Identifying the limitations of cardiopulmonary exercise testing prior to esophagectomy using a pooled analysis of patient-level data.

Preoperative cardiopulmonary exercise testing (CPET) provides an objective assessment of aerobic fitness in patients undergoing surgery. While peak oxygen uptake during exercise (VO2peak) and anaerobic threshold have demonstrated a moderate correlation with the development of complications following esophagectomy, no clinically useful threshold values have been defined. By pooling patient level data from existing studies, we aimed to define optimal thresholds for preoperative CPET parameters to predict patients at high risk of postoperative complications. Studies reporting on the relationship between preoperative CPET variables and post-esophagectomy complications were determined from a comprehensive literature search. Patient-level data were obtained from six contributing centers for pooled-analyses. Outcomes of interest included cardiopulmonary and non-cardiopulmonary complications, unplanned intensive care unit readmission, and 90-day and 12-month all-cause mortality. Receiver operating characteristic curves and logistic regression models estimated the predictive value of CPET parameters for each individual outcome of interest. This analysis comprised of 621 patients who underwent CPET prior to esophagectomy during the period from January 2004 to March 2017. For both anaerobic threshold and VO2peak, none of the receiver operating characteristic curves achieved an area under the curve value > 0.66 for the outcomes of interest. The discriminatory ability of CPET for determining high-risk patients was found to be poor in patients undergoing an esophagectomy. CPET may only carry an adjunct role to clinical decision-making.

Utilisation of tracheostomy in patients with COVID-19 in England: Patient characteristics, timing and outcomes.

OBJECTIVES: We aimed to characterise the use of tracheostomy procedures for all COVID-19 critical care patients in England and to understand how patient factors and timing of tracheostomy affected outcomes. DESIGN: A retrospective observational study using exploratory analysis of hospital administrative data. SETTING: All 500 National Health Service hospitals in England. PARTICIPANTS: All hospitalised COVID-19 patients aged ≥18 years in England between 1 March and 31 October 2020 were included. MAIN OUTCOMES AND MEASURES: This was a retrospective exploratory analysis using the Hospital Episode Statistics administrative data set. Multilevel modelling was used to explore the relationship between demographic factors, comorbidity and use of tracheostomy and the association between tracheostomy use, tracheostomy timing and the outcomes. RESULTS: In total, 2200 hospitalised COVID-19 patients had a tracheostomy. Tracheostomy utilisation varied across the study period, peaking in April-June 2020. In multivariable modelling, for those admitted to critical care, tracheostomy was most common in those aged 40-79 years, in males and in people of Black and Asian ethnic groups and those with a history of cerebrovascular disease. In critical care patients, tracheostomy was associated with lower odds of mortality (OR: 0.514 [95% CI 0.443 to 0.596], but greater length of stay OR: 41.143 [95% CI 30.979 to 54.642]). In patients that survived, earlier timing of tracheostomy (≤14 days post admission to critical care) was significantly associated with shorter length of stay. CONCLUSIONS: Tracheostomy is safe and advantageous for critical care COVID-19 patients. Early tracheostomy may be associated with better outcomes, such as shorter length of stay, compared to late tracheostomy.

Findings of a feasibility study of pre-operative pulmonary rehabilitation to reduce post-operative pulmonary complications in people with chronic obstructive pulmonary disease scheduled for major abdominal surgery.

