Further Insight into Extractable (Organo)fluorine Mass Balance Analysis of Tap Water from Shanghai, China.

The ubiquitous occurrence of per- and polyfluoroalkyl substances (PFAS) and the detection of unexplained extractable organofluorine (EOF) in drinking water have raised growing concerns. A recent study reported the detection of inorganic fluorinated anions in German river systems, and therefore, in some samples, EOF may include some inorganic fluorinated anions. Thus, it might be more appropriate to use the term "extractable fluorine (EF) analysis" instead of the term EOF analysis. In this study, tap water samples (n = 39) from Shanghai were collected to assess the levels of EF/EOF, 35 target PFAS, two inorganic fluorinated anions (tetrafluoroborate (BF4-) and hexafluorophosphate (PF6-)), and novel PFAS through suspect screening and potential oxidizable precursors through oxidative conversion. The results showed that ultra-short PFAS were the largest contributors to target PFAS, accounting for up to 97% of ΣPFAS. To the best of our knowledge, this was the first time that bis(trifluoromethanesulfonyl)imide (NTf2) was reported in drinking water from China, and p-perfluorous nonenoxybenzenesulfonate (OBS) was also identified through suspect screening. Small amounts of precursors that can be oxidatively converted to PFCAs were noted after oxidative conversion. EF mass balance analysis revealed that target PFAS could only explain less than 36% of EF. However, the amounts of unexplained extractable fluorine were greatly reduced when BF4- and PF6- were included. These compounds further explained more than 44% of the EF, indicating the role of inorganic fluorinated anions in the mass balance analysis.

Locus Reference Genomic: reference sequences for the reporting of clinically relevant sequence variants.

Locus Reference Genomic (LRG; http://www.lrg-sequence.org/) records contain internationally recognized stable reference sequences designed specifically for reporting clinically relevant sequence variants. Each LRG is contained within a single file consisting of a stable 'fixed' section and a regularly updated 'updatable' section. The fixed section contains stable genomic DNA sequence for a genomic region, essential transcripts and proteins for variant reporting and an exon numbering system. The updatable section contains mapping information, annotation of all transcripts and overlapping genes in the region and legacy exon and amino acid numbering systems. LRGs provide a stable framework that is vital for reporting variants, according to Human Genome Variation Society (HGVS) conventions, in genomic DNA, transcript or protein coordinates. To enable translation of information between LRG and genomic coordinates, LRGs include mapping to the human genome assembly. LRGs are compiled and maintained by the National Center for Biotechnology Information (NCBI) and European Bioinformatics Institute (EBI). LRG reference sequences are selected in collaboration with the diagnostic and research communities, locus-specific database curators and mutation consortia. Currently >700 LRGs have been created, of which >400 are publicly available. The aim is to create an LRG for every locus with clinical implications.

Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism.

BACKGROUND: Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine. RESULTS: We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome. CONCLUSIONS: We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

Ensembl 2012.

The Ensembl project (http://www.ensembl.org) provides genome resources for chordate genomes with a particular focus on human genome data as well as data for key model organisms such as mouse, rat and zebrafish. Five additional species were added in the last year including gibbon (Nomascus leucogenys) and Tasmanian devil (Sarcophilus harrisii) bringing the total number of supported species to 61 as of Ensembl release 64 (September 2011). Of these, 55 species appear on the main Ensembl website and six species are provided on the Ensembl preview site (Pre!Ensembl; http://pre.ensembl.org) with preliminary support. The past year has also seen improvements across the project.

Ensembl 2011.

The Ensembl project (http://www.ensembl.org) seeks to enable genomic science by providing high quality, integrated annotation on chordate and selected eukaryotic genomes within a consistent and accessible infrastructure. All supported species include comprehensive, evidence-based gene annotations and a selected set of genomes includes additional data focused on variation, comparative, evolutionary, functional and regulatory annotation. The most advanced resources are provided for key species including human, mouse, rat and zebrafish reflecting the popularity and importance of these species in biomedical research. As of Ensembl release 59 (August 2010), 56 species are supported of which 5 have been added in the past year. Since our previous report, we have substantially improved the presentation and integration of both data of disease relevance and the regulatory state of different cell types.

