RiboReport - benchmarking tools for ribosome profiling-based identification of open reading frames in bacteria.

Small proteins encoded by short open reading frames (ORFs) with 50 codons or fewer are emerging as an important class of cellular macromolecules in diverse organisms. However, they often evade detection by proteomics or in silico methods. Ribosome profiling (Ribo-seq) has revealed widespread translation in genomic regions previously thought to be non-coding, driving the development of ORF detection tools using Ribo-seq data. However, only a handful of tools have been designed for bacteria, and these have not yet been systematically compared. Here, we aimed to identify tools that use Ribo-seq data to correctly determine the translational status of annotated bacterial ORFs and also discover novel translated regions with high sensitivity. To this end, we generated a large set of annotated ORFs from four diverse bacterial organisms, manually labeled for their translation status based on Ribo-seq data, which are available for future benchmarking studies. This set was used to investigate the predictive performance of seven Ribo-seq-based ORF detection tools (REPARATION_blast, DeepRibo, Ribo-TISH, PRICE, smORFer, ribotricer and SPECtre), as well as IRSOM, which uses coding potential and RNA-seq coverage only. DeepRibo and REPARATION_blast robustly predicted translated ORFs, including sORFs, with no significant difference for ORFs in close proximity to other genes versus stand-alone genes. However, no tool predicted a set of novel, experimentally verified sORFs with high sensitivity. Start codon predictions with smORFer show the value of initiation site profiling data to further improve the sensitivity of ORF prediction tools in bacteria. Overall, we find that bacterial tools perform well for sORF detection, although there is potential for improving their performance, applicability, usability and reproducibility.

The locality dilemma of Sankoff-like RNA alignments.

MOTIVATION: Elucidating the functions of non-coding RNAs by homology has been strongly limited due to fundamental computational and modeling issues. While existing simultaneous alignment and folding (SA&F) algorithms successfully align homologous RNAs with precisely known boundaries (global SA&F), the more pressing problem of identifying new classes of homologous RNAs in the genome (local SA&F) is intrinsically more difficult and much less understood. Typically, the length of local alignments is strongly overestimated and alignment boundaries are dramatically mispredicted. We hypothesize that local SA&F approaches are compromised this way due to a score bias, which is caused by the contribution of RNA structure similarity to their overall alignment score. RESULTS: In the light of this hypothesis, we study pairwise local SA&F for the first time systematically-based on a novel local RNA alignment benchmark set and quality measure. First, we vary the relative influence of structure similarity compared to sequence similarity. Putting more emphasis on the structure component leads to overestimating the length of local alignments. This clearly shows the bias of current scores and strongly hints at the structure component as its origin. Second, we study the interplay of several important scoring parameters by learning parameters for local and global SA&F. The divergence of these optimized parameter sets underlines the fundamental obstacles for local SA&F. Third, by introducing a position-wise correction term in local SA&F, we constructively solve its principal issues. AVAILABILITY AND IMPLEMENTATION: The benchmark data, detailed results and scripts are available at https://github.com/BackofenLab/local_alignment. The RNA alignment tool LocARNA, including the modifications proposed in this work, is available at https://github.com/s-will/LocARNA/releases/tag/v2.0.0RC6. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The impact of various seed, accessibility and interaction constraints on sRNA target prediction- a systematic assessment.

BACKGROUND: Seed and accessibility constraints are core features to enable highly accurate sRNA target screens based on RNA-RNA interaction prediction. Currently, available tools provide different (sets of) constraints and default parameter sets. Thus, it is hard to impossible for users to estimate the influence of individual restrictions on the prediction results. RESULTS: Here, we present a systematic assessment of the impact of established and new constraints on sRNA target prediction both on a qualitative as well as computational level. This is done exemplarily based on the performance of IntaRNA, one of the most exact sRNA target prediction tools. IntaRNA provides various ways to constrain considered seed interactions, e.g. based on seed length, its accessibility, minimal unpaired probabilities, or energy thresholds, beside analogous constraints for the overall interaction. Thus, our results reveal the impact of individual constraints and their combinations. CONCLUSIONS: This provides both a guide for users what is important and recommendations for existing and upcoming sRNA target prediction approaches.We show on a large sRNA target screen benchmark data set that only by altering the parameter set, IntaRNA recovers 30% more verified interactions while becoming 5-times faster. This exemplifies the potential of seed, accessibility and interaction constraints for sRNA target prediction.

TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines.

In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.

Substantial reprogramming of the Eutrema salsugineum (Thellungiella salsuginea) transcriptome in response to UV and silver nitrate challenge.

