, even at the molecular level. PEPC C4-adaptive changes were traced and used to show multiple appearances of the C, pathway in clades where species tree inferences were unable to differentiate multiple C4 appearances and a single appearance followed by C4 to C3 reversion. Further investigations of genes involved in some of the C4 subtypes only (genes encoding decarboxylating enzymes NADP-malic enzyme and phosphoenolpyruvate carboxykinase) showed that these C4-enzymes also evolved through strong positive selection and underwent parallel genetic changes during the different Ca origins. The adaptive changes on these subtype-specific C4 genes were used to retrace the history of the C4-subtypes phenotypes, which revealed that the evolution of C4-PEPC and C4-decarboxylating enzymes was in several cases disconnected, emphasizing the multiplicity of the C4 trait and the gradual acquisition of the features that create the CO2-pump. Finally, phylogenetic analyses of a gene encoding the Rubisco (the enzyme responsible for the fixation of CO2 into organic compounds in all photosynthetic organisms) showed that C4 evolution switched the selective pressures on this gene. Five codons were recurrently mutated to adapt the enzyme kinetics to the high CO2 concentrations of C4 photosynthetic cells. This knowledge could be used to introgress C4-like Rubisco in C3 crops, which could lead to an increased yield under predicted future high CO2 atmosphere. Globally, the phylogenetic framework adopted during this thesis demonstrated the widespread occurrence of genetic convergence on C4-related enzymes. The genetic traces of C4 photosynthesis evolution allowed reconstructing events that happened during the last 30 million years and proved the usefulness of studying genes directly responsible for phenotype variations when inferring evolutionary history of a given trait. Résumé Durant la diversification évolutive des organismes, des pressions écologiques similaires ont amené à l'apparition récurrente de certains traits (phénotypes) dans des lignées distantes, un phénomène appelé évolution convergente. Dans la plupart des cas, l'origine génétique des traits convergents reste inconnue mais des études récentes ont montré qu'ils étaient dus dans certains cas à des changements répétés du même gène ou, dans de rares cas, à des changements génétiques identiques. Malgré tout, ces cas restent anecdotiques et il y a un réel besoin d'un système d'étude qui ait évolué indépendamment de nombreuses fois et dont le déterminisme génétique soit clairement identifié. La photosynthèse dite en Ça répond à ces critères. Cette adaptation aux environnements chauds, dont l'évolution a pu être encouragé par des baisses passées de la concentration atmosphérique en CO2, est constituée de nombreuses nouveautés morphologiques et biochimiques qui créent une pompe à CO2. La totalité des gènes codant les enzymes Ç4 étaient déjà présents dans les ancêtres C3. Leur recrutement pour la photosynthèse Ç4 est supposé s'être fait par le biais de duplications géniques suivies par une néo-fonctionnalisation pour leur conférer l'expression cellule-spécifique et les propriétés cinétiques qui caractérisent les enzymes C4. Ces prédictions n'ont jusqu'à présent été testées que dans des familles C4 contenant peu d'espèces et ayant un rôle écologique marginal. Les graminées (Poaceae), qui sont la famille C4 la plus importante, tant en termes de nombre d'espèces que de dominance écologique et d'importance économique, ont toujours été considérés comme un système d'étude peu adapté et ont fait le sujet de peu d'investigations évolutives. Le but de cette thèse était de comprendre l'évolution de la photosynthèse en C4 chez les graminées au niveau génétique et d'utiliser les gènes pour inférer l'évolution du phénotype C4 ainsi que les pressions de sélection responsables de son évolution. Une phylogénie moléculaire de la famille des graminées et des monocotylédones apparentés a identifié 17 à 18 acquisitions indépendantes de la photosynthèse chez les graminées. Grâce à une méthode d'horloge moléculaire relâchée, ces évènements ont été datés et la première apparition C4 a été estimée dans la sous-famille des Chloridoideae, il y a 32 à 25 millions d'années, à une période où les concentrations atmosphériques de CO2 ont décliné abruptement. Des modèles de maximum de vraisemblance ont montré qu'à la suite du déclin de CO2, la probabilité d'évoluer la photosynthèse C4 a fortement augmenté, confirmant ainsi qu'une faible concentration de CO2 est une cause potentielle de l'évolution de la photosynthèse C4. Afin d'identifier les mécanismes génétiques responsables des évolutions répétées de la photosynthèse C4, un segment des gènes codant pour la phosphoénolpyruvate carboxylase (PEPC), l'enzyme responsable de la fixation initiale du CO2 atmosphérique chez les plantes C4, ont été séquencés dans une centaine de graminées C3 et C4. Des analyses phylogénétiques ont permis de reconstituer l'histoire évolutive de la famille multigénique des PEPC et ont montré que l'évolution de PEPC spécifiques à la photosynthèse Ça a été causée par de la sélection positive agissant sur 21 codons, et ce simultanément dans huit lignées C4 différentes. Cette sélection positive a conduit à un grand nombre de changements génétiques convergents dans de nombreux clades différents, ce qui illustre la répétabilité de certains phénomènes évolutifs, et ce même au niveau génétique. Les changements sur la PEPC liés au C4 ont été utilisés pour confirmer des évolutions indépendantes du phénotype C4 dans des clades où l'arbre des espèces était incapable de différencier des apparitions indépendantes d'une seule apparition suivie par une réversion de C4 en C3. En considérant des gènes codant des protéines impliquées uniquement dans certains sous-types C4 (deux décarboxylases, l'enzyme malique à NADP et la phosphoénolpyruvate carboxykinase), des études ultérieures ont montré que ces enzymes C4 avaient elles-aussi évolué sous forte sélection positive et subi des changements génétiques parallèles lors des différentes origines de la photosynthèse C4. Les changements adaptatifs sur ces gènes liés seulement à certains sous-types C4 ont été utilisés pour retracer l'histoire des phénotypes de sous-types C4, ce qui a révélé que les caractères formant le trait C4 ont, dans certains cas, évolué de manière déconnectée. Ceci souligne la multiplicité du trait C4 et l'acquisition graduelle de composants participant à la pompe à CO2 qu'est la photosynthèse C4. Finalement, des analyses phylogénétiques des gènes codant pour la Rubisco (l'enzyme responsable de la fixation du CO2 en carbones organiques dans tous les organismes photosynthétiques) ont montré que l'évolution de la photosynthèse Ça a changé les pressions de sélection sur ce gène. Cinq codons ont été mutés de façon répétée afin d'adapter les propriétés cinétiques de la Rubisco aux fortes concentrations de CO2 présentes dans les cellules photosynthétiques des plantes C4. Globalement, l'approche phylogénétique adoptée durant cette thèse de doctorat a permis de démontré des phénomène fréquents de convergence génétique sur les enzymes liées à la photosynthèse C4. Les traces génétiques de l'évolution de la photosynthèse C4 ont permis de reconstituer des évènements qui se sont produits durant les derniers 30 millions d'années et ont prouvé l'utilité d'étudier des gènes directement responsables des variations phénotypiques pour inférer l'histoire évolutive d'un trait donné.

