Oxford nanopore MinION sequencing enables rapid whole genome assembly of rickettsia typhi in a resource-limited setting

The infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies’ portable MinION sequencer. However, the question remains open whether MinION-based bacterial whole genome sequencing is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate of Rickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR, using v9.5 chemistry, and in parallel, we sequenced the same isolate on the Illumina platform in a genomics laboratory in the United Kingdom. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 indel errors. The combined assembly represents the first complete genome sequence of a human R. typhi isolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, with no rearrangements. Filtering based on the known error profile of MinION data improved the accuracy of the nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. Although nanopore-only sequencing cannot yet recover phylogenetic signals in R. typhi, such an approach may be applicable for more diverse organism

Deconstructing Insight: EEG Correlates of Insightful Problem Solving

Background: Cognitive insight phenomenon lies at the core of numerous discoveries. Behavioral research indicates four salient features of insightful problem solving: (i) mental impasse, followed by (ii) restructuring of the problem representation, which leads to (iii) a deeper understanding of the problem, and finally culminates in (iv) an “Aha!” feeling of suddenness and obviousness of the solution. However, until now no efforts have been made to investigate the neural mechanisms of these constituent features of insight in a unified framework. Methodology/Principal Findings: In an electroencephalographic study using verbal remote associate problems, we identified neural correlates of these four features of insightful problem solving. Hints were provided for unsolved problems or after mental impasse. Subjective ratings of the restructuring process and the feeling of suddenness were obtained on trial-by-trial basis. A negative correlation was found between these two ratings indicating that sudden insightful solutions, where restructuring is a key feature, involve automatic, subconscious recombination of information. Electroencephalogram signals were analyzed in the space×time×frequency domain with a nonparametric cluster randomization test. First, we found strong gamma band responses at parieto-occipital regions which we interpreted as (i) an adjustment of selective attention (leading to a mental impasse or to a correct solution depending on the gamma band power level) and (ii) encoding and retrieval processes for the emergence of spontaneous new solutions. Secondly, we observed an increased upper alpha band response in right temporal regions (suggesting active suppression of weakly activated solution relevant information) for initially unsuccessful trials that after hint presentation led to a correct solution. Finally, for trials with high restructuring, decreased alpha power (suggesting greater cortical excitation) was observed in right prefrontal area. Conclusions/Significance: Our results provide a first account of cognitive insight by dissociating its constituent components and potential neural correlates

Genotypic and phenotypic variation among Staphylococcus saprophyticus from human and animal isolates

<p>Abstract</p> <p>Background</p> <p>The main aim of this study was to examine the genotypic and phenotypic diversity of <it>Staphylococcus saprophyticus </it>isolates from human and animal origin.</p> <p>Findings</p> <p>In total, 236 clinical isolates and 15 animal isolates of <it>S. saprophyticus </it>were characterized in respect of the occurrence of 9 potential virulence genes and four surface properties. All strains were PCR positive for the regulatory genes <it>agr</it>, <it>sar</it>>it>A and <it>rot </it>as well as for the surface proteins UafA and Aas. Nearly 90% of the clinical isolates were found to possess the gene for the surface-associated lipase Ssp and 10% for the collagen binding MSCRAMM SdrI. All animal isolates were negative for<it>sdrI</it>. Lipolytic activity could be detected in 66% of the clinical and 46% of the animal isolates. Adherence to collagen type I was shown of 20% of the clinical strains and 6% of the strains of animal origin. Most <it>S. saprophyticus </it>strains showed hydrophobic properties and only few could agglutinate sheep erythrocytes.</p> <p>Conclusions</p> <p>We described a broad analysis of animal and human <it>S. saprophyticus </it>isolates regarding virulence genes and phenotypic properties such as lipase activity, hydrophobicity, and adherence. While <it>S. saprophyticus </it>strains from animal sources have prerequisites for colonization of the urinary tract like the D-serine-deaminase, out findings suggested that they need to acquire new genes e.g. MSCRAMMS for adherence like sdrI and to modulate their existing properties e.g. increasing the lipase activity or reducing hydrophobicity. These apparently important new genes or properties for virulence have to be further analyzed.</p

