New and emerging drug treatments for severe asthma.

Asthma is a common chronic inflammatory condition of the airways affecting over 300 million people world-wide. In 5%-10% of cases, it is severe, with disproportionate healthcare resource utilization including costs associated with frequent exacerbations and the long-term health effects of systemic steroids. Characterization of inflammatory pathways in severe asthma has led to the development of targeted biological and small molecule therapies which aim to achieve disease control while minimizing corticosteroid-associated morbidity. Herein, we review currently licensed agents and those in development, and speculate how drug therapy for severe asthma might evolve and impact on clinical outcomes in the near future.

Died of wounds: a mortality review.

OBJECTIVES: Combat casualty care is a complex system involving multiple clinicians, medical interventions and casualty transfers. Improving the performance of this system requires examination of potential weaknesses. This study reviewed the cause and timing of death of casualties deemed to have died from their injuries after arriving at a medical treatment facility during the recent conflicts in Iraq and Afghanistan, in order to identify potential areas for improving outcomes. METHODS: This was a retrospective review of all casualties who reached medical treatment facilities alive, but subsequently died from injuries sustained during combat operations in Afghanistan and Iraq. It included all deaths from start to completion of combat operations. The UK military joint theatre trauma registry was used to identify cases, and further data were collected from clinical notes, postmortem records and coroner's reports. RESULTS: There were 71 combat-related fatalities who survived to a medical treatment facility; 17 (24%) in Iraq and 54 (76%) in Afghanistan. Thirty eight (54%) died within the first 24 h. Thirty-three (47%) casualties died from isolated head injuries, a further 13 (18%) had unsurvivable head injuries but not in isolation. Haemorrhage following severe lower limb trauma, often in conjunction with abdominal and pelvic injuries, was the cause of a further 15 (21%) deaths. CONCLUSIONS: Severe head injury was the most common cause of death. Irrespective of available medical treatment, none of this group had salvageable injuries. Future emphasis should be placed in preventative strategies to protect the head against battlefield trauma.

Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera.

The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids.

A qualitative study of the use of the four quadrant approach to assist ethical decision-making during deployment.

AIM: The aim of the study was to explore how useful clinicians deployed to the Field Hospital in Afghanistan found using the four quadrant approach (4QA) as a tool to aid ethical decision-making. In addition, the study aimed to determine whether the 4QA needed to be amended to make it more effective in assisting the ethical decision-making process for military health professionals on deployment. METHOD: A qualitative pilot study in two phases was undertaken between September 2012 and January 2013. In Phase I, senior deployed clinicians completed a pro forma of the 4QA on cases that potentially raised ethical issues. Thirteen pro formas were submitted on four cases; the Deployed Medical Director submitted a log of 14 cases that had involved using the 4QA. Phase II consisted of interviews with five senior clinicians who had recently returned from deployment in Afghanistan to discuss their experiences and perceptions of using the 4QA. RESULTS: Phase I identified a variation in the level of detail recorded and where that information was placed on the quadrant. Four themes were generated from Phase II. These included the characteristics of ethical decisions; the processes used to make ethical decisions; use, usefulness and limitations of the 4QA; and views about training in ethics. The findings suggested that amendments to the pro forma may improve its utility. CONCLUSIONS: The 4QA is a useful tool within an operational setting but amending its diagrammatic presentation could improve its effectiveness. Pre-deployment training should include practising using the quadrant as described in Clinical Guidelines for Operations. This is particularly important as the participants relied heavily on experience to help them make ethical decisions, and this experience may not be available in future operations outside Afghanistan.

The pattern of paediatric trauma on operations.

OBJECTIVES: Recent military campaigns in Iraq and Afghanistan have resulted in the treatment of children in British Medical facilities. In order to determine how care for children may develop in the future, it is necessary to understand the current situation. The aim of this article is to examine the pattern of paediatric trauma on recent operations in Iraq and Afghanistan. METHODS: Data was requested from the Joint Theatre Trauma Registry, held at the Royal Centre for Defence Medicine in Birmingham, on all trauma calls for patients aged under 16 between the dates 21/3/03 and 31/8/09. Data included age, gender, theatre of operation, injury mechanism and type, trauma scores and destination of the child. RESULTS: 176 children were identified with 16.5% from Iraq and 83.5% from Afghanistan. The overall survival rate was 88.6% with survival rates in Iraq of 89.7% and in Afghanistan of 88.4%. Males accounted for 66.5% of admissions and the commonest age group was age 6-8 years. In 59.1% of total admissions the mechanism of injury was related to explosives. This differed between theatres with explosive injury causing 27.6% of admissions in Iraq and 63.5% in Afghanistan. Injury Severity Scores (ISS) showed equal numbers of minor and severe injuries with fewer moderately injured patients. The median ISS of all data was nine. The median ISS from Iraq was 16 and the median ISS from Afghanistan was nine. CONCLUSIONS: The treatment of children in British medical facilities whilst deployed on operations is likely to continue. An assessment of the injury patterns of paediatric patients on current deployments allows development of training and an understanding of logistic requirements. Data collection will also need to be adapted to meet the needs of paediatric patients. These remain issues that are being addressed by the Defence Medical Services.

