DODGE: Automated point source bacterial outbreak detection using cumulative long term genomic surveillance.

SUMMARY: The reliable and timely recognition of outbreaks is a key component of public health surveillance for foodborne diseases. Whole genome sequencing (WGS) offers high resolution typing of foodborne bacterial pathogens and facilitates the accurate detection of outbreaks. This detection relies on grouping WGS data into clusters at an appropriate genetic threshold, however, methods and tools for selecting and adjusting such thresholds according to the required resolution of surveillance and epidemiological context are lacking. Here we present DODGE (Dynamic Outbreak Detection for Genomic Epidemiology), an algorithm to dynamically select and compare these genetic thresholds. DODGE can analyse expanding datasets over time and clusters that are predicted to correspond to outbreaks (or 'investigation clusters') can be named with the established genomic nomenclature systems to facilitate integrated analysis across jurisdictions. DODGE was tested in two real-world genomic surveillance datasets of different duration, two months from Australia and nine years from the UK. In both cases only a minority of isolates were identified as investigation clusters. Two known outbreaks in the UK dataset were detected by DODGE and were recognised at an earlier timepoint than the outbreaks were reported. These findings demonstrated the potential of the DODGE approach to improve the effectiveness and timeliness of genomic surveillance for foodborne diseases and the effectiveness of the algorithm developed. AVAILABILITY: DODGE is freely available at https://github.com/LanLab/dodge and can easily be installed using Conda. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DksA is a conserved master regulator of stress response in Acinetobacter baumannii.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Functional characterisation of substrate-binding proteins to address nutrient uptake in marine picocyanobacteria.

Marine cyanobacteria are key primary producers, contributing significantly to the microbial food web and biogeochemical cycles by releasing and importing many essential nutrients cycled through the environment. A subgroup of these, the picocyanobacteria (Synechococcus and Prochlorococcus), have colonised almost all marine ecosystems, covering a range of distinct light and temperature conditions, and nutrient profiles. The intra-clade diversities displayed by this monophyletic branch of cyanobacteria is indicative of their success across a broad range of environments. Part of this diversity is due to nutrient acquisition mechanisms, such as the use of high-affinity ATP-binding cassette (ABC) transporters to competitively acquire nutrients, particularly in oligotrophic (nutrient scarce) marine environments. The specificity of nutrient uptake in ABC transporters is primarily determined by the peripheral substrate-binding protein (SBP), a receptor protein that mediates ligand recognition and initiates translocation into the cell. The recent availability of large numbers of sequenced picocyanobacterial genomes indicates both Synechococcus and Prochlorococcus apportion >50% of their transport capacity to ABC transport systems. However, the low degree of sequence homology among the SBP family limits the reliability of functional assignments using sequence annotation and prediction tools. This review highlights the use of known SBP structural representatives for the uptake of key nutrient classes by cyanobacteria to compare with predicted SBP functionalities within sequenced marine picocyanobacteria genomes. This review shows the broad range of conserved biochemical functions of picocyanobacteria and the range of novel and hypothetical ABC transport systems that require further functional characterisation.

Myasthenia in pregnancy: best practice guidelines from a U.K. multispecialty working group.

A national U.K. workshop to discuss practical clinical management issues related to pregnancy in women with myasthenia gravis was held in May 2011. The purpose was to develop recommendations to guide general neurologists and obstetricians and facilitate best practice before, during and after pregnancy. The main conclusions were (1) planning should be instituted well in advance of any potential pregnancy to allow time for myasthenic status and drug optimisation; (2) multidisciplinary liaison through the involvement of relevant specialists should occur throughout pregnancy, during delivery and in the neonatal period; (3) provided that their myasthenia is under good control before pregnancy, the majority of women can be reassured that it will remain stable throughout pregnancy and the postpartum months; (4) spontaneous vaginal delivery should be the aim and actively encouraged; (5) those with severe myasthenic weakness need careful, multidisciplinary management with prompt access to specialist advice and facilities; (6) newborn babies born to myasthenic mothers are at risk of transient myasthenic weakness, even if the mother's myasthenia is well-controlled, and should have rapid access to neonatal high-dependency support.

