Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells.

Transcriptomic analysis is a powerful method in the utilization of New Approach Methods (NAMs) for identifying mechanisms of toxicity and application to hazard characterization. With this regard, mapping toxicological events to time of exposure would be helpful to characterize early events. Here, we investigated time-dependent changes in gene expression levels in iPSC-derived renal proximal tubular-like cells (PTL) treated with five diverse compounds using TempO-Seq transcriptomics with the aims to evaluate the application of PTL for toxicity prediction and to report on temporal effects for the activation of cellular stress response pathways. PTL were treated with either 50 µM amiodarone, 10 µM sodium arsenate, 5 nM rotenone, or 300 nM tunicamycin over a temporal time course between 1 and 24 h. The TGFß-type I receptor kinase inhibitor GW788388 (1 µM) was used as a negative control. Pathway analysis revealed the induction of key stress-response pathways, including Nrf2 oxidative stress response, unfolding protein response, and metal stress response. Early response genes per pathway were identified much earlier than 24 h and included HMOX1, ATF3, DDIT3, and several MT1 isotypes. GW788388 did not induce any genes within the stress response pathways above, but showed deregulation of genes involved in TGFß inhibition, including downregulation of CYP24A1 and SERPINE1 and upregulation of WT1. This study highlights the application of iPSC-derived renal cells for prediction of cellular toxicity and sheds new light on the temporal and early effects of key genes that are involved in cellular stress response pathways.

Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells.

Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

Initial Pain Assessment and Management in Pediatric Burn Patients Presenting to a Major Trauma Center in Saudi Arabia.

BACKGROUND: Pediatric burn injury is a traumatic experience for affected children and their families. Burn pain is frequently undertreated and may adversely affect patient experience and outcomes. The aim of this study was to investigate the current practice of initial pediatric burn pain assessment and management at a major trauma center in Riyadh, Kingdom of Saudi Arabia. METHODS: We conducted a retrospective cohort study that included children 14 years and younger who visited King Saud Medical City in the Kingdom of Saudi Arabia with a presenting complaint of burn injury from January 01, 2017 to August 30, 2018. Variables were reported using descriptive statistics as appropriate. RESULTS: The 309 patients who were analyzed were classified into 3 age groups ranging from 0 to younger than 3 years (61%), 3 to 7 years (24%), and older than 7 years (15%). They included 145 (47%) female and 164 (53%) male patients. Pain levels of 182 patients (59%) were documented using an age-appropriate tool. In 75 children (24%), pain levels were documented using an alternate tool, and the tool used was not defined for 44 children (14%). Pain assessment was not documented for 8 children. Of those with an age-appropriate tool, the median initial pain score was 4 (interquartile range [IQR], 2-4). Analgesia was recorded to have been administered to 139 patients (45%), within a median time of 50 minutes (IQR, 17-154 minutes) to first analgesia. Among patients who had appropriate assessment of pain, 92 (50.3%) received analgesia compared with 52 (41.3%) who did not have appropriate assessment (P = 0.12). Among patients who had appropriate pain assessment, time to analgesia was 42 minutes (IQR, 15-132 minutes) compared with 53 minutes (IQR, 17-189 minutes) among patients who did not have appropriate assessment (P = 0.48). DISCUSSION: Most pediatric patients presenting with burns had pain assessment, but a substantial proportion of children were not managed using recommended age-specific tools. The use of age-specific tools was not necessarily associated with delivery of analgesia. For pediatric burns, prompt delivery of analgesia should be prioritized with pain assessment using age-appropriate tools being recommended, but optional.

The Development of Helicopter Emergency Medical Services in the Republic of Ireland.

Helicopter emergency medical services (HEMS) have formed an integral component of the Irish health care system for the past decade; yet, the factors leading their commencement, their evolutions over this time, and the current model of service delivery have not been widely published. Aeromedical service provision may vary significantly from country to country and may also vary regionally within countries. A health system's necessities; capacity and maturity; the level of state, corporate, private, or community investment; and the capacity of the contracted service provider are all factors that influence the service provision. This research article describes the historic factors leading to a military and health system collaboration to HEMS during an era of health care reform. Over the past decade, the Irish health system has undergone significant reconfiguration and centralization of services, leading to increased demands on emergency medical ground and air medical services. Future advancements in aeromedical service provision require an innate understanding of the current model. This article adds to the knowledge base, informs policy makers, and supports decision making surrounding HEMS provision and the potential to explore military and health system collaborations and enhanced overall service provision.

"Commandeuring" Xenobiotic Metabolism: Advances in Understanding Xenobiotic Metabolism.

