Multiple introductions boosted genetic diversity in the invasive range of black cherry (Prunus serotina; Rosaceae).

BACKGROUND AND AIMS: Black cherry (Prunus serotina) is a North American tree that is rapidly invading European forests. This species was introduced first as an ornamental plant, then it was massively planted by foresters in many countries, but its origins and the process of invasion remain poorly documented. Based on a genetic survey of both native and invasive ranges, the invasion history of black cherry was investigated by identifying putative source populations and then assessing the importance of multiple introductions on the maintenance of gene diversity. METHODS: Genetic variability and structure of 23 populations from the invasive range and 22 populations from the native range were analysed using eight nuclear microsatellite loci and five chloroplast DNA regions. KEY RESULTS: Chloroplast DNA diversity suggests there were multiple introductions from a single geographic region (the north-eastern United States). A low reduction of genetic diversity was observed in the invasive range for both nuclear and plastid genomes. High propagule pressure including both the size and number of introductions shaped the genetic structure in Europe and boosted genetic diversity. Populations from Denmark, The Netherlands, Belgium and Germany showed high genetic diversity and low differentiation among populations, supporting the hypothesis that numerous introduction events, including multiple individuals and exchanges between sites, have taken place during two centuries of plantation. CONCLUSIONS: This study postulates that the invasive black cherry has originated from east of the Appalachian Mountains (mainly the Allegheny plateau) and its invasiveness in north-western Europe is mainly due to multiple introductions containing high numbers of individuals.

Hour of birth as a prognostic factor for perinatal death.

The analysis of the 220,540 births and 2152 perinatal deaths recorded in Switzerland between 1979 and 1981 showed a variation of perinatal mortality rates (PMR) according to the hour of birth. The PMR for babies born between 4 pm and 2 am was 12 per 1000, contrasting with a figure of 8.4 per 1000 for babies born between 2 am and 4 pm. This pattern, which was fairly constant throughout the week, was characterised by a slow and steady increase from the very early morning, reaching a maximum in the late evening. There was also an hour-to-hour variation in the proportion of babies born weighing less than 2500 g, with a maximum in the evening and a less pronounced peak in the morning: the mortality rates by birthweight were raised only in the evening. Since the availability of hospital staff and equipment also follows a circadian rhythm, the variation in PMR may be related to a circadian rhythm of quality of care or possibly to chronobiological or selection factors.

Study of isotopic variations in black powder: reflections on the use of stable isotopes in forensic science for source inference.

Isotope ratio mass spectrometry (IRMS) has recently made its appearance in the forensic community. This high-precision technology has already been applied to a broad range of forensic fields such as illicit drugs, explosives and flammable liquids, where current, routinely used techniques have limited powers of discrimination. The conclusions drawn from the majority of these IRMS studies appear to be very promising. Used in a comparative process, as in food or drug authentication, the measurement of stable isotope ratios is a new and remarkable analytical tool for the discrimination or the identification of a substance with a definite source or origin. However, the research consists mostly of preliminary studies. The significance of this 'new' piece of information needs to be evaluated in light of a forensic framework to assess the actual potential and validity of IRMS, considering the characteristics of each field. Through the isotopic study of black powder, this paper aims at illustrating the potential of the method and the limitations of current knowledge in stable isotopes when facing forensic problems.

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose.

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Low concentrations of 3-hydroxy glutarate leads to ammonium accumulation and non-apoptotic cell death in developing brain cells

We previously showed in a 3D rat brain cell in vitro model for glutaric aciduria type-I that repeated application of 1mM 3-hydroxy-glutarate (3-OHGA) caused ammonium accumulation, morphologic alterations and induction of non-apoptotic cell death in developing brain cells. Here, we performed a dose-response study with lower concentrations of 3- OHGA.We exposed our cultures to 0.1, 0.33 and 1mM 3-OHGA every 12h over three days at two developmental stages (DIV5-8 and DIV11-14). Ammonium accumulation was observed at both stages starting from 0.1mM 3-OHGA, in parallel with a glutamine decrease. Morphological changes started at 0.33mM with loss of MBP expression and loss of astrocytic processes. Neurons were not substantially affected. At DIV8, release of LDH in the medium and cellular TUNEL staining increased from 0.1mM and 0.33mM 3-OHGA exposure, respectively. No increase in activated caspase-3 was observed. We confirmed ammonium accumulation and non-apoptotic cell death of brain cells in our in vitro model at lower 3-OHGA concentrations thus strongly suggesting that the observed effects are likely to take place in the brain of affected patients. The concomitant glutamine decrease suggests a defect in the astrocyte ammonium buffering system. Ammonium accumulation might be the cause of non-apoptotic cell death.

