Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

Reply to the discussion by Karen Johannesson on “Rare earth element geochemistry of scleractinian coral skeleton during meteoric diagenesis: a sequence through neomorphism of aragonite to calcite” by Webb et al., Sedimentology, 56, 1433-1463

Webb et al. (2009) described a late Pleistocenecoral sample wherein the diagenetic stabilization of original coral aragonite to meteoric calcite was halted more or less mid-way through the process, allowing direct comparison of pre-diagenetic and post-diagenetic microstructure and trace element distributions. Those authors found that the rare earth elements (REEs) were relatively stable during meteoric diagenesis, unlike divalent cations such as Sr,and it was thus concluded that original, in this case marine, REE distributions potentially could be preserved through the meteoric carbonate stabilization process that must have affected many, if not most, ancient limestones. Although this was not the case in the analysed sample, they noted that where such diagenesis took place in laterally transported groundwater, trace elements derived from that groundwater could be incorporated into diagenetic calcite, thus altering the initial REE distribution (Banner et al., 1988). Hence, the paper was concerned with the diagenetic behaviour of REEs in a groundwater-dominated karst system. The comment offered by Johannesson (2011) does not question those research results, but rather, seeks to clarify an interpretation made by Webb et al. (2009) of an earlier paper, Johannesson et al. (2006).

Sensitivity of the NMR density matrix to pulse sequence parameters : a simplified analytic approach

We present a formalism for the analysis of sensitivity of nuclear magnetic resonance pulse sequences to variations of pulse sequence parameters, such as radiofrequency pulses, gradient pulses or evolution delays. The formalism enables the calculation of compact, analytic expressions for the derivatives of the density matrix and the observed signal with respect to the parameters varied. The analysis is based on two constructs computed in the course of modified density-matrix simulations: the error interrogation operators and error commutators. The approach presented is consequently named the Error Commutator Formalism (ECF). It is used to evaluate the sensitivity of the density matrix to parameter variation based on the simulations carried out for the ideal parameters, obviating the need for finite-difference calculations of signal errors. The ECF analysis therefore carries a computational cost comparable to a single density-matrix or product-operator simulation. Its application is illustrated using a number of examples from basic NMR spectroscopy. We show that the strength of the ECF is its ability to provide analytic insights into the propagation of errors through pulse sequences and the behaviour of signal errors under phase cycling. Furthermore, the approach is algorithmic and easily amenable to implementation in the form of a programming code. It is envisaged that it could be incorporated into standard NMR product-operator simulation packages.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson’s Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59–119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.

The use of virtual prototyping to rehearse the sequence of construction work involving mobile cranes

Purpose – Rehearsing practical site operations is without doubt one of the most effective methods for minimising planning mistakes, because of the learning that takes place during the rehearsal activity. However, real rehearsal is not a practical solution for on-site construction activities, as it not only involves a considerable amount of cost but can also have adverse environmental implications. One approach to overcoming this is by the use of virtual rehearsals. The purpose of this paper is to investigate an approach to simulation of the motion of cranes in order to test the feasibility of associated construction sequencing and generate construction schedules for review and visualisation. Design/methodology/approach – The paper describes a system involving two technologies, virtual prototyping (VP) and four-dimensional (4D) simulation, to assist construction planners in testing the sequence of construction activities when mobile cranes are involved. The system consists of five modules, comprising input, database, equipment, process and output, and is capable of detecting potential collisions. A real-world trial is described in which the system was tested and validated. Findings – Feedback from the planners involved in the trial indicated that they found the system to be useful in its present form and that they would welcome its further development into a fully automated platform for validating construction sequencing decisions. Research limitations/implications – The tool has the potential to provide a cost-effective means of improving construction planning. However, it is limited at present to the specific case of crane movement under special consideration. Originality/value – This paper presents a large-scale, real life case of applying VP technology in planning construction processes and activities.

An expressed sequence tag (EST) library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies

The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup.

Beyond barcoding : a mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

Background: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods. HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (∼1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/g Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice. © 2011 Valley-Omar et al; licensee BioMed Central Ltd.

Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions

Background. A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses in the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. Results. Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. Conclusion. Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity. © 2008 van der Walt et al; licensee BioMed Central Ltd.

Does teaching sequence matter when teaching high school chemistry with scientific visualisations?

Five Canadian high school Chemistry classes in one school, taught by three different teachers, studied the concepts of dynamic chemical equilibria and Le Chatelier’s Principle. Some students received traditional teacher-led explanations of the concept first and used an interactive scientific visualisation second, while others worked with the visualisation first and received the teacher-led explanation second. Students completed a test of their conceptual understanding of the relevant concepts prior to instruction, after the first instructional session and at the end of instruction. Data on students’ academic achievement (highest, middle or lowest third of the class on the mid-term exam) and gender were also collected to explore the relationship between these factors, conceptual development and instructional sequencing. Results show, within this context at least, that teaching sequence is not important in terms of students’ conceptual learning gains.

A description of the Mei2-like protein family; structure, phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a “mei2-dot” in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.

Phylodynamic analysis of the emergence and epidemiological impact of transmissible defective dengue viruses

Intra-host sequence data from RNA viruses have revealed the ubiquity of defective viruses in natural viral populations, sometimes at surprisingly high frequency. Although defective viruses have long been known to laboratory virologists, their relevance in clinical and epidemiological settings has not been established. The discovery of long-term transmission of a defective lineage of dengue virus type 1 (DENV-1) in Myanmar, first seen in 2001, raised important questions about the emergence of transmissible defective viruses and their role in viral epidemiology. By combining phylogenetic analyses and dynamical modelling, we investigate how evolutionary and ecological processes at the intra-host and inter-host scales shaped the emergence and spread of the defective DENV-1 lineage. We show that this lineage of defective viruses emerged between June 1998 and February 2001, and that the defective virus was transmitted primarily through co-transmission with the functional virus to uninfected individuals. We provide evidence that, surprisingly, this co-transmission route has a higher transmission potential than transmission of functional dengue viruses alone. Consequently, we predict that the defective lineage should increase overall incidence of dengue infection, which could account for the historically high dengue incidence reported in Myanmar in 2001-2002. Our results show the unappreciated potential for defective viruses to impact the epidemiology of human pathogens, possibly by modifying the virulence-transmissibility trade-off, or to emerge as circulating infections in their own right. They also demonstrate that interactions between viral variants, such as complementation, can open new pathways to viral emergence.