Fluid Optimisation in Emergency Laparotomy (FLO-ELA) Trial: study protocol for a multi-centre randomised trial of cardiac output-guided fluid therapy compared to usual care in patients undergoing major emergency gastrointestinal surgery.

INTRODUCTION: Postoperative morbidity and mortality in patients undergoing major emergency gastrointestinal surgery are a major burden on healthcare systems. Optimal management of perioperative intravenous fluids may reduce mortality rates and improve outcomes from surgery. Previous small trials of cardiac-output guided haemodynamic therapy algorithms in patients undergoing gastrointestinal surgery have suggested this intervention results in reduced complications and a modest reduction in mortality. However, this existing evidence is based mainly on elective (planned) surgery, with little evaluation in the emergency setting. There are fundamental clinical and pathophysiological differences between the planned and emergency surgical setting which may influence the effects of this intervention. A large definitive trial in emergency surgery is needed to confirm or refute the potential benefits observed in elective surgery and to inform widespread clinical practice. METHODS: The FLO-ELA trial is a multi-centre, parallel-group, open, randomised controlled trial. 3138 patients aged 50 and over undergoing major emergency gastrointestinal surgery will be randomly allocated in a 1:1 ratio using minimisation to minimally invasive cardiac output monitoring to guide protocolised administration of intra-venous fluid, or usual care without cardiac output monitoring. The trial intervention will be carried out during surgery and for up to 6 h postoperatively. The trial is funded through an efficient design call by the National Institute for Health and Care Research Health Technology Assessment (NIHR HTA) programme and uses existing routinely collected datasets for the majority of data collection. The primary outcome is the number of days alive and out of hospital within 90 days of randomisation. Participants and those delivering the intervention will not be blinded to treatment allocation. Participant recruitment started in September 2017 with a 1-year internal pilot phase and is ongoing at the time of publication. DISCUSSION: This will be the largest contemporary randomised trial examining the effectiveness of perioperative cardiac output-guided haemodynamic therapy in patients undergoing major emergency gastrointestinal surgery. The multi-centre design and broad inclusion criteria support the external validity of the trial. Although the clinical teams delivering the trial interventions will not be blinded, significant trial outcome measures are objective and not subject to detection bias. TRIAL REGISTRATION: ISRCTN 14729158. Registered on 02 May 2017.

Contemporary use of antimicrobial prophylaxis for surgical patients: An observational cohort study.

BACKGROUND: Antimicrobial prophylaxis is commonly used to prevent surgical site infection (SSI), despite concerns of overuse leading to antimicrobial resistance. However, it is unclear how often antimicrobials are used and whether guidelines are followed. OBJECTIVES: To describe contemporary clinical practice for antimicrobial prophylaxis including guideline compliance, the rate of postoperative infection and associated side effects. DESIGN: A prospective, multicentre, observational cohort study. SETTING: Twelve United Kingdom National Health Service hospitals. PARTICIPANTS: One thousand one hundred and sixteen patients, aged at least 18 years undergoing specific colo-rectal, obstetric, gynaecological, urological or orthopaedic surgical procedures. EXPOSURE: Compliance with guidelines for antimicrobial prophylaxis. OUTCOMES: The primary outcome was SSI within 30 days after surgery. Secondary outcomes were number of doses of antimicrobials for prophylaxis and to treat infection, incidence of antimicrobial-related side effects and mortality within 30 days after surgery. Data are presented as number with percentage (%) or median with interquartile range [IQR].Results of logistic regression analyses are presented as odds ratio/rate ratio (OR/RR) with 95% confidence intervals (95% CIs). RESULTS: 1102 out of 1106 (99.6%) patients received antimicrobial prophylaxis, which was compliant with local guidelines in 929 out of 1102 (84.3%) cases. 2169 out of 51 28 (42.3%) doses of antimicrobials were administered as prophylaxis (median 1 [1 to 2] dose) and 2959 out of 5128 (57.7%) were administered to treat an infection (median 21 [11 to 28] doses). 56 patients (5.2%) developed SSI. Antimicrobial prophylaxis administered according to local guidelines was not associated with a lower incidence of SSI compared with administration outside guidelines [OR 0.90 (0.35 to 2.29); P  = 0.823]. 23 out of 1072 (2.2%) patients experienced a side effect of antimicrobial therapy. 7 out of 1082 (0.6%) patients died. The median hospital stay was 3 [1 to 5] days. CONCLUSION: Antimicrobial prophylaxis was administered for almost all the surgical procedures under investigation. However, this was not always compliant with guidelines. Further research is required to determine whether the amount of prophylactic antimicrobials could be safely and effectively reduced without increasing the incidence of SSI.

