Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 2. Surgical results.

BACKGROUND: Longstanding uncertainty surrounds the selection of surgical protocols for unilateral cleft lip and palate, and randomised trials have only rarely been performed. The Scandcleft Project consists of three trials commenced in 1997 involving ten centres in Denmark, Finland, Norway, Sweden, and the UK. Three groups of centres tested a newly-defined common technique for palatal repair (Arm A) against their local protocols (Arms B, C, D). Arm A was familiar to most of the surgeons in Trial 1, but not to the surgeons in the other Trials. AIM: To evaluate surgical events and complications of the 448 (293 boys, 155 girls) patients with complete unilateral cleft lip and palate (UCLP) enrolled in the three trials. METHOD: The three trials were carried out in parallel in adherence with a fully developed, ethically approved protocol. Operative time, bleeding, complications, and major dehiscence during and after both primary surgeries were recorded by the surgeon. Rates of fistula and surgery for velopharyngeal incompetence (VPI) were assessed until the youngest patient of the study had reached the age of 9 years. Pearson Chi-square statistical analysis was used to compare the outcomes. RESULTS: No significant differences in bleeding, infection, anaesthetic complications or length of hospital stay between the different arms were found for Trial 1. However, in Trials 2 and 3 there were more airway problems in Arm A than with the traditional local protocols (Arms C or D). In Trial 3 fistula and VPI surgery rates were also higher in Arm A. CONCLUSIONS: The results do not provide statistical evidence that any technique is better than others, but indicate that surgery was more problematic for surgeons who were still gaining experience with an unfamiliar surgical protocol. TRIAL REGISTRATION: ISRCTN29932826.

A Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 1. Planning and management.

BACKGROUND AND AIMS: Longstanding uncertainty surrounds the selection of surgical protocols for the closure of unilateral cleft lip and palate, and randomised trials have only rarely been performed. This paper is an introduction to three randomised trials of primary surgery for children born with complete unilateral cleft lip and palate (UCLP). It presents the protocol developed for the trials in CONSORT format, and describes the management structure that was developed to achieve the long-term engagement and commitment required to complete the project. METHOD: Ten established national or regional cleft centres participated. Lip and soft palate closure at 3-4 months, and hard palate closure at 12 months served as a common method in each trial. Trial 1 compared this with hard palate closure at 36 months. Trial 2 compared it with lip closure at 3-4 months and hard and soft palate closure at 12 months. Trial 3 compared it with lip and hard palate closure at 3-4 months and soft palate closure at 12 months. The primary outcomes were speech and dentofacial development, with a series of perioperative and longer-term secondary outcomes. RESULTS: Recruitment of 448 infants took place over a 9-year period, with 99.8% subsequent retention at 5 years. CONCLUSION: The series of reports that follow this introductory paper include comparisons at age 5 of surgical outcomes, speech outcomes, measures of dentofacial development and appearance, and parental satisfaction. The outcomes recorded and the numbers analysed for each outcome and time point are described in the series. TRIAL REGISTRATION: ISRCTN29932826.