Previous microvascular decompression decreases the chances of pain free in patients treated with Gamma Knife radiosurgery for TIC

Object: The authors sought to establish whether the safety-efficacy of Gamma Knife radiosurgery (GKRS) as a second treatment for intractable trigeminal neuralgia (ITN) are influenced by prior microvascular decompression (MVD) which remains, for some of the authors, the reference technique. Methods: Between July 1992 and November 2010, 737 patients have been operated with GKRS for ITN and prospectively evaluated in Timone University Hospital in Marseille, France. Among these, 54 patients had a previous MVD history. Radiosurgery using a Gamma Knife (model B or C or Perfexion) was performed relying on both MR and CT targeting. A single 4 mm isocenter was positioned in the cisternal portion of the trigeminal nerve at a median distance of 7.6 mm (range 3.9- 11.9) anteriorly to the emergence of the nerve (retrogasserian target). A median maximum dose of 85 Gy (range 70-90) was delivered. Are further analyzed only 45 patients with previous MVD and a follow-up longer than one year (the patients with megadolichobasilar artery compression and multiple sclerosis were excluded). Results: The median age in this series was 56.75 years (range 28.09-82.39). The median follow-up period was 39.48 months (range 14.10-144.65). All the patients had a past history of surgery, with at least one previous failed MVD, but also a radiofrequency lesion (RFL) in 16 (35.6%) patients, balloon microcompression in 7 (15.6%) patients and glycerol rhizotomy in 1 case (2.2%). Thirty-five patients (77.8%) were initially pain free in a median time of 14 days (range 0, 180). Patients from this group had less probability of being pain free compared to our global population of essential trigeminal neuralgia without previous MVD history (p=0.010, hazard ratio of 0.64). Their probability of remaining pain free at 3, 5, 7 and 10 years was 66.5%, 59.1%, 59.1% and 44.3%, respectively. Twelve patients (34.3%) initially pain free experienced a recurrence with a median delay of 31.21 months (range 3.40-89.93). The hypoesthesia actuarial rate at 1 year was 9.1% and remained stable till 12 years with a median delay of onset of 8 months (range 8-8). Conclusions: Retrogasserian GKRS proofed to be safe and effective on the long-term basis even after failed previous MVD. Even if the initial result of pain free was of only 77.8%, the toxicity was low with only 9.1% hypoesthesia. No patient reported a bothersome hypoesthesia. The probability of maintaining pain relief in long-term was of 44.3% at 10 years.