Background: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk of complications and death following surgery. Pulmonary complications are particularly prominent.  Pulmonary rehabilitation is a course of physical exercise and education that helps people with COPD manage their condition.  Although proven to improve health outcomes in patients with stable COPD, it has never been formally tested as a pre-surgical intervention in patients scheduled for non-cardiothoracic surgery.  If a beneficial effect were to be demonstrated, pulmonary rehabilitation for pre-surgical patients with COPD might be rapidly implemented across the National Health Service, as pulmonary rehabilitation courses are already well established across much of the United Kingdom (UK). Methods: We performed a feasibility study to test study procedures and barriers to identification and recruitment to a randomised controlled trial testing whether pulmonary rehabilitation, delivered before major abdominal surgery in a population of people with COPD, would reduce the incidence of post-operative pulmonary complications.  This study was run in two UK centres (Oxford and Newcastle upon Tyne). Results:  We determined that a full randomised controlled trial would not be feasible, due to failure to identify and recruit participants.  We identified an unmet need to identify more effectively patients with COPD earlier in the surgical pathway.  Service evaluations suggested that barriers to identification and recruitment would likely be the same across other UK hospitals. Conclusions:  Although pulmonary rehabilitation is a potentially beneficial intervention to prevent post-operative pulmonary complications, a randomised controlled trial is unlikely to recruit sufficient participants to answer our study question conclusively at the present time, when spirometry is not automatically conducted in all patients planned for surgery.  As pulmonary rehabilitation is a recommended treatment for all people with COPD, alternative study methods combined with earlier identification of candidate patients in the surgical pathway should be considered. Trial registration: ISRCTN29696295, 31/08/2017.

Preoperative behavioural intervention to reduce drinking before elective orthopaedic surgery: the PRE-OP BIRDS feasibility RCT.

BACKGROUND: Heavy alcohol consumption is associated with an increased risk of postoperative complications and extended hospital stay. Alcohol consumption therefore represents a modifiable risk factor for surgical outcomes. Brief behavioural interventions have been shown to be effective in reducing alcohol consumption among increased risk and risky drinkers in other health-care settings and may offer a method of addressing preoperative alcohol consumption. OBJECTIVES: To investigate the feasibility of introducing a screening process to assess adult preoperative drinking levels and to deliver a brief behavioural intervention adapted for the target population group. To conduct a two-arm (brief behavioural intervention plus standard preoperative care vs. standard preoperative care alone), multicentre, pilot randomised controlled trial to assess the feasibility of proceeding to a definitive trial. To conduct focus groups and a national web-based survey to establish current treatment as usual for alcohol screening and intervention in preoperative assessment. DESIGN: A single-centre, qualitative, feasibility study was followed by a multicentre, two-arm (brief behavioural intervention vs. treatment as usual), individually randomised controlled pilot trial with an embedded qualitative process evaluation. Focus groups and a quantitative survey were employed to characterise treatment as usual in preoperative assessment. SETTING: The feasibility study took place at a secondary care hospital in the north-east of England. The pilot trial was conducted at three large secondary care centres in the north-east of England. PARTICIPANTS: Nine health-care professionals and 15 patients (mean age 70.5 years, 86.7% male) participated in the feasibility study. Eleven health-care professionals and 68 patients (mean age 66.2 years, 80.9% male) participated in the pilot randomised trial. An additional 19 health-care professionals were recruited to one of three focus groups, while 62 completed an electronic survey to characterise treatment as usual. INTERVENTIONS: The brief behavioural intervention comprised two sessions. The first session, delivered face to face in the preoperative assessment clinic, involved 5 minutes of structured brief advice followed by 15-20 minutes of behaviour change counselling, including goal-setting, problem-solving and identifying sources of social support. The second session, an optional booster, took place approximately 1 week before surgery and offered the opportunity to assess progress and boost self-efficacy. MAIN OUTCOME MEASURES: Feasibility was assessed using rates of eligibility, recruitment and retention. The progression criteria for a definitive trial were recruitment of ≥ 40% of eligible patients and retention of ≥ 70% at 6-month follow-up. Acceptability was assessed using themes identified in qualitative data. RESULTS: The initial recruitment of eligible patients was low but improved with the optimisation of recruitment processes. The recruitment of eligible participants to the pilot trial (34%) fell short of the progression criteria but was mitigated by very high retention (96%) at the 6-month follow-up. Multimethod analyses identified the methods as acceptable to the patients and professionals involved and offers recommendations of ways to further improve recruitment. CONCLUSIONS: The evidence supports the feasibility of a definitive trial to assess the effectiveness of brief behavioural intervention in reducing preoperative alcohol consumption and for secondary outcomes of surgical complications if recommendations for further improvements are adopted. TRIAL REGISTRATION: Current Controlled Trials ISRCTN36257982. FUNDING: This project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 12. See the National Institute for Health Research Journals Library website for further project information.