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria.

Functional genomics techniques, such as transposon insertion sequencing and RNA-sequencing, are key to studying relative differences in bacterial mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy numbers along the chromosome. This can lead to artifacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control. Further, relative differences in gene-wise read counts may result from alterations in chromosomal replication dynamics, rather than selection or direct gene regulation. We term this artifact "chromosomal location bias" and implement a principled statistical approach to correct it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated into statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analyzing a ciprofloxacin-treated transposon insertion sequencing data set in Escherichia coli as a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress.IMPORTANCEAltered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.

Chromosome-scale assembly of the streamlined picoeukaryote Picochlorum sp. SENEW3 genome reveals Rabl-like chromatin structure and potential for C4 photosynthesis.

Genome sequencing and assembly of the photosynthetic picoeukaryotic Picochlorum sp. SENEW3 revealed a compact genome with a reduced gene set, few repetitive sequences, and an organized Rabl-like chromatin structure. Hi-C chromosome conformation capture revealed evidence of possible chromosomal translocations, as well as putative centromere locations. Maintenance of a relatively few selenoproteins, as compared to similarly sized marine picoprasinophytes Mamiellales, and broad halotolerance compared to others in Trebouxiophyceae, suggests evolutionary adaptation to variable salinity environments. Such adaptation may have driven size and genome minimization and have been enabled by the retention of a high number of membrane transporters. Identification of required pathway genes for both CAM and C4 photosynthetic carbon fixation, known to exist in the marine mamiellale pico-prasinophytes and seaweed Ulva, but few other chlorophyte species, further highlights the unique adaptations of this robust alga. This high-quality assembly provides a significant advance in the resources available for genomic investigations of this and other photosynthetic picoeukaryotes.

Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin.

Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are ß-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. IMPORTANCE Due to the growing antibiotic resistance crisis, there is a critical need to develop new antibiotics, particularly compounds capable of targeting high-priority antibiotic-resistant Gram-negative pathogens. In order to develop new compounds capable of overcoming resistance a greater understanding of how Gram-negative bacteria are able to prevent the uptake and accumulation of many antibiotics is required. This study used a novel genome wide approach to investigate the significance of a primary amine group as a chemical feature that promotes the uptake and accumulation of compounds in the Gram-negative model organism Escherichia coli. The results support previous biochemical observations that the primary amine promotes passage through the outer membrane porin OmpF, but also highlight active efflux as a major resistance factor.

Proteomics dataset from 26th Dynasty Egyptian mummified remains sampled using minimally invasive skin sampling tape strips.

Paleoproteomics typically involves the destructive sampling of precious bioarchaeological materials. This analysis aims to investigate the proteins identifiable via nanoLC-MS/MS from highly degraded 26th Dynasty Egyptian mummified human remains (NMR.29.1-8) after non-destructive sampling with commercially available dermatology-grade skin sampling tape strips. A collection of cranial and other bone fragments were sampled with the tape strips then subsequently analysed using a shotgun proteomics approach. The number of proteins identified using this method ranged from 18 to 437 at a peptide FDR of <1%. Deamidation ratios were assessed using an in-house R script, with asparagine deamidation averaging ∼20-30% and glutamine deamidation averaging ∼15-25%.

Perioperative fasting in adults and children: guidelines from the European Society of Anaesthesiology.