BACKGROUND: Cruciferous plants synthesize a large variety of tryptophan-derived phytoalexins in response to pathogen infection, UV irradiation, or high dosages of heavy metals. The major phytoalexins of Eutrema salsugineum (Thellungiella salsuginea), which has recently been established as an extremophile model plant, are probably derivatives of indole glucosinolates, in contrast to Arabidopsis, which synthesizes characteristic camalexin from the glucosinolate precursor indole-3-acetaldoxime. RESULTS: The transcriptional response of E. salsugineum to UV irradiation and AgNO3 was monitored by RNAseq and microarray analysis. Most transcripts (respectively 70% and 78%) were significantly differentially regulated and a large overlap between the two treatments was observed (54% of total). While core genes of the biosynthesis of aliphatic glucosinolates were repressed, tryptophan and indole glucosinolate biosynthetic genes, as well as defence-related WRKY transcription factors, were consistently upregulated. The putative Eutrema WRKY33 ortholog was functionally tested and shown to complement camalexin deficiency in Atwrky33 mutant. CONCLUSIONS: In E. salsugineum, UV irradiation or heavy metal application resulted in substantial transcriptional reprogramming. Consistently induced genes of indole glucosinolate biosynthesis and modification will serve as candidate genes for the biosynthesis of Eutrema-specific phytoalexins.

Sustained exposure to abscisic acid enhances the colonization potential of the mutualist fungus Piriformospora indica on Arabidopsis thaliana roots.

Root colonization by the beneficial fungus Piriformospora indica is controlled by plant innate immunity, but factors that channel this interaction into a mutualistic relationship are not known. We have explored the impact of abscisic acid (ABA) and osmotic stress on the P. indica interaction with Arabidopsis thaliana. The activation of plant innate immunity in roots was determined by measuring the concentration of the phytoalexin camalexin and expression of transcription factors regulating the biosynthesis of tryptophan-related defence metabolites. Furthermore, the impact of the fungus on the content of ABA, salicylic acid, jasmonic acid (JA) and JA-related metabolites was examined. We demonstrated that treatment with exogenous ABA or the ABA analogue pyrabactin increased fungal colonization efficiency without impairment of plant fitness. Concomitantly, ABA-deficient mutants of A. thaliana (aba1-6 and aba2-1) were less colonized, while plants exposed to moderate stress were more colonized than corresponding controls. Sustained exposure to ABA attenuated expression of transcription factors MYB51, MYB122 and WRKY33 in roots upon P. indica challenge or chitin treatment, and prevented an increase in camalexin content. The results indicate that ABA can strengthen the interaction with P. indica as a consequence of its impact on plant innate immunity. Consequently, ABA will be relevant for the establishment and outcome of the symbiosis under stress conditions.

CheRRI-Accurate classification of the biological relevance of putative RNA-RNA interaction sites.

BACKGROUND: RNA-RNA interactions are key to a wide range of cellular functions. The detection of potential interactions helps to understand the underlying processes. However, potential interactions identified via in silico or experimental high-throughput methods can lack precision because of a high false-positive rate. RESULTS: We present CheRRI, the first tool to evaluate the biological relevance of putative RNA-RNA interaction sites. CheRRI filters candidates via a machine learning-based model trained on experimental RNA-RNA interactome data. Its unique setup combines interactome data and an established thermodynamic prediction tool to integrate experimental data with state-of-the-art computational models. Applying these data to an automated machine learning approach provides the opportunity to not only filter data for potential false positives but also tailor the underlying interaction site model to specific needs. CONCLUSIONS: CheRRI is a stand-alone postprocessing tool to filter either predicted or experimentally identified potential RNA-RNA interactions on a genomic level to enhance the quality of interaction candidates. It is easy to install (via conda, pip packages), use (via Galaxy), and integrate into existing RNA-RNA interaction pipelines.

[Development of an indicator set for the evaluation of the quality of routine ambulatory health care for common disorders in children and adolescents]. / Entwicklung eines Indikatorensets zur Evaluation der Versorgungsqualität in der ambulanten Routineversorgung häufiger Erkrankungen des Kindes- und Jugendalters.

