Phage ϕC2 mediates transduction of Tn6215, encoding erythromycin resistance, between Clostridium difficile strains

UNLABELLED: In this work, we show that Clostridium difficile phage ϕC2 transduces erm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10(-6) per PFU. The transductants were lysogenic for ϕC2 and contained the erm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating. IMPORTANCE: Clostridium difficile is a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics. C. difficile is potentially zoonotic and has been detected in animals, food, and environmental samples. C. difficile genomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element in C. difficile. Transduction may also be a useful tool for the genetic manipulation of C. difficile.Peer reviewe

Using oligonucleotide signatures to build a system for effective detection of pathogenic bacteria in metagenomic samples

Pathogenic bacteria are responsible for millions of deaths every year with an estimated mortality of 70 million people by 2010 for Mycobacterium tuberculosis alone. Novel methods for identification of bacterial species in hosts, urban environments, water sources and food stuffs are required to advance diagnosis and preventative medicine. Detection of bacterial species in environmental samples is a complex task since large numbers of bacteria are present and are resistant to culturing. Therefore, the genetic content of the entire sample has to be analysed simultaneously and this constitutes a metagenomic sample. Commonly-used methods of bacterial identification focus on detection of specific genomic regions to determine species. Currently only one percent of a metagenomic sample can be used for identification employing phylogenetic markers. This method is highly inefficient. The search for more widespread markers within each genome is essential to improve detection methods. Also, modern sequencing technologies used in these environments have short read lengths which prove difficult to assemble e.g. repeats can lead to incorrect assembly. The use of overrepresented oligonucleotides provides a solution to both of these difficulties. Overrepresented oligonucleotides (8-14bp in length) are utilised to differentiate between species based on observed frequency of occurrence rather than presence or absence. They occur throughout the genome thereby increasing genomic coverage. Furthermore, overrepresented oligonucleotides can be easily identified in a raw metagenomic sample, bypassing the need for sequence assembly. Raw oligonucleotide data was filtered, analysed and imported into a structured database. A program, Oligosignatures, allowed for creation of species and phylogenetic lineage specific oligonucleotide markers dependent on the selection of species specified by the user. For the purposes of this study, the context of bacterial identification in an unknown environment was selected. A similarity trial was then executed to determine if strains of the same species can be separated from each other using overrepresented oligonucleotides. Outcomes of this test provided a guideline for the creation of species and lineage specific oligonucleotide markers. Each species and lineage was therefore described by a marker profile which consisted of representative oligonucleotide markers. These marker profiles were then tested against artificial and experimental data to determine their effectivity. Two approaches were used for testing, namely Oligonucleotide frequency analysis and Sequence read analysis. Oligonucleotide frequency analysis focused on the identification of species dependent on the global frequencies of marker oligonucleotides within each marker profile. Sequence read analysis attempted to assign metagenomic reads to a specific species dependent on the number of marker oligonucleotides present within the read. The final database contained 439 bacterial genomes from 22 different phylogenetic lineages. Interpretation of the results obtained after strain similarity testing showed that strains of the same species had highly similar markers and were not separable using this approach. All strains of a species that conformed to this premise were reduced to a single representative member. Similarly, species marker profiles demonstrated that closely related species remained difficult to separate. Twenty-one of the 22 lineages showed sufficient lineage specific markers for use in testing. This provides support for the abundance of overrepresented oligonucleotides and their potential for use as a detection method. In general, metagenomic testing of marker profiles showed that species specific determination was prone to interference, specifically, in closely related species. However, more distantly related species could be separated using both methods. Lineage discrimination generated more reliable results proving that lineage determination was possible in both artificial and experimental datasets. Oligonucleotide frequency analysis, the most sensitive approach, showed the best results for lineage determination but poorer results for species identification. Sequence read analysis provided a more effective method of determining confidence using different thresholds for read classification. In conclusion, the use of overrepresented oligonucleotides holds promise as a novel method for bacterial identification in a metagenomic context. Although several obstacles still prevent optimal utilization of these oligonucleotides, with further research the classification and identification of species and phylogenetic lineages from metagenomic samples can become a reality. CopyrightDissertation (MSc)--University of Pretoria, 2009.Biochemistryunrestricte

Individual Ability to Learn a Parallel Processing Technique and Musical Aptitude.

