Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 30 end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode

Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear.ObjectiveWe sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin.MethodsWe applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set.ResultsTwo thousand four hundred thirty differentially expressed genes (false discovery rate, P < .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response.ConclusionThese analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development

Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium. Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faecium. IMPORTANCE Enterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.Peer reviewe

X-ray Spectral Survey with XMM--Newton of a Complete Sample of Nearby Seyfert Galaxies

Results obtained from an X-ray spectral survey of nearby Seyfert galaxies using XMM--Newton are reported. The sample was optically selected, well defined, complete in B mag, and distance limited: it consists of the nearest (D<22 Mpc) 27 Seyfert galaxies (9 of type 1, 18 of type 2) taken from the Ho et al. (1997) sample. This is one of the largest atlases of hard X-ray spectra of low-L active galaxies ever assembled. All nuclear sources except two Sey 2s are detected between 2-10 keV, half for the first time ever, and average spectra are obtained for all of them. Nuclear luminosities reach values down to 10**38 erg/s. The shape of the distribution of X-ray parameters is affected by the presence of Compton-thick objects (> 30% among type 2s). The latter have been identified either directly from their intense FeK line and flat X-ray spectra, or indirectly with flux diagnostic diagrams which use isotropic indicators. After taking into account these highly absorbed sources, we find that (i) the intrinsic X-ray spectral properties (i.e., spectral shapes and luminosities above 2 keV) are consistent between type 1 and type 2 Sey, as expected from ``unified models'', (ii) Sey galaxies as a whole are distributed fairly continuously over the entire range of Nh, between 10**20 and 10**25 cm**-2, and (iii) while Sey 1s tend to have lower Nh and Sey 2s tend to have the highest, we find 30% and 10% exceptions, respectively. Overall the sample well represents the average intrinsic X-ray spectral properties of nearby AGN, including a proper estimate of the distribution of their absorbing columns. Finally, we conclude that, with the exception of a few cases, the present study agrees with predictions of unified models of Sey galaxies, and extends their validity down to very low luminosities.Comment: 23 pages, 4 tables, 4 figures, 2 Appendices with 27 source spectra and notes, to be published in the Astronomy & Astrophysics Journa

Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-seq and ESTs

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct annotation is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3-prime untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3-prime polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3-prime UTR re-annotation (including extension of one 3-prime UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental dataComment: 44 pages, 9 figure

The XMM-SSC survey of hard-spectrum XMM-Newton sources 1: optically bright sources

We present optical and X-ray data for a sample of serendipitous XMM-Newton sources that are selected to have 0.5-2 keV vs 2-4.5 keV X-ray hardness ratios which are harder than the X-ray background. The sources have 2-4.5 keV X-ray flux >= 10^-14 cgs, and in this paper we examine a subsample of 42 optically bright (r < 21) sources; this subsample is 100 per cent spectroscopically identified. All but one of the optical counterparts are extragalactic, and we argue that the single exception, a Galactic M star, is probably a coincidental association. The X-ray spectra are consistent with heavily absorbed power laws (21.8 < log NH < 23.4), and all of them appear to be absorbed AGN. The majority of the sources show only narrow emission lines in their optical spectra, implying that they are type-2 AGN. Only a small fraction of the sources (7/42) show broad optical emission lines, and all of these have NH < 10^23 cm^-2. This implies that ratios of X-ray absorption to optical/UV extinction equivalent to > 100 times the Galactic gas-to-dust ratio are rare in AGN absorbers (at most a few percent of the population), and may be restricted to broad absorption-line QSOs. Seven objects appear to have an additional soft X-ray component in addition to the heavily absorbed power law. We consider the implications of our results in the light of the AGN unified scheme. We find that the soft components in narrow-line objects are consistent with the unified scheme provided that > 4 per cent of broad-line AGN have ionised absorbers that attenuate their soft X-ray flux by >50 per cent. In at least one of the X-ray absorbed, broad-line AGN in our sample the X-ray spectrum requires an ionised absorber, consistent with this picture.Comment: accepted for publication in MNRA