Prevalence of PErioperAtive CHildhood obesitY in children undergoing general anaesthesia in the UK: a prospective, multicentre, observational cohort study.

BACKGROUND: Childhood obesity has become a serious global healthcare challenge. No UK data currently define its anaesthetic and perioperative implications. We aimed to determine obesity prevalence amongst UK children undergoing general anaesthesia and the incidence of predefined adverse perioperative events, and to compare perioperative obesity rates with National Child Measurement Programme (NCMP) data. METHODS: During a site-selected consecutive 7-day study period, all children (2-16 yr) undergoing general anaesthesia were included. Anonymised hospital, surgical, and procedural details; demographic data; and adverse perioperative events were collected prospectively by Paediatric Anaesthesia Trainee Research Network (PATRN) collaborators. RESULTS: For this study, 102 UK hospitals participated and 4232 cases were included in the final analysis; 76% of hospitals did not routinely calculate BMI. In addition, 3030 (71.6%; 95% confidence interval [CI]: 70.2-73.0%) children of healthy weight were compared with 537 (12.7%; 11.7-13.7%) children who were overweight and 478 (11.3%; 10.3-12.2%) children with obesity. Children with obesity (n=71; 14.9%) more commonly underwent (adeno)tonsillectomy than children of healthy weight (n=282; 9.3%; P<0.001; odds ratio [OR] 2.15; 95% CI: 1.58-2.92). Fewer children with obesity (n=365; 77% vs n=2552; 85%) were anaesthetised by consultant anaesthetists (OR 0.62; 95% CI: 0.48-0.79). Mask ventilation was difficult for 3.7% of children with obesity vs 0.6% of children of healthy weight (difference 3.0%; 95% CI: 1.3-4.7%; P<0.001). Comparison with NCMP data demonstrated an over-representation of obesity amongst the paediatric surgical population. CONCLUSIONS: This large multicentre cohort study suggests a concerning prevalence of children with obesity presenting for anaesthesia. These results should be used to inform optimal provision of care for this population and support perioperative healthcare initiatives to address the burden of childhood obesity. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03994419.

Myosteatosis is associated with poor physical fitness in patients undergoing hepatopancreatobiliary surgery.

BACKGROUND: Body composition assessment, measured using single-slice computed tomography (CT) image at L3 level, and aerobic physical fitness, objectively measured using cardiopulmonary exercise testing (CPET), are each independently used for perioperative risk assessment. Sarcopenia (i.e. low skeletal muscle mass), myosteatosis [i.e. low skeletal muscle radiation attenuation (SM-RA)], and impaired objectively measured aerobic fitness (reduced oxygen uptake) have been associated with poor post-operative outcomes and survival in various cancer types. However, the association between CT body composition and physical fitness has not been explored. In this study, we assessed the association of CT body composition with selected CPET variables in patients undergoing hepatobiliary and pancreatic surgery. METHODS: A pragmatic prospective cohort of 123 patients undergoing hepatobiliary and pancreatic surgery were recruited. All patients underwent preoperative CPET. Preoperative CT scans were analysed using a single-slice CT image at L3 level to assess skeletal muscle mass, adipose tissue mass, and muscle radiation attenuation. Multivariate linear regression was used to test the association between CPET variables and body composition. Main outcomes were oxygen uptake at anaerobic threshold ( V̇ O2 at AT), oxygen uptake at peak exercise ( V̇ O2 peak), skeletal muscle mass, and SM-RA. RESULTS: Of 123 patients recruited [77 men (63%), median age 66.9 ± 11.7, median body mass index 27.3 ± 5.2], 113 patients had good-quality abdominal CT scans available and were included. Of the CT body composition variables, SM-RA had the strongest correlation with V̇ O2 peak (r = 0.57, P < 0.001) and V̇ O2 at AT (r = 0.45, P < 0.001) while skeletal muscle mass was only weakly associated with V̇ O2 peak (r = 0.24, P < 0.010). In the multivariate analysis, only SM-RA was associated with V̇ O2 peak (B = 0.25, 95% CI 0.15-0.34, P < 0.001, R2  = 0.42) and V̇ O2 at AT (B = 0.13, 95% CI 0.06-0.18, P < 0.001, R2  = 0.26). CONCLUSIONS: There is a positive association between preoperative CT SM-RA and preoperative physical fitness ( V̇ O2 at AT and at peak). This study demonstrates that myosteatosis, and not sarcopenia, is associated with reduced aerobic physical fitness. Combining both myosteatosis and physical fitness variables may provide additive risk stratification accuracy and guide interventions during the perioperative period.

