Neonatal emergency transport teams and general emergency departments: Who will intubate the neonate?

OBJECTIVE: Confidence treating critically ill infants presenting to general ED may be limited by inexperience, with procedures deferred until specialised transport teams arrive. METHODS: This retrospective cohort study analysed critical procedures performed by referring ED physicians, compared with a neonatal emergency transport service, on infants transferred over a 12-month period. RESULTS: All 150 eligible infants were included, with median (interquartile range) age 28 (16-43) days. Forty critical procedures were performed in this cohort. Of 26 intubations, 17 (65%) were performed by local ED physicians. CONCLUSION: Referring ED physicians perform the majority of critical procedures where infants require inter-hospital transfer by neonatal emergency transport service.

Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells.

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

Frequent exchange of the DNA polymerase during bacterial chromosome replication.

The replisome is a multiprotein machine that carries out DNA replication. In Escherichia coli, a single pair of replisomes is responsible for duplicating the entire 4.6 Mbp circular chromosome. In vitro studies of reconstituted E. coli replisomes have attributed this remarkable processivity to the high stability of the replisome once assembled on DNA. By examining replisomes in live E. coli with fluorescence microscopy, we found that the Pol III* subassembly frequently disengages from the replisome during DNA synthesis and exchanges with free copies from solution. In contrast, the DnaB helicase associates stably with the replication fork, providing the molecular basis for how the E. coli replisome can maintain high processivity and yet possess the flexibility to bypass obstructions in template DNA. Our data challenges the widely-accepted semi-discontinuous model of chromosomal replication, instead supporting a fully discontinuous mechanism in which synthesis of both leading and lagging strands is frequently interrupted.

Implications of a negative abdominal CT in the management of pediatric blunt abdominal trauma.

BACKGROUND: No consensus exists on management of children with a negative trauma CT following blunt abdominal trauma (BAT). Asymptomatic children are frequently "admitted for observation" following negative CT owing to concerns about missing an intraabdominal injury (IAI) without evidence for this practice. We aimed to investigate the feasibility of discharge following a negative CT scan in children sustaining blunt abdominal trauma. METHODS: Retrospective audit at a UK paediatric major trauma center and review of the literature. RESULTS: 108 patients were included (median age 11y; 60% male). Commonest mechanisms of injury: road traffic collisions (61 patients; 56%) and falls from a height (37; 34%). 40 (37%) had a normal CT scan, of whom 6 (15%) were discharged from ED. The remaining 34 patients were admitted, of whom 14 (41%) were discharged within 24h. The other 20 children were admitted for other specialty input. None of the 108 children had a missed IAI or reattended with suspicion of IAI. The NPV for CT to detect IAI was 100% (95% CI: 96%-100%). The literature search identified 3 observational cohort studies and 2 patient groups contained in a systematic review (total of 9149 patients with normal CT abdomen after BAT). Only 9 (<0.1%) patients required operative intervention for missed IAI. The NPV for CT to detect IAI was 99.6%-99.8% (95% CI 99%-100%). CONCLUSION: Our study and literature review demonstrate that asymptomatic children with a normal abdominal CT scan in the ED are very unlikely to have IAI and that the NPV of CT is very high (96%-100%). Direct discharge from the ED is possible for asymptomatic children with a negative CT following blunt abdominal trauma, as long as no other reasons for admission exist and should be accompanied by safety-net advice. LEVEL OF EVIDENCE STATEMENT: This is a level II evidence study. In itself it is a retrospective study, with the literature review including one large, high-quality prospective cohort study, and further prospective cohort studies of ordinary quality.

A Replisome's journey through the bacterial chromosome.

Genome duplication requires the coordinated activity of a multi-component machine, the replisome. In contrast to the background of metabolic diversity across the bacterial domain, the composition and architecture of the bacterial replisome seem to have suffered few changes during evolution. This immutability underlines the replisome's efficiency in copying the genome. It also highlights the success of various strategies inherent to the replisome for responding to stress and avoiding problems during critical stages of DNA synthesis. Here we summarize current understanding of bacterial replisome architecture and highlight the known variations in different bacterial taxa. We then look at the mechanisms in place to ensure that the bacterial replisome is assembled appropriately on DNA, kept together during elongation, and disassembled upon termination. We put forward the idea that the architecture of the replisome may be more flexible that previously thought and speculate on elements of the replisome that maintain its stability to ensure a safe journey from origin to terminus.

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus.

DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNA-polymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.