Critical decision-making and timelines in the emergency department.

The resuscitation of severely injured patients has evolved during the last decade. Patients are now surviving injuries that previously were thought to be unsurvivable. Systems have been put in place, that reflect the intensity of workload and severity of injury of patients presenting to deployed medical treatment facilities. This paper aims to describe some of the elements that may differ from routine practice in the resuscitation room, and in particular the role of timelines in the management of such patients.

Antigen-induced locomotor responses in lymphocytes.

The effect of protein antigens on the locomotion of lymphocytes from the lymph nodes draining the site of antigenic challenge in immunized mice, and from the same nodes in control mice, was studied in filters using a checkerboard assay in which the absolute concentration and the concentration gradient of attractant was varied in a series of chambers. Serum albumin (HSA or BSA) was chemokinetic for unimmunized lymphocytes inasmuch as the distance migrated into filters by cells in its presence varied with the absolute concentration of albumin, but not with the concentration gradient, indicating an influence of the serum albumin on the rate but not on the direction of locomotion. Ovalbumin and nonalbumin proteins did not show this effect. Using the same assay, the migration of primed lymphocytes in the presence of the priming antigen was shown to be influenced by the antigen gradient in a way that suggested a positive chemotactic response of the lymphocytes to antigen. This response was only shown clearly when the cells were in a chemokinetic medium containing serum albumin.

British military experience of pre-hospital paediatric trauma in Afghanistan.

INTRODUCTION: Trauma is a leading cause of death in children. Life support courses have been developed to reduce the mortality and morbidity of children suffering trauma; differences in anatomy and physiology may produce different injury patterns to adults when children are exposed to trauma, challenging the care providers. METHODS: A retrospective analysis of all paediatric patients transported by the helicopter-borne MERT between 01 May 2006 and 31 December 2007 in Helmand Province, Afghanistan. RESULTS: 78 children were brought in over the study period by the MERT team representing 7.3% of MERT casualties and 2.2% of the total seen in the Emergency Department. Breakdown by demographics, triage category, mechanism of injury, and treatment is given. CONCLUSION: A significant number of paediatric patients are treated by the deployed pre-hospital team. All military pre-hospital care providers should gain training and experience in the care of the seriously injured child prior to deployment.

Biofilm problems in dental unit water systems and its practical control.

Dental chair units (DCUs) contain integrated systems that provide the instruments and services for a wide range of dental procedures. DCUs use water to cool and irrigate DCU-supplied instruments and tooth surfaces during dental treatment. Water is supplied to these instruments by a network of interconnected narrow-bore (2-3 mm) plastic tubes called dental unit waterlines (DUWLs). Many studies over the last 40 years demonstrated that DUWL output water is often contaminated with high densities of micro-organisms, predominantly Gram-negative aerobic heterotropic environmental bacteria, including Legionella and Pseudomonas species. Untreated DUWLs host biofilms that permit micro-organisms to multiply and disperse through the water network and which are aerosolized by DCU instrument use, thus exposing patients and staff to these micro-organisms, to fragments of biofilm and bacterial endotoxins. This review concentrates on how practical developments and innovations in specific areas can contribute to effective DUWL biofilm control. These include the use of effective DUWL treatment agents, improvements to DCU supply water quality, DCU design changes, development of automated DUWL treatment procedures that are effective at controlling biofilm in the long-term and require minimal human intervention, are safe for patients and staff, and which do not cause deterioration of DCU components following prolonged use.

Hydrolysis of individual isomers of fluorogenic pyrethroid analogs by mutant carboxylesterases from Lucilia cuprina.