The fixation of locally beneficial alleles in a metapopulation.

Extinction, recolonization, and local adaptation are common in natural spatially structured populations. Understanding their effect upon genetic variation is important for systems such as genetically modified organism management or avoidance of drug resistance. Theoretical studies on the effect of extinction and recolonization upon genetic variance started appearing in the 1970s, but the role of local adaptation still has no good theoretical basis. Here we develop a model of a haploid species in a metapopulation in which a locally adapted beneficial allele is introduced. We study the effect of different spatial patterns of local adaptation, and different metapopulation dynamics, upon the fixation probability of the beneficial allele. Controlling for the average selection pressure, we find that a small area of positive selection can significantly increase the global probability of fixation. However, local adaptation becomes less important as extinction rate increases. Deme extinction and recolonization have a spatial smoothing effect that effectively reduces spatial variation in fitness.

Fitness and the level of homozygosity in a social insect.

To date very few studies have addressed the effects of inbreeding in social Hymenoptera, perhaps because the costs of inbreeding are generally considered marginal owing to male haploidy whereby recessive deleterious alleles are strongly exposed to selection in males. Here, we present one of the first studies on the effects of queen and worker homozygosity on colony performance. In a wild population of the ant Formica exsecta, the relative investment of single-queen colonies in sexual production decreased with increased worker homozygosity. This may either stem from increased homozygosity decreasing the likelihood of diploid brood to develop into queens or a lower efficiency of more homozygous workers at feeding larvae and thus a lower proportion of the female brood developing into queens. There was also a significant negative association between colony age and the level of queen but not worker homozygosity. This association may stem from inbreeding affecting queen lifespan and/or their fecundity, and thus colony survival. However, there was no association between queen homozygosity and colony size, suggesting that inbreeding affects colony survival as a result of inbred queens having a shorter lifespan rather than a lower fecundity. Finally, there was no significant association between either worker or queen homozygosity and the probability of successful colony founding, colony size and colony productivity, the three other traits studied. Overall, these results indicate that inbreeding depression may have important effects on colony fitness by affecting both the parental (queen) and offspring (worker)generations cohabiting within an ant colony.

Quantitative monitoring of tamoxifen in human plasma extended to 40 metabolites using liquid-chromatography high-resolution mass spectrometry: new investigation capabilities for clinical pharmacology.

Liquid-chromatography (LC) high-resolution (HR) mass spectrometry (MS) analysis can record HR full scans, a technique of detection that shows comparable selectivity and sensitivity to ion transitions (SRM) performed with triple-quadrupole (TQ)-MS but that allows de facto determination of "all" ions including drug metabolites. This could be of potential utility in in vivo drug metabolism and pharmacovigilance studies in order to have a more comprehensive insight in drug biotransformation profile differences in patients. This simultaneous quantitative and qualitative (Quan/Qual) approach has been tested with 20 patients chronically treated with tamoxifen (TAM). The absolute quantification of TAM and three metabolites in plasma was realized using HR- and TQ-MS and compared. The same LC-HR-MS analysis allowed the identification and relative quantification of 37 additional TAM metabolites. A number of new metabolites were detected in patients' plasma including metabolites identified as didemethyl-trihydroxy-TAM-glucoside and didemethyl-tetrahydroxy-TAM-glucoside conjugates corresponding to TAM with six and seven biotransformation steps, respectively. Multivariate analysis allowed relevant patterns of metabolites and ratios to be associated with TAM administration and CYP2D6 genotype. Two hydroxylated metabolites, α-OH-TAM and 4'-OH-TAM, were newly identified as putative CYP2D6 substrates. The relative quantification was precise (<20 %), and the semiquantitative estimation suggests that metabolite levels are non-negligible. Metabolites could play an important role in drug toxicity, but their impact on drug-related side effects has been partially neglected due to the tremendous effort needed with previous MS technologies. Using present HR-MS, this situation should evolve with the straightforward determination of drug metabolites, enlarging the possibilities in studying inter- and intra-patients drug metabolism variability and related effects.

