Phylogenetic analysis of plastid and nuclear DNA sequences reveals a rapid late Miocene radiation of the temperate bamboo tribe Arundinarieae (Poaceae, Bambusoideae)

Background: Arundinarieae are a large tribe of temperate woody bamboos for which phylogenetics are poorly understood because of limited taxon sampling and lack of informative characters. Aims: This study assessed phylogenetic relationships, origins and classification of Arundinarieae. Methods: DNA sequences (plastid trnL-F; nuclear ITS) were used for parsimony and Bayesian inference including 41 woody bamboo taxa. Divergence dates were estimated using a relaxed Bayesian clock. Results: Arundinarieae were monophyletic but their molecular divergence was low compared to the tropical Bambuseae. Ancestors of the Arundinarieae lineage were estimated to have diverged from the other bamboos 23 (15-30) million years ago (Mya). However, the Arundinarieae radiation occurred 10 (6-16) Mya compared to 18 (11-25) Mya for the tropical Bambuseae. Some groups could be defined within Arundinarieae, but they do not correspond to recognised subtribes such as Arundinariinae or Shibataeinae. Conclusions: Arundinarieae are a relatively ancient bambusoid lineage that underwent a rapid radiation in the late Miocene. The radiation coincides with the continental collision of the Indo-Australian and Eurasian Plates. Arundinarieae are distributed primarily in East Asia and the Himalayas to northern Southeast Asia. It is unknown whether they were present in Asia long before their radiation, but we believe recent dispersal is a more likely scenario. Keywords: Arundinarieae; Bambuseae; internal transcribed spacer (ITS); molecular clock; phylogenetics; radiation; temperate bamboos; Thamnocalaminae; trnL-F

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.

New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples

Given the low sensitivity of amoebal coculture, we developed a specific real-time PCR for the detection of Parachlamydia. The analytical sensitivity was high, and the inter- and intrarun variabilities were low. When the PCR was applied to nasopharyngeal aspirates, it was positive for six patients with bronchiolitis. Future studies should assess the role of Parachlamydia in bronchiolitis.

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA-binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

Plastid DNA variation in Prunus serotina var. serotina (Rosaceae), a North American tree invading Europe

Black cherry (Prunus serotina) is a tree from North America, where it is often used for economical purposes, whereas it is widespread and invasive in Europe. Plastid DNA variation was Wrst investigated in both its native and invasive ranges using microsatellite loci and sequences of three intergenic spacers (trnT-trnL, trnD-trnT and trnS-trnG). This analysis was focused on P. serotina var. serotina, with the inclusion of samples of closely related taxa. Length variation at a microsatellite locus (ccmp5) and a few sequence polymorphisms were identi- Wed among P. serotina samples. Four new primer pairs were then designed to speciWcally amplify variable regions and a combination of Wve markers was Wnally proposed for phylogeographic studies in P. serotina. These loci allow identiWcation of six chlorotypes in P. serotina var. serotina, which may be particularly useful to depict the maternal origins of European invasive populations

Complementary DNA cloning of complement C8 beta and its sequence homology to C9.

The complete amino acid sequence of mature C8 beta has been derived from the DNA sequence of a cDNA clone identified by expression screening of a human liver cDNA library. Comparison with the amino acid sequence of C9 shows an overall homology with few deletions and insertions. In particular, the cysteine-rich domains and membrane-inserting regions of C9 are well conserved. These findings are discussed in relation to a possible mechanism of membrane attack complex formation.

Efeito da aplicação de enzimas pectinolíticas no rendimento da extração de polpa de cupuaçu

O Brasil apresenta uma diversidade de frutas regionais, principalmente nas regiões Norte e Nordeste. Dentre estas, destaca-se o cupuaçu, que é uma fruta considerada exótica e de grande potencial comercial nos mercados dos estados do Sudeste do Brasil e dos países europeus. A polpa e a semente são importantes para o desenvolvimento de produtos industriais. A polpa tem sido estudada como matéria-prima para néctar; entretanto, é usada em sorvetes, geléias, purês e polpa enlatada. Atualmente, o processo de extração da polpa ainda é feito de maneira empírica, utilizando-se de acessórios como facas, colheres ou tesouras de uso doméstico, devido à forte aderência da mesma ao caroço, causando baixo rendimento no produto final. Com o objetivo de aumentar o rendimento durante o processo de extração da polpa de cupuaçu, adicionaram-se à polpa bruta dois tipos de preparações enzimáticas de ação pectolítica (Citrozym-L e Rohament PL). As concentrações estabelecidas foram de 100; 300; 500 e 750 ppm. Após todo o processo, obteve-se a polpa, e o rendimento foi comparado com a polpa-controle. O experimento foi realizado nos laboratórios de Tecnologia de Alimentos da Embrapa Agroindústria Tropical. Pelos resultados obtidos, observou-se que o rendimento da polpa com adição de Citrozym-L foi superior a 60% nas concentrações de 300; 500 e 750 ppm, enquanto, com a utilização de Rohament-PL, o rendimento variou de 43 a 57% nas mesmas concentrações. O rendimento da polpa-controle foi de 44 e 41 %, para os experimentos realizados com a primeira e segunda preparação enzimática, respectivamente. Conclui-se que a utilização de enzimas é uma alternativa para melhorar o processo de extração da polpa; adicionando-se acima de 300ppm, obtém-se um rendimento próximo a 60%.