Genomic, transcriptomic and epigenomic sequencing data of the B-cell leukemia cell line REH.

OBJECTIVES: The aim of this data paper is to describe a collection of 33 genomic, transcriptomic and epigenomic sequencing datasets of the B-cell acute lymphoblastic leukemia (ALL) cell line REH. REH is one of the most frequently used cell lines for functional studies of pediatric ALL, and these data provide a multi-faceted characterization of its molecular features. The datasets described herein, generated with short- and long-read sequencing technologies, can both provide insights into the complex aberrant karyotype of REH, and be used as reference datasets for sequencing data quality assessment or for methods development. DATA DESCRIPTION: This paper describes 33 datasets corresponding to 867 gigabases of raw sequencing data generated from the REH cell line. These datasets include five different approaches for whole genome sequencing (WGS) on four sequencing platforms, two RNA sequencing (RNA-seq) techniques on two different sequencing platforms, DNA methylation sequencing, and single-cell ATAC-sequencing.

Sporulation in mycobacteria.

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette-Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.

Arteria: An automation system for a sequencing core facility.

BACKGROUND: In recent years, nucleotide sequencing has become increasingly instrumental in both research and clinical settings. This has led to an explosive growth in sequencing data produced worldwide. As the amount of data increases, so does the need for automated solutions for data processing and analysis. The concept of workflows has gained favour in the bioinformatics community, but there is little in the scientific literature describing end-to-end automation systems. Arteria is an automation system that aims at providing a solution to the data-related operational challenges that face sequencing core facilities. FINDINGS: Arteria is built on existing open source technologies, with a modular design allowing for a community-driven effort to create plug-and-play micro-services. In this article we describe the system, elaborate on the underlying conceptual framework, and present an example implementation. Arteria can be reduced to 3 conceptual levels: orchestration (using an event-based model of automation), process (the steps involved in processing sequencing data, modelled as workflows), and execution (using a series of RESTful micro-services). This creates a system that is both flexible and scalable. Arteria-based systems have been successfully deployed at 3 sequencing core facilities. The Arteria Project code, written largely in Python, is available as open source software, and more information can be found at https://arteria-project.github.io/ . CONCLUSIONS: We describe the Arteria system and the underlying conceptual framework, demonstrating how this model can be used to automate data handling and analysis in the context of a sequencing core facility.

Communicating Intent of Automated Vehicles to Pedestrians.

While traffic signals, signs, and road markings provide explicit guidelines for those operating in and around the roadways, some decisions, such as determinations of "who will go first," are made by implicit negotiations between road users. In such situations, pedestrians are today often dependent on cues in drivers' behavior such as eye contact, postures, and gestures. With the introduction of more automated functions and the transfer of control from the driver to the vehicle, pedestrians cannot rely on such non-verbal cues anymore. To study how the interaction between pedestrians and automated vehicles (AVs) might look like in the future, and how this might be affected if AVs were to communicate their intent to pedestrians, we designed an external vehicle interface called automated vehicle interaction principle (AVIP) that communicates vehicles' mode and intent to pedestrians. The interaction was explored in two experiments using a Wizard of Oz approach to simulate automated driving. The first experiment was carried out at a zebra crossing and involved nine pedestrians. While it focused mainly on assessing the usability of the interface, it also revealed initial indications related to pedestrians' emotions and perceived safety when encountering an AV with/without the interface. The second experiment was carried out in a parking lot and involved 24 pedestrians, which enabled a more detailed assessment of pedestrians' perceived safety when encountering an AV, both with and without the interface. For comparison purposes, these pedestrians also encountered a conventional vehicle. After a short training course, the interface was deemed easy for the pedestrians to interpret. The pedestrians stated that they felt significantly less safe when they encountered the AV without the interface, compared to the conventional vehicle and the AV with the interface. This suggests that the interface could contribute to a positive experience and improved perceived safety in pedestrian encounters with AVs - something that might be important for general acceptance of AVs. As such, this topic should be further investigated in future studies involving a larger sample and more dynamic conditions.

Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi.

We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.

Novel non-coding RNAs in Dictyostelium discoideum and their expression during development.

The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.

Gender does not influence prepubertal growth velocity during standard growth hormone therapy--analysis of United States KIGS data.

BACKGROUND: Gender is an important determinant that affects the ultimate dose of growth hormone (GH) used for replacement in adult GH deficiency (GHD). Women require larger doses of GH per body weight to achieve comparable age-adjusted serum IGF-I concentrations than do men. OBJECTIVE: To test whether this is entirely a sex steroid effect or biologically inherent in gender. PATIENTS AND METHODS: We examined growth response to GH (0.25-0.35 mg/kg/week) during the first 2 years of therapy in 147 children (44 girls), and in the first 3 years of therapy in 83 of these children (23 girls). Children were aged 3-8 years at onset of therapy, had peak stimulated GH <10 microg/l, and were reported to be prepubertal during the period of analysis. RESULTS: In the relative absence of sex steroid, there was no gender difference in growth velocity SDS or gain in height SDS during 2 or 3 years of GH therapy. CONCLUSIONS: Inherent gender differences in linear growth response to GH prior to puberty may exist, but are not evident in the first years of GH therapy at this GH dose.

Using Sound to Reduce Visual Distraction from In-vehicle Human-Machine Interfaces.

OBJECTIVE: Driver distraction and inattention are the main causes of accidents. The fact that devices such as navigation displays and media players are part of the distraction problem has led to the formulation of guidelines advocating various means for minimizing the visual distraction from such interfaces. However, although design guidelines and recommendations are followed, certain interface interactions, such as menu browsing, still require off-road visual attention that increases crash risk. In this article, we investigate whether adding sound to an in-vehicle user interface can provide the support necessary to create a significant reduction in glances toward a visual display when browsing menus. METHODS: Two sound concepts were developed and studied; spearcons (time-compressed speech sounds) and earcons (musical sounds). A simulator study was conducted in which 14 participants between the ages of 36 and 59 took part. Participants performed 6 different interface tasks while driving along a highway route. A 3 × 6 within-group factorial design was employed with sound (no sound /earcons/spearcons) and task (6 different task types) as factors. Eye glances and corresponding measures were recorded using a head-mounted eye tracker. Participants' self-assessed driving performance was also collected after each task with a 10-point scale ranging from 1 = very bad to 10 = very good. Separate analyses of variance (ANOVAs) were conducted for different eye glance measures and self-rated driving performance. RESULTS: It was found that the added spearcon sounds significantly reduced total glance time as well as number of glances while retaining task time as compared to the baseline (= no sound) condition (total glance time M = 4.15 for spearcons vs. M = 7.56 for baseline, p =.03). The earcon sounds did not result in such distraction-reducing effects. Furthermore, participants ratings of their driving performance were statistically significantly higher in the spearcon conditions compared to the baseline and earcon conditions (M = 7.08 vs. M = 6.05 and M = 5.99 respectively, p =.035 and p =.002). CONCLUSIONS: The spearcon sounds seem to efficiently reduce visual distraction, whereas the earcon sounds did not reduce distraction measures or increase subjective driving performance. An aspect that must be further investigated is how well spearcons and other types of auditory displays are accepted by drivers in general and how they work in real traffic.

Growth hormone treatment in Aarskog syndrome: analysis of the KIGS (Pharmacia International Growth Database) data.