Multiscale determinants of tree frog (Hyla arborea L.) calling ponds in western Switzerland

A tree frog (Hyla arborea L., 1758) metapopulation in western Switzerland was studied during spring 2001. All potential calling ponds in an area of 350 km(2) were searched for tree frog calling males. Twenty-nine out of 111 ponds sheltered between 1 and 250 callers. Most ponds were occupied by less than 12 males. Pond parameters were measured at three different levels using field analysis and a Geographical Information System (GIS). The first level was water chemistry and pond-associated measures. The second level was the surrounding land use in a 30 m buffer around the pond. The third level consisted of landscape indices on a broader scale (up to 2 km). Logistic regression was applied to identify parameters that can predict the presence of calling males in a pond. Response variable was the presence or absence of callers. Four significant parameters allowed us to explain about 40% of the total deviance of the observed occupational pattern. Urbanization around the pond had a highly negative impact on the probability of presence of calling males. Hours of direct sunlight on the pond was positively correlated with callers. Higher water conductivity was associated with a lesser probability of species presence. Finally, the further the closest two-lane road, the higher the probability of callers presence. Our results show that presence or absence of callers is influenced by factors acting at various geographical scales.

Use of long-term suppressive acyclovir after hematopoietic stem-cell transplantation: impact on herpes simplex virus (HSV) disease and drug-resistant HSV disease.

The effect that long-term use of suppressive acyclovir (ACV) has on both overall herpes simplex virus (HSV) disease and ACV-resistant HSV disease was examined in 3 consecutive cohorts of hematopoietic stem-cell transplant (HCT) recipients (n=2049); cohort 1 received ACV for 30 days after HCT, cohort 2 received it for 1 year after HCT, and cohort 3 received it for an extended period (i.e., >1 year) if the patient's immunosuppression continued after 1 year. The 2-year probability of HSV disease was 31.6% (95% confidence interval [CI], 28.0%-35%) in cohort 1, 3.9% (95% CI, 2.7%-5.2%) in cohort 2, and 0% in cohort 3 (P<.001). ACV-resistant HSV disease developed in 10 patients in cohort 1 (2-year probability, 1.3% [95% CI, 0.8%-2.7%]), in 2 patients in cohort 2 (2-year probability, 0.2% [95% CI, 0%-0.8%]; P=.006), and in 0 patients in cohort 3 (cohort 2 vs. cohort 3, P=.3). Long-term use of suppressive prophylactic ACV appears to prevent the emergence of drug-resistant HSV disease in HCT.

Understanding informal payments in health care: motivation of health workers in Tanzania

BACKGROUND: There is growing evidence that informal payments for health care are fairly common in many low- and middle-income countries. Informal payments are reported to have a negative consequence on equity and quality of care; it has been suggested, however, that they may contribute to health worker motivation and retention. Given the significance of motivation and retention issues in human resources for health, a better understanding of the relationships between the two phenomena is needed. This study attempts to assess whether and in what ways informal payments occur in Kibaha, Tanzania. Moreover, it aims to assess how informal earnings might help boost health worker motivation and retention. METHODS: Nine focus groups were conducted in three health facilities of different levels in the health system. In total, 64 health workers participated in the focus group discussions (81% female, 19% male) and where possible, focus groups were divided by cadre. All data were processed and analysed by means of the NVivo software package. RESULTS: The use of informal payments in the study area was confirmed by this study. Furthermore, a negative relationship between informal payments and job satisfaction and better motivation is suggested. Participants mentioned that they felt enslaved by patients as a result of being bribed and this resulted in loss of self-esteem. Furthermore, fear of detection was a main demotivating factor. These factors seem to counterbalance the positive effect of financial incentives. Moreover, informal payments were not found to be related to retention of health workers in the public health system. Other factors such as job security seemed to be more relevant for retention. CONCLUSION: This study suggests that the practice of informal payments contributes to the general demotivation of health workers and negatively affects access to health care services and quality of the health system. Policy action is needed that not only provides better financial incentives for individuals but also tackles an environment in which corruption is endemic.