Graphitized carbon black in quartz tubes for the sampling of indoor air nicotine and analysis by microwave thermal desorption-capillary gas chromatography

Nicotine in a smoky indoor air environment can be determined using graphitized carbon black as a solid sorbent in quartz tubes. The temperature stability, high purity, and heat absorption characteristics of the sorbent, as well as the permeability of the quartz tubes to microwaves, enable the thermal desorption by means of microwaves after active sampling. Permeation and dynamic dilution procedures for the generation of nicotine in the vapor phase at low and high concentrations are used to evaluate the performances of the sampler. Tube preparation is described and the microwave desorption temperature is measured. Breakthrough volume is determined to allow sampling at 0.1-1 L/min for definite periods of time. The procedure is tested for the determination of gas and paticulate phase nicotine in sidestream smoke produced in an experimental chamber.

More than cell death: caspases and caspase inhibitors on the move.

It is becoming clear that "apoptotic" caspases can effect cellular processes other than cell death. A recent paper in Cell points to a novel role of the Drosophila caspase inhibitor DIAP1 as a determinant of cell migration.

Contribution of isotope ratio mass spectrometry to the investigation of improvised explosives: isotopic study of black powders ans ammonium nitrates

The establishment of legislative rules about explosives in the eighties has reduced the illicit use of military and civilian explosives. However, bomb-makers have rapidly taken advantage of substances easily accessible and intended for licit uses to produce their own explosives. This change in strategy has given rise to an increase of improvised explosive charges, which is moreover assisted by the ease of implementation of the recipes, widely available through open sources. While the nature of the explosive charges has evolved, instrumental methods currently used in routine, although more sensitive than before, have a limited power of discrimination and allow mostly the determination of the chemical nature of the substance. Isotope ratio mass spectrometry (IRMS) has been applied to a wide range of forensic materials. Conclusions drawn from the majority of the studies stress its high power of discrimination. Preliminary studies conducted so far on the isotopic analysis of intact explosives (pre-blast) have shown that samples with the same chemical composition and coming from different sources could be differentiated. The measurement of stable isotope ratios appears therefore as a new and remarkable analytical tool for the discrimination or the identification of a substance with a definite source. However, much research is still needed to assess the validity of the results in order to use them either in an operational prospect or in court. Through the isotopic study of black powders and ammonium nitrates, this research aims at evaluating the contribution of isotope ratio mass spectrometry to the investigation of explosives, both from a pre-blast and from a post-blast approach. More specifically, the goal of the research is to provide additional elements necessary to a valid interpretation of the results, when used in explosives investigation. This work includes a fundamental study on the variability of the isotopic profile of black powder and ammonium nitrate in both space and time. On one hand, the inter-variability between manufacturers and, particularly, the intra-variability within a manufacturer has been studied. On the other hand, the stability of the isotopic profile over time has been evaluated through the aging of these substances exposed to different environmental conditions. The second part of this project considers the applicability of this high-precision technology to traces and residues of explosives, taking account of the characteristics specific to the field, including their sampling, a probable isotopic fractionation during the explosion, and the interferences with the matrix of the site.

Alleged Tax Competition: The Mysterious Death of Inheritance Taxes in Switzerland

Interjurisdictional competition over mobile tax bases is an easily understood mechanism, but actual tax-base elasticities are difficult to estimate. Political pressure for reducing tax rates could therefore be based on erroneous estimates of the mobility of tax bases. We show that tax competition provided the overwhelmingly dominant argument in the policy debates leading to a succession of reforms of bequest taxation by Swiss cantons. Yet, we find only very weak statistical evidence of a relationship between tax burdens on bequests and the concerned tax base of wealthy elderly individuals. Moreover, inheritance tax revenues are found to increase in inheritance tax rates even in the long run, and actual tax rates lie well below the revenue-maximising levels throughout. The alleged pressures of tax competition did not seem in reality to exist.

Specific targeting of pro-death NMDA receptor signals with differing reliance on the NR2B PDZ ligand.

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca(2+), and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

In conditions of limited chromophore supply rods entrap 11-cis-retinal leading to loss of cone function and cell death.

RPE65 is a retinoid isomerase required for the production of 11-cis-retinal, the chromophore of both cone and rod visual pigments. We recently established an R91W knock-in mouse strain as homologous animal model for patients afflicted by this mutation in RPE65. These mice have impaired vision and can only synthesize minute amounts of 11-cis-retinal. Here, we investigated the consequences of this chromophore insufficiency on cone function and pathophysiology. We found that the R91W mutation caused cone opsin mislocalization and progressive geographic cone atrophy. Remnant visual function was mostly mediated by rods. Ablation of rod opsin corrected the localization of cone opsin and improved cone retinal function. Thus, our analyses indicate that under conditions of limited chromophore supply rods and cones compete for 11-cis-retinal that derives from regeneration pathway(s) which are reliant on RPE65. Due to their higher number and the instability of cone opsin, rods are privileged under this condition while cones suffer chromophore deficiency and degenerate. These findings reinforce the notion that in patients any effective gene therapy with RPE65 needs to target the cone-rich macula directly to locally restore the cones' chromophore supply outside the reach of rods.