Perioperative Quality Initiative consensus statement on the physiology of arterial blood pressure control in perioperative medicine.

BACKGROUND: Perioperative arterial blood pressure management is a physiologically complex challenge influenced by multiple factors. METHODS: A multidisciplinary, international working subgroup of the Third Perioperative Quality Initiative (POQI) consensus meeting reviewed the (patho)physiology and measurement of arterial pressure as applied to perioperative medicine. We addressed predefined questions by undertaking a modified Delphi analysis, in which primary clinical research and review articles were identified using MEDLINE. Strength of recommendations, where applicable, were graded by National Institute for Health and Care Excellence (NICE) guidelines. RESULTS: Multiple physiological factors contribute to the perioperative physiological importance of arterial pressure: (i) arterial pressure is the input pressure to organ blood flow, but is not the sole determinant of perfusion pressure; (ii) blood flow is often independent of changes in perfusion pressure because of autoregulatory changes in vascular resistance; (iii) microvascular dysfunction uncouples microvascular blood flow from arterial pressure (haemodynamic incoherence). From a practical clinical perspective, we identified that: (i) ambulatory measurement is the optimal method to establish baseline arterial pressure; (ii) automated and invasive arterial pressure measurements have inherent physiological and technical limitations; (iii) individualised arterial pressure targets may change over time, especially in the perioperative period. There remains a need for research in non-invasive, continuous arterial pressure measurements, macro- and micro-circulatory control, regional perfusion pressure measurement, and the development of sensitive, specific, and continuous measures of cellular function to evaluate blood pressure management in a physiologically coherent manner. CONCLUSION: The multivariable, complex physiology contributing to dynamic changes in perioperative arterial pressure may be underappreciated clinically. The frequently unrecognised dissociation between arterial pressure, organ blood flow, and microvascular and cellular function requires further research to develop a more refined, contextualised clinical approach to this routine perioperative measurement.

Perioperative Quality Initiative consensus statement on intraoperative blood pressure, risk and outcomes for elective surgery.

BACKGROUND: Intraoperative mortality is now rare, but death within 30 days of surgery remains surprisingly common. Perioperative myocardial infarction is associated with a remarkably high mortality. There are strong associations between hypotension and myocardial injury, myocardial infarction, renal injury, and death. Perioperative arterial blood pressure management was thus the basis of a Perioperative Quality Initiative consensus-building conference held in London in July 2017. METHODS: The meeting featured a modified Delphi process in which groups addressed various aspects of perioperative arterial pressure. RESULTS: Three consensus statements on intraoperative blood pressure were established. 1) Intraoperative mean arterial pressures below 60-70 mm Hg are associated with myocardial injury, acute kidney injury, and death. Injury is a function of hypotension severity and duration. 2) For adult non-cardiac surgical patients, there is insufficient evidence to recommend a general upper limit of arterial pressure at which therapy should be initiated, although pressures above 160 mm Hg have been associated with myocardial injury and infarction. 3) During cardiac surgery, intraoperative systolic arterial pressure above 140 mm Hg is associated with increased 30 day mortality. Injury is a function of arterial pressure severity and duration. CONCLUSIONS: There is increasing evidence that even brief durations of systolic arterial pressure <100 mm Hg and mean arterial pressure <60-70 mm Hg are harmful during non-cardiac surgery.

Perioperative Quality Initiative consensus statement on preoperative blood pressure, risk and outcomes for elective surgery.

BACKGROUND: A multidisciplinary international working subgroup of the third Perioperative Quality Initiative consensus meeting appraised the evidence on the influence of preoperative arterial blood pressure and community cardiovascular medications on perioperative risk. METHODS: A modified Delphi technique was used, evaluating papers published in MEDLINE on associations between preoperative numerical arterial pressure values or cardiovascular medications and perioperative outcomes. The strength of the recommendations was graded by National Institute for Health and Care Excellence guidelines. RESULTS: Significant heterogeneity in study design, including arterial pressure measures and perioperative outcomes, hampered the comparison of studies. Nonetheless, consensus recommendations were that (i) preoperative arterial pressure measures may be used to define targets for perioperative management; (ii) elective surgery should not be cancelled based solely upon a preoperative arterial pressure value; (iii) there is insufficient evidence to support lowering arterial pressure in the immediate preoperative period to minimise perioperative risk; and (iv) there is insufficient evidence that any one measure of arterial pressure (systolic, diastolic, mean, or pulse) is better than any other for risk prediction of adverse perioperative events. CONCLUSIONS: Future research should define which preoperative arterial pressure values best correlate with adverse outcomes, and whether modifying arterial pressure in the preoperative setting will change the perioperative morbidity or mortality. Additional research should define optimum strategies for continuation or discontinuation of preoperative cardiovascular medications.

