Protein function prediction and annotation in an integrated environment powered by web services (AFAWE)

Many sequenced genes are mainly annotated through automatic transfer of annotation from similar sequences. Manual comparison of results or intermediate results from different tools can help avoid wrong annotations and give hints to the function of a gene even if none of the automated tools could return any result. AFAWE simplifies the task of manual functional annotation by running different tools and workflows for automatic function prediction and displaying the results in a way that facilitates comparison. Because all programs are executed as web services, AFAWE is easily extensible and can directly query primary databases, thereby always using the most up-to-date data sources. Visual filters help to distinguish trustworthy results from non-significant results. Furthermore, an interface to add detailed manual annotation to each gene is provided, which can be displayed to other users.     

Gene flow at the margin of Lake Matano’s adaptive sailfin silverside radiation: Telmatherinidae of River Petea in Sulawesi

Classical speciation concepts focus almost exclusively on the evolution of strict reproductive isolation as a prerequisite for speciation. However, there is a growing body of evidence indicating that speciation is possible despite or even triggered by gene flow among populations or species. Previous findings indicate that introgressive hybridization is a dominant phenomenon in the adaptive radiation of sailfin silversides (Telmatherinidae) endemic to Lake Matano (Sulawesi). In this study, we investigate patterns of genotypic and phenotypic variation of “sharpfin” sailfin silversides in the outlet area of L. Matano and six locations along River Petea, which is the only connection between L. Matano and other lakes and streams of the Malili Lakes system. Fieldwork revealed no hints for a previously cited major waterfall in River Petea, which was thought to separate L. Matano’s sailfin silverside radiation from the diversity of the downstream lake drainages. Likewise, genomic (AFLP) and morphometric data suggest high levels of gene flow between upper and lower stretches of this river, as well as between riverine Petea and lacustrine Matano populations. Increasing levels of genotypic and phenotypic dissimilarity are correlated with distance over a remarkably short geographic range.     

2��-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m⁷GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.     

P-bodies directly regulate MARF1-mediated mRNA decay in human cells

Processing bodies (P-bodies) are ribonucleoprotein granules that contain mRNAs, RNA-binding proteins and effectors of mRNA turnover. While P-bodies have been reported to contain translationally repressed mRNAs, a causative role for P-bodies in regulating mRNA decay has yet to be established. Enhancer of decapping protein 4 (EDC4) is a core P-body component that interacts with multiple mRNA decay factors, including the mRNA decapping (DCP2) and decay (XRN1) enzymes. EDC4 also associates with the RNA endonuclease MARF1, an interaction that antagonizes the decay of MARF1-targeted mRNAs. How EDC4 interacts with MARF1 and how it represses MARF1 activity is unclear. In this study, we show that human MARF1 and XRN1 interact with EDC4 using analogous conserved short linear motifs in a mutually exclusive manner. While the EDC4–MARF1 interaction is required for EDC4 to inhibit MARF1 activity, our data indicate that the interaction with EDC4 alone is not sufficient. Importantly, we show that P-body architecture plays a critical role in antagonizing MARF1-mediated mRNA decay. Taken together, our study suggests that P-bodies can directly regulate mRNA turnover by sequestering an mRNA decay enzyme and preventing it from interfacing with and degrading targeted mRNAs.     

Teaching to Relax: Development of a Program to Potentiate Stress—Results of a Feasibility Study with Medical Undergraduate Students

Medical students are a population at risk for the development of stress-related risk states (e.g. burnout) and manifest mental disorders (e.g. depression). Still the learning of coping mechanisms against stress is not an integral part of the medical curriculum. In a pilot study we developed an elective course for learning relaxation techniques (Relacs) which was geared to the clinical practice of autogenic training (AT) with psychiatric patients. The course focussed on an innovative and mostly communicative transfer of knowledge about AT, progressive muscle relaxation and medical hypnosis and stressed the principle of repeated and supervised exercises in small student groups alongside self-administered exercise. 42 students took part in this course and showed a very high acceptance for the topic and positive evaluation. Moreover, we found a distinct improvement of the participants’ mental parameters (burnout, anxiety) and a good knowledge about the course’s contents within the final exams at the end of the semester. The structure and realisation of the course is easily adaptable and very effective regarding the improvement of the students’ mental health. Due to our results and the commonly known prevalence of stress-related disorders in medical students we postulate the integration of courses on relaxation strategies in the medical curriculum.     