BACKGROUND: In Germany, no consented quality indicator set (QI set) exists to date that can be used to assess the quality of pediatric care. Therefore, the aim of the project "Assessment of the quality of routine ambulatory health care for common disorders in children and adolescents" (QualiPäd) funded by the Innovation Committee of the Federal Joint Committee (grant no.: 01VSF19035) was to develop a QI set for the diseases asthma, atopic eczema, otitis media, tonsillitis, attention-deficit hyperactivity disorder (ADHD), depression and conduct disorder. METHODS: For the observation period 2018/2019, quality indicators (QIs) were searched in indicator databases, guidelines and literature databases and complemented in part by newly formulated QIs (e.g., derived from guideline recommendations). The QIs were then assigned to content categories and dimensions according to Donabedian and OECD and reduced by removing duplicates. Finally, a panel of experts consulted the QIs using the modified RAND-UCLA Appropriateness Method (RAM). RESULTS: The search resulted in a preliminary QI set of 2324 QIs. After the reduction steps and the evaluation of the experts, 282 QIs were included in the QI set (asthma: 72 QIs, atopic eczema: 25 QIs, otitis media: 31 QIs, tonsillitis: 12 QIs, ADHD: 53 QIs, depression: 43 QIs, conduct disorder: 46 QIs). The QIs are distributed among the following different categories: Therapy (138 QIs), Diagnostics (95 QIs), Patient-reported outcome measures/Patient-reported experience measures (PROM/PREM) (45 QIs), Practice management (31 QIs), and Health reporting (4 QIs). In the Donabedian model, 89% of the QIs capture process quality, 9% outcome quality, and 2% structural quality; according to the OECD classification, 61% measure effectiveness, 23% patient-centeredness, and 16% safety of care. CONCLUSION: The consented QI set is currently being tested and can subsequently be used (possibly modified) to measure the quality of routine outpatient care for children and adolescents in Germany, in order to indicate the status quo and potential areas for improvement in outpatient care.

Process over outcome quality in paediatrics? An analysis of outpatient healthcare quality indicators for seven common diseases.

PURPOSE: The purpose of this study was to examine the scope, quality dimensions and treatment aspects covered by existing quality indicators (QIs) for the somatic diseases bronchial asthma, atopic eczema, otitis media and tonsillitis as well as the psychiatric disorders attention deficit hyperactivity disorder (ADHD), depression and conduct disorder in paediatrics. METHODS: QIs were identified through an analysis of the guidelines and a systematic search of literature and indicator databases. Subsequently, two researchers independently assigned the QIs to the quality dimensions according to Donabedian and Organisation for Economic Cooperation and Development (OECD) and to the content categories covering the treatment process. RESULTS: We found 1268 QIs for bronchial asthma, 335 QIs for depression, 199 QIs for ADHD, 115 QIs for otitis media, 72 QIs for conduct disorder, 52 QIs for tonsillitis and 50 QIs for atopic eczema. Of these, 78% focused on process quality, 20% on outcome quality and 2% on structural quality. Using OECD criteria, 72% of the QIs were assigned to effectiveness, 17% to patient-centredness, 11% to patient safety and 1% to efficiency. The QIs covered the following categories: diagnostics (30%), therapy (38%), patient-reported outcome measures/ observer-reported outcome measures/patient-reported experience measures (in sum 11%), health monitoring (11%) and office management (11%). CONCLUSION: Most QIs focused on the dimensions of effectiveness and process quality, and on the categories of diagnostics and therapy, with outcome-focused and patient-focused QIs being under-represented. Possible reasons for this striking imbalance could be the easier measurability and clearer assignment of accountability in comparison to the QIs of outcome quality, patient-centredness and patient safety. To produce a more balanced picture of the quality of healthcare, the future development of QIs should prioritise the currently under-represented dimensions.

Revealing the small proteome of Haloferax volcanii by combining ribosome profiling and small-protein optimized mass spectrometry.

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon Haloferax volcanii. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence in vivo for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in Haloferax species and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.

What's in a wine? - A spot check of the integrity of European wine sold in China based on anthocyanin composition, stable isotope and glycerol impurity analysis.

The international wine market has been repeatedly hit by cases of fraud in recent decades. While several studies attested a special vulnerability of the fast growing wine business in China, reports on chemical analyses of commercial wine samples are rare. We examined 50 predominantly red wines with European labelling, which were purchased on the Chinese market, for fraud-relevant parameters. More than 20% of the tested samples revealed anomalies in relation to the stable isotope ratios of D/H, 18O/16O and 13C/12C, contents of technical glycerol by-products or anthocyanin composition. These results strongly suggested watering of the wines, chaptalisation, glycerol addition or the use of non-Vitis anthocyanin sources, respectively. Some of these samples also showed suspicious spelling errors or other irregularities in the labelling, but the majority appeared genuine to the eye. Hence, this spot check demonstrates the importance of chemical authenticity analysis of market samples in order to detect fraudulent products. Moreover, we used the same sample set for an evaluation of the Chinese standard method for carbon stable isotope determination of wine ethanol in comparison to the current OIV (International Organisation of Vine and Wine) standard method. The results of a Bland-Altman analysis indicated that the methods can be applied interchangeably. As the two methods differ in their workflow and in the requested equipment, this might eventually enable more laboratories to perform 13C/12C analysis of wine and spirits.