Detection of strongly processed ice in the central starburst of NGC4945

The composition of ice grains provides an important tool for the study of the molecular environment of star forming regions. Using ISAAC at the VLT to obtain spectra around 4.65 microns we have detected for the first time `XCN' and CO ice in an extragalactic environment: the nuclear region of the nearby dusty starburst/AGN galaxy NGC4945. The profile of the solid CO band reveals the importance of thermal processing of the ice while the prominence of the XCN band attests to the importance of energetic processing of the ice by FUV radiation and/or energetic particles. In analogy to the processing of ices by embedded protostars in our Galaxy, we attribute the processing of the ices in the center of NGC4945 to ongoing massive star formation. Our M-band spectrum also shows strong HI Pfund-beta and H2 0-0 S(9) line emission and gas phase CO absorption lines. The HI, H2, PAH, gas phase CO and the ices seem to be embedded in a rotating molecular disk which is undergoing vigorous star formation. Recently, strong OCN- absorption has been detected in the spectrum of the Galactic center star GC:IRS19. The most likely environment for the OCN- absorption is the strongly UV-exposed GC molecular ring. The presence of processed ice in the center of NGC4945 and our Galactic center leads us to believe that processed ice may be a common characteristic of dense molecular material in star forming galactic nuclei.Comment: 9 pages, 8 figures. Accepted for publication in A&A. Also available at this http://www.astro.rug.nl/~spoon/publications.htm

X-ray Absorption and Reflection in Active Galactic Nuclei

X-ray spectroscopy offers an opportunity to study the complex mixture of emitting and absorbing components in the circumnuclear regions of active galactic nuclei, and to learn about the accretion process that fuels AGN and the feedback of material to their host galaxies. We describe the spectral signatures that may be studied and review the X-ray spectra and spectral variability of active galaxies, concentrating on progress from recent Chandra, XMM-Newton and Suzaku data for local type 1 AGN. We describe the evidence for absorption covering a wide range of column densities, ionization and dynamics, and discuss the growing evidence for partial-covering absorption from data at energies > 10 keV. Such absorption can also explain the observed X-ray spectral curvature and variability in AGN at lower energies and is likely an important factor in shaping the observed properties of this class of source. Consideration of self-consistent models for local AGN indicates that X-ray spectra likely comprise a combination of absorption and reflection effects from material originating within a few light days of the black hole as well as on larger scales. It is likely that AGN X-ray spectra may be strongly affected by the presence of disk-wind outflows that are expected in systems with high accretion rates, and we describe models that attempt to predict the effects of radiative transfer through such winds, and discuss the prospects for new data to test and address these ideas.Comment: Accepted for publication in the Astronomy and Astrophysics Review. 58 pages, 9 figures. V2 has fixed an error in footnote

Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1: 10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities

X-ray bright sources in the Chandra Small Magellanic Cloud Wing Survey - detection of two new pulsars

We investigate the X-ray and optical properties of a sample of X-ray bright sources from the Small Magellanic Cloud (SMC) Wing Survey. We have detected two new pulsars with pulse periods of 65.8 s (CXOU J010712.6-723533) and 700 s (CXOU J010206.6-714115), and present observations of two previously known pulsars RX J0057.3-7325 (SXP101) and SAX J0103.2-7209 (SXP348). Our analysis has led to three new optical identifications for the detected pulsars. We find long-term optical periods for two of the pulsars, CXOU J010206.6-714115 and SXP101, of 267 and 21.9 d, respectively. Spectral analysis of a sub-set of the sample shows that the pulsars have harder spectra than the other sources detected. By employing a quantile-based colour-colour analysis we are able to separate the detected pulsars from the rest of the sample. Using archival catalogues we have been able to identify counterparts for the majority of the sources in our sample. Combining this with our results from the temporal analysis of the Chandra data and archival optical data, the X-ray spectral analysis, and by determining the X-ray to optical flux ratios we present preliminary classifications for the sources. In addition to the four detected pulsars, our sample includes two candidate foreground stars, 12 probable active galactic nuclei, and five unclassified sources.Comment: 13 pages, 7 figues, 4 tables, accepted for publication in MNRA