A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry.

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17ß-estradiol and estriol). •SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

ATP-binding cassette family C member 1 constrains metabolic responses to high-fat diet in male mice.

Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Plasma steroid concentrations reflect acute disease severity and normalise during recovery in people hospitalised with COVID-19.

OBJECTIVE: Endocrine systems are disrupted in acute illness, and symptoms reported following coronavirus disease 2019 (COVID-19) are similar to those found with clinical hormone deficiencies. We hypothesised that people with severe acute COVID-19 and with post-COVID symptoms have glucocorticoid and sex hormone deficiencies. DESIGN/PATIENTS: Samples were obtained for analysis from two UK multicentre cohorts during hospitalisation with COVID-19 (International Severe Acute Respiratory Infection Consortium/World Health Organisation [WHO] Clinical Characterization Protocol for Severe Emerging Infections in the UK study), and at follow-up 5 months after hospitalisation (Post-hospitalisation COVID-19 study). MEASUREMENTS: Plasma steroids were quantified by liquid chromatography-mass spectrometry. Steroid concentrations were compared against disease severity (WHO ordinal scale) and validated symptom scores. Data are presented as geometric mean (SD). RESULTS: In the acute cohort (n = 239, 66.5% male), plasma cortisol concentration increased with disease severity (cortisol 753.3 [1.6] vs. 429.2 [1.7] nmol/L in fatal vs. least severe, p < .001). In males, testosterone concentrations decreased with severity (testosterone 1.2 [2.2] vs. 6.9 [1.9] nmol/L in fatal vs. least severe, p < .001). In the follow-up cohort (n = 198, 62.1% male, 68.9% ongoing symptoms, 165 [121-192] days postdischarge), plasma cortisol concentrations (275.6 [1.5] nmol/L) did not differ with in-hospital severity, perception of recovery, or patient-reported symptoms. Male testosterone concentrations (12.6 [1.5] nmol/L) were not related to in-hospital severity, perception of recovery or symptom scores. CONCLUSIONS: Circulating glucocorticoids in patients hospitalised with COVID-19 reflect acute illness, with a marked rise in cortisol and fall in male testosterone. These findings are not observed 5 months from discharge. The lack of association between hormone concentrations and common post-COVID symptoms suggests steroid insufficiency does not play a causal role in this condition.

Systematic review and longitudinal analysis of implementing Artificial Intelligence to predict clinical deterioration in adult hospitals: what is known and what remains uncertain.

OBJECTIVE: To identify factors influencing implementation of machine learning algorithms (MLAs) that predict clinical deterioration in hospitalized adult patients and relate these to a validated implementation framework. MATERIALS AND METHODS: A systematic review of studies of implemented or trialed real-time clinical deterioration prediction MLAs was undertaken, which identified: how MLA implementation was measured; impact of MLAs on clinical processes and patient outcomes; and barriers, enablers and uncertainties within the implementation process. Review findings were then mapped to the SALIENT end-to-end implementation framework to identify the implementation stages at which these factors applied. RESULTS: Thirty-seven articles relating to 14 groups of MLAs were identified, each trialing or implementing a bespoke algorithm. One hundred and seven distinct implementation evaluation metrics were identified. Four groups reported decreased hospital mortality, 1 significantly. We identified 24 barriers, 40 enablers, and 14 uncertainties and mapped these to the 5 stages of the SALIENT implementation framework. DISCUSSION: Algorithm performance across implementation stages decreased between in silico and trial stages. Silent plus pilot trial inclusion was associated with decreased mortality, as was the use of logistic regression algorithms that used less than 39 variables. Mitigation of alert fatigue via alert suppression and threshold configuration was commonly employed across groups. CONCLUSIONS: : There is evidence that real-world implementation of clinical deterioration prediction MLAs may improve clinical outcomes. Various factors identified as influencing success or failure of implementation can be mapped to different stages of implementation, thereby providing useful and practical guidance for implementers.

The serotonin transporter sustains human brown adipose tissue thermogenesis.

Activation of brown adipose tissue (BAT) in humans is a strategy to treat obesity and metabolic disease. Here we show that the serotonin transporter (SERT), encoded by SLC6A4, prevents serotonin-mediated suppression of human BAT function. RNA sequencing of human primary brown and white adipocytes shows that SLC6A4 is highly expressed in human, but not murine, brown adipocytes and BAT. Serotonin decreases uncoupled respiration and reduces uncoupling protein 1 via the 5-HT2B receptor. SERT inhibition by the selective serotonin reuptake inhibitor (SSRI) sertraline prevents uptake of extracellular serotonin, thereby potentiating serotonin's suppressive effect on brown adipocytes. Furthermore, we see that sertraline reduces BAT activation in healthy volunteers, and SSRI-treated patients demonstrate no 18F-fluorodeoxyglucose uptake by BAT at room temperature, unlike matched controls. Inhibition of BAT thermogenesis may contribute to SSRI-induced weight gain and metabolic dysfunction, and reducing peripheral serotonin action may be an approach to treat obesity and metabolic disease.