Dissection of the network of indolic defence compounds in Arabidopsis thaliana by multiple mutant analysis.

Characteristic for cruciferous plants is the synthesis of a complex array of defence-related indolic compounds. In Arabidopsis, these include indol-3-ylmethyl glucosinolates (IMGs), as well as stress-inducible indole-3-carbaldehyde (ICHO)/indole-3-carboxylic acid (ICOOH) derivatives and camalexin. Key enzymes in the biosynthesis of the inducible metabolites are the cytochrome P450 enzymes CYP71A12, CYP71A13 and CYP71B6 and Arabidopsis Aldehyde Oxidase 1 (AAO1). Multiple mutants in the corresponding genes were generated and their metabolic phenotypes were comprehensively analysed in untreated, UV exposed and silver nitrate-treated leaves. Most strikingly, ICOOH and ICHO derivatives synthesized in response to UV exposure were not metabolically related. While ICHO concentrations correlated with IMGs, ICOOH derivatives were anti-correlated with IMGs and partially dependent on CYP71B6. The AAO1 genotype was shown to not only be important for ICHO metabolism but also for the accumulation of 4-pyridoxic acid, suggesting a dual role of AAO1 in vitamin B6 metabolism and IMG degradation in Arabidopsis.

Suppress to Forget: The Effect of a Mindfulness-Based Strategy during an Emotional Item-Directed Forgetting Paradigm.

Forgetting is a common phenomenon in everyday life. Although it often has negative connotations, forgetting is an important adaptive mechanism to avoid loading the memory storage with irrelevant information. A very important aspect of forgetting is its interaction with emotion. Affective events are often granted special and priority treatment over neutral ones with regards to memory storage. As a consequence, emotional information is more resistant to extinction than neutral information. It has been suggested that intentional forgetting serves as a mechanism to cope with unwanted or disruptive emotional memories and the main goal of this study was to assess forgetting of emotional auditory material using the item-method directed forgetting (DF) paradigm using a forgetting strategy based on mindfulness as a means to enhance DF. Contrary to our prediction, the mindfulness-based strategy not only did not improve DF but reduced it for neutral material. These results suggest that an interaction between processes such as response inhibition and attention is required for intentional forgetting to succeed.

Electrospun Polyhydroxybutyrate/Poly(ε-caprolactone)/58S Sol-Gel Bioactive Glass Hybrid Scaffolds with Highly Improved Osteogenic Potential for Bone Tissue Engineering.

Electrospinning of biopolymer and inorganic substances is one of the efficient ways to combine various advantageous properties in one single fibrous structure with potential for tissue engineering applications. In the present study, to integrate the high stiffness of polyhydroxybutyrate (PHB), the flexibility of poly(ε-caprolactone) (PCL) and the bioactivity of 58S bioactive glass, PHB/PCL/58S sol-gel bioactive glass hybrid scaffolds were fabricated using combined electrospinning and sol-gel method. Physical features such as fiber diameter distribution, mechanical strength and Young's modulus were characterized thoroughly. FTIR analysis demonstrated the successful incorporation of 58S bioactive glass into the blend polymers, which greatly improved the hydrophilicity of PHB/PCL fibermats. The primary biological response of MG-63 osteoblast-like cells on the prepared fibrous scaffolds was evaluated, proving that the 58S glass sol containing hybrid scaffold were not only favorable to MG-63 cell adhesion but also slightly enhanced cell viability and significantly increased alkaline phosphate activity .

Neonicotinoids interfere with specific components of navigation in honeybees.

Three neonicotinoids, imidacloprid, clothianidin and thiacloprid, agonists of the nicotinic acetylcholine receptor in the central brain of insects, were applied at non-lethal doses in order to test their effects on honeybee navigation. A catch-and-release experimental design was applied in which feeder trained bees were caught when arriving at the feeder, treated with one of the neonicotinoids, and released 1.5 hours later at a remote site. The flight paths of individual bees were tracked with harmonic radar. The initial flight phase controlled by the recently acquired navigation memory (vector memory) was less compromised than the second phase that leads the animal back to the hive (homing flight). The rate of successful return was significantly lower in treated bees, the probability of a correct turn at a salient landscape structure was reduced, and less directed flights during homing flights were performed. Since the homing phase in catch-and-release experiments documents the ability of a foraging honeybee to activate a remote memory acquired during its exploratory orientation flights, we conclude that non-lethal doses of the three neonicotinoids tested either block the retrieval of exploratory navigation memory or alter this form of navigation memory. These findings are discussed in the context of the application of neonicotinoids in plant protection.