Within-host evolution of Burkholderia pseudomallei over a twelve-year chronic carriage infection

UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host.

IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Nestor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation

Background: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Nestor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure.

Results: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope.

Conclusions: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

Arene cis-Diol Dehydrogenase-Catalysed Regio- and Stereoselective Oxidation of Arene-, Cycloalkane- and Cycloalkene-cis-diols to Yield Catechols and Chiral α-Ketols

Benzene cis-diol dehydrogenase and naphthalene cis-diol dehydrogenase enzymes, expressed in Pseudomonas putida wild-type and Escherichia coli recombinant strains, were used to investigate regioselectivity and stereoselectivity during dehydrogenations of arene, cyclic alkane and cyclic alkene vicinal cis-diols. The dehydrogenase-catalysed production of enantiopure cis-diols, α-ketols and catechols, using benzene cis-diol dehydrogenase and naphthalene cis-diol dehydrogenase, involved both kinetic resolution and asymmetric synthesis methods. The chemoenzymatic production and applications of catechol bioproducts in synthesis were investigated.

Diagnostic accuracy of loop-mediated isothermal amplification as a near-patient test for meningococcal disease in children: An observational cohort study

Background: Diagnosis of meningococcal disease relies on recognition of clinical signs and symptoms that are notoriously non-specific, variable, and often absent in the early stages of the disease. Loop-mediated isothermal amplification (LAMP) has previously been shown to be fast and effective for the molecular detection of meningococcal DNA in clinical specimens. We aimed to assess the diagnostic accuracy of meningococcal LAMP as a near-patient test in the emergency department.

Methods: For this observational cohort study of diagnostic accuracy, children aged 0-13 years presenting to the emergency department of the Royal Belfast Hospital for Sick Children (Belfast, UK) with suspected meningococcal disease were eligible for inclusion. Patients underwent a standard meningococcal pack of investigations testing for meningococcal disease. Respiratory (nasopharyngeal swab) and blood specimens were collected from patients and tested with near-patient meningococcal LAMP and the results were compared with those obtained by reference laboratory tests (culture and PCR of blood and cerebrospinal fluid).

Findings: Between Nov 1, 2009, and Jan 31, 2012, 161 eligible children presenting at the hospital underwent the meningococcal pack of investigations and were tested for meningococcal disease, of whom 148 consented and were enrolled in the study. Combined testing of respiratory and blood specimens with use of LAMP was accurate (sensitivity 89% [95% CI 72-96], specificity 100% [97-100], positive predictive value 100% [85-100]; negative predictive value 98% [93-99]) and diagnostically useful (positive likelihood ratio 213 [95% CI 13-infinity] and negative likelihood ratio 0·11 [0·04-0·32]). The median time required for near-patient testing from sample to result was 1 h 26 min (IQR 1 h 20 min-1 h 32 min).

Interpretation: Meningococcal LAMP is straightforward enough for use in any hospital with basic laboratory facilities, and near-patient testing with this method is both feasible and effective. By contrast with existing UK National Institute of Health and Care Excellence guidelines, we showed that molecular testing of non-invasive respiratory specimens from children is diagnostically accurate and clinically useful.

Exendin-4 protects against post-myocardial infarction remodelling via specific actions on inflammation and the extracellular matrix

Glucagon-like peptide-1 (GLP-1) is an insulin-releasing hormone clinically exploited for glycaemic control in diabetes, which also confers acute cardioprotection and benefits in experimental/clinical heart failure. We specifically investigated the role of the GLP-1 mimetic, exendin-4, in post-myocardial infarction (MI) remodelling, which is a key contributor to heart failure. Adult female normoglycaemic mice underwent coronary artery ligation/sham surgery prior to infusion with exendin-4/vehicle for 4 weeks. Metabolic parameters and infarct sizes were comparable between groups. Exendin-4 protected against cardiac dysfunction and chamber dilatation post-MI and improved survival. Furthermore, exendin-4 modestly decreased cardiomyocyte hypertrophy/apoptosis but markedly attenuated interstitial fibrosis and myocardial inflammation post-MI. This was associated with altered extracellular matrix (procollagen IαI/IIIαI, connective tissue growth factor, fibronectin, TGF-β3) and inflammatory (IL-10, IL-1β, IL-6) gene expression in exendin-4-treated mice, together with modulation of both Akt/GSK-3β and Smad2/3 signalling. Exendin-4 also altered macrophage response gene expression in the absence of direct actions on cardiac fibroblast differentiation, suggesting cardioprotective effects occurring secondary to modulation of inflammation. Our findings indicate that exendin-4 protects against post-MI remodelling via preferential actions on inflammation and the extracellular matrix independently of its established actions on glycaemic control, thereby suggesting that selective targeting of GLP-1 signalling may be required to realise its clear therapeutic potential for post-MI heart failure.

Susceptibility to Invasive Meningococcal Disease: Polymorphism of Complement System Genes and Neisseria meningitidis Factor H Binding Protein

BACKGROUND: Neisseria meningitidis can cause severe infection in humans. Polymorphism of Complement Factor H (CFH) is associated with altered risk of invasive meningococcal disease (IMD). We aimed to find whether polymorphism of other complement genes altered risk and whether variation of N. meningitidis factor H binding protein (fHBP) affected the risk association.

METHODS: We undertook a case-control study with 309 European cases and 5,200 1958 Birth Cohort and National Blood Service cohort controls. We used additive model logistic regression, accepting P<0.05 as significant after correction for multiple testing. The effects of fHBP subfamily on the age at infection and severity of disease was tested using the independent samples median test and Student's T test. The effect of CFH polymorphism on the N. meningitidis fHBP subfamily was investigated by logistic regression and Chi squared test.

RESULTS: Rs12085435 A in C8B was associated with odds ratio (OR) of IMD (0.35 [95% CI 0.19-0.67]; P = 0.03 after correction). A CFH haplotype tagged by rs3753396 G was associated with IMD (OR 0.56 [95% CI 0.42-0.76], P = 1.6x10-4). There was no bacterial load (CtrA cycle threshold) difference associated with carriage of this haplotype. Host CFH haplotype and meningococcal fHBP subfamily were not associated. Individuals infected with meningococci expressing subfamily A fHBP were younger than those with subfamily B fHBP meningococci (median 1 vs 2 years; P = 0.025).

DISCUSSION: The protective CFH haplotype alters odds of IMD without affecting bacterial load for affected heterozygotes. CFH haplotype did not affect the likelihood of infecting meningococci having either fHBP subfamily. The association between C8B rs12085435 and IMD requires independent replication. The CFH association is of interest because it is independent of known functional polymorphisms in CFH. As fHBP-containing vaccines are now in use, relationships between CFH polymorphism and vaccine effectiveness and side-effects may become important.

Reactions of nitrogen nucleophiles with enantiopure cyclohexenyl electrophiles:a stereo- and regio- selective study

The reactions of enantiopure cyclohexene epoxides and trans-1,2-bromoacetates, derived from the corresponding substituted benzene cis-dihydrodiol metabolites, with nitrogen nucleophiles, were examined and possible mechanisms proposed. An initial objective was the synthesis of new 1,2-aminoalcohol enantiomers as potential chiral ligands and synthetic scaffolds for library generation. These apparently simple substitution reactions proved to be more complex than initially anticipated and were found to involve a combination of different reaction mechanisms. Allylic trans-1,2-azidohydrins were prepared by Lewis acid-catalysed ring-opening of cyclic vinyl epoxides with sodium azide via an S(N)2 mechanism. On heating, these trans-1,2-azidohydrins isomerized to the corresponding trans-1,4-azidohydrins via a suprafacial allyl azide [3,3]-sigmatropic rearrangement mechanism. Conversion of a 1,2-azidohydrin to a 1,2-azidoacetate moved the equilibrium position in favour of the 1,4-substitution product. Allylic trans-1,2-bromoacetates reacted with sodium azide at room temperature to give C-2 and C-4 substituted products. A clean inversion of configuration at C-2 was found, as expected, from a concerted S(N)2-pathway. However, substitution at C-4 was not stereoselective and resulted in mixtures of 1,4-cis and 1,4-trans products. This observation can be rationalized in terms of competitive S(N)2 and S(N)2 reactions allied to a [3,3]-sigmatropic rearrangement. cis-1,2-Azidohydrins and cis-1,2-azidoacetates were much more prone to rearrange than the corresponding trans-isomers. Reaction of the softer tosamide nucleophile with trans-1,2-bromoacetates resulted, predominantly, in C-4 substitution via a syn-S(N)2 mechanism. One application of the reaction of secondary amines with allylic cyclohexene epoxides, to give trans-1,2-aminoalcohols, is in the synthesis of the anticholinergic drug vesamicol, via an S(N)2 mechanism. Copyright (c) 2013 John Wiley & Sons, Ltd.

"'Nothing but a printer's devil ever drives me into finishing': the Genesis of Thomas Moore's Irish Melodies'

This chapter outlines the working methods of the prolific writer and lyricist Thomas Moore—which were characterized by the unfortunate combination of a perfectionist streak, a tendency to release material to the publishers while still in the creative mode, and a tendency to re-visit previously-published material. The Gibson-Massie Moore collection at Queen's University Belfast teaches us a great deal about Moore’s creative processes, and also records the nineteenth-century publishing industry’s response to one of its most prolific and popular creative artists. This chapter is illustrated by an online Exhibition, the 'Thomas Moore Project', Digital Collections, Special Collections, McClay library (see URL below).

Toluene Dioxygenase Catalysed synthesis and reactions of cis-diol metabolites derived from 2 and 3-methoxy phenols

Using toluene dioxygenase as biocatalyst, enantiopure cisdihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone tautomers, were obtained from meta and ortho methoxyphenols. Although these isomeric phenol substrates are structurally similar, the major bioproducts from each of these biotransformations were found at different oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from meta methoxyphenol was isolated, while the corresponding metabolite from ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol. The chemistry of the 3-methoxycyclohexenone cis-diol product was investigated and elimination, aromatization, hydrogenation, regioselective O-exchange, Stork−Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis also established the previously unassigned absolute configuration.