Perioperative Quality Initiative consensus statement on postoperative blood pressure, risk and outcomes for elective surgery.

BACKGROUND: Postoperative hypotension and hypertension are frequent events associated with increased risk of adverse outcomes. However, proper assessment and management is often poorly understood. As a part of the PeriOperative Quality Improvement (POQI) 3 workgroup meeting, we developed a consensus document addressing this topic. The target population includes adult, non-cardiac surgical patients in the postoperative phase outside of the ICU. METHODS: A modified Delphi technique was used, evaluating papers published in MEDLINE examining postoperative blood pressure monitoring, management, and outcomes. Practice recommendations were developed in line with National Institute for Health and Care Excellence guidelines. RESULTS: Consensus recommendations were that (i) there is evidence of harm associated with postoperative systolic arterial pressure <90 mm Hg; (ii) for patients with preoperative hypertension, the threshold at which harm occurs may be higher than a systolic arterial pressure of 90 mm Hg; (iii) there is insufficient evidence to precisely define the level of postoperative hypertension above which harm will occur; (iv) a greater frequency of postoperative blood pressure measurement is likely to identify risk of harm and clinical deterioration earlier; and (v) there is evidence of harm from withholding beta-blockers, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors in the postoperative period. CONCLUSIONS: Despite evidence of associations with postoperative hypotension or hypertension with worse postoperative outcome, further research is needed to define the optimal levels at which intervention is beneficial, to identify the best methods and timing of postoperative blood pressure measurement, and to refine the management of long-term antihypertensive treatment in the postoperative phase.

Optimisation of Perioperative Cardiovascular Management to Improve Surgical Outcome II (OPTIMISE II) trial: study protocol for a multicentre international trial of cardiac output-guided fluid therapy with low-dose inotrope infusion compared with usual care in patients undergoing major elective gastrointestinal surgery.

INTRODUCTION: Postoperative morbidity and mortality in older patients with comorbidities undergoing gastrointestinal surgery are a major burden on healthcare systems. Infections after surgery are common in such patients, prolonging hospitalisation and reducing postoperative short-term and long-term survival. Optimal management of perioperative intravenous fluids and inotropic drugs may reduce infection rates and improve outcomes from surgery. Previous small trials of cardiac-output-guided haemodynamic therapy algorithms suggested a modest reduction in postoperative morbidity. A large definitive trial is needed to confirm or refute this and inform widespread clinical practice. METHODS: The Optimisation of Perioperative Cardiovascular Management to Improve Surgical Outcome II (OPTIMISE II) trial is a multicentre, international, parallel group, open, randomised controlled trial. 2502 high-risk patients undergoing major elective gastrointestinal surgery will be randomly allocated in a 1:1 ratio using minimisation to minimally invasive cardiac output monitoring to guide protocolised administration of intravenous fluid combined with low-dose inotrope infusion, or usual care. The trial intervention will be carried out during and for 4 hours after surgery. The primary outcome is postoperative infection of Clavien-Dindo grade II or higher within 30 days of randomisation. Participants and those delivering the intervention will not be blinded to treatment allocation; however, outcome assessors will be blinded when feasible. Participant recruitment started in January 2017 and is scheduled to last 3 years, within 50 hospitals worldwide. ETHICS/DISSEMINATION: The OPTIMISE II trial has been approved by the UK National Research Ethics Service and has been approved by responsible ethics committees in all participating countries. The findings will be disseminated through publication in a widely accessible peer-reviewed scientific journal. TRIAL REGISTRATION NUMBER: ISRCTN39653756.

Ethanol Stimulates Locomotion via a Gαs-Signaling Pathway in IL2 Neurons in Caenorhabditis elegans.