Low back pain in the paediatric athlete.

BACKGROUND: This study was designed to provide an overview of the epidemiology and clinical findings in children presenting to a sports injury clinic with 'low back pain' (LBP). OBJECTIVES: The aim of this study was to determine the pattern of presentation, management and outcome of children and adolescents presenting with back pain to a specialist paediatric sports injury clinic. MATERIALS AND METHODS: A retrospective descriptive review of patients aged 8-16 years presenting with LBP to a specialist at sports injury clinic between January 2004 and December 2010 was performed. Epidemiological variables, historical points and examination features, including several 'red flags', were evaluated in each patient and the findings related to the diagnosis made from the consultant radiologist reported imaging at that time. RESULTS: A total of 174 patients were analysed. LBP constituted 30% of presentations to the clinic. The median patient age in the study group was 14.0 years, with the male to female ratio of the population analysed being almost 1 : 1. For males, the most prevalent primary sport was rugby and for females, swimming was the most prevalent primary sport. Biomechanical back pain was most frequently diagnosed with spondylolysis, the most prevalent radiological diagnosis. CONCLUSION: No consistent demonstrable association was established between clinical presentation and final diagnosis. It was found that red flags could not be relied upon for the inclusion or the exclusion of a significant radiological finding. This study therefore suggests that, in this population group, a significant diagnosis cannot always be reliably excluded from clinical assessment alone.

Tools for detection of Mycoplasma amphoriforme: a primary respiratory pathogen?

Mycoplasma amphoriforme is a recently described organism isolated from the respiratory tracts of patients with immunodeficiency and evidence of chronic infection. Novel assays for the molecular detection of the organism by real-time quantitative PCRs (qPCRs) targeting the uracil DNA glycosylase gene (udg) or the 23S rRNA gene are described here. The analytical sensitivities are similar to the existing conventional M. amphoriforme 16S rRNA gene PCR, with the advantage of being species specific, rapid, and quantitative. By using these techniques, we demonstrate the presence of this organism in 17 (19.3%) primary antibody-deficient (PAD) patients, 4 (5%) adults with lower respiratory tract infection, 1 (2.6%) sputum sample from a patient attending a chest clinic, and 23 (0.21%) samples submitted for viral diagnosis of respiratory infection, but not in normal adult control subjects. These data show the presence of this microorganism in respiratory patients and suggest that M. amphoriforme may infect both immunocompetent and immunocompromised people. Further studies to characterize this organism are required, and this report provides the tools that may be used by other research groups to investigate its pathogenic potential.

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation.

Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.

Molecular machines in archaeal DNA replication.

The archaeal DNA replication apparatus is a simplified version of that of eukaryotes and has attracted attention as a tractable model system for the orthologous, but significantly more complex eukaryal machinery. A variety of archaeal model organisms have been investigated with strong emphasis on structural and biochemical analyses of replication-associated proteins. In this review we will describe recent advances in understanding the properties of the replicative helicase, the MCM complex, and the role of the sliding clamp, PCNA, in mediating a range of protein-DNA transactions. Although both complexes form ring shaped assemblies, they play very distinct roles at the leading and trailing edges of the replication fork machinery respectively.

The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes.

Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5'-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.

The epidemiology of fractures in children.

A retrospective study of all paediatric fractures presenting to hospital in Edinburgh, Scotland in 2000 was undertaken. It showed that the incidence of fractures was 20.2/1000/year and that 61% of children's fractures occurred in males. Analysis of paediatric fractures shows that there are six basic fracture distribution curves with six fractures showing a bimodal distribution but most having a unimodal distribution affecting younger or older children. The incidence of fractures increases with age with falls from below bed height (<1m) being the commonest cause of fracture. The majority of fractures in children involve the upper limb. Lower limb fractures are mainly caused by twisting injuries and road traffic accidents. The incidence of fractures in cyclists and pedestrians remains relatively high whereas the incidence in vehicle occupants is low suggesting that road safety programs have been successful. Similar programs should be instituted for young cyclists. The importance of accident prevention programmes in the home is also highlighted.

The impact of national guidelines on the assessment and management of acute paediatric asthma presenting at a tertiary children's emergency department.