Unlimited multistability in multisite phosphorylation systems

Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins (1, 2). The number of phosphorylated sites on a protein (n) shows a significant increase from prokaryotes, with n less than or equal to 7 sites, to eukaryotes, with examples having n greater than or equal to 150 sites (3). Multisite phosphorylation has many roles (4, 5) and site conservation indicates that increasing numbers of sites cannot be due merely to promiscuous phosphorylation. A substrate with n sites has an exponential number (2^n) of phospho-forms and individual phospho-forms may have distinct biological effects (6, 7). The distribution of these phospho-forms and how this distribution is regulated have remained unknown. Here we show that, when kinase and phosphatase act in opposition on a multisite substrate, the system can exhibit distinct stable phospho-form distributions at steady state and that the maximum number of such distributions increases with n. Whereas some stable distributions are focused on a single phospho-form, others are more diffuse, giving the phospho-proteome the potential to behave as a fluid regulatory network able to encode information and flexibly respond to varying demands. Such plasticity may underlie complex information processing in eukaryotic cells (8) and suggests a functional advantage in having many sites. Our results follow from the unusual geometry of the steady-state phospho-form concentrations, which we show to constitute a rational algebraic curve, irrespective of n. We thereby reduce the complexity of calculating steady states from simulating 3 times 2^n differential equations to solving two algebraic equations, while treating parameters symbolically. We anticipate that these methods can be extended to systems with multiple substrates and multiple enzymes catalysing different modifications, as found in posttranslational modification 'codes' (9) such as the histone code (10, 11). Whereas simulations struggle with exponentially increasing molecular complexity, mathematical methods of the kind developed here can provide a new language in which to articulate the principles of cellular information processing (12)

Bats host major mammalian paramyxoviruses

The large virus family Paramyxoviridae includes some of the most significant human and livestock viruses, such as measles-, distemper-, mumps-, parainfluenza-, Newcastle disease-, respiratory syncytial virus and metapneumoviruses. Here we identify an estimated 66 new paramyxoviruses in a worldwide sample of 119 bat and rodent species (9,278 individuals). Major discoveries include evidence of an origin of Hendra- and Nipah virus in Africa, identification of a bat virus conspecific with the human mumps virus, detection of close relatives of respiratory syncytial virus, mouse pneumonia- and canine distemper virus in bats, as well as direct evidence of Sendai virus in rodents. Phylogenetic reconstruction of host associations suggests a predominance of host switches from bats to other mammals and birds. Hypothesis tests in a maximum likelihood framework permit the phylogenetic placement of bats as tentative hosts at ancestral nodes to both the major Paramyxoviridae subfamilies (Paramyxovirinae and Pneumovirinae). Future attempts to predict the emergence of novel paramyxoviruses in humans and livestock will have to rely fundamentally on these data

Phase I trial of intravesical Suramin in recurrent superficial transitional cell bladder carcinoma

Suramin is an antitrypanosomal agent with antineoplastic activity, but with serious systemic side effects. We administered Suramin intravesically to determine a concentration with low toxicity but with evidence of a pharmacodynamic effect, to recommend a dose level for phase II trials. This was an open-labelled, nonrandomised dose-escalation phase I study. In all, 12 patients with a history of recurrent superficial bladder cancer were grouped into four dose levels (10–150 mg ml−1 in 60 ml saline). Six catheter instillations at weekly intervals were used. Cystoscopy and biopsy were performed before and 3 months after the start of treatment. Suramin was assayed using high-performance liquid chromatography, vascular endothelial growth factor (VEGF) using ELISA (enzyme-linked immunosorbent assay), and urinary protein profile using surface-enhanced laser desorption ionisation mass spectroscopy (SELDI). Minimal systemic absorption of Suramin was found at the highest dose of 150 mg ml−1. Urinary VEGF was affected by Suramin at doses above 50 mg ml−1, corresponding to the estimated threshold of saturation of Suramin binding to urine albumin. SELDI showed a specific disappearance of urinary protein peaks during treatment. Intravesical Suramin shows lack of toxicity and low systemic absorption. The results of this phase I trial support expanded clinical trials of efficacy at a dose of 100 mg ml−1 intravesically

Microevolutionary analysis of Clostridium difficile genomes to investigate transmission

Background The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing. Results Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions. Conclusions Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens

A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity

Including gaming disorder in the ICD-11: the need to do so from a clinical and public health perspective

The proposed introduction of gaming disorder (GD) in the 11th revision of the International Classification of Diseases (ICD-11) developed by the World Health Organization (WHO) has led to a lively debate over the past year. Besides the broad support for the decision in the academic press, a recent publication by van Rooij et al. (2018) repeated the criticism raised against the inclusion of GD in ICD-11 by Aarseth et al. (2017). We argue that this group of researchers fails to recognize the clinical and public health considerations, which support the WHO perspective. It is important to recognize a range of biases that may influence this debate; in particular, the gaming industry may wish to diminish its responsibility by claiming that GD is not a public health problem, a position which maybe supported by arguments from scholars based in media psychology, computer games research, communication science, and related disciplines. However, just as with any other disease or disorder in the ICD-11, the decision whether or not to include GD is based on clinical evidence and public health needs. Therefore, we reiterate our conclusion that including GD reflects the essence of the ICD and will facilitate treatment and prevention for those who need it

Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells

Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens