Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions

Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small-subunit rDNA) of an intertidal protistan model community, using the Roche GS-FLX and the most recent GS-FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2039 unique tags (from 3879 V4 GS-FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS-FLX). Pyrosequencing error rates were higher for V4 fragments compared with the V9 domain and for the GS-FLX Titanium compared with the GS-FLX system. Furthermore, this experiment revealed that error rates are taxon-specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data-processing pipelines.     

Genetic erosion in habitat specialist shows need to protect large peat bogs

The specialized fauna of peat bogs declines strongly both in species numbers and population density and becomes fragmented because of anthropogenic land use changes. We investigated 15 populations of the stenotopic ground beetle species Agonum ericeti in south Sweden to address the question of reduced genetic variability in populations inhabiting smaller habitat patches. Our results reveal a generally low differentiation and a significant positive relationship between habitat size and allelic richness.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting

In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.     

Dating the arthropod tree based on large-scale transcriptome data

Molecular sequences do not only allow the reconstruction of phylogenetic relationships among species, but also provide information on the approximate divergence times. Whereas the fossil record dates the origin of most multicellular animal phyla during the Cambrian explosion less than 540 million years ago(mya), molecular clock calculations usually suggest much older dates. Here we used a large multiple sequence alignment derived from Expressed Sequence Tags and genomes comprising 129genes (37,476 amino acid positions) and 117 taxa, including 101 arthropods. We obtained consistent divergence time estimates applying relaxed Bayesian clock models with different priors and multiple calibration points. While the influence of substitution rates, missing data, and model priors were negligible, the clock model had significant effect. A log-normal autocorrelated model was selected on basis of cross-validation. We calculated that arthropods emerged ~600 mya. Onychophorans (velvet worms) and euarthropods split ~590 mya, Pancrustacea and Myriochelata ~560 mya, Myriapoda and Chelicerata ~555 mya, and 'Crustacea' and Hexapoda ~510 mya. Endopterygote insects appeared ~390 mya. These dates are considerably younger than most previous molecular clock estimates and in better agreement with the fossil record. Nevertheless, a Precambrian origin of arthropods and other metazoan phyla is still supported. Our results also demonstrate the applicability of large datasets of random nuclear sequences for approximating the timing of multicellular animal evolution.     

Historical ecology meets conservation and evolutionary genetics: a secondary contact zone between Carabus violaceus (Coleoptera, Carabidae) populations inhabiting ancient and recent woodlands in north-western Germany

Only very few cases have documented that an increase in connectivity after a period of fragmentation in ecological time has had an effect on the distribution, genetic structure and morphology of stenotopic species. In this study we present an example of clinal variability in a woodland ground beetle as a result of changes in the connectivity of a landscape during the last two centuries. The study area hosts both the nominate form Carabus violaceus s. str. and the subspecies Carabus violaceus purpurascens, which is ranked as a distinct species by some authors. We studied 12 Carabus violaceus populations from a 30 km transect of ancient and recent forests in north-western Germany. We analyzed three polymorphic enzyme loci, classified the elytron sculpture and measured the shape of the aedeagus tip of the specimens. Carabus violaceus showed secondary gradients both in allozyme markers and morphometric characters in our study area. A genetic differentiation of 16% between the populations is high but lies within the range of intraspecific variability in habitat specialists of the genus Carabus. Populations had no significant deficit of heterozygotes. We found many hybrid populations in terms of morphological properties. This study highlights the conservation value of ancient woodland and the consequences of landscape connectivity and defragmentation on the genetic setting of a ground beetle. Moreover, it shows that differences in the external shape of male genitalia do not prevent gene flow within the genus Carabus. Thus, the establishment of species status should not exclusively be based on this property.     

Eucamaragnathus desenderi, a new ground beetle species from Africa (Coleoptera, Carabidae)

Eucamaragnathus desenderisp. n., a new ground beetle species of the tribe Hiletini, is described from eastern and southern Africa and dedicated to the recently deceased Belgian carabidologist Konjev Desender. The new taxon is known so far from localities in Zambia (Mukuku, southeast of Mansa) and in South Africa (Bothaville, south of Klerksdorp). The new species belongs to the Eucamaragnathus castelnaui group and is characterized by shape of pronotum, smooth or sparsely punctate pronotal transverse impression, characters of male genitalia and elytral striae continued to the apex. Illustrations of the habitus, the median lobe and its internal sac and several other morphological features are presented. An updated identification key to the African Eucamaragnathus species is given.     

GeneReporter--sequence-based document retrieval and annotation

GeneReporter is a web tool that reports functional information and relevant literature on a protein-coding sequence of interest. Its purpose is to support both manual genome annotation and document retrieval. PubMed references corresponding to a sequence are detected by the extraction of query words from UniProt entries of homologous sequences. Data on protein families, domains, potential cofactors, structure, function, cellular localization, metabolic contribution and corresponding DNA binding sites complement the information on a given gene product of interest. Availability and implementation: GeneReporter is available at http://www.genereporter.tu-bs.de. The web site integrates databases and analysis tools as SOAP-based web services from the EBI (European Bioinformatics Institute) and NCBI (National Center for Biotechnology Information).     

Ultrastructural changes of the intracellular surfactant pool in a rat model of lung transplantation-related events

Background

Ischemia/reperfusion (I/R) injury, involved in primary graft dysfunction following lung transplantation, leads to inactivation of intra-alveolar surfactant which facilitates injury of the blood-air barrier. The alveolar epithelial type II cells (AE2 cells) synthesize, store and secrete surfactant; thus, an intracellular surfactant pool stored in lamellar bodies (Lb) can be distinguished from the intra-alveolar surfactant pool. The aim of this study was to investigate ultrastructural alterations of the intracellular surfactant pool in a model, mimicking transplantation-related procedures including flush perfusion, cold ischemia and reperfusion combined with mechanical ventilation.

Methods

Using design-based stereology at the light and electron microscopic level, number, surface area and mean volume of AE2 cells as well as number, size and total volume of Lb were determined in a group subjected to transplantation-related procedures including both I/R injury and mechanical ventilation (I/R group) and a control group.

Results

After I/R injury, the mean number of Lb per AE2 cell was significantly reduced compared to the control group, accompanied by a significant increase in the luminal surface area per AE2 cell in the I/R group. This increase in the luminal surface area correlated with the decrease in surface area of Lb per AE2. The number-weighted mean volume of Lb in the I/R group showed a tendency to increase.

Conclusion

We suggest that in this animal model the reduction of the number of Lb per AE2 cell is most likely due to stimulated exocytosis of Lb into the alveolar space. The loss of Lb is partly compensated by an increased size of Lb thus maintaining total volume of Lb per AE2 cell and lung. This mechanism counteracts at least in part the inactivation of the intra-alveolar surfactant.     

A call for standardized naming and reporting of human ESC and iPSC lines

Human embryonic and induced pluripotent stem cell lines are being generated at a rapid pace and now number in the thousands. We propose a standard nomenclature and suggest the use of a centralized database for all cell line names and a minimum set of information for reporting new derivations.