Structural insights into peptidoglycan glycosidase EtgA binding to the inner rod protein EscI of the type III secretion system via a designed EscI-EtgA fusion protein.

Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51-87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72-87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56-71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66-87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria.

School age outcome of hospitalisation with respiratory syncytial virus infection of prematurely born infants.

BACKGROUND: Hospitalisation due to respiratory syncytial virus (RSV) infection in the first 2 years after birth has been associated with increased healthcare utilisation and associated costs up to 5 years of age in children born prematurely at less than 32 weeks of gestation who developed bronchopulmonary dysplasia (BPD). A study was undertaken to determine whether hospitalisation due to RSV infection in the first 2 years was associated with increased morbidity and lung function abnormalities in such children at school age, and if any effects were influenced by age. METHODS: Healthcare utilisation and cost of care in years 5-7 were reviewed in 147 children and changes in healthcare utilisation between 0 and 8 years were assessed also using results from two previous studies. At age 8-10 years, 77 children had their lung function assessed and bronchial hyper-responsiveness determined. RESULTS: Children hospitalised with RSV infection (n = 25) in the first 2 years had a greater cost of care related to outpatient attendance than those with a non-respiratory or no admission (n = 72) when aged 5-7 years (p = 0.008). At 8-10 years of age, children hospitalised with RSV infection (n = 14) had lower forced expiratory volume in 0.75 s (FEV(0.75)) (p = 0.015), FEV(0.75)/forced vital capacity (p = 0.027) and flows at 50% (p = 0.034) and 75% (p = 0.006) of vital capacity than children hospitalised for non-RSV causes (n = 63). Healthcare utilisation decreased with increasing age regardless of RSV hospitalisation status. CONCLUSIONS: In prematurely born children who had BPD, hospitalisation due to RSV infection in the first 2 years is associated with reduced airway calibre at school age.

Preoperative planning for DIEP breast reconstruction: early experience of the use of computerised tomography angiography with VoNavix 3D software for perforator navigation.

The deep inferior epigastric perforator (DIEP) flap is normally the first choice in breast reconstruction; however, due to the considerable vascular anatomical variation and the learning curve for the procedure, muscle-sparing transverse rectus abdominis musculocutaneous (TRAM) flaps are still frequently performed to reduce the rate of complications. Accurate preoperative investigation of the perforators would allow better operative preparation and possibly shorten the learning curve. In an effort to increase accuracy of preoperative planning and to aid preoperative decision-making in free abdominal flap breast reconstruction, we have acquired the use of VoNavix, software that creates three dimensional images from computerised tomography angiography (CTA) data. The use of the VoNavix software for analysis of CTA provides superior imaging that can be viewed in theatre. It, together with CTA, enables decisions to be made preoperatively, including: which side to raise the flap; whether to aim for a medial or lateral row perforator; whether to take a segment of muscle and whether to expect an easy or difficult dissection. We have now performed over 60 free abdominal flap breast reconstructions aided with CTA, and 10 of these cases also used VoNavix technology. This paper presents our initial experience with the use of this software, illustrated with three patient examples. The advantages and disadvantages are discussed.

The long-term effect of unilateral t-tube insertion in patients undergoing cleft palate repair: 20-year follow-up of a randomised controlled trial.

OBJECTIVES: Between July 1984 and March 1987, all children that underwent repair for primary cleft palate at the Queen Victoria Hospital were enrolled in a clinical trial. Those found to have otitis media with effusion at time of surgery had a t-tube inserted into one randomised ear, whilst the other ear received no treatment. The object of the study was to reassess the patients from the original trial to discover the impact of the unilateral t-tube, twenty years later analysed on an intention to treat basis. DESIGN: Children that underwent primary palatal closure during the study dates were identified from the cleft palate database. Patients that were involved in the original trial were identified, contacted and invited to take part in the follow-up study. SETTING: Tertiary referral Specialist unit. PARTICIPANTS: Twenty-two patients were identified as potential study participants. Of this group, fourteen were contactable and seven participants agreed to take part in our follow-up study. MAIN OUTCOME MEASURES: Persistent symptomatology, otoscopy, pure tone audiometry and tympanometry. RESULTS: Follow-up results were compared within the original treatment groups from the primary study, on an intention to treat basis. Otoscopically the ears were normal in 2 of the 7 treated ears compared with four of the seven non-treated ears. All the other ear ears had various types of chronic otitis media. Four of the seven had hearing of >10 dB in the treated ear compared with the non-treated ear. CONCLUSIONS: These findings would indicate need for caution in the use of t-tubes in the cleft population and raises the question of long-term follow-up to assess for secondary cholesteatoma.

Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria.

