The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli Clone

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage : genome analysis and virulence mechanisms

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Old mastheads and new media : newspapers striving to adapt in Australia and South-east Asia

As a key element in their response to new media forcing transformations in mass media and media use, newspapers have deployed various strategies to not only establish online and mobile products, and develop healthy business plans, but to set out to be dominant portals. Their response to change was the subject of an early investigation by one of the present authors (Keshvani 2000). That was part of a set of short studies inquiring into what impact new software applications and digital convergence might have on journalism practice (Tickle and Keshvani 2000), and also looking for demonstrations of the way that innovations, technologies and protocols then under development might produce a “wireless, streamlined electronic news production process (Tickle and Keshvani 2001).” The newspaper study compared the online products of The Age in Melbourne and the Straits Times in Singapore. It provided an audit of the Singapore and Australia Information and Communications Technology (ICT) climate concentrating on the state of development of carrier networks, as a determining factor in the potential strength of the two services with their respective markets. In the outcome, contrary to initial expectations, the early cable roll-out and extensive ‘wiring’ of the city in Singapore had not produced a level of uptake of Internet services as strong as that achieved in Melbourne by more ad hoc and varied strategies. By interpretation, while news websites and online content were at an early stage of development everywhere, and much the same as one another, no determining structural imbalance existed to separate these leading media participants in Australia and South-east Asia. The present research revisits that situation, by again studying the online editions of the two large newspapers in the original study, and one other, The Courier Mail, (recognising the diversification of types of product in this field, by including it as a representative of Newscorp, now a major participant). The inquiry works through the principle of comparison. It is an exercise in qualitative, empirical research that establishes a comparison between the situation in 2000 as described in the earlier work, and the situation in 2014, after a decade of intense development in digital technology affecting the media industries. It is in that sense a follow-up study on the earlier work, although this time giving emphasis to content and style of the actual products as experienced by their users. It compares the online and print editions of each of these three newspapers; then the three mastheads as print and online entities, among themselves; and finally it compares one against the other two, as representing a South-east Asian model and Australian models. This exercise is accompanied by a review of literature on the developments in ICT affecting media production and media organisations, to establish the changed context. The new study of the online editions is conducted as a systematic appraisal of the first level, or principal screens, of the three publications, over the course of six days (10-15.2.14 inclusive). For this, categories for analysis were made, through conducting a preliminary examination of the products over three days in the week before. That process identified significant elements of media production, such as: variegated sourcing of materials; randomness in the presentation of items; differential production values among media platforms considered, whether text, video or stills images; the occasional repurposing and repackaging of top news stories of the day and the presence of standard news values – once again drawn out of the trial ‘bundle’ of journalistic items. Reduced in this way the online artefacts become comparable with the companion print editions from the same days. The categories devised and then used in the appraisal of the online products have been adapted to print, to give the closest match of sets of variables. This device, to study the two sets of publications on like standards -- essentially production values and news values—has enabled the comparisons to be made. This comparing of the online and print editions of each of the three publications was set up as up the first step in the investigation. In recognition of the nature of the artefacts, as ones that carry very diverse information by subject and level of depth, and involve heavy creative investment in the formulation and presentation of the information; the assessment also includes an open section for interpreting and commenting on main points of comparison. This takes the form of a field for text, for the insertion of notes, in the table employed for summarising the features of each product, for each day. When the sets of comparisons as outlined above are noted, the process then becomes interpretative, guided by the notion of change. In the context of changing media technology and publication processes, what substantive alterations have taken place, in the overall effort of news organisations in the print and online fields since 2001; and in their print and online products separately? Have they diverged or continued along similar lines? The remaining task is to begin to make inferences from that. Will the examination of findings enforce the proposition that a review of the earlier study, and a forensic review of new models, does provide evidence of the character and content of change --especially change in journalistic products and practice? Will it permit an authoritative description on of the essentials of such change in products and practice? Will it permit generalisation, and provide a reliable base for discussion of the implications of change, and future prospects? Preliminary observations suggest a more dynamic and diversified product has been developed in Singapore, well themed, obviously sustained by public commitment and habituation to diversified online and mobile media services. The Australian products suggest a concentrated corporate and journalistic effort and deployment of resources, with a strong market focus, but less settled and ordered, and showing signs of limitations imposed by the delay in establishing a uniform, large broadband network. The scope of the study is limited. It is intended to test, and take advantage of the original study as evidentiary material from the early days of newspaper companies’ experimentation with online formats. Both are small studies. The key opportunity for discovery lies in the ‘time capsule’ factor; the availability of well-gathered and processed information on major newspaper company production, at the threshold of a transformational decade of change in their industry. The comparison stands to identify key changes. It should also be useful as a reference for further inquiries of the same kind that might be made, and for monitoring of the situation in regard to newspaper portals on line, into the future.