Most patients undergoing knee and hip replacements are over 65 years old. Older patients have an increased risk of complications following surgery. Heavy alcohol consumption in the weeks before surgery increases the risk of complications after surgery, which can extend recovery times. Advice that helps patients reduce their alcohol consumption before surgery may have benefits for recovery. The PRE-OP BIRDS study had two parts: a feasibility study followed by a pilot randomised controlled trial with focus groups and an electronic survey used to characterise usual care in the preoperative assessment clinic. The feasibility study took place at one hospital. It aimed to develop materials that help health-care professionals provide brief advice to patients on how to reduce alcohol consumption before surgery. This brief advice was delivered to eligible patients and the acceptability to staff and patients was assessed in interviews. The pilot trial took place in three hospitals. Patients who agreed to take part were placed, by equal chance, into either a group that received usual care or a group that received usual care plus brief advice about reducing alcohol use. The aim was to count how many people agreed to take part and how many also agreed to complete a follow-up 6 months later. Interviews were carried out with patients and staff to explore their views on the intervention and the trial as a whole. All of this information was collected to help decide if a future larger trial was possible. This work found that the tools used were acceptable to both patients and staff. Although the number of people who agreed to take part was smaller than hoped, almost all of those who took part also completed the 6-month follow-up. Therefore, a future larger trial was found to be possible, but some changes could be made to encourage more people to take part.

Mitigating Respiratory Motion in Radiation Therapy: Rapid, Shallow, Non-invasive Mechanical Ventilation for Internal Thoracic Targets.

PURPOSE: Reducing respiratory motion during the delivery of radiation therapy reduces the volume of healthy tissues irradiated and may decrease radiation-induced toxicity. The purpose of this study was to assess the potential for rapid shallow non-invasive mechanical ventilation to reduce internal anatomy motion for radiation therapy purposes. METHODS AND MATERIALS: Ten healthy volunteers (mean age, 38 years; range, 22-54 years; 6 female and 4 male) were scanned using magnetic resonance imaging during normal breathing and at 2 ventilator-induced frequencies: 20 and 25 breaths per minute for 3 minutes. Sagittal and coronal cinematic data sets, centered over the right diaphragm, were used to measure internal motions across the lung-diaphragm interface. Repeated scans assessed reproducibility. Physiologic parameters and participant experiences were recorded to quantify tolerability and comfort. RESULTS: Physiologic observations and experience questionnaires demonstrated that rapid shallow non-invasive ventilation technique was tolerable and comfortable. Motion analysis of the lung-diaphragm interface demonstrated respiratory amplitudes and variations reduced in all subjects using rapid shallow non-invasive ventilation compared with spontaneous breathing: mean amplitude reductions of 56% and 62% for 20 and 25 breaths per minute, respectively. The largest mean amplitude reductions were found in the posterior of the right lung; 40.0 mm during normal breathing to 15.5 mm (P < .005) and 15.2 mm (P < .005) when ventilated with 20 and 25 breaths per minute, respectively. Motion variations also reduced with ventilation; standard deviations in the posterior lung reduced from 14.8 mm during normal respiration to 4.6 mm and 3.5 mm at 20 and 25 breaths per minute, respectively. CONCLUSIONS: To our knowledge, this study is the first to measure internal anatomic motion using rapid shallow mechanical ventilation to regularize and minimize respiratory motion over a period long enough to image and to deliver radiation therapy. Rapid frequency and shallow, non-invasive ventilation both generate large reductions in internal thoracic and abdominal motions, the clinical application of which could be profound-enabling dose escalation (increasing treatment efficacy) or high-dose ablative radiation therapy.