This guideline aims to provide an overview of the present knowledge on aspects of perioperative fasting with assessment of the quality of the evidence. A systematic search was conducted in electronic databases to identify trials published between 1950 and late 2009 concerned with preoperative fasting, early resumption of oral intake and the effects of oral carbohydrate mixtures on gastric emptying and postoperative recovery. One study on preoperative fasting which had not been included in previous reviews and a further 13 studies published since the most recent review were identified. The searches also identified 20 potentially relevant studies of oral carbohydrates and 53 on early resumption of oral intake. Publications were classified in terms of their evidence level, scientific validity and clinical relevance. The Scottish Intercollegiate Guidelines Network scoring system for assessing level of evidence and grade of recommendations was used. The key recommendations are that adults and children should be encouraged to drink clear fluids up to 2 h before elective surgery (including caesarean section) and all but one member of the guidelines group consider that tea or coffee with milk added (up to about one fifth of the total volume) are still clear fluids. Solid food should be prohibited for 6 h before elective surgery in adults and children, although patients should not have their operation cancelled or delayed just because they are chewing gum, sucking a boiled sweet or smoking immediately prior to induction of anaesthesia. These recommendations also apply to patients with obesity, gastro-oesophageal reflux and diabetes and pregnant women not in labour. There is insufficient evidence to recommend the routine use of antacids, metoclopramide or H2-receptor antagonists before elective surgery in non-obstetric patients, but an H2-receptor antagonist should be given before elective caesarean section, with an intravenous H2-receptor antagonist given prior to emergency caesarean section, supplemented with 30 ml of 0.3 mol l(-1) sodium citrate if general anaesthesia is planned. Infants should be fed before elective surgery. Breast milk is safe up to 4 h and other milks up to 6 h. Thereafter, clear fluids should be given as in adults. The guidelines also consider the safety and possible benefits of preoperative carbohydrates and offer advice on the postoperative resumption of oral intake.

How antibiotics work together: molecular mechanisms behind combination therapy.

Antibiotics used in combination are an effective strategy for combatting numerous infectious diseases in clinical and veterinary settings, particularly as a last-line therapy for difficult-to-treat cases. Combination therapy can either increase or slow the rate of killing, broaden the antibiotic spectrum, reduce dosage and unwanted side-effects, and even control the emergence of resistance. The administration of antibiotics in combination has been used effectively against bacterial infections for >70 years, first used to treat tuberculosis. However, effective antibiotic combinations and their dosage regimes have been largely determined empirically in the clinic, and the molecular mechanisms underpinning how these combinations work remains surprisingly elusive. This review focuses on studies that have outlined the genetics and molecular mechanisms of action underlying antibiotic combinations, as well as those that examine how resistance develops. We highlight the need for further experimentation and genetic validation to fully realise the potential of combination therapy.

The MEK inhibitor selumetinib reduces spinal neurofibroma burden in patients with NF1 and plexiform neurofibromas.

BACKGROUND: Spinal neurofibromas (SNFs) in neurofibromatosis type 1 (NF1) can cause progressive spinal cord compression and neurological dysfunction. The MEK inhibitor selumetinib shrinks the majority of plexiform neurofibromas (PNs) in patients with NF1. We assessed the effect of selumetinib on SNF. METHODS: Pediatric and adult patients with NF1 and inoperable PN participating in phase 2 studies of selumetinib for PN were included in this analysis if they had SNF and serial spine magnetic resonance imaging (MRI). Selumetinib was administered orally at the recommended dose of 25 mg/m2/dose twice daily (max 50 mg b.i.d.; 1 cycle = 28 days). We qualitatively assessed the effect of selumetinib on SNF-related spinal canal distortion, cerebrospinal fluid distribution, and spinal cord deformity on MRI. RESULTS: Twenty-four patients (18 male), median age 16.9 years (range, 6.2-60.3), had SNF, 22 of which were associated with the same nerves as the target PN assessed on the clinical trial. Twenty patients had spinal cord deformity. Twenty-three patients completed at least 12 treatment cycles to date. Eighteen patients showed subtle to a marked improvement in SNF burden, 5 remained stable, and no worsening was observed during treatment. CONCLUSIONS: This is the first study describing the effect of selumetinib on SNF. Of 24 patients, 18 exhibited some improvement of SNF burden on imaging. These findings suggest that selumetinib may prevent the worsening of cord compression, potentially reducing the need for surgical interventions in select patients or benefitting patients who do not have a surgical option. Prospective evaluation of the clinical benefit of selumetinib for SNF is warranted.

