Supporting doctors as healthcare quality and safety advocates: Recommendations from the Association of Surgeons in Training (ASiT).

BACKGROUND: As front-line healthcare staff, doctors in surgical training occupy a unique organisational space rotating through hospitals and services in which they witness first hand both good and bad practice. This puts trainees in a clear position to identify and raise patient safety issues, and to contribute to discussions regarding quality and safety improvement. However, there are a number of real and perceived barriers to trainees doing so. These include concerns about the impact on training assessments and career progression, and uncertainty about the appropriate route. METHODS: Paper-based survey of delegates attending the Association of Surgeons in Training (ASiT) conference (response rate 73%; 479/652). RESULTS: 288 (60%) of trainees reported previous concerns over practices and behaviour of colleagues that might pose risks to patient care including concerns over poor performance (n = 243; 84%), bullying (n = 45; 16%), alcohol and drug abuse (n = 15; 5%) and mental health problems (n = 8; 3%). However, 53% (n = 153) did not escalate these concerns. 178 (37%) of trainees also reported concerns over hospital policies, protocols or systems that might pose a risk to patient care, with 46% (n = 82) not escalating such concerns. Respondents highlighted fear of personal vilification or reprisal (n = 224; 47%), fear of impact on career (n = 206; 43%) and a lack of confidence in the process (n = 170; 36%) as barriers to whistleblowing. More senior trainees were significantly more likely to raise concerns than more junior grades (p < 0.0001). CONCLUSION: These results highlight worrying issues around reporting concerns, with trainees often "silent witnesses" to poor performance in healthcare. Adverse events must provide opportunities for learning to improve future outcomes. Herein, ASiT proposes 14 recommendations to improve protection for trainees in raising patient safety concerns. These include the creation of a positive workplace culture, promoting the active involvement of trainees in quality improvement discussions, with clear mechanisms for trainees to raise concerns.

A systematic review of the incidence of and risk factors for postoperative atrial fibrillation following general surgery.

Atrial fibrillation is a common cardiac arrhythmia and can occur de novo following a surgical procedure. It is associated with increased inpatient and long-term mortality. There is limited evidence concerning new-onset atrial fibrillation following abdominal surgery. This study aimed to identify the prevalence of and risk factors for postoperative atrial fibrillation in the general surgical population. A systematic search of the Embase, MEDLINE and Cochrane (CENTRAL) databases was conducted. Studies were included in the review if they reported cases of new-onset atrial fibrillation within 30 days of the index operation. Results were evaluated qualitatively due to substantial clinical heterogeneity. Incidence rates were pooled using a weighted random-effects meta-analysis model. A total of 835 records were initially identified, from which 32 full texts were retrieved. Following review, 13 studies were included that involved 52,959 patients, of whom 10.94% (95%CI 7.22-15.33) developed atrial fibrillation. Five studies of patients undergoing oesophagectomy (n = 376/1923) had a weighted average rate of 17.66% (95%CI 12.16-21.47), compared with 7.63% (95%CI 4.39-11.98) from eight studies of non-oesophageal surgery (n = 2927/51,036). Identified risk factors included: increasing age; history of cardiac disease; postoperative complications, particularly, sepsis, pneumonia and pleural effusions. New-onset postoperative atrial fibrillation is common, and is more frequent after surgery involving the thorax. Future work should focus on stratifying risk to allow targeted prophylaxis of atrial fibrillation and other peri-operative complications.

Undermining and bullying in surgical training: A review and recommendations by the Association of Surgeons in Training.

The 2012 General Medical Council National Trainees' Survey found that 13% of UK trainees had experienced undermining or bullying in the workplace. The Association of Surgeons in Training subsequently released a position statement raising concerns stemming from these findings, including potential compromise to patient safety. This article considers the impact of such behaviour on the NHS, and makes recommendations for creating a positive learning environment within the NHS at national, organisational, and local levels. The paper also discusses the nature of issues within the UK, and pathways through which trainees can seek help.

Emergency cross-cover of surgical specialties: consensus recommendations by the Association of Surgeons in Training.

In recent years, working time restrictions and a restructuring of postgraduate surgical training have resulted in increased reliance on emergency cross-cover (ECC)--the provision of emergency care by a doctor trained or training in a different specialty to that which they are requested to assess or manage. There are increasing concerns surrounding the provision of ECC, particularly regarding appropriate supervision of trainees and in turn their competence, experience and confidence in dealing with surgical problems of outside their own specialty. Surgical training has failed to keep pace with workforce changes and in this document we outline the key principles of providing safe ECC. In particular this includes the medico-legal implications of providing such cover outside a surgical trainee's normal area of practice, particularly without previous experience or means for regular skills practice and up-dating. We report the findings of an ASiT snapshot survey that demonstrates concerns surrounding existing cross-cover arrangements. Variable access to senior support, together with varied willingness to provide this, and a paucity of specific training opportunities for trainees required to provide cross-cover were highlighted. These have the potential to promote variability in patient care and resource use by those providing care outside of their usual specialty. This document provides consensus recommendations to address these issues, including clarification of curricula and improved provision of training for, and supervision of, trainees who are expected to deliver cross-cover.