Parent feeding interactions and practices during childhood cancer treatment: A qualitative investigation

In the general population it is evident that parent feeding practices can directly shape a child’s life long dietary intake. Young children undergoing childhood cancer treatment may experience feeding difficulties and limited food intake, due to the inherent side effects of their anti-cancer treatment. What is not clear is how these treatment side effects are influencing the parent–child feeding relationship during anti-cancer treatment. This retrospective qualitative study collected telephone based interview data from 38 parents of childhood cancer patients who had recently completed cancer treatment (child’s mean age: 6.98 years). Parents described a range of treatment side effects that impacted on their child’s ability to eat, often resulting in weight loss. Sixty-one percent of parents (n = 23) reported high levels of stress in regard to their child’s eating and weight loss during treatment. Parents reported stress, feelings of helplessness, and conflict and/or tension between parent and the child during feeding/eating interactions. Parents described using both positive and negative feeding practices, such as: pressuring their child to eat, threatening the insertion of a nasogastric feeding tube, encouraging the child to eat and providing home cooked meals in hospital. Results indicated that parent stress may lead to the use of coping strategies such as positive or negative feeding practices to entice their child to eat during cancer treatment. Future research is recommended to determine the implication of parent feeding practice on the long term diet quality and food preferences of childhood cancer survivors.

A qualitative analysis of how parents assess acute pain in young children

An accurate and comprehensive pain assessment is crucial for adequate pain management in pre- and early verbal children during painful medical procedures. This study used an inductive approach to explore the processes involved in parental pain assessment and to develop a new model of Parental Assessment of Acute Child Pain. Participants were 19 parents of children aged under 3 years who had previously or were potentially about to experience an intravenous cannula or nasogastric tube insertion. Parental affect regulation, while witnessing their child in acute pain/distress, appeared to be critical to the processes involved in assessing their child’s pain.

Mechanistic details of the membrane perforation and passive translocation of TAT peptides

The trans-activator of transcription (TAT) peptide is regarded as the “gold standard” for cell-penetrating peptides, capable of traversing a mammalian membrane passively into the cytosolic space. This characteristic has been exploited through conjugation of TAT for applications such as drug delivery. However, the process by which TAT achieves membrane penetration remains ambiguous and unresolved. Mechanistic details of TAT peptide action are revealed herein by using three complementary methods: quartz crystal microbalance with dissipation (QCM-D), scanning electrochemical microscopy (SECM) and atomic force microscopy (AFM). When combined, these three scales of measurement define that the membrane uptake of the TAT peptide is by trans-membrane insertion using a “worm-hole” pore that leads to ion permeability across the membrane layer. AFM data provided nanometre-scale visualisation of TAT punctuation using a mammalian-mimetic membrane bilayer. The TAT peptide does not show the same specificity towards a bacterial mimetic membrane and QCM-D and SECM showed that the TAT peptide demonstrates a disruptive action towards these membranes. This investigation supports the energy-independent uptake of the cationic TAT peptide and provides empirical data that clarify the mechanism by which the TAT peptide achieves its membrane activity. The novel use of these three biophysical techniques provides valuable insight into the mechanism for TAT peptide translocation, which is essential for improvements in the cellular delivery of TAT-conjugated cargoes including therapeutic agents required to target specific intracellular locations.

Insertions in the OCL1 locus of Acinetobacter baumannii lead to shortened lipooligosaccharides

Genomes of 82 Acinetobacter baumannii global clones 1 (GC1) and 2 (GC2) isolates were sequenced and different forms of the locus predicted to direct synthesis of the outer core (OC) of the lipooligosaccharide were identified. OCL1 was in all GC2 genomes, whereas GC1 isolates carried OCL1, OCL3 or a new locus, OCL5. Three mutants in which an insertion sequence (ISAba1 or ISAba23) interrupted OCL1 were identified. Isolates with OCL1 intact produced only lipooligosaccharide, while the mutants produced lipooligosaccharide of reduced molecular weight. Thus, the assignment of the OC locus as that responsible for the synthesis of the OC is correct.

Comparative analysis reveals loss of the appetite-regulating peptide hormone ghrelin in falcons

Ghrelin and leptin are key peripherally secreted appetite-regulating hormones in vertebrates. Here we consider the ghrelin gene (GHRL) of birds (class Aves), where it has been reported that ghrelin inhibits rather than augments feeding. Thirty-one bird species were compared, revealing that most species harbour a functional copy of GHRL and the coding region for its derived peptides ghrelin and obestatin. We provide evidence for loss of GHRL in saker and peregrine falcons, and this is likely to result from the insertion of an ERVK retrotransposon in intron 0. We hypothesise that the loss of anorexigenic ghrelin is a predatory adaptation that results in increased food-seeking behaviour and feeding in falcons.