Aarskog syndrome is an X-linked disorder characterized by faciogenital dysplasia and short stature. The present study set out to determine the effect of growth hormone (GH) therapy in patients with Aarskog syndrome enrolled in KIGS--the Pharmacia International Growth Database. Twenty-one patients (20 males) were evaluated. Median age at start of treatment was 8.3 years (10-90th percentiles, 5.1-14.1 years) and median height SDS was -2.8 (10-90th percentiles, -2.1 to -3.7). The median dose of GH was 0.22 mg/kg/week (10-90th percentiles, 0.15-0.30 mg/kg/week) given at a median frequency of six (4-7) times per week. Prepubertal patients were followed longitudinally for 1 year (n = 13) or 3 years (n = 7). After 1 year, the median height SDS had improved from -2.8 to -2.3 in 13 patients. After 3 years, height SDS had improved significantly (p <0.05) to -1.8 (10-90th percentiles, -2.1 to -1.1) in the seven patients. No adverse events were noted. Although final height data for these patients are still awaited, the present results support the use of GH to promote growth in children with Aarskog syndrome.

Emotion and auditory virtual environments: affect-based judgments of music reproduced with virtual reverberation times.

Emotions are experienced both in real and virtual environments (VEs). Most research to date have focused on the content that causes emotional reactions, but noncontent features of a VE (such as the realism and quality of object rendering) may also influence emotional reactions to the mediated object. The present research studied how noncontent features (different reverberation times) of an auditory VE influenced 76 participants' ratings of emotional reactions and expressed emotional qualities of the sounds. The results showed that the two emotion dimensions of pleasantness and arousal were systematically affected if the same musical piece was rendered with different reverberation times. Overall, it was found that high reverberation time was perceived as most unpleasant. Taken together, the results suggested that noncontent features of a VE influence emotional reactions to mediated objects. Moreover, the study suggests that emotional reactions may be a important aspect of the VE experience that can help complementing standard presence questionnaires and quality evaluations.

When room size matters: acoustic influences on emotional responses to sounds.

When people hear a sound (a "sound object" or a "sound event") the perceived auditory space around them might modulate their emotional responses to it. Spaces can affect both the acoustic properties of the sound event itself and may also impose boundaries to the actions one can take with respect to this event. Virtual acoustic rooms of different sizes were used in a subjective and psychophysiological experiment that evaluated the influence of the auditory space perception on emotional responses to various sound sources. Participants (N = 20) were exposed to acoustic spaces with sound source positions and room acoustic properties varying across the experimental conditions. The results suggest that, overall, small rooms were considered more pleasant, calmer, and safer than big rooms, although this effect of size seems to disappear when listening to threatening sound sources. Sounds heard behind the listeners tended to be more arousing, and elicited larger physiological changes than sources in front of the listeners. These effects were more pronounced for natural, compared to artificial, sound sources, as confirmed by subjective and physiological measures.

Locus Reference Genomic sequences: an improved basis for describing human DNA variants.

As our knowledge of the complexity of gene architecture grows, and we increase our understanding of the subtleties of gene expression, the process of accurately describing disease-causing gene variants has become increasingly problematic. In part, this is due to current reference DNA sequence formats that do not fully meet present needs. Here we present the Locus Reference Genomic (LRG) sequence format, which has been designed for the specific purpose of gene variant reporting. The format builds on the successful National Center for Biotechnology Information (NCBI) RefSeqGene project and provides a single-file record containing a uniquely stable reference DNA sequence along with all relevant transcript and protein sequences essential to the description of gene variants. In principle, LRGs can be created for any organism, not just human. In addition, we recognize the need to respect legacy numbering systems for exons and amino acids and the LRG format takes account of these. We hope that widespread adoption of LRGs - which will be created and maintained by the NCBI and the European Bioinformatics Institute (EBI) - along with consistent use of the Human Genome Variation Society (HGVS)-approved variant nomenclature will reduce errors in the reporting of variants in the literature and improve communication about variants affecting human health. Further information can be found on the LRG web site: http://www.lrg-sequence.org.

De novo search for non-coding RNA genes in the AT-rich genome of Dictyostelium discoideum: performance of Markov-dependent genome feature scoring.

Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.

Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs.

Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.

U1-like snRNAs lacking complementarity to canonical 5' splice sites.

We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5' splice site (5'SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA-protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes.