Padrões temporais e grau de diversificação cariotípica em espécies atlânticas da família Acanthuridae (Perciformes)

The Acanthuridae family is a representative group from the marine fish that plays a key role in ecological dynamics of coral reefs. Three species are common along coastal reefs of Western Atlantic: Acanthurus coeruleus, Acanthurus bahianus and Acanthurus chirurgus. In the present study, cytogenetic data are presented for these three species Acanthurus based on classical cytogenetic methods and mapping of repetitive sequences such as ribosomal 18S and 5S rDNA and telomeric repeats to improve their karyotype evolutionary analyses. The cytogenetic pattern of these species indicated sequential steps of chromosomal rearrangements dating back 19 to 5 millions of years ago (M.a.) that accounted for their interspecific differences. A. coeruleus (2n=48; 2sm+4st+42a), A. bahianus (2n=36; 12m+2sm+4st+18a) and A. chirurgus (2n=34; 12m+2sm+4st+16a) share an older set of three chromosomal pairs that were originated through pericentric inversions. A set of six large metacentric pairs formed by Robertsonian (Rb) translocations found in A. bahianus and A. chirurgus and a putative in tandem fusion found in A. chirurgus are more recent events. The lack of interstitial telomeric sequences (ITS) in spite of several centric fusions in A. bahianus and A. chirurgus might be related to the long period of time after their occurrence (estimated in 5 M.a.). Furthermore, the homeologies among the chromosome pairs bearing ribosomal genes, in addition to other structural features, highlight large conserved chromosomal regions in the three species. Our findings indicate that macrostructural changes occurred during the cladogenesis of these species were not followed by conspicuous microstructural rearrangements in the karyotypes.

Validação de espécies de Centropomus (Centropomidae, Perciformes) do litoral do Rio Grande do Norte através de caracterização citogenética e molecular

The Centropomidae family consists of three genera, Centropomus, Lates and Psammoperca. Centropomus is the most diverse group, with six Centropomus species occur in the Western Atlantic Ocean C. poeyi Chávez, 1961, C. parallelus Poey, 1860, C. mexicanus Bocourt, 1868, C. pectinatus Poey, 1860 and C. ensiferus Poey, 1860. Some of these species are considered cryptic, because of its morphological traits showed low resolution for identification purposes. Despite showing great interest as a natural resource and fish culture, aspects of their diversity and karyotypic patterns are poorly understood. In this work morphological identification and comparison of mitochondrial 16S gene sequence were used to identify the species of the genus Centropomus occurring in Rio Grande do Norte, northeastern Brazil. Two sepecies were identified, C. undecimalis and C. mexicanus, which had the chromosomal aspects analyzed, through Classical cytogenetic method analyzes (conventional staining, C-banding, Ag-NORs), fluorochrome staining AT- and GC-specific, replication bands by incorporating of the base analog 5-Bromo-2’-deoxyuridine (5-BrdU), in situ chromosomal mapping of (TTAGGG)n sequences and in situ chromosome mapping 18S and 5S rRNA genes. Both species show 2n=48 acrocentric chromosomes, with ribosomal sites (Ag-NOR/18S rDNA/ Mitramycin+) in second chromosomal pair, in telomeric position on the long arm in C. mexicanus and interstitial in C. undecimalis. The nuclear organization pair (pair 2) shown a resolutive cytotaxonomic marker for these two species. The generated data reveal a lower species diversity than previously believed, suggesting that greater attention should be paid in taxonomic identification of the species, in view of optimize commercial actions exploitation, biological conservation and cultivation.