Effectiveness of electrocardiographic guidance in CVAD tip placement

Background International standard practice for the correct confirmation of the central venous access device is the chest X-ray. The intracavitary electrocardiogram-based insertion method is radiation-free, and allows real-time placement verification, providing immediate treatment and reduced requirement for post-procedural repositioning. Methods Relevant databases were searched for prospective randomised controlled trials (RCTs) or quasi RCTs that compared the effectiveness of electrocardiogram-guided catheter tip positioning with placement using surface-anatomy-guided insertion plus chest X-ray confirmation. The primary outcome was accurate catheter tip placement. Secondary outcomes included complications, patient satisfaction and costs. Results Five studies involving 729 participants were included. Electrocardiogram-guided insertion was more accurate than surface anatomy guided insertion (odds ratio: 8.3; 95% confidence interval (CI) 1.38; 50.07; p=0.02). There was a lack of reporting on complications, patient satisfaction and costs. Conclusion The evidence suggests that intracavitary electrocardiogram-based positioning is superior to surface-anatomy-guided positioning of central venous access devices, leading to significantly more successful placements. This technique could potentially remove the requirement for post-procedural chest X-ray, especially during peripherally inserted central catheter (PICC) line insertion.

Updates on sorting of fully homomorphic encrypted data

In this paper, we show implementation results of various algorithms that sort data encrypted with Fully Homomorphic Encryption scheme based on Integers. We analyze the complexities of sorting algorithms over encrypted data by considering Bubble Sort, Insertion Sort, Bitonic Sort and Odd-Even Merge sort. Our complexity analysis together with implementation results show that Odd-Even Merge Sort has better performance than the other sorting techniques. We observe that complexity of sorting in homomorphic domain will always have worst case complexity independent of the nature of input. In addition, we show that combining different sorting algorithms to sort encrypted data does not give any performance gain when compared to the application of sorting algorithms individually.

Nitrogen-doped graphene films from chemical vapor deposition of pyridine: Influence of process parameters on the electrical and optical properties

Graphene films were produced by chemical vapor deposition (CVD) of pyridine on copper substrates. Pyridine-CVD is expected to lead to doped graphene by the insertion of nitrogen atoms in the growing sp2 carbon lattice, possibly improving the properties of graphene as a transparent conductive film. We here report on the influence that the CVD parameters (i.e., temperature and gas flow) have on the morphology, transmittance, and electrical conductivity of the graphene films grown with pyridine. A temperature range between 930 and 1070 °C was explored and the results were compared to those of pristine graphene grown by ethanol-CVD under the same process conditions. The films were characterized by atomic force microscopy, Raman and X-ray photoemission spectroscopy. The optical transmittance and electrical conductivity of the films were measured to evaluate their performance as transparent conductive electrodes. Graphene films grown by pyridine reached an electrical conductivity of 14.3 × 105 S/m. Such a high conductivity seems to be associated with the electronic doping induced by substitutional nitrogen atoms. In particular, at 930 °C the nitrogen/carbon ratio of pyridine-grown graphene reaches 3%, and its electrical conductivity is 40% higher than that of pristine graphene grown from ethanol-CVD.

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line

This study aimed to investigate the effects of arsenic trioxide (As2O3) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 μmol/L As2O3in vitro, and the primary APL cells were treated with 2.0 μmol/L As2O3in vitro and 0.16 mg kg-1 d-1 As2O3in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As2O3 use, but the mutation spots were remarkably increased after As2O3 treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: rNB4-As2O3=0.973818, and rAPL-As2O3=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As2O3 aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As2O3 in APL treatment.

Generalized Query-By-Rule: a heterogeneous database query language

This paper describes the design and implementation of a high-level query language called Generalized Query-By-Rule (GQBR) which supports retrieval, insertion, deletion and update operations. This language, based on the formalism of database logic, enables the users to access each database in a distributed heterogeneous environment, without having to learn all the different data manipulation languages. The compiler has been implemented on a DEC 1090 system in Pascal.

Morphological Characteristics of Genetically-Modified Pineapple Fruit

Genetic engineering is an attractive method for changing a single characteristic of ‘Smooth Cayenne’ pineapple, without altering its other desirable attributes. Techniques used in pineapple transformation, however, such as tissue culture and biolistic-mediated or Agrobacterium-mediated gene insertion are prone to somaclonal variation, resulting in the production of several morphological mutations (Smith et al., 2002). Fruit mutations can include distortion in fruit shape (round ball, conical, fan-shaped), reduced fruit size, multiple crowns, crownless fruit, fruitless crowns, and spiny crown leaves (Dalldorf, 1975; Sanewski et al., 1992). The present paper describes the variability in fruit-shape mutations between transgenic and non-transgenic fruit, and its subsequent impact on organoleptic characteristics.