Subconjunctival Injection of XG-102, a JNK Inhibitor Peptide, in Patients with Intraocular Inflammation: A Safety and Tolerability Study.

Abstract Purpose: We aimed to investigate the safety, tolerability, and systemic diffusion of a single escalating dose of XG-102 (a 31-D-amino-acid peptide inhibiting JNK pathway activation), administered subconjunctivally in the treatment of post-surgery or post-trauma intraocular inflammation. Methods: This is a dose-escalating, tolerance Phase Ib study. Twenty patients with post-surgery or post-traumatic intraocular inflammation were assigned to 1 of the 4 dose escalating (45, 90, 450, or 900 μg XG-102) groups of 5 patients each. Patients were evaluated at 24, 48 h, 8, and 28 days following the administration of XG-102, including laboratory tests, standard eye examinations, vital signs, and occurrence of adverse events. A single plasma quantification of XG-102 was performed 30 min after administration, according to previous pharmacokinetics studies performed on volunteers. Results: A total of 17 non-serious adverse events, considered unrelated to the study treatment, were reported for 10 patients. The adverse event incidence was not related to the drug dose. All patients experienced a decrease in intraocular inflammation as of 24 h post-administration and this decrease was sustained up to 28 days thereafter. No patient required local injection or systemic administration of corticoids following the administration of XG-102. XG-102 was undetectable in the first 3 dose groups. In the fourth-dose group (900 μg) the XG-102 plasma levels were above the limit of detection for 3 patients and above the limit of quantification for 1 patient. Conclusions: In this first clinical trial using XG-102, administered as a single subconjunctival injection as adjunct therapy, in patients with recent post-surgery or post-trauma intraocular inflammation is safe and well tolerated. Further studies are required to evaluate its efficacy.

Treatment of penile carcinoma: to cut or not to cut?

PURPOSE: The aim of this study was to assess the outcome in patients with penile cancer. METHODS AND MATERIALS: A total of 60 patients with penile carcinoma were included. Of the patients, 45 (n = 27) underwent surgery, and 51 underwent definitive (n = 29) or postoperative (n = 22) radiotherapy (RT). Median follow-up was 62 months. RESULTS: Median time to locoregional relapse was 14 months. Local failure was observed in 3 of 23 patients (13%) treated with surgery with or without postoperative RT vs. in 19 of 33 patients (56%) given organ-sparing treatment (p = 0.0008). Of 22 local failures, 16 (73%) were salvaged with surgery. Of the 33 patients treated with definitive RT (n = 29) and the 4 patients refusing RT after excisional biopsy, local control was obtained with organ preservation in 13 (39%). In the remaining 20, 4 patients with local failure underwent salvage conservatively, resulting in an ultimate penis preservation rate of 17 of 33 (52%) patients treated with definitive RT. The 5-year and 10-year probability of surviving with an intact penis was 43% and 26%, respectively. There was no survival difference between the patients treated with definitive RT and primary surgery (56% vs. 53%; p = 0.16). In multivariate analysis, independent factors influencing survival were N-classification and pathologic grade. Surgery was the only independent predictor for better local control. CONCLUSION: Based on our study findings, in patients with penile cancer, local control is superior with surgery. However, there is no difference in survival between patients treated with surgery and those treated with definitive RT, with 52% organ preservation.

Performance of panel-based criteria to evaluate the appropriateness of colonoscopy: a prospective study.