Targeting Refractory Sarcomas and Malignant Peripheral Nerve Sheath Tumors in a Phase I/II Study of Sirolimus in Combination with Ganetespib (SARC023).

PURPOSE: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas. Combining Hsp90 inhibitors to enhance endoplasmic reticulum stress with mTOR inhibition results in dramatic MPNST shrinkage in a genetically engineered MPNST mouse model. Ganetespib is an injectable potent small molecule inhibitor of Hsp90. Sirolimus is an oral mTOR inhibitor. We sought to determine the safety, tolerability, and recommended dose of ganetespib and sirolimus in patients with refractory sarcomas and assess clinical benefits in patients with unresectable/refractory MPNSTs. Patients and Methods. In this multi-institutional, open-label, phase 1/2 study of ganetespib and sirolimus, patients ≥16 years with histologically confirmed refractory sarcoma (phase 1) or MPNST (phase 2) were eligible. A conventional 3 + 3 dose escalation design was used for phase 1. Pharmacokinetic and pharmacodynamic measures were evaluated. Primary objectives of phase 2 were to determine the clinical benefit rate (CBR) of this combination in MPNSTs. Patient-reported outcomes assessed pain. RESULTS: Twenty patients were enrolled (10 per phase). Toxicities were manageable; most frequent non-DLTs were diarrhea, elevated liver transaminases, and fatigue. The recommended dose of ganetespib was 200 mg/m2 intravenously on days 1, 8, and 15 with sirolimus 4 mg orally once daily with day 1 loading dose of 12 mg. In phase 1, one patient with leiomyosarcoma achieved a sustained partial response. In phase 2, no responses were observed. The median number of cycles treated was 2 (1-4). Patients did not meet the criteria for clinical benefit as defined per protocol. Pain ratings decreased or were stable. CONCLUSION: Despite promising preclinical rationale and tolerability of the combination therapy, no responses were observed, and the study did not meet parameters for further evaluation in MPNSTs. This trial was registered with (NCT02008877).

Complete Genome Sequence of Pseudomonas aeruginosa Reference Strain PAK.

We report the complete genome of Pseudomonas aeruginosa strain PAK, a strain which has been instrumental in the study of a range of P. aeruginosa virulence and pathogenesis factors and has been used for over 50 years as a laboratory reference strain.

Effect of food intake during labour on obstetric outcome: randomised controlled trial.

OBJECTIVE: To investigate the effect of feeding during labour on obstetric and neonatal outcomes. DESIGN: Prospective randomised controlled trial. SETTING: Birth centre in London teaching hospital. PARTICIPANTS: 2426 nulliparous, non-diabetic women at term, with a singleton cephalic presenting fetus and in labour with a cervical dilatation of less than 6 cm. INTERVENTION: Consumption of a light diet or water during labour. MAIN OUTCOME MEASURES: The primary outcome measure was spontaneous vaginal delivery rate. Other outcomes measured included duration of labour, need for augmentation of labour, instrumental and caesarean delivery rates, incidence of vomiting, and neonatal outcome. RESULTS: The spontaneous vaginal delivery rate was the same in both groups (44%; relative risk 0.99, 95% confidence interval 0.90 to 1.08). No clinically important differences were found in the duration of labour (geometric mean: eating, 597 min v water, 612 min; ratio of geometric means 0.98, 95% confidence interval 0.93 to 1.03), the caesarean delivery rate (30% v 30%; relative risk 0.99, 0.87 to 1.12), or the incidence of vomiting (35% v 34%; relative risk 1.05, 0.9 to 1.2). Neonatal outcomes were also similar. CONCLUSIONS: Consumption of a light diet during labour did not influence obstetric or neonatal outcomes in participants, nor did it increase the incidence of vomiting. Women who are allowed to eat in labour have similar lengths of labour and operative delivery rates to those allowed water only. TRIAL REGISTRATION: Current Controlled Trials ISRCTN33298015.

NPO during labor. Is there any scientific validation?

