REPARATION : ribosome profiling assisted (re-)annotation of bacterial genomes

Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence composition and often underestimate the complexity of the proteome. We developed RibosomeE Profiling Assisted (re-)AnnotaTION (REPARATION), a de novo machine learning algorithm that takes advantage of experimental protein synthesis evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation (https://github.com/Biobix/ REPARATION). REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds based on a growth curve model to screen for spurious ORFs. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel (small) ORFs including variants of previously annotated ORFs and >70% of all (variants of) annotated protein coding ORFs were predicted by REPARATION to be translated. Our predictions are supported by matching mass spectrometry proteomics data, sequence composition and conservation analysis. REPARATION is unique in that it makes use of experimental translation evidence to intrinsically perform a de novo ORF delineation in bacterial genomes irrespective of the sequence features linked to open reading frames

Holistic engineering design : a combined synchronous and asynchronous approach

To aid the creation and through-life support of large, complex engineering products, organizations are placing a greater emphasis on constructing complete and accurate records of design activities. Current documentary approaches are not sufficient to capture activities and decisions in their entirety and can lead to organizations revisiting and in some cases reworking design decisions in order to understand previous design episodes. Design activities are undertaken in a variety of modes; many of which are dichotomous, and thus each require separate documentary mechanisms to capture information in an efficient manner. It is possible to identify the modes of learning and transaction to describe whether an activity is aimed at increasing a level of understanding or whether it involves manipulating information to achieve a tangible task. The dichotomy of interest in this paper is that of synchronous and asynchronous working, where engineers may work alternately as part of a group or as individuals and where different forms of record are necessary to adequately capture the processes and rationale employed in each mode. This paper introduces complimentary approaches to achieving richer representations of design activities performed synchronously and asynchronously, and through the undertaking of a design based case study, highlights the benefit of each approach. The resulting records serve to provide a more complete depiction of activities undertaken, and provide positive direction for future co-development of the approaches

Ribosome signatures aid bacterial translation initiation site identification

Background: While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information, developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. Results: Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. Conclusions: Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites

Directional Next-Generation RNA Sequencing and Examination of Premature Termination Codon Mutations in Endoglin/Hereditary Haemorrhagic Telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is a disease characterised by abnormal vascular structures, and most commonly caused by mutations in ENG, ACVRL1 or SMAD4 encoding endothelial cell-expressed proteins involved in TGF-β superfamily signalling. The majority of mutations reported on the HHT mutation database are predicted to lead to stop codons, either due to frameshifts or direct nonsense substitutions. The proportion is higher for ENG (67%) and SMAD4 (65%) than for ACVRL1 (42%), p < 0.0001. Here, by focussing on ENG, we report why conventional views of these mutations may need to be revised. Of the 111 stop codon-generating ENG mutations, on ExPASy translation, all except one were premature termination codons (PTCs), sited at least 50-55 bp upstream of the final exon-exon boundary of the main endoglin isoform, L-endoglin. This strongly suggests that the mutated RNA species will undergo nonsense-mediated decay. We provide new in vitro expression data to support dominant negative activity of stable truncated endoglin proteins but suggest these will not generate HHT: the single natural stop codon mutation in L-endoglin (sited within 50-55 nucleotides of the final exon-exon boundary) is unlikely to generate functional protein since it replaces the entire transmembrane domain, as would 8 further natural stop codon mutations, if the minor S-endoglin isoform were implicated in HHT pathogenesis. Finally, next-generation RNA sequencing data of 7 different RNA libraries from primary human endothelial cells demonstrate that multiple intronic regions of ENG are transcribed. The potential consequences of heterozygous deletions or duplications of such regions are discussed. These data support the haploinsufficiency model for HHT pathogenesis, explain why final exon mutations have not been detected to date in HHT, emphasise the potential need for functional examination of non-PTC-generating mutations, and lead to proposals for an alternate stratification system of mutational types for HHT genotype-phenotype correlations

Epstein Barr virus shedding in multiple sclerosis: similar frequencies of EBV in saliva across separate patient cohorts

Background: Epstein Barr Virus (EBV) infection is closely associated with multiple sclerosis (MS), but the relationship between viral load and disease activity is unclear. This study tested the observed levels of salivary EBV in MS, as a first step in investigating this relationship. Methods: Real-time quantitative PCR (qPCR) was used to measure EBV DNA levels in saliva samples from three separate Multiple Sclerosis (MS) patient cohorts. Results: The qPCR assay was used to delineate EBV shedding, defined here as a reliably detectable level of extracellular EBV DNA in saliva. Frequency of EBV shedding was found to be similar across the groups, with 20–25% of subjects releasing virus on any given sampling date. Diurnal variation in EBV count was tested in one of the cohorts, in which 26% of subjects showed more than a 10-fold difference between the highest and lowest EBV levels on a single day. In the same cohort, elevated viral levels at one time point did not predict elevated viral levels at a subsequent time point. Conclusions: These results indicate that EBV lytic activity in a subject cannot be inferred from a single measure of EBV in saliva. Also, subjects do not appear to be behave constantly as “EBV shedders” or “non-shedders”. The assay is useful in giving a clear indication of salivary gland EBV lytic activity across a patient cohort – for example, in testing anti-viral drugs in M

Uncovering convolutional neural network decisions for diagnosing multiple sclerosis on conventional MRI using layer-wise relevance propagation

Machine learning-based imaging diagnostics has recently reached or even superseded the level of clinical experts in several clinical domains. However, classification decisions of a trained machine learning system are typically non-transparent, a major hindrance for clinical integration, error tracking or knowledge discovery. In this study, we present a transparent deep learning framework relying on convolutional neural networks (CNNs) and layer-wise relevance propagation (LRP) for diagnosing multiple sclerosis (MS). MS is commonly diagnosed utilizing a combination of clinical presentation and conventional magnetic resonance imaging (MRI), specifically the occurrence and presentation of white matter lesions in T2-weighted images. We hypothesized that using LRP in a naive predictive model would enable us to uncover relevant image features that a trained CNN uses for decision-making. Since imaging markers in MS are well-established this would enable us to validate the respective CNN model. First, we pre-trained a CNN on MRI data from the Alzheimer's Disease Neuroimaging Initiative (n = 921), afterwards specializing the CNN to discriminate between MS patients and healthy controls (n = 147). Using LRP, we then produced a heatmap for each subject in the holdout set depicting the voxel-wise relevance for a particular classification decision. The resulting CNN model resulted in a balanced accuracy of 87.04% and an area under the curve of 96.08% in a receiver operating characteristic curve. The subsequent LRP visualization revealed that the CNN model focuses indeed on individual lesions, but also incorporates additional information such as lesion location, non-lesional white matter or gray matter areas such as the thalamus, which are established conventional and advanced MRI markers in MS. We conclude that LRP and the proposed framework have the capability to make diagnostic decisions of..

Melting behaviour of waste glass cullet briquettes in soda-lime-silica container glass batch

The melting behaviour of representative container glass batch with and without the addition of 15wt % briquettes produced from waste cullet fine particles was investigated in the context of reducing both waste and glass melting energies. Carbonate raw material decomposition and reactions during melting were studied by DTA-TGA-MS. The decomposition kinetics of two batches, representing typical container glass batches with 0% and 15% briquette additions, were calculated by transformation degree based on the Ginstling-Brounstein and Arrhenius equations. High temperature phase transitions and fractions of silica reaction in each batch were obtained from X-ray diffractometry (XRD). The briquette additions accelerated the decomposition reactions and the silicate reaction kinetics by decreasing the activation energy for carbonate decomposition. Silica sand in the batch was shown to melt at lower temperatures with the addition of briquettes. Batch melting processes at different temperatures and briquette melting on top of the molten glass at high temperatures, were investigated by macroscopic investigations of sample cross-sections post-melting. The positive effects of briquette additions to container glass batches, in terms of increased melting rate and reduced batch reaction and decomposition temperatures, are supported by the results of this study

7 Tesla MRI of Balo's concentric sclerosis versus multiple sclerosis lesions

Background: Baló’s concentric sclerosis (BCS) is a rare condition characterized by concentrically layered white matter lesions. While its pathogenesis is unknown, hypoxia-induced tissue injury and chemotactic stimuli have been proposed as potential causes of BCS lesion formation. BCS has been suggested to be a variant of multiple sclerosis (MS). Here, we aimed to elucidate similarities and differences between BCS and MS by describing lesion morphology and localization in high-resolution 7 Tesla (7 T) magnetic resonance imaging (MRI) scans. Methods: Ten patients with Baló-type lesions underwent 7 T MRI, and 10 relapsing remitting MS patients served as controls. The 7 T MR imaging protocol included 3D T1-weighted (T1w) magnetization-prepared rapid gradient echo, 2D high spatial resolution T2*-weighted (T2*w) fast low-angle shot and susceptibility-weighted imaging. Results: Intralesional veins were visible in the center of all but one Baló-type lesion. Four Baló-type lesions displayed inhomogeneous intralesional T2*w signal intensities, which are suggestive of microhemorrhages or small ectatic venules. Eight of 10 BCS patients presented with 97 additional lesions, 36 of which (37%) had a central vein. Lesions involving the cortical gray matter and the U-fibers were not detected in BCS patients. Conclusion: Our findings support the hypothesis that BCS and MS share common pathogenetic mechanisms but patients present with different lesion phenotypes

Socioeconomic status and global physical self-concept of adolescents: a multilevel structural equation modeling approach

The global physical self-concept (GPSC) is a central part of one’s self-definition in adolescence and plays an important role in adolescents’ physical and psychological health. Socioeconomic status (SES) can be assumed to have an impact on GPSC, but this relationship has received little attention thus far. We investigated, therefore, the direct and indirect relationships between SES factors, such as parental educational level, occupational status, and family income, and adolescents’ GPSC. A sample of 966 adolescents from 47 9th-grade classes was examined. Multilevel structural equation modeling was used for the analyses. The results revealed an indirect positive effect of parental educational level on adolescents’ global physical self-concept. The effect was completely mediated by parental occupational status, parental sport participation, adolescents’ social resources, and adolescents’ physical exercise and sport activity. The tested model explained 28% of the variance in adolescents’ global physical self-concept. Implications for the development of suitable interventions to improve the global physical self-concept of adolescents are derived

Extracting information from manufacturing data using data mining methods

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