Aminoguanidine administered during the induction of oral tolerance alters the systemic response of the tolerised rats

Herewith we investigated the role of nitric oxide synthase (NOS)-II in the establishment of oral tolerance induced by low antigen dose. To accomplish this, we used a rat model of oral tolerance induced by intragastric administration of low doses of ovalbumin (OVA). NOS-II was inhibited in vivo during the onset of tolerance by intraperitoneal (i.p.) treatment with aminoguanidine (AMG), a selective NOS-II inhibitor. Four experimental groups were generated: (TOL), tolerised rats, receiving OVA but no AMG; (TAG), rats tolerised with OVA and simultaneously receiving AMG i.p.; (CAG), controls treated with AMG but no oral antigen; and (CONT), controls receiving neither OVA nor AMG treatment. The state of oral tolerance was evaluated in all groups by analysing several immune parameters upon subcutaneous administration of OVA in Freund’s complete adjuvant. First, we were able to determine that NOS-II inhibition altered the TH1/TH2 balance in tolerised rats, driving the TH2 anti-OVA response in TOL rats towards TH1 in TAG animals, which showed enhanced delayed hypersensitivity responses. Second, splenocyte cultures from TAG rats showed lower levels of IL-10 production compared to TOL samples as determined by ELISA analysis. Last, we detected the presence of a functional distinct Tr1 regulatory T cell population in spleen samples recovered from TAG animals. Contrary to what happened with TOL Tr1 cells, the levels of Tr1 cells in TAG samples were modified by in vitro stimulation with OVA.All together, these data indicate a preponderant role for NOS-II in the process of oral tolerance induced by low antigen dose.     

Opioid-Induced Nausea Involves a Vestibular Problem Preventable by Head-Rest

Background and Aims

Opioids are indispensable for pain treatment but may cause serious nausea and vomiting. The mechanism leading to these complications is not clear. We investigated whether an opioid effect on the vestibular system resulting in corrupt head motion sensation is causative and, consequently, whether head-rest prevents nausea.

Methods

Thirty-six healthy men (26.6±4.3 years) received an opioid remifentanil infusion (45 min, 0.15 μg/kg/min). Outcome measures were the vestibulo-ocular reflex (VOR) gain determined by video-head-impulse-testing, and nausea. The first experiment (n = 10) assessed outcome measures at rest and after a series of five 1-Hz forward and backward head-trunk movements during one-time remifentanil administration. The second experiment (n = 10) determined outcome measures on two days in a controlled crossover design: (1) without movement and (2) with a series of five 1-Hz forward and backward head-trunk bends 30 min after remifentanil start. Nausea was psychophysically quantified (scale from 0 to 10). The third controlled crossover experiment (n = 16) assessed nausea (1) without movement and (2) with head movement; isolated head movements consisting of the three axes of rotation (pitch, roll, yaw) were imposed 20 times at a frequency of 1 Hz in a random, unpredictable order of each of the three axes. All movements were applied manually, passively with amplitudes of about ± 45 degrees.

Results

The VOR gain decreased during remifentanil administration (p<0.001), averaging 0.92±0.05 (mean±standard deviation) before, 0.60±0.12 with, and 0.91±0.05 after infusion. The average half-life of VOR recovery was 5.3±2.4 min. 32/36 subjects had no nausea at rest (nausea scale 0.00/0.00 median/interquartile range). Head-trunk and isolated head movement triggered nausea in 64% (p<0.01) with no difference between head-trunk and isolated head movements (nausea scale 4.00/7.25 and 1.00/4.5, respectively).

Conclusions

Remifentanil reversibly decreases VOR gain at a half-life reflecting the drug’s pharmacokinetics. We suggest that the decrease in VOR gain leads to a perceptual mismatch of multisensory input with the applied head movement, which results in nausea, and that, consequently, vigorous head movements should be avoided to prevent opioid-induced nausea.