We previously showed that wild-type E3 carboxylesterase of Lucilia cuprina has high activity against Type 1 pyrethroids but much less for the bulkier, alpha-cyano containing Type 2 pyrethroids. Both Types have at least two optical centres and, at least for the Type 1 compounds, we found that wild-type E3 strongly prefers the less insecticidal configurations of the acyl group. However, substitutions to smaller residues at two sites in the acyl pocket of the enzyme substantially increased overall activity, particularly for the more insecticidal isomers. Here we extend these analyses to Type 2 pyrethroids by using fluorogenic analogs of all the diastereomers of cypermethrin and fenvalerate. Wild-type E3 hydrolysed some of these appreciably, but, again, not those corresponding to the most insecticidal isomers. Mutations in the leaving group pocket or oxyanion hole were again generally neutral or deleterious. However, the two sets of mutants in the acyl pocket again improved activity for the more insecticidal acyl group arrangements as well as for the more insecticidal configuration of the cyano moiety on the leaving group. The activities of the best mutant enzyme against the analogs of the most insecticidal isomers of cypermethrin and fenvalerate were more than ten and a hundred fold higher, respectively, than those of wild-type. The implications for resistance development are discussed.

Jhe in Gryllus assimilis: cloning, sequence-activity associations and phylogeny.

The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identified after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purified from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918bp) introns. No coding DNA sequence variation was observed over a 1293bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F(2) progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade.

The role of manufacturers in reducing biofilms in dental chair waterlines.

OBJECTIVES: This paper reviews how dental chair unit (DCU) manufacturers can contribute practically to resolving the problem of biofilm formation in dental unit waterlines (DUWs). STUDY SELECTION: The review concentrates on how novel developments and changes in a range of specific areas have, and might contribute to DUW biofilm control. These include (i) DCU engineering and design changes; (ii) improvements to DCU supply water quality; (iii) development of automated DUW treatment procedures that are effective at controlling biofilm in the long-term, safe for patients and dental staff, environmentally friendly and which do not exhibit adverse effects on DCU components after prolonged use. SOURCES: The majority of the material contained in this review is based on, or supported by the peer-reviewed literature. DATA: The current consensus from the literature reveals that the emphasis on DUW biofilm and its control has focused on describing the problem and its control using a range of periodic and residual DUW treatment agents. Unfortunately, until recently, DCU manufacturers have provided very little specific guidance in this regard. Indeed, ensuring that DCUs provide good quality output water has generally been regarded to be the responsibility of dental practitioners. Some recent studies have shown that novel DCUs with integral semi-automated or automated DUW cleaning systems can effectively control DUW biofilm in the long-term. However, there are other potential DCU engineering and design changes that DCU manufacturers could undertake to further improve DUW biofilm control. CONCLUSIONS: DCU manufacturers can significantly contribute to controlling the problem of DUW biofilm.

Optimisation of the long-term efficacy of dental chair waterline disinfection by the identification and rectification of factors associated with waterline disinfection failure.

UNLABELLED: Although many studies have highlighted the problem of biofilm growth in dental chair unit waterlines (DUWs), no long-term studies on the efficacy of DUW disinfection using a large number of dental chair units (DCUs) have been reported. OBJECTIVES: To investigate the long-term (21 months) efficacy of the Planmeca Waterline Cleaning System (WCS) to maintain the quality of DUW output water below the American Dental Association (ADA) recommended standard of < or =200cfu/mL of aerobic heterotrophic bacteria using once weekly disinfection with the hydrogen peroxide-and silver ion-containing disinfectant Planosil. METHODS: Microbiological quality of DUW output water was monitored by culture on R2A agar for 10 DCUs fitted with the WCS. The presence of biofilm in DUWs was examined by electron microscopy. RESULTS: During the first 9 months a high prevalence (28/300 disinfection cycles; 9.3%) of intermittent DUW disinfection failure occurred in 8/10 DCUs due to operator omission to disinfect all DUWs (10/28 failed cycles), incorrect compressed air pressure failing to distribute the disinfectant properly (4/28 failed cycles) and physical blockage of disinfectant intake valves due to corrosion effects of Planosil (14/28 failed cycles). On rectification of these faults through engineering redesign and procedural changes, no further cases of intermittent DUW disinfection failure were observed. Independently of these factors, a rapid and consistent decline in efficacy of DUW disinfection occurred in 4/10 DCUs following the initial 9 months of once weekly disinfection. There was a highly significant difference (P<0.0001) in the prevalence of strongly catalase-positive Novosphingobium and Sphingomonas bacterial species (mean average prevalence of 37.1%) in DUW output water from these 4 DCUs compared to the other 6 DCUs and DCU supply water (prevalence <1%), which correlated with biofilm presence in the DUWs and indicated selective pressure for maintenance of these species by prolonged disinfectant usage. Planosil was reformulated to a more concentrated form (Planosil Forte) and when used once weekly was found to maintain bacterial density in output water below the ADA standard for all 10 DCUs. CONCLUSIONS: A variety of factors can contribute to failure of DUW disinfection in the long-term, including human error, disinfectant corrosion of equipment and natural selection of naturally disinfectant-tolerant bacterial species.