Alcohol is a potent pharmacological agent when consumed acutely at sufficient quantities and repeated overuse can lead to addiction and deleterious effects on health. Alcohol is thought to modulate neuronal function through low-affinity interactions with proteins, in particular with membrane channels and receptors. Paradoxically, alcohol acts as both a stimulant and a sedative. The exact molecular mechanisms for the acute effects of ethanol on neurons, as either a stimulant or a sedative, however remain unclear. We investigated the role that the heat shock transcription factor HSF-1 played in determining a stimulatory phenotype of Caenorhabditis elegans in response to physiologically relevant concentrations of ethanol (17 mM; 0.1% v/v). Using genetic techniques, we demonstrate that either RNA interference of hsf-1 or use of an hsf-1(sy441) mutant lacked the enhancement of locomotion in response to acute ethanol exposure evident in wild-type animals. We identify that the requirement for HSF-1 in this phenotype was IL2 neuron-specific and required the downstream expression of the α-crystallin ortholog HSP-16.48 Using a combination of pharmacology, optogenetics, and phenotypic analyses we determine that ethanol activates a Gαs-cAMP-protein kinase A signaling pathway in IL2 neurons to stimulate nematode locomotion. We further implicate the phosphorylation of a specific serine residue (Ser322) on the synaptic protein UNC-18 as an end point for the Gαs-dependent signaling pathway. These findings establish and characterize a distinct neurosensory cell signaling pathway that determines the stimulatory action of ethanol and identifies HSP-16.48 and HSF-1 as novel regulators of this pathway.

Metabolic dysfunction in lymphocytes promotes postoperative morbidity.

Perioperative lymphopenia has been linked with an increased risk of postoperative infectious complications, but the mechanisms remain unclear. We tested the hypothesis that bioenergetic dysfunction is an important mechanism underlying lymphopenia, impaired functionality and infectious complications. In two cohorts of patients (61-82 years old) undergoing orthopaedic joint replacement (n=417 and 328, respectively), we confirmed prospectively that preoperative lymphopenia (≤1.3 x 10(9)·l(-1); <20% white cell count; prevalence 15-18%) was associated with infectious complications (relative risk 1.5 (95% confidence interval 1.1-2.0); P=0.008) and prolonged hospital stay. Lymphocyte respirometry, mitochondrial bioenergetics and function were assessed (n=93 patients). Postoperative lymphocytes showed a median 43% fall (range: 26-65%; P=0.029; n=13 patients) in spare respiratory capacity, the extra capacity available to produce energy in response to stress. This was accompanied by reduced glycolytic capacity. A similar hypometabolic phenotype was observed in lymphocytes sampled preoperatively from chronically lymphopenic patients (n=21). This hypometabolic phenotype was associated with functional lymphocyte impairment including reduced T-cell proliferation, lower intracellular cytokine production and excess apoptosis induced by a range of common stressors. Glucocorticoids, which are ubiquitously elevated for a prolonged period postoperatively, generated increased levels of mitochondrial reactive oxygen species, activated caspase-1 and mature interleukin (IL)-1ß in human lymphocytes, suggesting inflammasome activation. mRNA transcription of the NLRP1 inflammasome was increased in lymphocytes postoperatively. Genetic ablation of the murine NLRP3 inflammasome failed to prevent glucocorticoid-induced lymphocyte apoptosis and caspase-1 activity, but increased NLRP1 protein expression. Our findings suggest that the hypometabolic phenotype observed in chronically lymphopenic patients and/or acquired postoperatively increases the risk of postoperative infection through glucocorticoid activation of caspase-1 via the NLRP1 inflammasome.

Fluid therapy in critical illness.

Major surgery and critical illnesses such as sepsis and trauma all disturb normal physiological fluid handling. Intravenous fluid therapy for resuscitation and fluid maintenance is a central part of medical care during these conditions, yet the evidence base supporting practice in this area lacks answers to a number of important questions. Recent research developments include a refinement of our knowledge of the endothelial barrier structure and function and a focus on the potential harm that may be associated with intravenous fluid therapy. Here, we briefly describe the contemporary view of fluid physiology and how this may be disrupted by pathological processes. The important themes in critical illness fluid research are discussed, with a particular focus on two emerging ideas: firstly, that individualising fluid treatment to the patient, their underlying disease state and the phase of that illness may be key to improving clinical outcomes using fluid interventions and, secondly, that fluids should be considered to be drugs, with specific indications and contraindications, dose ranges and potential toxicities.

Cardiopulmonary exercise capacity and preoperative markers of inflammation.