Association of Surgeons in Training Conference: Cardiff 2012.

The Association of Surgeons in Training (ASiT) is a professional body and registered charity working to promote excellence in surgical training for the benefit of junior doctors and patients alike. With a membership of over 2000 surgical trainees from all ten surgical specialities, the association provides support at both regional and national levels throughout the United Kingdom and Republic of Ireland. Originally founded in 1976, ASiT is independent of the National Health Service (NHS), Surgical Royal Colleges, and speciality associations. The 2012 Annual Conference in Cardiff City Hall brought together nearly 700 delegates for an educational weekend programme with expert guest speakers. Clinical updates were complimented by debates on current training in surgery, and the weekend included 6 pre-conference courses covering a diverse range of topics including laparoscopic skills, surgical drawing and a masterclass in journal club. A record number of 1168 abstract submissions were received and those successful competed for 18 awards representing £3500 in trainee prizes and bursaries. As the only national surgical trainee meeting for all specialities, ASiT continues to grow and we look forward to an even larger and more successful conference next year.

Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.

Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.

The influence of external environment fluctuations on the signal formation of microbiosensors.

This part of theoretical analysis describes the fluctuations of output signal of microbiosensors when the number of accessible molecular recognition elements (enzymes, receptors, antibodies, etc.) fluctuated under external environmental influences. The mean electric current, dispersion correlating function, as well as spectral density of output current fluctuation are analyzed, and it is shown that a comparison of theoretically calculated mean current and correlation function with experimental data allow a determination of the kinetic parameters of substrate binding reaction with the molecular recognition element of biosensor.

Allosteric signal transmission involves synergy between discrete structural units of the regulatory subunit of aspartate transcarbamoylase.

Previous studies have shown that the S5' beta-strand (r93-r97) of the regulatory polypeptides of the aspartate transcarbamoylases (ATCases) from Serratia marcescens and Escherichia coli are responsible for their diverged allosteric regulatory patterns, including conversion of CTP from an inhibitor in E. coli to an activator in S. marcescens. Similarly, mutation of residues located in the interface between the allosteric and the zinc domains resulted in conversion of the ATP responses of the E. coli enzyme from activation to inhibition, suggesting that this interface not only mediates but also discriminates the allosteric responses of ATP and CTP. To further decipher the roles and the interrelationships of these regions in allosteric communication, allosteric-zinc interface mutations (Y77F and V106A) have been introduced into both the native and the S5' beta-strand chimeric backgrounds. While the significance of this interface in the allosteric regulation has been confirmed, there is no direct evidence supporting the presence of distinct pathways for the ATP and CTP signals through this interface. The analysis of the mutational effects reported here suggested that the S5' beta-strand transmits the allosteric signal by modulating the hydrophobic allosteric-zinc interface rather than disturbing the allosteric ligand binding. Intragenic suppression by substitutions in the hydrophobic interface between the allosteric and the zinc domains of the regulatory chains resulted in the partial recovery of allosteric responses in the EC:rS5'sm chimera and reduced the activation by ATP in the Sm:rS5'ec chimera. Thus, it seems that there is a synergy between these two structural units.

Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola.

The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified.

Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase.

The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity for a new evaluation of the common paradigm involving allosteric control of ATCase.

Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins.

A simple approach is described for preparing poly-(ethylene glycol) hydrogel materials with encapsulated seminapthofluorescein (SNAFL)-organophosphorus hydrolase enzyme conjugates. Direct determination of enzyme-catalyzed neurotoxin hydrolysis is provided by the self-referencing, pH-sensitive dye SNAFL-1, whose emission spectrum changes at lambda = 550 in response to pH. Using spectrofluorimetry and paraoxon as a model organophosphate, paraoxon concentrations as low as 8 x 10(-7) M could be readily detected. On the basis of the signal-to-noise ratio, a detection limit of 16 nM was determined. The materials demonstrated high stability against enzyme-denaturing, leaching, and photobleaching when stored under ambient conditions.

Rational design of organophosphorus hydrolase for altered substrate specificities.

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a broad variety of OP neurotoxins, including chemical warfare agents and many widely used pesticides. OPH has extremely high hydrolytic efficiency with different phosphotriester and phophothiolester pesticides (k(cat) = 50-15,000 s(-1)) as well as phosphorofluorates such as DFP and the chemical warfare agents sarin and soman (k(cat) = 50-11,000 s(-1)). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl aminoethyl) methylphosphonothioate] (k(cat) = 0.3-20 s(-1)). Different metal-associated forms of the enzyme have demonstrated varying hydrolytic capabilities for each of the OP neurotoxins, and the activity of OPH (Co2+) is consistently higher than that of OPH (Zn2+) by five- to 20-fold. Protein engineering strategies have exploited these metal-induced catalytic differences, and other slight modifications to the opd gene have resulted in significant enhancement of the rates of detoxification of the thioate pesticides and chemical warfare agents. In order to develop practical applications of OPH, other experiments have focused on improvement of enzyme production, localization, stability, and shelf-life, as well as efficient catalysis of substrates of interest.