Preoperative Behavioural Intervention versus standard care to Reduce Drinking before elective orthopaedic Surgery (PRE-OP BIRDS): protocol for a multicentre pilot randomised controlled trial.

BACKGROUND: Evidence suggests that increased preoperative alcohol consumption increases the risk of postoperative complications; therefore, a reduction or cessation in alcohol intake before surgery may reduce perioperative risk. Preoperative assessment presents an opportunity to intervene to optimise patients for surgery. This multicentre, two-arm, parallel group, individually randomised controlled trial will investigate whether a definitive trial of a brief behavioural intervention aimed at reducing preoperative alcohol consumption is feasible and acceptable to healthcare professionals responsible for its delivery and the preoperative elective orthopaedic patient population. METHODS: Screening will be conducted by trained healthcare professionals at three hospitals in the North East of England. Eligible patients (those aged 18 or over, listed for elective hip or knee arthroplasty surgery and scoring 5 or more or reporting consumption of six or more units on a single occasion at least weekly on the alcohol screening tool) who enrol in the trial will be randomised on a one-to-one non-blinded basis to either treatment as usual or brief behavioural intervention delivered in the pre-assessment clinic. Patients will be followed up 1-2 days pre-surgery, 1-5 days post-surgery (as an in-patient), 6 weeks post-surgery, and 6 months post intervention. Feasibility will be assessed through rates of screening, eligibility, recruitment, and retention to 6-month follow-up. An embedded qualitative study will explore the acceptability of study methods to patients and staff. DISCUSSION: This pilot randomised controlled trial will establish the feasibility and acceptability of trial procedures reducing uncertainties ahead of a definitive randomised controlled trial to establish the effectiveness of brief behavioural intervention to reduce alcohol consumption in the preoperative period and the potential impact on perioperative complications. TRIAL REGISTRATION: Reference number ISRCTN36257982.

A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp.

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.

Impaired cardiopulmonary reserve in an elderly population is related to postoperative morbidity and length of hospital stay after radical cystectomy.

OBJECTIVE: To determine the relationship of preoperatively measured cardiorespiratory function, to the development of postoperative complications and length of hospital stay (LOS) in a cohort of patients undergoing radical cystectomy (RC), as RC and conduit formation is curative but is associated with significant postoperative morbidity and mortality. PATIENTS AND METHODS: Consecutive patients planned to have RC underwent cardiopulmonary exercise testing (CPET) to a standardised protocol. The results of the CPET were 'blinded' from the clinicians involved in the care of the patients. Patients were prospectively monitored for the primary outcome of postoperative complications, as defined by a validated classification (Clavien-Dindo). Secondary outcome included LOS and mortality. RESULTS: In all, 82 patients underwent CPET before RC. Eight patients did not subsequently undergo RC and a further five did not exercise sufficiently to allow for appropriate determination of the cardiopulmonary variables of interest. There was a significant difference in LOS between those patients who had a major perioperative complication (Clavien score > 3) and those that did not (16 vs 30 days; P < 0.001; hazard ratio [HR] 3.6, 95% confidence interval [CI] 2.1-6.3). The anaerobic threshold (AT) remained as the only significant independent predictor variable for the presence or absence of major postoperative complications (odds ratio 0.74, 95% CI 0.57-0.97; P = 0.03). When the optimal predictive value of AT of 12 mL/min/kg was used as a fitness marker, there was a significant relationship between fitness and LOS (median LOS: 'unfit' 22 days vs 'fit' 16 days; HR 0.47, 95% CI 0.28-0.80; P = 0.006) CONCLUSION: Impaired preoperative cardiopulmonary reserve was related to major morbidity, prolonged LOS and increased use of critical care resource after RC. This has important health and economic implications for risk assessment, rationalisation of postoperative resource and the potential for therapeutic preoperative intervention with exercise therapy.

Preoperative optimization: rationale and process: is it economic sense?