Explanatory mechanisms for the association between poor exercise capacity and infections following surgery are underexplored. We hypothesized that aerobic fitness-assessed by cardiopulmonary exercise testing (CPET)-would be associated with circulating inflammatory markers, as quantified by the neutrophil-lymphocyte ratio (NLR) and monocyte subsets. The association between cardiopulmonary reserve and inflammation was tested by multivariable regression analysis with covariates including anaerobic threshold (AT) and malignancy. In a first cohort of 240 colorectal patients, AT was identified as the sole factor associated with higher NLR (P = 0.03) and absolute and relative lymphopenia (P = 0.01). Preoperative leukocyte subsets and monocyte CD14(+) expression (downregulated by endotoxin and indicative of chronic inflammation) were also assessed in two further cohorts of age-matched elective gastrointestinal and orthopaedic surgical patients. Monocyte CD14(+) expression was lower in gastrointestinal patients (n = 43) compared to age-matched orthopaedic patients (n = 31). The circulating CD14(+)CD16(-) monocyte subset was reduced in patients with low cardiopulmonary reserve. Poor exercise capacity in patients without a diagnosis of heart failure is independently associated with markers of inflammation. These observations suggest that preoperative inflammation associated with impaired cardiorespiratory performance may contribute to the pathophysiology of postoperative outcome.

PKC-2 phosphorylation of UNC-18 Ser322 in AFD neurons regulates temperature dependency of locomotion.

Diacylglycerol (DAG)/protein kinase C (PKC) signaling plays an integral role in the regulation of neuronal function. This is certainly true in Caenorhabditis elegans and in particular for thermosensory signaling and behavior. Downstream molecular targets for transduction of this signaling cascade remain, however, virtually uncharacterized. We investigated whether PKC phosphorylation of Munc18-1, an essential protein in vesicle trafficking and exocytosis, was the downstream effector for DAG regulation of thermosensory behavior. We demonstrate here that the C. elegans ortholog of Munc18-1, UNC-18, was phosphorylated in vitro at Ser322. Transgenic rescue of unc-18-null worms with Ser322 phosphomutants displayed altered thermosensitivity. C. elegans expresses three DAG-regulated PKCs, and blocking UNC-18 Ser322 phosphorylation was phenocopied only by deletion of calcium-activated PKC-2. Expression of nonphosphorylatable UNC-18 S322A, either pan-neuronally or specifically in AFD thermosensory neurons, converted wild-type worms to a pkc-2-null phenotype. These data demonstrate that an individual DAG-dependent thermosensory behavior of an organism is effected specifically by the downstream PKC-2 phosphorylation of UNC-18 on Ser322 in AFD neurons.

Chemical labelling of active serum thioester proteins for quantification.

The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum.

Presynaptic targets for acute ethanol sensitivity.

Acute exposure to ethanol is known to modulate signalling within the nervous system. Physiologically these effects are both presynaptic and postsynaptic in origin; however, considerably more research has focused primarily on postsynaptic targets. Recent research using the model organism Caenorhabditis elegans has determined a role for specific proteins (Munc18-1 and Rab3) and processes (synaptic vesicle recruitment and fusion) in transducing the presynaptic effects of ethanol. In the present paper, we review these results, identifying the proteins and protein interactions involved in ethanol sensitivity and discuss their links with mammalian studies of alcohol abuse.

UNC-18 modulates ethanol sensitivity in Caenorhabditis elegans.

Acute ethanol exposure affects the nervous system as a stimulant at low concentrations and as a depressant at higher concentrations, eventually resulting in motor dysfunction and uncoordination. A recent genetic study of two mouse strains with varying ethanol preference indicated a correlation with a polymorphism (D216N) in the synaptic protein Munc18-1. Munc18-1 functions in exocytosis via a number of discrete interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1. We report that the mutation affects binding to syntaxin but not through either a closed conformation mode of interaction or through binding to the syntaxin N terminus. The D216N mutant instead has a specific impairment in binding the assembled SNARE complex. Furthermore, the mutation broadens the duration of single exocytotic events. Expression of the orthologous mutation (D214N) in the Caenorhabditis elegans UNC-18 null background generated transgenic rescues with phenotypically similar locomotion to worms rescued with the wild-type protein. Strikingly, D214N worms were strongly resistant to both stimulatory and sedative effects of acute ethanol. Analysis of an alternative Munc18-1 mutation (I133V) supported the link between reduced SNARE complex binding and ethanol resistance. We conclude that ethanol acts, at least partially, at the level of vesicle fusion and that its acute effects are ameliorated by point mutations in UNC-18.