Effect of ocean warming and acidification on the early life stages of subtropical Acropora spicifera

This study investigated the impacts of acidified seawater (pCO2 900 µatm) and elevated water temperature (+3 °C) on the early life history stages of Acropora spicifera from the subtropical Houtman Abrolhos Islands (28°S) in Western Australia. Settlement rates were unaffected by high temperature (27 °C, 250 µatm), high pCO2 (24 °C, 900 µatm), or a combination of both high temperature and high pCO2 treatments (27 °C, 900 µatm). There were also no significant differences in rates of post-settlement survival after 4 weeks of exposure between any of the treatments, with survival ranging from 60 to 70 % regardless of treatment. Similarly, calcification, as determined by the skeletal weight of recruits, was unaffected by an increase in water temperature under both ambient and high pCO2 conditions. In contrast, high pCO2 significantly reduced early skeletal development, with mean skeletal weight in the high pCO2 and combined treatments reduced by 60 and 48 %, respectively, compared to control weights. Elevated temperature appeared to have a partially mitigative effect on calcification under high pCO2; however, this effect was not significant. Our results show that rates of settlement, post-settlement survival, and calcification in subtropical corals are relatively resilient to increases in temperature. This is in marked contrast to the sensitivity to temperature reported for the majority of tropical larvae and recruits in the literature. The subtropical corals in this study appear able to withstand an increase in temperature of 3 °C above ambient, indicating that they may have a wider thermal tolerance range and may not be adversely affected by initial increases in water temperature from subtropical 24 to 27 °C. However, the reduction in skeletal weight with high pCO2 indicates that early skeletal formation will be highly vulnerable to the changes in ocean pCO2 expected to occur over the twenty-first century, with implications for their longer-term growth and resilience.

Topoisomerase 1 (Top1)-associated Genome Instability in Yeast: Effects of Persistent Cleavage Complexes or Increased Top1 Levels

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.

Strategies for Coping with Multiple Biological Safety Regulations in High-Containment Laboratories

The increase in biological safety regulations and/or guidelines regarding personnel and facilities in high containment laboratories demands constant vigilance by biological safety professionals responsible for safety in these environments. Safety professionals have been faced with legislative compliance issues in the past and have developed effective management methods to cope with the demands of these requirements. Examples include the impact of the National Institutes of Health (NIH) recombinant DNA (rDNA) Guidelines and the Occupational Safety and Health Administration's (OSHA) Bloodborne Pathogens Standard. This chapter will attempt to describe seven successful strategies for management of regulatory compliance in research that are based on an overall philosophy of developing a “culture of safety”. Strategies range from interactive involvement with administration and research staff to biological safety professional development.

Sampling information, length measurements, DNA alignments, models and phylogenetics trees for Oithona similis s.l.

Traditionally, many small-sized copepod species are considered to be widespread, bipolar or cosmopolitan. However, these large-scale distribution patterns need to be re-examined in view of increasing evidence of cryptic and pseudo-cryptic speciation in pelagic copepods. Here, we present a phylogeographic study of Oithona similis s.l. populations from the Arctic Ocean, the Southern Ocean and its northern boundaries, the North Atlantic and the Mediterrranean Sea. O. similis s.l. is considered as one of the most abundant species in temperate to polar oceans and acts as an important link in the trophic network between the microbial loop and higher trophic levels such as fish larvae. Two gene fragments were analysed: the mitochondrial cytochrome oxidase c subunit I (COI), and the nuclear ribosomal 28S genetic marker. Seven distinct, geographically delimitated, mitochondrial lineages could be identified, with divergences among the lineages ranging from 8 to 24 %, thus representing most likely cryptic or pseudocryptic species within O. similis s.l. Four lineages were identified within or close to the borders of the Southern Ocean, one lineage in the Arctic Ocean and two lineages in the temperate Northern hemisphere. Surprisingly the Arctic lineage was more closely related to lineages from the Southern hemisphere than to the other lineages from the Northern hemisphere, suggesting that geographic proximity is a rather poor predictor of how closely related the clades are on a genetic level. Molecular clock application revealed that the evolutionary history of O. similis s.l. is possibly closely associated with the reorganization of the ocean circulation in the mid Miocene and may be an example of allopatric speciation in the pelagic zone.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

The identification of a novel role for BRCA1 in regulating RNA Polymerase I transcription