PURPOSE OF REVIEW: The development of preoperative clinics and the increasing importance of the anaesthetist in the management of surgical risk have expanded the concept of preoperative optimization. This review will focus on the rationale and process for cost-effective preoperative optimization. RECENT FINDINGS: Postoperative morbidity, rather than mortality, is the most important surgical outcome in economic terms. Since preoperative comorbidity, in association with surgical complexity, is more predictive of hospital costs than the subsequent treatment of postoperative complications per se, preoperative optimization represents an appropriate economic target. Process management, including guidelines to reduce unnecessary investigations and specialist referrals and enhancing perioperative recovery, makes economical sense in the majority of patients who undergo noncardiac surgery with few complications. Preoperative optimization of a minority of high-risk surgical patients is also important given limited critical care resource. However, the evidence for specific optimization strategies in this latter group continues to evolve and requires further clarification in well designed trials. SUMMARY: The requirement for appropriate methods of risk stratification of surgical patients targeted at the reduction of postoperative morbidity, underpins the development of cost-effective preoperative optimization. Specific process-based and clinical measures may then be applied to the development of individualized perioperative care packages.

A double-blind randomized controlled clinical trial to assess the effect of Doppler optimized intraoperative fluid management on outcome following radical cystectomy.

PURPOSE: Cardiovascular optimization via esophageal Doppler can minimize gastrointestinal hypoperfusion, reducing the risk of multiple organ dysfunction and postoperative complications during major surgery. We assessed the effect of esophageal Doppler guided cardiovascular optimization in patients undergoing radical cystectomy. MATERIALS AND METHODS: We conducted a prospective, randomized, double-blind controlled trial at a United Kingdom teaching hospital between 2006 and 2009. A total of 66 patients were randomized to a control arm (34) and an intervention arm (32). The control group received standard intraoperative fluids. The intervention group received (additional) Doppler guided fluid. Primary outcomes were markers of gastrointestinal morbidity such as ileus, flatus and bowel opening. Secondary outcomes were postoperative nausea and vomiting, wound infection and operative intravenous fluid volumes (total and hourly). RESULTS: There were significant reductions in the control and intervention arms in the incidence of ileus (18 vs 7, p <0.001), flatus (5.36 vs 3.55 days, p <0.01) and bowel opening (9.79 vs 6.53 days, p = 0.02), respectively. Nausea and vomiting were significantly reduced in the study group at 24 and 48 hours postoperatively (11 vs 3, p <0.01 and 13 vs 1, p <0.0001). Wound infection rates were significantly reduced (8 vs 1 superficial, p <0.01 and 10 vs 2 combined, p <0.01). Study patients received significantly higher volumes (ml/kg per minute) of intravenous fluid (0.19 vs 0.23, p <0.01) related to a significantly higher volume (ml/kg) in the first hour of surgery (14.1 vs 21.0, p = 0.0001). CONCLUSIONS: Cardiovascular optimization using esophageal Doppler significantly improved postoperative markers of gastrointestinal function.

Acute lung injury after ruptured abdominal aortic aneurysm repair: the effect of excluding donations from females from the production of fresh frozen plasma.

OBJECTIVES: Transfusion-related acute lung injury may contribute to the development of acute lung injury in the critically ill, due to plasma from female donors containing antileukocyte antibodies. In July 2003, the U.K. National Blood Service stopped using female donor plasma for the production of fresh frozen plasma. Patients undergoing repair of a ruptured abdominal aortic aneurysm receive large amounts of fresh frozen plasma and often develop acute lung injury. We investigated whether the change to male fresh frozen plasma was associated with a change in the frequency of acute lung injury in these patients. DESIGN: A retrospective, before and after, observational, single-center study. SETTING: Tertiary care center and a regional blood center. PATIENTS: The study included 211 patients undergoing open repair of a ruptured abdominal aortic aneurysm between 1998 and 2006. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Primary outcome was the development of acute lung injury in the first 6 hrs after surgery. Secondary outcomes were significant hypoxia (PaO2/FiO2 ratio <300), time to extubation, and survival at 30 days. Groups were well matched and received similar volumes of intravenous fluids and blood components. There was significantly less acute lung injury following the change to male fresh frozen plasma (36% before vs. 21% after, p = .04). At 6 hrs after surgery, fewer patients were hypoxic (87% before vs. 62% after, p < .01). In multivariate analysis, the change in donor policy was associated with a decreased risk of developing acute lung injury (odds ratio 0.39; 95% confidence interval, 0.16-0.90). Time to extubation and survival at 30 days were not statistically different between groups. CONCLUSIONS: The policy to exclude female donors from the production of fresh frozen plasma was associated with a decrease in the frequency of acute lung injury in patients undergoing repair of a ruptured abdominal aortic aneurysm.