Surgical outcome improvement by shared decision-making: value of a preoperative multidisciplinary target clinic for the elderly in colorectal surgery.

PURPOSE: Frailty and comorbidities increase the risk of postoperative complications and raise treatment costs. Perioperative optimisation is shown to improve surgical outcomes for the elderly. The aim of this study was to assess the impact of introducing a multidisciplinary preoperative clinic for older patients (Colchester Older Persons' Evaluation for Surgery (COPES) clinic) undergoing major colorectal surgery. METHODS: This 5-year single centre study included patients >65 years with ≥3 comorbidities undergoing major colorectal surgery. From October 2018, patients with these characteristics were evaluated and optimised in the COPES clinic by a geriatrician and an anaesthetist. Outcomes were compared to high-risk patients operated on prior to COPES (pre-COPES group). The primary outcomes were postoperative morbidity at discharge and 6 months measured by the Comprehensive Complication Index. Patients were matched on age and number of comorbidities. RESULTS: A total of 54 patients were enrolled in the pre-COPES and 18 in the COPES group. After matching, the results were comparable for both groups. The length of stay was shorter in the COPES group and the recurrence rate was higher; however, it did not reach statistical significance in both findings. CONCLUSION: This clinic intends to improve treatment quality, placing emphasis on shared decision-making. More focus should be put on patient-reported outcomes and experiences. Especially for elderly patients, quality of life and maintaining independence are often their priority. To determine the true value of a preoperative multidisciplinary clinic targeting elderly comorbid patients, a prospective study with larger cohort is needed, focusing not only on objective outcomes but also on patient-reported outcomes.

Analysis of AT7519 as a pro-resolution compound in an acetaminophen-induced mouse model of acute inflammation by UPLC-MS/MS.

BACKGROUND: Uncontrolled inflammation contributes to the progression of organ damage in acute conditions, such as acetaminophen-induced acute liver injury (APAP-ALI) and there are limited treatments for this condition. AT7519, a cyclic-dependent kinase inhibitor (CDKI), has been used successfully in several conditions, to resolve inflammation and return tissue homeostatic functions. AT7519 has not been assessed in APAP-ALI and its effect on APAP metabolism is unknown. Targeted chromatography and mass spectrometry can be used to assess multiple compounds simultaneously and this approach has not been applied yet to measure APAP and AT7519 in a mouse model. RESULTS: We show an optimised simple and sensitive LC-MS/MS method for determining concentrations of AT7519 and APAP in low volumes of mouse serum. Using positive ion mode electrospray ionisation, separation of AT7519 and APAP and their corresponding isotopically labelled internal standards [2H]8-AT16043M (d8-AT7519) and [2H]8-APAP (d4-APAP), was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm; 1.7µm). A gradient mobile phase system of water and methanol was delivered at a flow rate of 0.5 mL/min with a run time of 9 min. Calibration curves were linear, intra-day and inter-day precision and accuracy were acceptable and the covariates of all standards and quality control replicates were less than 15%. The method was successfully applied to evaluate AT7519 and APAP levels 20 h post AT7519 (10 mg/mg) in C57Bl6J wild type mouse serum treated with either vehicle or APAP. Serum AT7519 was significantly higher in mice that had received APAP compared to control, but there was no correlation between APAP and AT7519 quantification. There was also no correlation of AT7519 and hepatic damage or proliferation markers. CONCLUSION: We optimised an LC-MS/MS method to quantify both AT7519 and APAP in mouse serum (50 µL), using labelled internal standards. Application of this method to a mouse model of APAP toxicity proved effective in accurately measuring APAP and AT7519 concentrations after i.p. dosing. AT7519 was significantly higher in mice with APAP toxicity, indicating hepatic metabolism of this CDKI, but there was no correlation with markers of hepatic damage or proliferation, demonstrating that this dose of AT7519 (10 mg/kg) does not contribute to hepatic damage or repair. This optimised method can be used for future investigations of AT7519 in APAP in mice.

Using LC-MS/MS to Determine Salivary Steroid Reference Intervals in a European Older Adult Population.