The unrestrained proliferation of cancer cells requires a high level of ribosome biogenesis. The first stage of ribosome biogenesis is the transcription of the large ribosomal RNAs (rRNAs); the structural and functional components of the ribosome. Transcription of rRNA is carried out by RNA Polymerase I (Pol-I) and its associated holoenzyme complex. Here we report that BRCA1, a nuclear phosphoprotein, and a known tumour suppressor involved in variety of cellular processes such as DNA damage response, transcriptional regulation, cell cycle control and ubiquitylation, is associated with rDNA repeats, in particular with the regulatory regions of the rRNA gene. We demonstrate that BRCA1 interacts directly with the basal Pol-I transcription factors; upstream binding factor (UBF), selectivity factor-1 (SL1) as well as interacting with RNA Pol-I itself. We show that in response to DNA damage, BRCA1 occupancy at the rDNA repeat is decreased and the observed BRCA1 interactions with the Pol-I transcription machinery are weakened. We propose, therefore, that there is a rDNA associated fraction of BRCA1 involved in DNA damage dependent regulation of Pol-I transcription, regulating the stability and formation of the Pol-I holoenzyme during initiation and/or elongation in response to DNA damage.

Future(s) Perfect: uchronian mapping as a research and visualisation tool in the fringes of the Olympic Park

This article explores and discusses the development of a mapping tool inspired by Charles Renouvier’s philosophical novel Uchronie (l’utopie dans l’histoire) (1876). The article explains the research and design process of creating a uchronian map of a formerly empty site in Fish Island in East London and describes a participatory workshop titled ‘Hackney Wick and Fish Island: Future Perfect(s)’ (25 April 2015) that used uchronian mapping to explore past and future development imaginaries of two sites in the neighbourhood. Given a uchronian mapping template, a protocol and a dossier of planning and other documents, participants were encouraged to develop their own uchronian map of each site, and in doing so test and question the process of visualizing ‘what was supposed to happen’, ‘what actually happened’ and ‘what could have happened’. The article concludes with a reflection on uchronian mapping as a tool for researching, analysing and making visible urban alternatives.

Messenger ribonucleic acid of cerebral nuclei.

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.

Sources of antibiotic resistance in the environment

The discovery of antibiotics was a major breakthrough in medicine. However, short after their introduction in clinical practice resistant bacteria were detected. Nowadays, antibiotic resistance constitutes a serious public health problem. In hospital settings, with high resistance levels, reducing drastically the therapeutic options. Carbapenems are last-resort antibiotics used in Portugal, only in hospitals, to treat serious infections. Bacterial resistance towards this class of antibiotics has increased during last years. In Gram-negative bacteria the production of carbapenemases is a common resistance mechanism. OXA-48 is a carbapenemase of Ambler class D and represents a major concern for human health. It is frequently detected in clinical isolates of Enterobacteriaceae. There are few studies suggesting that genes encoding for OXA-48 variants originated from genes present in the chromosome of members of genus Shewanella, and have disseminated to Enterobacteriaceae members, associated with mobile genetic elements. The aim of this study was to characterize strains from different sources of Shewanella to confirm its role as OXA-48 progenitor. For this, the phylogenetic affiliation of 33 strains of Shewanella was performed by 16SrDNA and gyrB sequencing. The most common species were S. hafniensis and S. xiamenensis, but also S. aestuarii, S. baltica, S. indica, S. haliotis, S. putrefaciens, S. algidipiscicola, S. irciniae, S. algae and S. fodinae were identified. blaOXA-48-like genes were detected in 21 isolates: S. hafniensis (8/8), S. xiamenensis (5/5), S. baltica (4/4), S. algae (1/1), S. fodinae (1/1), S. putrefaciens (1/2) and S. algidipiscicola (1/2). Sequence analysis revealed that genes encoded enzymes identical to OXA-48, OXA-181 and OXA-204 but also new variants differing from OXA-48 from 2 to 81 aminoacids. Genetic context analysis revealed the C15 gene upstream and lysR gene downstream, identical to what has been identified so far flanking blaOXA-48-like genes in Shewanella spp. The assessment of antibiotic susceptibility was performed for all isolates using the disk diffusion method. In general, it was observed a great sensitivity for all antibiotics except to amoxicillin and aztreonam. Multidrug resistance was detected in only 1 isolate. Other resistance genes and the presence of integrons were not identified. Plasmids were detected in 30.3% isolates (10/ 33). These results reinforce the role of Shewanella spp. as origin of blaOXA-48-like genes.