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midges was carried out between 2001 and 2003, at 119 sites within a 50 x 50-km grid distributed across Bulgaria, using light trap collections around the time of peak adult midge abundance. Sentinel and ad hoc serum surveillance of hosts susceptible to bluetongue infection was carried out at around 300 sites between 1999 and 2003. Following the initial incursion of bluetongue virus 9 (BTV-9) into Bourgas province in 1999, affecting 85 villages along the southern border, a further 76 villages were affected along the western border in 2001, with outbreaks extending as far north as 43.6 degrees N. The BTV-9 strain in circulation was found to have a low pathogenicity for Bulgarian sheep populations, with less than 2% of susceptible individuals becoming sick and seroconversions detected up to 30 km from recorded outbreaks in the south. The major Old World vector Culicoides imicola Kieffer was not detected among over 70,000 Culicoides identified in summer collections, suggesting that BTV-9 transmission in Bulgaria was primarily carried out by indigenous European vectors. The most likely candidates, the Palaearctic species complexes - the Culicoides obsoletus Meigen and C. pulicaris L. complexes - were widespread and abundant across the whole country. The C. obsoletus complex represented 75% of all individuals trapped in summer and occurred in high catch sizes (up to 15,000 individuals per night) but was not found across all outbreak sites, indicating that both Palearctic complexes probably played a role in transmission. Within the C. pulicaris complex, only C. pulicaris s.s., C. punctatus Meigen and C. newsteadi Austen were sufficiently abundant and prevalent to have been widely involved in transmission, whilst within the C. obsoletus complex most trapped males were C. obsoletus s.s. Adult vectors were found to be largely absent from sites in west Bulgaria for a period of at least 3 months over winter, which, taken along with the spatiotemporal pattern of outbreaks in the region between years, indicates the virus may be overwintering here by an alternative mechanism - either by covert persistence in the vertebrate host or possibly by persistence in larval stages of the vector.

Preschool healthcare utilisation related to home oxygen status.

OBJECTIVE: To determine, in prematurely born children who had bronchopulmonary dysplasia (BPD), if respiratory morbidity, healthcare utilisation, and cost of care during the preschool years were influenced by use of supplementary oxygen at home after discharge from the neonatal intensive care unit. DESIGN: Observational study. SETTING: Four tertiary neonatal intensive care units. PATIENTS: 190 children, median gestational age 27 weeks (range 22-31), 70 of whom received supplementary oxygen when discharged home. INTERVENTIONS: Review of hospital and general practitioner records together with a parent completed respiratory questionnaire. MAIN OUTCOME MEASURES: Healthcare utilisation, cost of care, cough, wheeze, and use of an inhaler. RESULTS: Seventy children had supplementary oxygen at home (home oxygen group), but only one had a continuous requirement for home oxygen beyond 2 years of age. There were no significant differences in the gestational age or birth weight of the home oxygen group compared with the rest of the cohort. However, between 2 and 4 years of age inclusive, the home oxygen group had more outpatient attendances (p = 0.0021) and specialist attendances (p = 0.0023), and, for respiratory problems, required more prescriptions (p<0.0001). Their total cost of care was higher (p<0.0001). In addition, more of the home oxygen group wheezed more than once a week (p = 0.0486) and were more likely to use an inhaler (p<0.0001). CONCLUSIONS: Children with BPD who have supplementary oxygen at home after discharge have increased respiratory morbidity and healthcare utilisation in the preschool years.

Health care utilisation of prematurely born, preschool children related to hospitalisation for RSV infection.

BACKGROUND: In prematurely born infants with chronic lung disease (CLD), RSV hospitalisation is associated with increased health service utilisation and costs in the first two years after birth. AIMS: To determine whether RSV hospitalisation in the first two years was associated with chronic respiratory morbidity during the preschool years in prematurely born children who had had CLD. METHODS: Retrospective review of readmissions, outpatient attendances, and community care in years 2-4 and, at age 5 years, assessment of the children's respiratory status and their health related quality of life. Comparison was made of the results of children who had had at least one hospitalisation in the first two years after birth for RSV infection (RSV group) to those of the rest of the cohort. Participants were 190 of an original cohort of 235 infants with CLD and a median gestational age 27 (range 22-33) weeks. RESULTS: The 33 children in the RSV group, compared to the rest of the cohort, had a greater duration of hospital stay and more outpatient appointments. The RSV group had required more prescriptions for all treatments and respiratory medications, and more had used an inhaler. The cost of care of the RSV group was higher (median 2630 pounds sterling [4000 Euros, US4800 dollars], range 124-18,091 pounds sterling versus 1360 pounds sterling [2500 Euros, US3000 dollars], range 5-18 929 pounds sterling ) and their health related quality of life was lower. CONCLUSION: In prematurely born children who had developed CLD, RSV hospitalisation in the first two years was associated with chronic respiratory morbidity and increased cost of care.

