High-Throughput Sequencing—The Key to Rapid Biodiversity Assessment of Marine Metazoa?

The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in pyrosequencing datasets is complicated by potential PCR-based biases and that the V1-V2 marker is poorly resolved for some taxa.     

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2^−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2^−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2^−/−mammary fat pad showed a decrease in the content of myristic acid (C₁₄), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2^−/− mice.     

Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.     

Autosomal-Dominant Striatal Degeneration Is Caused by a Mutation in the Phosphodiesterase 8B Gene

Autosomal-dominant striatal degeneration (ADSD) is an autosomal-dominant movement disorder affecting the striatal part of the basal ganglia. ADSD is characterized by bradykinesia, dysarthria, and muscle rigidity. These symptoms resemble idiopathic Parkinson disease, but tremor is not present. Using genetic linkage analysis, we have mapped the causative genetic defect to a 3.25 megabase candidate region on chromosome 5q13.3-q14.1. A maximum LOD score of 4.1 (Θ = 0) was obtained at marker D5S1962. Here we show that ADSD is caused by a complex frameshift mutation (c.94G>C+c.95delT) in the phosphodiesterase 8B (PDE8B) gene, which results in a loss of enzymatic phosphodiesterase activity. We found that PDE8B is highly expressed in the brain, especially in the putamen, which is affected by ADSD. PDE8B degrades cyclic AMP, a second messenger implied in dopamine signaling. Dopamine is one of the main neurotransmitters involved in movement control and is deficient in Parkinson disease. We believe that the functional analysis of PDE8B will help to further elucidate the pathomechanism of ADSD as well as contribute to a better understanding of movement disorders.     

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

Electrical signals and their physiological significance in plants

Electrical excitability and signalling, frequently associated with rapid responses to environmental stimuli, are well known in some algae and higher plants. The presence of electrical signals, such as action potentials (AP), in both animal and plant cells suggested that plant cells, too, make use of ion channels to transmit information over long distances. In the light of rapid progress in plant biology during the past decade, the assumption that electrical signals do not only trigger rapid leaf movements in 'sensitive' plants such as Mimosa pudica or Dionaea muscipula, but also physiological processes in ordinary plants proved to be correct. Summarizing recent progress in the field of electrical signalling in plants, the present review will focus on the generation and propagation of various electrical signals, their ways of transmission within the plant body and various physiological effects.     

Enrichment strategies for siRNA-transfected cells in an untransfected background

Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.