Acclimatization of rats after ground transportation to a new animal facility.

This study aimed to assess the time needed by rats, which had not been previously transported, to acclimate to a new environment after 5 h of van transport, using physiological parameters as measures of acclimatization. Animal shipping boxes and transport van conditions were standardized to minimize stress factors that could be associated with transport. Heart rate (HR), body temperature and activity levels were measured in the rats before and after transport using previously implanted radio-telemetry transmitters. Body weight was also recorded. All parameters were changed significantly except for body temperature. Results suggest that rats take three days to acclimate to a new environment, as measured by the physiological parameters of body weight, HR and activity.

The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum.

BACKGROUND: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the thermophilic Archaea tolerate temperatures in the range 55-110 degrees C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 degrees C) may provide important information on the structural basis of protein thermostability. We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies. RESULTS: We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). CONCLUSIONS: The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.

Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium.

BACKGROUND: The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric citrate synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55 degrees C and 100 degrees C having previously been determined. Here the study is extended to include a citrate synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exit. RESULTS: We report here the first crystal structure of a cold-active enzyme, citrate synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 A. In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain. Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs. CONCLUSIONS: Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency. For hyperthermophiles, thermal denaturation of the citrate synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins. For the cold-active citrate synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme. Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain.

Elimination of biofilm and microbial contamination reservoirs in hospital washbasin U-bends by automated cleaning and disinfection with electrochemically activated solutions.

BACKGROUND: Washbasin U-bends are reservoirs of microbial contamination in healthcare environments. U-Bends are constantly full of water and harbour microbial biofilm. AIM: To develop an effective automated cleaning and disinfection system for U-bends using two solutions generated by electrochemical activation of brine including the disinfectant anolyte (predominantly hypochlorous acid) and catholyte (predominantly sodium hydroxide) with detergent properties. METHODS: Initially three washbasin U-bends were manually filled with catholyte followed by anolyte for 5min each once weekly for five weeks. A programmable system was then developed with one washbasin that automated this process. This U-bend had three cycles of 5min catholyte followed by 5min anolyte treatment per week for three months. Quantitative bacterial counts from treated and control U-bends were determined on blood agar (CBA), R2A, PAS, and PA agars following automated treatment and on CBA and R2A following manual treatment. FINDINGS: The average bacterial density from untreated U-bends throughout the study was >1×10(5) cfu/swab on all media with Pseudomonas aeruginosa accounting for ∼50% of counts. Manual U-bend electrochemically activated (ECA) solution treatment reduced counts significantly (<100cfu/swab) (P<0.01 for CBA; P<0.005 for R2A). Similarly, counts from the automated ECA-treatment U-bend were significantly reduced with average counts for 35 cycles on CBA, R2A, PAS, and PA of 2.1±4.5 (P<0.0001), 13.1±30.1 (P<0.05), 0.7±2.8 (P<0.001), and 0 (P<0.05) cfu/swab, respectively. P. aeruginosa was eliminated from all treated U-bends. CONCLUSION: Automated ECA treatment of washbasin U-bends consistently minimizes microbial contamination.

Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase.

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation.

Crystallization and preliminary crystallographic study of citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum.

Single crystals of citrate synthase from the Archaeon Thermoplasma acidophilum were obtained in two forms using the hanging drop vapour diffusion method and polyethylene glycol 3350 as precipitant. Type 1 crystals belong to the orthorhombic space group P222(1), with unit cell dimensions a = 80.9 A, b = 103.8 A, c = 98.3 A and one dimer in the asymmetric unit. Type 2 crystals belong to the monoclinic space group P2(1), with unit cell dimensions a = 53.8 A, b = 173.8 A, c = 86.7 A and beta = 97.1 degrees and two dimers in the asymmetric unit.

Engineering thermostability: lessons from thermophilic proteins.

As several groups begin to tap the rich pickings found in the Archaea--a vast kingdom that stretches the concept of life as we know it--the structures of proteins from hyperthermophiles are being elucidated. Certain features are beginning to emerge, such as compactness and hydrophobic clustering, but the ability to engineer these features into temperature-intolerant proteins is still some way off.