Disruption of cleft palate repair following the use of the laryngeal mask airway.

A 55-year-old man was admitted for routine examination of ears with insertion of grommets under general anaesthesia. At 2 years of age he had undergone successful repair of cleft lip and palate. A reinforced laryngeal mask airway was employed to maintain the airway. Postoperatively, it was evident he had suffered complete disruption of the soft palate repair, leading to velopharyngeal insufficiency with nasal regurgitation of fluids. We discuss the possible aetiology, having found no such reported injury pattern documented in the literature.

Seasonal abundance of four Culicoides spp. (Diptera: Ceratopogonidae) at Al-Ahsa oasis, Eastern Province, Saudi Arabia.

This report constitutes the first study of Culicoides spp. and their seasonal abundance at Al-Ahsa, the largest oasis in the Eastern Province of Saudi Arabia. New Jersey light traps were used to collect the midges at Mastock farm and Al-Mansura village. The mean monthly abundance was determined from October 1993 to October 1994. The mean monthly number per trap reached its minimum value during January 1994, increasing gradually from February to reach its maximum value during September 1994. During the study period, the following species were collected: Culicoides schultzei group (September), non-spotted group of Culicoides (September), Culicoides imicola (May) and Culicoides newstaedi (March). The potential importance of the Culicoides spp. in relation to arboviral activity in Saudi Arabia is discussed.

Post-burn breast resurfacing using an abdominal full-thickness skin graft.

We describe a case of delayed resurfacing of the right breast using the abdominal skin as a full-thickness graft in a 38-year-old woman who sustained a scald injury in childhood. The postoperative results are shown.

Home oxygen status and rehospitalisation and primary care requirements of infants with chronic lung disease.

OBJECTIVES: To determine whether the rehospitalisation and primary care requirements of infants with chronic lung disease (CLD) during the first two years after birth were influenced by a requirement for supplementary oxygen after discharge from the neonatal intensive care unit. METHODS: Review of records from both the hospital and general practitioner. PATIENTS: 235 infants, median gestational age 27 (range 22-31) weeks, 88 of whom were receiving supplementary oxygen when discharged home. RESULTS: Overall, the infants required a median of 2 (range 0-20) admissions per patient, 8 (0-41) outpatient attendances, 13 (0-76) contacts with the general practitioner, and 17 (0-169) consultations with other primary healthcare professionals. The home oxygen patients required significantly more and longer admissions (p < 0.01) and more outpatient attendances (p < 0.05). The total cost of care per infant of the home oxygen group was greater (p < 0.001); this reflected higher costs for hospital stay (p < 0.01), total inpatient care (p < 0.01), and primary care drugs (p < 0.01). CONCLUSION: Despite routine use of antenatal steroids and postnatal surfactant, certain patients with CLD, particularly those who receive home oxygen treatment, show high rates of utilisation of health service resources after discharge from the neonatal care unit.

Health care utilisation of infants with chronic lung disease, related to hospitalisation for RSV infection.

AIMS: To compare the use of health care resources and associated costs between infants with chronic lung disease (CLD) who had or had not an admission with a proven respiratory syncytial virus (RSV) infection. METHODS: Review of community care, outpatient attendances, and readmissions in the first two years after birth. PATIENTS: 235 infants (median gestational age 27 weeks) evaluated in four groups: 45 infants with a proven RSV admission (RSV proven); 24 with a probable bronchiolitis admission; 60 with other respiratory admissions; and 106 with non-respiratory or no admissions. RESULTS: The RSV proven compared to the other groups required more frequent and longer admissions to general paediatric wards and intensive care units, more outpatient attendances and GP consultations for respiratory related disorders, and had a higher total cost of care. CONCLUSION: RSV hospitalisation in patients with CLD is associated with increased health service utilisation and costs in the first two years after birth.

Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach.

1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, alpha5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3beta4alpha5 receptors expressed in oocytes. 2. As previously reported, the main indication of the presence of alpha5 in alpha3beta4alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3beta4 receptors). If the alpha3beta4alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, alpha5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4alpha5 vs. alpha3(LT)beta4 and by 40 fold for alpha3beta4(LT)alpha5 vs. alpha3beta4(LT)). The much greater effect observed in alpha3beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3beta4alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4. Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC(50) for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3beta4alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.

Failure to thrive in babies with cleft lip and palate.