Quorum Sensing Is Accompanied by Global Metabolic Changes in the Opportunistic Human Pathogen Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa uses N-acyl-homoserine lactone (AHL)-dependent quorum sensing (QS) systems to control the expression of secreted effectors. These effectors can be crucial to the ecological fitness of the bacterium, playing roles in nutrient acquisition, microbial competition, and virulence. In this study, we investigated the metabolic consequences of AHL-dependent QS by monitoring the metabolic profile(s) of a lasI rhlI double mutant (unable to make QS signaling molecules) and its wild-type progenitor as they progressed through the growth curve. Analysis of culture supernatants by (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy revealed that at the point where AHL concentrations peaked in the wild type, the metabolic footprints (i.e., extracellular metabolites) of the wild-type and lasI rhlI mutant diverged. Subsequent gas chromatography-mass spectrometry (GC-MS)-based analysis of the intracellular metabolome revealed QS-dependent perturbations in around one-third of all identified metabolites, including altered concentrations of tricarboxylic acid (TCA) cycle intermediates, amino acids, and fatty acids. Further targeted fatty acid methyl ester (FAME) GC-MS-based profiling of the cellular total fatty acid pools revealed that QS leads to changes associated with decreased membrane fluidity and higher chemical stability. However, not all of the changes we observed were necessarily a direct consequence of QS; liquid chromatography (LC)-MS analyses revealed that polyamine levels were elevated in the lasI rhlI mutant, perhaps a response to the absence of QS-dependent adaptations. Our data suggest that QS leads to a global readjustment in central metabolism and provide new insight into the metabolic changes associated with QS during stationary-phase adaptation. IMPORTANCE: Quorum sensing (QS) is a transcriptional regulatory mechanism that allows bacteria to coordinate their gene expression profile with the population cell density. The opportunistic human pathogen Pseudomonas aeruginosa uses QS to control the production of secreted virulence factors. In this study, we show that QS elicits a global "metabolic rewiring" in P. aeruginosa. This metabolic rerouting of fluxes is consistent with a variety of drivers, ranging from altered QS-dependent transcription of "metabolic genes" through to the effect(s) of global "metabolic readjustment" as a consequence of QS-dependent exoproduct synthesis, as well as a general stress response, among others. To our knowledge, this is the first study of its kind to assess the global impact of QS on the metabolome.

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

Analysis of chloroformate-derivatised amino acids, dipeptides and polyamines by LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed which, with sample preparation using a commercially available kit, allows rapid quantitation of 39 chloroformate-derivatised amino acids (AAs), polyamines (PAs) and dipeptides (DPs) in complex biological matrices. Lower limits of quantitation (LOQ) were 20-150nM for putrescine, spermine, spermidine, cadaverine, agmatine, and below 5µM for all analytes. Responses were linear for all analytes between 0.5 and 50µM. Quantitative measurements of all 39 metabolites were achieved within a 15min runtime. The method was evaluated with a Pseudomonas aeruginosa cell extract study (n=24) and a larger human urine study (n=308). Batch effects were observed in the urine study and an investigation of instrument and sample stability showed a wave-like pattern in the MS responses. Both the run order and inter-batch variation were successfully corrected by normalising to pooled urine quality control data. Thus, this method should be suitable for diverse biological matrices and for large as well as small sample sets.

Moraxella catarrhalis: an unrecognized pathogen of the oral cavity?

OBJECTIVE: We investigated the effect of the bacterial flora of the nose and throat on the outcome of the initial repairs of the cleft palate in the presence of prophylactic antibiotics. DESIGN: A retrospective review of 90 procedures in 66 patients who had cleft palate repair between April 2005 and June 2007 was conducted at Booth Hall Children's Hospital, Manchester, U.K. Both isolated cleft palate and cleft lip and palate patients were included. Exclusion criteria included syndromic cases, other medical disorders, and revisions of previous cleft palate repairs. Nose and throat swabs were taken on admission. Benzyl penicillin and flucloxacillin were given perioperatively. The occurrence of oronasal fistulas was correlated with the bacteria grown on culture. RESULTS: The oronasal fistula rate was 15.9%. The highest fistula rate in procedures with positive swabs was seen with Moraxella catarrhalis. CONCLUSIONS: M. catarrhalis has not been previously recognized as a pathogen in cleft palate repairs. This study demonstrates a higher fistula rate in procedures positive for M. catarrhalis. Other factors that may have contributed to the fistula formation include the severity of the initial cleft and technical factors. Further study is required before a definitive link can be established.

Mutation of nfxB causes global changes in the physiology and metabolism of Pseudomonas aeruginosa.