A number of steroids, including glucocorticoids and sex hormones, have been associated with neurodegenerative and cardiovascular conditions common in aging populations. The application of liquid chromatography tandem mass spectrometry (LC-MS/MS) steroid analysis offers an opportunity to conduct simultaneous multiplex steroid analysis within a given sample. In this paper, we describe the application of an LC-MS/MS steroid analysis method for the assessment of reference ranges of steroids in human saliva samples (200 µL) collected from older adults (age 50 years and above) enrolled in a European cohort investigating the risk for Alzheimer's dementia. Saliva samples were prepared using supported liquid extraction (SLE) along with a calibration curve and analysed using a Waters I-Class UPLC (Ultra Performance Liquid Chromatography) and a Sciex QTrap 6500+ mass spectrometer. Mass spectrometry parameters of steroids were optimised for each steroid and a method for the chromatographic separation of 19 steroids was developed. Lower limits of quantitation (LLOQs), linearity and other method criteria were assessed. In total, data from 125 participants (500 samples) were analysed and assessed for reference ranges (64 male, 61 female). A total of 19 steroids were detected in saliva within the range of the method. There were clear diurnal patterns in most of the steroid hormones detected. Sex differences were observed for androstenedione (A4), testosterone (T), cortisone (E) and aldosterone (Aldo). In the first sample of the day, dehydroepiandrosterone (DHEA) was significantly higher in healthy volunteers compared to those with Alzheimer's disease biomarkers. This LC-MS/MS method is suitable for the analysis of 19 steroids in saliva in adults.

Frequency and nature of medical emergency team afferent limb failure in patients with documented vital sign abnormalities: A retrospective point prevalence study.

BACKGROUND: Medical emergency team (MET) afferent limb failure is the presence of MET triggers and the absence of a documented MET call. OBJECTIVES: The aim of this study was to measure and understand the frequency and nature of MET afferent limb failure in patients with documented vital sign abnormalities in an Australian major teaching hospital. METHODS: A retrospective point prevalence study was conducted at a 600-bed teaching hospital in Melbourne, Australia. Data were collected for all adult inpatients (aged ≥18 years) on 13 wards (three general medicine, three surgical, and seven specialist wards) during a randomly selected 24-h period. Data were extracted from the electronic medical record. RESULTS: There were 357 patients included in the study, with a median age of 72 y. Of the 9716 vital sign measures extracted, 0.9% fulfilled patient-specific MET activation criteria. There were 93 MET triggers documented in 36 patients: 25 patients experienced MET afferent limb failure. The major issues related to MET afferent limb failure were MET trigger modification processes, resolution of vital sign abnormalities, alternative escalation of care, and limitations of medical treatment orders without specific modifications to MET triggers. CONCLUSIONS: Mandating MET activation for one aberrant vital sign at a single point in time warrants further assessment: lack of timely vital sign resolution may be a more appropriate trigger for MET calls and should be formally tested in future research. The frequency and effectiveness of alternative escalation pathways and local management of patients with MET triggers also warrant further investigation.

Dimethyl fumarate reduces hepatocyte senescence following paracetamol exposure.

Liver disease is a major cause of premature death. Oxidative stress in the liver represents a key disease driver. Compounds, such as dimethyl fumarate (DMF), can activate the antioxidant response and are used clinically to treat disease. In this study, we tested the protective properties of DMF before or after paracetamol exposure. Following DMF administration, Nrf2 nuclear translocation was tracked at the single-cell level and target gene transactivation confirmed. Next, the protective properties of DMF were examined following paracetamol exposure. Transcriptomic and biochemical analysis revealed that DMF rescue was underpinned by reduced Nf-kB and TGF-ß signaling and cell senescence. Following on from these studies, we employed a Zebrafish model to study paracetamol exposure in vivo. We combined a genetically modified Zebrafish model, expressing green fluorescent protein exclusively in the liver, with automated microscopy. Pre-treatment with DMF, prior to paracetamol exposure, led to reduced liver damage in Zebrafish demonstrating protective properties.

Derivatization with 2-hydrazino-1-methylpyridine enhances sensitivity of analysis of 5α-dihydrotestosterone in human plasma by liquid chromatography tandem mass spectrometry.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of DHT by LC-MS/MS. Derivatization of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of DHT, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies.

Synaptic signalling in a network of dopamine neurons: what prevents proper intercellular crosstalk?