We established the frequency of failure to thrive (FTT) in children undergoing primary cleft procedures by using growth charts and standard-deviation scores. Initially, 147 babies with cleft lip and/or palate undergoing 186 primary lip-and-palate repairs were studied between 1993 and 1996. Rates of FTT were categorised according to cleft type. There was an increasing rate of FTT from 32% for unilateral cleft lip and palate to 38% for bilateral cleft lip and palate to 49% for cleft palate. There was a high incidence of FTT in palatal clefts, especially if these were associated with a syndrome or anomaly (P= 0.001). The incidence of FTT with the Pierre Robin sequence was 100%. In view of the high rates of FTT, two changes were instituted: a feeding-support nurse was appointed to supervise and monitor patients at risk and all patients with the Pierre Robin sequence had supervised airway management. Thereafter, the incidence of FTT was prospectively studied in 68 babies undergoing 84 primary procedures between 1997 and 1999. There was a decrease in the incidence of FTT in comparison with the earlier cohort (9% for unilateral cleft lip and palate, 20% for bilateral cleft lip and palate, 26% for cleft palate). There was a significant decrease in the incidence of FTT in the group with the Pierre Robin sequence, from 100% to 40%. As a result of the provision of a feeding-support nurse and airway management of patients with the Pierre Robin sequence, the incidence of FTT was reduced and the audit loop closed.

Waterproofing in hypospadias: a refinement of the two-stage reconstruction.

A new technique for 'waterproofing' during the second stage of a two-stage hypospadias repair is described. It is simpler and involves less tissue dissection than other waterproofing techniques. The senior author has used it over a period of 6 years as standard practice. The technique has been used in a total of 24 cases, in which the fistula rate was 8% (two cases).

Velopharyngeal incompetence and chromosome 22q11 deletion.

Chromosome 22q11 deletion gives rise to various phenotypes, including cardiac malformations, velopharyngeal abnormalities, absent thymus, and neurological defects. We assessed, in a prospective study, chromosome 22q11 deletion in 50 of 144 patients with velopharyngeal incompetence in the absence of overt clefting. 18 (12.5% of the whole cohort and 36% of patients tested for the deletion) had the 22q11 deletion. This frequency differs from an estimated population prevalence of 0.025% and suggests a need for screening for the 22q11 deletion in these patients.

Patient satisfaction with cleft lip and palate services in a regional centre.

We sent a questionnaire to the parents of 478 children aged between 3 and 14 years who are under the care of the cleft team at the Queen Victoria Hospital, East Grinstead, and received 341 replies. A wide variety of questions were asked about aspects of patient satisfaction, and the results are reported and discussed. There was a high level of satisfaction with the service provided but 30% of parents would like to be more involved in treatment-planning decisions; 33% thought they had either not enough or no knowledge about cleft lip and palate and its treatment. Only 8% of parents would rather have seen the specialists separately than together in the joint clinic.

Stoichiometry of human recombinant neuronal nicotinic receptors containing the b3 subunit expressed in Xenopus oocytes.

The neuronal nicotinic subunit beta3 forms functional receptors when co-expressed with both an alpha and a beta subunit, such as alpha3 and beta4. We examined the subunit stoichiometry of these 'triplet' alpha3beta4beta3 receptors by expression in Xenopus oocytes of the alpha3, beta4 and beta3 subunits, either in wild-type form or after insertion of a reporter mutation. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9THORN ) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated. In alpha3beta4beta3 receptors, the ACh EC50 was decreased by approximately 17-fold when this mutation was in alpha3 alone and only by fourfold when beta3 alone was mutated. Mutating beta4 was equivalent to mutating alpha3, suggesting that the 'triplet' receptor contains one copy of beta3 and two copies each of alpha3 and beta4. Mutating beta3 and alpha3 or beta3 and beta4 reduced the ACh EC50 further, to values two- to threefold lower than those seen when only alpha3 or beta4 carried the mutation. In 'pair' alpha3beta4 receptors (known to contain two alpha and three beta subunits), mutating beta4 had a greater effect on the ACh EC50 than mutating alpha3, in agreement with an alpha:beta ratio of 2:3 and a constant and independent effect of each copy of the mutation. Our results suggest that alpha3beta4beta3 neuronal nicotinic receptors contain one copy of beta3 and two copies each of alpha3 and beta4 and confirm that in pair alpha3beta4 receptors the alpha/beta subunits are present in a 2:3 ratio.

Use of cartilage grafts for closure of cleft palate fistulae.

We describe the results of using a free cartilage graft in the closure of cleft palate fistulae in 14 patients with a mean follow-up of 8.6 months. Complete closure of the fistula was achieved in 11 patients (79%), with partial closure in the remaining three patients. This technique is simple, causes relatively little discomfort, involves little tissue dissection and can be performed as a day-case procedure. The success rate is comparable with or better than other methods, and we consider it the treatment of choice for small cleft palate fistulae.