BACKGROUND: Prospective data describing the appropriateness of use of colonoscopy based on detailed panel-based clinical criteria are not available. METHODS: In a cohort of 553 consecutive patients referred for colonoscopy to two university-based Swiss outpatient clinics, the percentage of patients who underwent colonoscopy for appropriate, equivocal, and inappropriate indications and the relationship between appropriateness of use and the presence of relevant endoscopic lesions was prospectively assessed. This assessment was based on criteria of the American Society for Gastrointestinal Endoscopy and explicit American and Swiss criteria developed in 1994 by a formal panel process using the RAND/UCLA appropriateness method. RESULTS: The procedures were rated appropriate or equivocal in 72.2% by criteria of the American Society for Gastrointestinal Endoscopy, in 68.5% by explicit American criteria, and in 74.4% by explicit Swiss criteria (not statistically significant, NS). Inappropriate use (overuse) of colonoscopy was found in 27.8%, 31.5%, and 25.6%, respectively (NS). The proportion of appropriate procedures was higher with increasing age. Almost all reasons for using colonoscopy could be assessed by the two explicit criteria sets, whereas 28.4% of reasons for using colonoscopy could not be evaluated by the criteria of the American Society for Gastrointestinal Endoscopy (p < 0.0001). The probability of finding a relevant endoscopic lesion was distinctly higher in the procedures rated appropriate or equivocal than in procedures judged inappropriate. CONCLUSIONS: The rate of inappropriate use of colonoscopy is substantial in Switzerland. Explicit criteria allow assessment of almost all indications encountered in clinical practice. In this study, all sets of appropriateness criteria significantly enhanced the probability of finding a relevant endoscopic lesion during colonoscopy.

Melanin-based coloration is a nondirectionally selected sex-specific signal of offspring development in the Alpine swift

Two mutually exclusive hypotheses have been put forward to explain the evolution and adaptive function of melanin-based color traits. According to sexual selection theory melanism is a directionally selected signal of individual quality, whereas theory on the maintenance of genetic polymorphism proposes that alternative melanin-based variants achieve equal fitness. Alpine swift (Apus melba) males and females have a conspicuous patch of white feathers on the breast with their rachis varying continuously from white to black, and hence the breast varies from white to striated. If this trait is a sexually selected signal of quality, its expression should be condition dependent and the degree of melanism directionally selected. If variation in melanism is a polymorphism, its expression should be genetically determined and fitness of melanin-based variants equal. We experimentally tested these predictions by exchanging eggs or hatchlings between randomly chosen nests and by estimating survival and reproduction in relation to melanism. We found that breast melanism is heritable and that the environment and body condition do not significantly influence its expression. Between 5 and 50 days of age nestlings were heavier and their wings longer when breast feathers of their biological father were blacker, and they also fledged at a younger age. This shows that aspects of offspring quality covary positively with the degree of melanism. However, this did not result in directional selection because nestling survival and recruitment in the local breeding population were not associated with father breast melanism. Furthermore, adult survival, age at first reproduction and probability of skipping reproduction did not covary with the degree of melanism. Genetic variation in breast melanism is therefore maintained either because nonmelanic males achieve fitness similar to melanic males via a different route than producing fast-growing offspring, or because the advantage of producing fast-growing offspring is not sufficiently pronounced to result in directional selection.

Ruin Theory with Excess of Loss Reinsurance and Reinstatements

The present paper studies the probability of ruin of an insurer, if excess of loss reinsurance with reinstatements is applied. In the setting of the classical Cramer-Lundberg risk model, piecewise deterministic Markov processes are used to describe the free surplus process in this more general situation. It is shown that the finite-time ruin probability is both the solution of a partial integro-differential equation and the fixed point of a contractive integral operator. We exploit the latter representation to develop and implement a recursive algorithm for numerical approximation of the ruin probability that involves high-dimensional integration. Furthermore we study the behavior of the finite-time ruin probability under various levels of initial surplus and security loadings and compare the efficiency of the numerical algorithm with the computational alternative of stochastic simulation of the risk process. (C) 2011 Elsevier Inc. All rights reserved.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up.