Human embryonic stem cell (hESC)-derived midbrain dopamine (DA) neurons stand out as a cell source for transplantation with their sustainability and consistency superior to the formerly used fetal tissues. However, multiple studies of DA neurons in culture failed to register action potential (AP) generation upon synaptic input. To test whether this is due to deficiency of NMDA receptor (NMDAR) coagonists released from astroglia, we studied the functional properties of neural receptors in hESC-derived DA neuronal cultures. We find that, apart from an insufficient amount of coagonists, lack of interneuronal crosstalk is caused by hypofunction of synaptic NMDARs due to their direct inhibition by synaptically released DA. This inhibitory tone is independent of DA receptors and affects the NMDAR coagonist binding site.

Ionisation bias undermines the use of matrix-assisted laser desorption/ionisation for estimating peptide deamidation: Synthetic peptide studies demonstrate electrospray ionisation gives more reliable response ratios.

RATIONALE: Although mass spectrometry (MS) is routinely used to determine deamination in peptide mixtures, the effects of the choice of ionisation source have not yet been investigated. In particular, matrix-assisted laser desorption/ionisation (MALDI) has become a popular tool with which to measure levels of glutamine deamidation in ancient proteins. Here we use model synthetic peptides to rigorously compare MALDI and electrospray ionisation (ESI). METHODS: We used two synthetic peptides, with glutamine (Q) in one substituted for glutamic acid (E) in the other, to investigate the suitability of MALDI and ESI sources for the assessment of deamidation in peptides using MS. We also compared measurements of the same Q- and E-containing peptide mixtures using two different mass analysers (time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR)). RESULTS: When standard mixtures of the Q- and E-containing peptides were analysed using MALDI, under-representation of the E-containing peptide was observed. This observation was consistent between analyses carried out using either TOF or FT-ICR-MS. When the same mixtures were analysed using ESI FT-ICR-MS, no ionisation bias was observed. CONCLUSIONS: MALDI may not be a suitable ionisation method for the determination of deamidation in peptide mixtures. However, ESI was successfully used to determine the ratio in known mixtures of Q- and E-containing peptides. These preliminary observations warrant further investigation into ionisation bias when measuring deamidation in other peptide sequences.

Results of a multicentre randomised controlled trial of cochlear-sparing intensity-modulated radiotherapy versus conventional radiotherapy in patients with parotid cancer (COSTAR; CRUK/08/004).

PURPOSE: About 40-60% of patients treated with post-operative radiotherapy for parotid cancer experience ipsilateral sensorineural hearing loss. Intensity-modulated radiotherapy (IMRT) can reduce radiation dose to the cochlea. COSTAR, a phase III trial, investigated the role of cochlear-sparing IMRT (CS-IMRT) in reducing hearing loss. METHODS: Patients (pT1-4 N0-3 M0) were randomly assigned (1:1) to 3-dimensional conformal radiotherapy (3DCRT) or CS-IMRT by minimisation, balancing for centre and radiation dose of 60Gy or 65Gy in 30 daily fractions. The primary end-point was proportion of patients with sensorineural hearing loss in the ipsilateral cochlea of ≥10 dB bone conduction at 4000 Hz 12 months after radiotherapy compared using Fisher's exact test. Secondary end-points included hearing loss at 6 and 24 months, balance assessment, acute and late toxicity, patient-reported quality of life, time to recurrence and survival. RESULTS: From Aug 2008 to Feb 2013, 110 patients (54 3DCRT; 56 CS-IMRT) were enrolled from 22 UK centres. Median doses to the ipsilateral cochlea were 3DCRT: 56.2Gy and CS-IMRT: 35.7Gy (p < 0.0001). 67/110 (61%) patients were evaluable for the primary end-point; main reasons for non-evaluability were non-attendance at follow-up or incomplete audiology assessment. At 12 months, 14/36 (39%) 3DCRT and 11/31 (36%) CS-IMRT patients had ≥10 dB loss (p = 0.81). No statistically significant differences were observed in hearing loss at 6 or 24 months or in other secondary end-points including patient-reported hearing outcomes. CONCLUSION: CS-IMRT reduced the radiation dose below the accepted tolerance of the cochlea, but this did not lead to a reduction in the proportion of patients with clinically relevant hearing loss.

The carbon chain-selective adenylation enzyme TamA: the missing link between fatty acid and pyrrole natural product biosynthesis.

The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4'-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6-C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.

Efficient multi-scale 3D CNN with fully connected CRF for accurate brain lesion segmentation.