BACKGROUND: Recent Scottish Intercollegiate Guidelines Network/British Thoracic Society guidelines have highlighted best practice for asthma management. This study examines asthma management in a paediatric emergency setting before and after the publication of these guidelines. OBJECTIVES: To assess the impact of Scottish Intercollegiate Guidelines Network/British Thoracic Society guidelines on asthma management. METHODS: Retrospective review of patient notes over two equivalent 2-month periods in 2002 and 2003. Main outcomes were documentation of clinical history, examination, investigation, treatment and discharge; and also the use of various treatment modalities in each case. RESULTS: One hundred and sixty-four children presented with asthma, 100 in 2002 and 64 in 2003. Documentation was adequate throughout, though better when nursing staff were responsible. Completeness of documentation was not related to seniority or discipline of medical staff. Measurement of peak flow was poor in both years. The 'doubling up' of inhaled steroid dose for acute episodes was the only aspect of management affected by publication of the guidelines, with significantly fewer patients receiving this in 2003 (P<0.0001). CONCLUSIONS: Documentation within the centre is good but has potential for improvement. Guidelines have not impacted on this except when explicit statements are made regarding treatment.

Acute lymphoblastic leukaemia presenting with atraumatic intermittent limp: a case report.

We report a case of a 34-month-old girl who presented with acute atraumatic intermittent limp. History and examination at this stage were not suggestive of an underlying serious disorder, but routine blood testing revealed abnormal results and prompted further investigations. She was subsequently diagnosed with acute lymphoblastic leukaemia. This case illustrates how routine phlebotomy lead to early diagnosis and facilitated instant therapy resulting in a significantly improved outcome for the child.

Clinical diagnosis of fractures in a paediatric population.

Diagnosing fractures in the paediatric population is a problematical process for which there are currently no accepted clinical criteria. We studied the physical signs sought by accident and emergency staff in 126 children with suspected fractures. We found a significant correlation between 'point tenderness' and fracture, as demonstrated by plain radiograph. 'Swelling' and 'redness' approached significance. These results correlate well with previous work in this area, but further research using a larger sample is required as confirmation.

Is measurement of capillary refill time useful as part of the initial assessment of children?

OBJECTIVE: The purpose of this study was to establish the usefulness of capillary refill time when measured during the initial assessment of children. METHODS: All children with spontaneous illness attending a paediatric accident and emergency department over a 7-month period were eligible for entry into the study. Capillary refill time was measured at the fingertip, using a standard technique, as part of the initial assessment. Each child was then followed up to ascertain clinical progress, including the need for admission, intravenous fluids, length of stay and diagnosis, as well as the white cell count when this was available. The value of capillary refill time as a predictor of the markers of illness severity was then assessed. RESULTS: Capillary refill time measurements were recorded on 4878 children. There was no significant association of capillary refill time with meningococcal disease, other significant bacterial illness or the white cell count. A prolonged capillary refill time was associated with a more urgent triage category, the administration of a fluid bolus and the length of hospital stay (P<0.0001). The best performance was obtained when a capillary refill time of 3s or more is taken to be 'prolonged'. However, this gave positive predictive values of only 9% for a triage category of 1 or 2 (negative predictive value 97%), 11% for requiring a fluid bolus (negative predictive value 99%), 55% for hospital admission (negative predictive value 65%) and 22% for stay over 2 days/death (negative predictive value 91%). CONCLUSION: The prolongation of capillary refill time is a poor predictor of the need for intravenous fluid bolus or hospital admission.

Pain assessment in the paediatric Emergency Department: whose view counts?

OBJECTIVES: To compare patient, guardian and professional assessment of acute pain in children presenting to an Emergency Department, and to examine whether there was a correlation between the scores obtained using the Faces and linear scales for each group. METHODS: A prospective, observational cohort study of 73 children aged 4-14 years attending a paediatric hospital Emergency Department between March and April 2002 with pain caused by an acute injury. The child's pain on admission, as estimated by the child, their guardian and a healthcare professional (nurse/doctor/emergency nurse practitioner) was recorded using a Faces scale and a linear scale. RESULTS: Professionals consistently score pain lower [median linear scale score 3.1; interquartile range (IQR) 1.6-5.3] than do patients (6.6; 4.9-7.4) or guardians (6.0; 3.9-7.1) using both linear and Faces scales. There is a significant correlation between pain scores obtained using the two scales for professionals [Spearman R value 0.88; 95% confidence interval (CI) 0.82-0.93], guardians (0.83; 0.74-0.89) and patients (0.42; 0.21-0.59). CONCLUSION: Professionals score pain lower than do children or guardians. Similar pain scores are obtained using both a Faces and a linear scale. This study offers no support for the introduction of a uniform pain assessment tool in a paediatric Emergency Department setting.