Maternal death from pulmonary aspiration of gastric contents has virtually disappeared in the United Kingdom. The one case documented in the most recent triennial report was a woman with multiorgan failure in intensive care and is probably not relevant to the current debate [1]. Although not so well documented, other Western countries seem to be experiencing the same decline in maternal death from this cause. At the same time, the burden of proof is falling increasingly on obstetric anesthesiologists as obstetricians and midwives demand that NPO policies should be rejected, unless anesthesiologists can prove that they are necessary. Without any proof of benefit, many midwives actively encourage eating in women who do not really want to eat. A hospital manager who wants to divert money to other areas of health care might make the same argument about employing less experienced--and therefore cheaper--anesthesiologists or nurse anesthetists on the labor floor. Although no self-respecting obstetric anesthesiologist would accept such a situation, there is still no randomized controlled trial that proves that experienced anesthesiologists reduce maternal mortality. Similarly it is difficult for a mother to comprehend the negligible risk of pulmonary aspiration during labor while her care providers insist that it would be more dangerous for her to cross a busy road! Against a background of conflicting advice from midwives and medical practitioners, the mother is likely to eat if she feels so inclined. Pulmonary aspiration is a rare complication, so even if a light diet in labor became acceptable, it is likely that it would take many years for a subsequent increase in maternal mortality to become apparent. It would be disappointing if mistakes made by a previous generation had to be relearned in the twenty-first century. Increasingly, media-controlled pressure groups dictate health fashions, and the physicians frequently can only stand on the sidelines and advise. Most obstetric anesthesiologists agree that a rigid NPO policy in labor is no longer appropriate and that at least water or ice chips should be allowed. Current evidence suggests that solids and semi-solids should be avoided once a woman is in active labor or requests analgesia. The appropriate advice is to allow a carefully audited introduction of isotonic drinks. These drinks seem to be an effective medium for providing calories while minimizing any increase in gastric volume, and such a policy would be unlikely to reverse the reduction in aspiration that has been achieved over the past 50 years.

An evaluation of isotonic "sport drinks" during labor.

UNLABELLED: We compared the metabolic effects of allowing women isotonic "sport drinks" rather than water to drink during labor. The effect of these drinks on gastric residual volume was also evaluated. Sixty women in early labor (cervical dilation <5 cm) were randomized to receive either isotonic sport drinks or water only. Plasma beta-hydroxybutyrate, nonesterified fatty acids, and glucose were measured in early labor and at the end of the first stage of labor. Residual gastric volume was assessed within 45 min of delivery by use of an ultrasound scanner. The incidence and volume of vomiting was recorded. At the end of the first stage of labor, plasma beta-hydroxybutyrate (P = 0.000) and nonesterified fatty acids (P = 0.000) had increased and plasma glucose (P = 0.007) had decreased significantly in the Water-Only group. Gastric antral cross-sectional area after delivery was similar in the two groups. The incidence of vomiting and the volume vomited during labor and within the hour of delivery were also similar. There was no difference between the groups in any maternal or neonatal outcome of labor. In conclusion, isotonic drinks reduce maternal ketosis in labor without increasing gastric volume. IMPLICATIONS: Solid foods may endanger a woman's life if consumed during labor. Isotonic sport fluids were evaluated as a nutritional alternative. Results demonstrate that mothers who have not received parenteral opioids can safely drink isotonic drinks in active labor.

Síndrome de Prader-Willi: presentación de un caso / Prader Willi` syndrome: presentation of an case

La diabetes mellitus se ha convertido en una pandemia. En forma inusual se manifiesta asociada a condiciones con una causa genética establecida, que causan trastornos metabólicos específicos y que son dubdiagnosticadas y por lo tanto tratadas parcialmente o con el enfoque incorrecto. Una de ellas es el Síndrome de Prader Willi, una rara entidad causada por un trastorno genético puntual, con una incidencia anual de 1:10.000-16.000 nacidos vivos. Presentamos un caso de síndrome de Prader Willi estudiado por el Servicio de Medicina 2 del Hospital Vargas de Caracas, Venezuela