Correlations between music training and psychosocial skills, sensory abilities, and aspects of intelligence, are sorted into primary or secondary effects. Correlations between these areas of human development and music training lack support pertaining to the underlying cognitive networks that these processes rely on. Thus, this study was based on the work of Baddeley and Hitch\u27s model of working memory, and implemented a test of parallel processing (Articulatory Suppression Task, AST), which measures proficiency of working memory systems. Individual differences therein, were compared with music aptitude. Participants were gathered throughout urban and rural regions of the state of Oregon. Half the participants received specific training on how to excel on AST, the other half received no training. The training was based on research showing musicians to be more proficient in rhythm, the phonological loop, and mental imagery. Group AST pretest/posttest scores and the Drake Musical Aptitude test scores were analyzed using 2-tailed t test and regression models for within-group and between-group variation. No significant difference between musical aptitude and participant ability to increase proficiency with parallel processing was found, however, the results indicated that music training influences proficiency with parallel processing in general, and there were indicators that a ceiling effect may have confounded the pretest-posttest range in scores. This supports findings of previous research that musical training has beneficial influences on mathematics, socio-emotional awareness, motor skills, language, and general intelligence, highlighting that positive social change may result if music were a core class in K-12 education

Land degradation neutrality: testing the indicator in a temperate agricultural landscape

Land degradation directly affects around 25% of land globally, undermining progress on most of the UN Sustainable Development Goals (SDG), particularly target 15.3. To assess land degradation, SDG indicator 15.3.1 combines sub-indicators of productivity, soil carbon and land cover. Over 100 countries have set Land Degradation Neutrality (LDN) targets. Here, we demonstrate application of the indicator for a well-established agricultural landscape using the case study of Great Britain. We explore detection of degradation in such landscapes by: 1) transparently evaluating land cover transitions; 2) comparing assessments using global and national data; 3) identifying misleading trends; and 4) including extra sub-indicators for additional forms of degradation. Our results demonstrate significant impacts on the indicator both from the land cover transition evaluation and choice or availability of data. Critically, we identify a misleading improvement trend due to a trade-off between improvement detected by the productivity sub-indicator, and 30-year soil carbon loss trends in croplands (11% from 1978 to 2007). This carbon loss trend would not be identified without additional data from Countryside Survey (CS). Thus, without incorporating field survey data we risk overlooking the degradation of regulating and supporting ecosystem services (linked to soil carbon), in favour of signals from improving provisioning services (productivity sub-indicator). Relative importance of these services will vary between socioeconomic contexts. Including extra sub-indicators for erosion or critical load exceedance, as additional forms of degradation, produced a switch from net area improving (9%) to net area degraded (58%). CS data also identified additional degradation for soil health, including 44% arable soils exceeding bulk density thresholds and 35% of CS squares exceeding contamination thresholds for metals

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. \uc2\ua9 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research

Exon junction complex shapes the transcriptome by repressing recursive splicing

Recursive splicing (RS) starts by defining an “RS-exon,” which is then spliced to the preceding exon, thus creating a recursive 5′ splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases

Heteromeric RNP Assembly at LINEs Controls Lineage-Specific RNA Processing.

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT

Clinical, radiologic, pathologic, and molecular characteristics of long-term survivors of diffuse intrinsic pontine glioma (DIPG): a collaborative report from the International and European Society for Pediatric Oncology DIPG registries

Purpose Diffuse intrinsic pontine glioma (DIPG) is a brainstem malignancy with a median survival of < 1 year. The International and European Society for Pediatric Oncology DIPG Registries collaborated to compare clinical, radiologic, and histomolecular characteristics between short-term survivors (STSs) and long-term survivors (LTSs). Materials and Methods Data abstracted from registry databases included patients from North America, Australia, Germany, Austria, Switzerland, the Netherlands, Italy, France, the United Kingdom, and Croatia. Results Among 1,130 pediatric and young adults with radiographically confirmed DIPG, 122 (11%) were excluded. Of the 1,008 remaining patients, 101 (10%) were LTSs (survival ≥ 2 years). Median survival time was 11 months (interquartile range, 7.5 to 16 months), and 1-, 2-, 3-, 4-, and 5-year survival rates were 42.3% (95% CI, 38.1% to 44.1%), 9.6% (95% CI, 7.8% to 11.3%), 4.3% (95% CI, 3.2% to 5.8%), 3.2% (95% CI, 2.4% to 4.6%), and 2.2% (95% CI, 1.4% to 3.4%), respectively. LTSs, compared with STSs, more commonly presented at age < 3 or > 10 years (11% v 3% and 33% v 23%, respectively; P < .001) and with longer symptom duration ( P < .001). STSs, compared with LTSs, more commonly presented with cranial nerve palsy (83% v 73%, respectively; P = .008), ring enhancement (38% v 23%, respectively; P = .007), necrosis (42% v 26%, respectively; P = .009), and extrapontine extension (92% v 86%, respectively; P = .04). LTSs more commonly received systemic therapy at diagnosis (88% v 75% for STSs; P = .005). Biopsies and autopsies were performed in 299 patients (30%) and 77 patients (10%), respectively; 181 tumors (48%) were molecularly characterized. LTSs were more likely to harbor a HIST1H3B mutation (odds ratio, 1.28; 95% CI, 1.1 to 1.5; P = .002). Conclusion We report clinical, radiologic, and molecular factors that correlate with survival in children and young adults with DIPG, which are important for risk stratification in future clinical trials