Loss-of-function mutations in nfxB lead to up-regulation of mexCD-oprJ expression and, consequently, increased resistance to fluoroquinolone antibiotics. Such nfxB mutants have also been reported to exhibit altered virulence profiles, diminished type III secretion system-dependent cytotoxicity, and impaired fitness. However, it is not clear whether these phenotypes are directly linked to NfxB activity or whether inappropriate expression of the MexCD-OprJ pump has pleiotropic effects, thereby impacting indirectly on the phenotype of the cells. The aim of the current work is to investigate which of these possibilities is correct. We isolated a novel type of nfxB mutant generated by a spontaneous polygenic deletion and show that this mutant is rapidly out-competed when grown in a mixed culture with the wild-type progenitor. This competitive fitness defect only manifested itself during the stationary phase of growth. The endoproteome of the nfxB mutant, assessed using 2D-DiGE (difference gel electrophoresis), showed major alterations compared with the wild-type. Consistent with this, we found that the nfxB mutant was impaired in all forms of motility (swimming, swarming, and twitching) as well as in the production of siderophores, rhamnolipid, secreted protease, and pyocyanin. Further investigation showed that the exoproteome, endometabolome, and exometabolome of the nfxB mutant were all globally different compared with the wild-type. The exometabolome of the nfxB mutant was enriched in a selection of long chain fatty acids raising the possibility that these might be substrates for the MexCD-OprJ pump. The nfxB mutant metabotype could be complemented by expression of nfxB in trans and was abolished in an nfxB mexD double mutant, suggesting that inappropriate overexpression of a functional MexCD-OprJ efflux pump causes pleiotropic changes. Taken together, our data suggest that many of the nfxB mutant phenotypes are not caused by the direct effects of the NfxB regulator, but instead by inappropriate mexCD-oprJ expression. Furthermore, the pleiotropic nature of the phenotypes indicate that these may simply reflect the globally dysregulated physiology of the strain.

Comparative microarray analysis reveals that the core biofilm-associated transcriptome of Pseudomonas aeruginosa comprises relatively few genes.

Pseudomonas aeruginosa is a model organism for the study of intercellular communication and biofilm formation. As such, P. aeruginosa has been the subject of several microarray analyses comparing gene expression in biofilms and planktonic cultures. In the current work, we carried out a meta-analysis of these data sets to try and identify genes that are generically associated with biofilm formation in all of the conditions examined. Although the total number of transcripts modulated in the biofilms was large within the individual studies, the overlap between the data sets was small. Indeed, only five transcripts were upregulated and six transcripts were downregulated by more than twofold in the three data sets analysed. However, when the threshold modulation was relaxed to less than twofold, the overlap between the data sets increased, revealing a set of transcripts common to all of the studies. Transcriptional fusions and quantitative real-time PCR were used to independently confirm a selection of the observed modulations. Notably, we found that the expression profile of genes encoding the catabolic pathways for branched chain and aromatic amino acids was altered in biofilms, and that these alterations correlated with the onset of anaerobic growth. These findings were confirmed by quantitative amino acid analysis of culture supernatants. A mutant in one of the genes that we identified showed diminished biofilm formation in an attachment assay. The relatively small number of common biofilm-specific endpoint transcripts throws doubt on the suggestion that biofim formation proceeds through a pre-determined developmental pathway.

Variations on a theme: diverse N-acyl homoserine lactone-mediated quorum sensing mechanisms in gram-negative bacteria.

Many Gram-negative bacteria employ a mechanism of cell-cell communication known as quorum sensing (QS). The role of QS is to enable the cells in a culture to coordinate their gene expression profile with changes in the population cell density. The best characterized mechanisms of QS employ N-acylated homoserine lactones (AHLs) as signalling molecules. These AHLs are made by enzymes known as LuxI homologs, and accumulate in the culture supernatant at a rate proportional to the increase in cell density. Once the AHL concentration exceeds a certain threshold value, these ligands bind to intracellular receptors known as LuxR homologs. The latter are transcriptional regulators, whose activity alters upon binding the AHL ligand, thereby eliciting a change in gene transcription. Over the last five years, it has become increasingly obvious that this is a rather simplistic view of AHL-dependent QS, and that in fact, there is considerable diversity in the way in which LuxI-R homologs operate. The aim of the current review is to describe these variations on the basic theme, and to show how functional genomics is revolutionizing our understanding of QS-controlled regulons.