BACKGROUND: Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up. METHODS: BIG 1-98 is a randomised, phase 3, double-blind trial of postmenopausal women with hormone receptor-positive early breast cancer that compares 5 years of tamoxifen or letrozole monotherapy, or sequential treatment with 2 years of one of these drugs followed by 3 years of the other. Randomisation was done with permuted blocks, and stratified according to the two-arm or four-arm randomisation option, participating institution, and chemotherapy use. Patients, investigators, data managers, and medical reviewers were masked. The primary efficacy endpoint was disease-free survival (events were invasive breast cancer relapse, second primaries [contralateral breast and non-breast], or death without previous cancer event). Secondary endpoints were overall survival, distant recurrence-free interval (DRFI), and breast cancer-free interval (BCFI). The monotherapy comparison included patients randomly assigned to tamoxifen or letrozole for 5 years. In 2005, after a significant disease-free survival benefit was reported for letrozole as compared with tamoxifen, a protocol amendment facilitated the crossover to letrozole of patients who were still receiving tamoxifen alone; Cox models and Kaplan-Meier estimates with inverse probability of censoring weighting (IPCW) are used to account for selective crossover to letrozole of patients (n=619) in the tamoxifen arm. Comparison of sequential treatments to letrozole monotherapy included patients enrolled and randomly assigned to letrozole for 5 years, letrozole for 2 years followed by tamoxifen for 3 years, or tamoxifen for 2 years followed by letrozole for 3 years. Treatment has ended for all patients and detailed safety results for adverse events that occurred during the 5 years of treatment have been reported elsewhere. Follow-up is continuing for those enrolled in the four-arm option. BIG 1-98 is registered at clinicaltrials.govNCT00004205. FINDINGS: 8010 patients were included in the trial, with a median follow-up of 8·1 years (range 0-12·4). 2459 were randomly assigned to monotherapy with tamoxifen for 5 years and 2463 to monotherapy with letrozole for 5 years. In the four-arm option of the trial, 1546 were randomly assigned to letrozole for 5 years, 1548 to tamoxifen for 5 years, 1540 to letrozole for 2 years followed by tamoxifen for 3 years, and 1548 to tamoxifen for 2 years followed by letrozole for 3 years. At a median follow-up of 8·7 years from randomisation (range 0-12·4), letrozole monotherapy was significantly better than tamoxifen, whether by IPCW or intention-to-treat analysis (IPCW disease-free survival HR 0·82 [95% CI 0·74-0·92], overall survival HR 0·79 [0·69-0·90], DRFI HR 0·79 [0·68-0·92], BCFI HR 0·80 [0·70-0·92]; intention-to-treat disease-free survival HR 0·86 [0·78-0·96], overall survival HR 0·87 [0·77-0·999], DRFI HR 0·86 [0·74-0·998], BCFI HR 0·86 [0·76-0·98]). At a median follow-up of 8·0 years from randomisation (range 0-11·2) for the comparison of the sequential groups with letrozole monotherapy, there were no statistically significant differences in any of the four endpoints for either sequence. 8-year intention-to-treat estimates (each with SE ≤1·1%) for letrozole monotherapy, letrozole followed by tamoxifen, and tamoxifen followed by letrozole were 78·6%, 77·8%, 77·3% for disease-free survival; 87·5%, 87·7%, 85·9% for overall survival; 89·9%, 88·7%, 88·1% for DRFI; and 86·1%, 85·3%, 84·3% for BCFI. INTERPRETATION: For postmenopausal women with endocrine-responsive early breast cancer, a reduction in breast cancer recurrence and mortality is obtained by letrozole monotherapy when compared with tamoxifen montherapy. Sequential treatments involving tamoxifen and letrozole do not improve outcome compared with letrozole monotherapy, but might be useful strategies when considering an individual patient's risk of recurrence and treatment tolerability. FUNDING: Novartis, United States National Cancer Institute, International Breast Cancer Study Group.

How patch configuration affects the impact of disturbances on metapopulation persistence.

Disturbances affect metapopulations directly through reductions in population size and indirectly through habitat modification. We consider how metapopulation persistence is affected by different disturbance regimes and the way in which disturbances spread, when metapopulations are compact or elongated, using a stochastic spatially explicit model which includes metapopulation and habitat dynamics. We discover that the risk of population extinction is larger for spatially aggregated disturbances than for spatially random disturbances. By changing the spatial configuration of the patches in the system--leading to different proportions of edge and interior patches--we demonstrate that the probability of metapopulation extinction is smaller when the metapopulation is more compact. Both of these results become more pronounced when colonization connectivity decreases. Our results have important management implication as edge patches, which are invariably considered to be less important, may play an important role as disturbance refugia.