Biodegradation of neutralized sarin.

This research investigated the biotransformation of IMPA, the neutralization product of the nerve agent Sarin, by a microbial consortia. As mandated by the Chemical Weapons Convention signed by 132 countries in 1993, all chemical warfare agents are to be destroyed within ten years of ratification. Technologies must be developed to satisfy this commitment. This paper presents data from a biodegradation kinetics study and background information on the biological transformation of IMPA. Microbial transformation of organophosphate nerve agents and organophosphate pesticide intermediates can be incorporated into a treatment process for the fast and efficient destruction of these similar compounds. Sarin (isopropyl methylphosphonofluoridate), also known as GB, is one of several highly neurotoxic chemical warfare agents that have been developed over the past 50 to 60 years. Four mixed cultures were acclimated to the Sarin hydrolysis product, isopropyl methylphosphonic acid (IMPA). Two of these cultures, APG microorganisms and SX microorganisms, used IMPA as the sole phosphorus source. Extended exposure to IMPA improved the cultures' abilities to degrade IMPA to form methylphosphonic acid (MPA) and inorganic phosphate. The presence of free phosphate in the reactor suppressed the degradation of IMPA. IMPA did not inhibit either cultural consortia within the tested concentration range (0 to 1250 mg/L). The numax was 120.9 mg/L/day for the SX microorganisms and 118.3 mg/L/day for the APG microorganisms. Initial IMPA concentrations of 85 to 90 mg/L were degraded to nondetectable levels within 75 h. These results demonstrate the potential for biodegradation to serve as a complementary treatment process for the destruction of stockpiled Sarin.

Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.

Temperature effects on the allosteric responses of native and chimeric aspartate transcarbamoylases.

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli have distinct allosteric regulatory patterns. It has been reported that a S. marcescens chimera, SM : rS5'ec, in which five divergent residues (r93 to r97) of the regulatory polypeptide were replaced with their Escherichia coli counterparts, possessed E. coli-like regulatory characteristics. The reverse chimera EC:rS5'sm, in which the same five residues of E. coli have been replaced with their S. marcescens counterpart, lost both heterotrophic and homotropic responses. These results indicate that the r93-r97 region is critical in defining the ATCase allosteric character. Molecular modeling of the regulatory polypeptides has suggested that the replacement of the S5' beta-strand resulted in disruption of the allosteric-zinc interface. However, the structure-function relationship could be indirect, and the disruption of the interface could influence allostery by altering the global energy of the enzyme. Studies of the temperature-sensitivity of the CTP response demonstrate that it is possible to convert CTP inhibition of the SM:rS5'ec chimera at high temperature to activation below 10 degreesC. Nonetheless, the temperature response of the native S. marcescens ATCase suggests a strong entropic effect that counteracts the CTP activation. Therefore, it is suggested that the entropy component of the coupling free energy plays a significant role in the determination of both the nature and magnitude of the allosteric effect in ATCase.

Role of allosteric: zinc interdomain region of the regulatory subunit in the allosteric regulation of aspartate transcarbamoylase from Escherichia coli.

The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme. The present work shows that this interface also affects CTP and CTP-UTP inhibition and proposes a structural explanation for the effects. Mutant enzymes derived from nonselective mutagenesis of residues r101-r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects. The cooperative behavior of the enzymes was affected, as indicated by reduced aspartate S0.5 values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C. In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants. The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP. Finally, the I103T mutant enzyme had an increased S0.5 value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced. Most of these allosteric variations can be explained in terms of perturbations to the "tongue and groove" hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface ("reg1:cat4") between regulatory and catalytic subunits.

Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria.

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp. , and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.

Dramatically stabilized phosphotriesterase-polymers for nerve agent degradation.

Phosphotriesterase (EC 3.1.8.1) was immobilized within a polyurethane foam matrix during polymer synthesis using a prepolymer synthesis strategy. In addition to retaining greater than 50% of the enzyme specific activity, numerous benefits were incurred upon immobilization. Orders of magnitude increases in storage and thermal stability (net stabilization energy = 12.5 kJ/mol) were observed without the need for enzyme premodification. The immobilized enzyme system was protease resistant and seemed to display no adverse effects from immobilization, such as an alteration of enzyme function. The organic solvent, dimethyl sulfoxide, also exhibited a stabilizing effect on phosphotriesterase enzyme systems over a range of intermediate concentrations. We attribute these effects in part to direct interaction between the aprotic solvent and metal containing residues present at the enzyme's active site. Our data demonstrate that just 2.5 kg of immobilized enzyme may be sufficient to degrade 30,000 tons of nerve agent in just 1 year.