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells.

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated.

In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain.

The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. Unexpectedly, expression of a mutant BiP with impaired ligand binding also interfered with secretory protein production. Although mutant BiP lacking its ATPase domain had no detrimental effect on ER function, expression of an isolated ATPase domain interfered with secretory protein synthesis. Interestingly, the inhibitory effect of the isolated ATPase was alleviated by the T46G mutation and aggravated by the G235D mutation. We propose that in addition to its role in ligand release, the ATPase domain can interact with other components of the protein translocation and folding machinery to influence secretory protein synthesis.

Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway.

Quality control in the endoplasmic reticulum (ER) prevents the arrival of incorrectly or incompletely folded proteins at their final destinations and targets permanently misfolded proteins for degradation. Such proteins have a high affinity for the ER chaperone BiP and are finally degraded via retrograde translocation from the ER lumen back to the cytosol. This ER-associated protein degradation (ERAD) is currently thought to constitute the main disposal route, but there is growing evidence for a vacuolar role in quality control. We show that BiP is transported to the vacuole in a wortmannin-sensitive manner in tobacco (Nicotiana tabacum) and that it could play an active role in this second disposal route. ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus, where it competes with other HDEL receptor ligands. When HDEL-mediated retrieval from the Golgi fails, BiP is transported to the lytic vacuole via multivesicular bodies, which represent the plant prevacuolar compartment. We also demonstrate that a subset of BiP-ligand complexes is destined to the vacuole and differs from those likely to be disposed of via the ERAD pathway. Vacuolar disposal could act in addition to ERAD to maximize the efficiency of quality control in the secretory pathway.

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting.

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.

Renal tubular injury after infrarenal aortic aneurysm repair.

OBJECTIVE: To investigate markers of tubular injury (glutathione-S-transferase [GST] isoforms) as early markers for renal damage in patients undergoing abdominal aortic aneurysm repair. DESIGN: Prospective study. SETTING: Regional teaching hospital. PARTICIPANTS: Eight consecutive patients undergoing elective infrarenal abdominal aortic aneurysm repair. INTERVENTIONS: All patients received a standard anesthetic technique including a dopamine infusion (3 microg/kg/min) but without supplemental renoprotective agents. Urine and blood samples were taken at induction, at 1 hour and 3 hours after limb reperfusion, and on days 1 and 2 postoperatively. Urine microalbumin and creatinine concentrations were measured using standard assays, and urine pi-GST and alpha-GST enzyme measurements were performed by a commercial immunoassay (Biotrin, Biotrin International Ltd., Co., Dublin, Ireland). MEASUREMENTS AND MAIN RESULTS: Five patients (63%) showed a postoperative elevation of serum creatinine (median increase from baseline, 35.4%; range, 8.3% to 50.6%) that was associated with significant elevations of urinary microalbumin-to-creatinine, alpha-GST-to-creatinine, and pi-GST-to-creatinine ratios soon after clamp removal. The remaining 3 patients showed no increase in serum creatinine or urine proteins. Peak alpha-GST-to-creatinine levels were different between the 2 groups. The peak levels of GST enzymes were significantly (r(2) > 80%) associated with the percent increase in serum creatinine from baseline. CONCLUSION: Urinary GST-to-creatinine ratios are a sensitive early biomarker for renal injury after infrarenal abdominal aortic aneurysm repair.