We propose a dual pathway, 11-layers deep, three-dimensional Convolutional Neural Network for the challenging task of brain lesion segmentation. The devised architecture is the result of an in-depth analysis of the limitations of current networks proposed for similar applications. To overcome the computational burden of processing 3D medical scans, we have devised an efficient and effective dense training scheme which joins the processing of adjacent image patches into one pass through the network while automatically adapting to the inherent class imbalance present in the data. Further, we analyze the development of deeper, thus more discriminative 3D CNNs. In order to incorporate both local and larger contextual information, we employ a dual pathway architecture that processes the input images at multiple scales simultaneously. For post-processing of the network's soft segmentation, we use a 3D fully connected Conditional Random Field which effectively removes false positives. Our pipeline is extensively evaluated on three challenging tasks of lesion segmentation in multi-channel MRI patient data with traumatic brain injuries, brain tumours, and ischemic stroke. We improve on the state-of-the-art for all three applications, with top ranking performance on the public benchmarks BRATS 2015 and ISLES 2015. Our method is computationally efficient, which allows its adoption in a variety of research and clinical settings. The source code of our implementation is made publicly available.

Rhabdomyolysis and acute kidney injury: creatine kinase as a prognostic marker and validation of the McMahon Score in a 10-year cohort: A retrospective observational evaluation.

BACKGROUND: High-volume fluid resuscitation and the administration of sodium bicarbonate and diuretics have a theoretical renoprotective role in patients at high risk of acute kidney injury (AKI) following rhabdomyolysis. Abnormally elevated creatine kinase has previously been used as a biological marker for the identification of patients at high risk of AKI following rhabdomyolysis. OBJECTIVE: To assess the sensitivity and specificity of plasma creatine kinase (admission and peak values) for the prediction of AKI requiring renal replacement therapy (RRT) or of death in patients with confirmed rhabdomyolysis. To compare the diagnostic performance of creatine kinase with the McMahon score. DESIGN: Retrospective observational study. Data collection included McMahon and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores; daily creatine kinase; daily creatinine and electrolytes; ICU length of stay and mortality. SETTING: Neurosciences and Trauma Critical Care Unit (Cambridge, UK). PATIENTS: In total, 232 adults with confirmed rhabdomyolysis (creatine kinase > 1000 Ul) admitted to Neurosciences and Trauma Critical Care Unit between 2002 and 2012. MAIN OUTCOME MEASURES: AKI, RRT and mortality. RESULTS: Forty-five (19%) patients developed AKI and 29 (12.5%) patients required RRT. Mortality was significantly higher in patients who developed AKI (62 vs. 18%, P < 0.001). Average creatine kinase on admission was 5009 (range 69-157 860) Ul. Creatine kinase peaked between the day of admission and day 3 in 91% of cases. PEAK creatine kinase of at least 5000 Ul is 55% specific and 83% sensitive for the prediction of AKI requiring RRT. A McMahon Score of at least 6 calculated on admission is 68% specific and 86% sensitive for RRT. CONCLUSIONS: Creatine kinase is not a specific or early predictor of AKI in patients with rhabdomyolysis. Although a PEAK creatine kinase of at least 5000 Ul has sensitivity acceptable for screening purposes, this is often a delayed finding. A McMahon score of at least 6 calculated on admission allows for a more sensitive, specific and timely identification of patients who may benefit from high-volume fluid resuscitation.

Introduction to the postanaesthetic care unit.

High-risk, noncardiac surgery represents only 12.5% of surgical procedures, but 83.3% of deaths. The postanaesthetic care unit (PACU) addresses the need for an improved level of care for these patients by providing postoperative high-dependency or intensive care (Level 2 or 3). The PACU aims to improve the structure of care provision for high-risk surgical patients. By maintaining 24-hour cover at the same staffing level, the risk of poorer 'out-of- hours' care is reduced. In a PACU, whose remit is solely postoperative care, evidence-based protocols can be established to standardize the care given. The aim is to provide 24 hours of postoperative optimized care, thus targeting the period when these patients are most vulnerable, to reduce the risk of complications developing and identify complications promptly, should they occur. The PACU is set up to facilitate certain processes to aid optimized care in the postoperative period. These include invasive and noninvasive ventilation, goal-directed haemodynamic management, invasive monitoring and optimal pain management. Identification of high-risk patients who might benefit from PACU care is not always straightforward. However, tools are available to aid the clinician, supplementing clinical assessment and basic investigations. These include clinical prediction rules and cardiopulmonary exercise testing. Both the setting up and the running of a PACU clearly have cost implications. However, the reduction in postoperative morbidity, and thus patients' length of stay, should, overall, reduce costs. The benefits of a PACU should therefore be seen in terms of improved surgical outcomes, reducing postoperative morbidity and mortality, and cost savings.