The understanding of how exogenous chemicals (xenobiotics) are metabolized, distributed, and eliminated is critical to determine the impact of the chemical and its metabolites to the (human) organism. This is part of the research and educational discipline ADMET (absorption, distribution, metabolism, elimination, and toxicity). Here, we review the work of Jan Commandeur and colleagues who have not only made a significant impact in understanding of phase I and phase II metabolism of several important compounds but also contributed greatly to the development of experimental techniques for the study of xenobiotic metabolism. Jan Commandeur's work has covered a broad area of research, such as the development of online screening methodologies, the use of a combination of enzyme mutagenesis and molecular modeling for structure-activity relationship (SAR) studies, and the development of novel probe substrates. This work is the bedrock of current activities and brings the field closer to personalized (cohort-based) pharmacology, toxicology, and hazard/risk assessment.

Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation.

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.

Temporal changes in blood pressure following prehospital rapid sequence intubation.

BACKGROUND: Rapid Sequence intubation (RSI) is an airway procedure that uses sedative and paralytic drugs to facilitate endotracheal intubation. It is known that RSI could impact blood pressure in the peri-intubation period. However, little is known about blood pressure changes in longer time frames. Therefore, this analysis aims to describe the changes in systolic blood pressure in a large cohort of paramedic-led RSI cases over the whole prehospital timespan. METHODS: Intensive Care Paramedics in Victoria, Australia, are authorised to use RSI in medical or trauma patients with a Glasgow Coma Scale <10. This retrospective cohort study analysed data from patientcare records for patients aged 12 years and above that had received RSI, from 1 January 2008 to 31 December 2019. This study quantifies the systolic blood pressure changes using regression with fractional polynomial terms. The analysis is further stratified by high versus Low Shock Index (LSI). The shock index is calculated by dividing pulse rate by systolic blood pressure. RESULTS: During the study period RSI was used in 8613 patients. The median number of blood pressure measurements was 5 (IQR 3-8). Systolic blood pressure rose significantly by 3.4 mm Hg (p<0.001) and then returned to baseline in the first 5 min after intubation for LSI cases. No initial rise in blood pressure is apparent in High Shock Index (HSI) cases. Across the whole cohort, systolic blood pressure decreased by 7.1 mm Hg (95% CI 7.9 to 6.3 mm Hg; p<0.001) from the first to the last blood pressure measured. CONCLUSIONS: Our study shows that in RSI patients a small transient elevation in systolic blood pressure in the immediate postintubation period is found in LSI, but this elevation is not apparent in HSI. Blood pressure decreased over the prehospital phase in RSI patients with LSI, but increased for HSI cases.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors.

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

Endotracheal Tube Intracuff Pressure Changes in Patients Transported by a Helicopter Emergency Medical Service: A Prospective Observational Study.

OBJECTIVE: The pressure within an endotracheal tube cuff is of particular importance in helicopter emergency medical services (HEMS) transport because the unpressurized cabin is subjected to decreases in atmospheric pressure. This can cause the cuff to overinflate and may be associated with clinical complications. We sought to evaluate endotracheal tube cuff pressure changes among intubated patients during HEMS transport. METHODS: A prospective observational study was performed including adult patients who were intubated and transported by helicopter between April 2017 and October 2018. Cuff pressures were measured before, during, and after HEMS transport with a commercial manometer. RESULTS: A total of 208 patients were included. The median maximum flight altitude was 3,000 (interquartile range [IQR], 2,000-5,000) ft. The median initial cuff pressure before takeoff was 35 (IQR, 24-50) cm H2O, which increased to 50 (IQR, 35-70) cm H2O at maximum altitude. A total of 169 (81.3%) patients had a cuff pressure > 30 cm H2O at maximum altitude. There was a moderate correlation between altitude and cuff pressure (r = 0.532, P < .001). CONCLUSIONS: Cuff pressure increased during HEMS transport, demonstrating the need for routine cuff pressure monitoring during flight. Further research is required to determine if exposure to transient increases in cuff pressure for short durations is clinically significant.

Correction to: Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

In the original publication of the article.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

The utility of the brain trauma evidence to inform paramedic rapid sequence intubation in out-of-hospital stroke.

BACKGROUND: Rapid sequence intubation (RSI) is used to secure the airway of stroke patients. Randomized controlled trial evidence exists to support the use of paramedic RSI for traumatic brain injury (TBI), but cannot necessarily be applied to stroke RSI because of differences between the stroke and TBI patient. To understand if the TBI evidence can be used for stroke RSI, we analysed a retrospective cohort of TBI and strokes to compare how survival is impacted differently by RSI when comparing strokes and TBI. METHODS: This study was a retrospective analysis of 10 years of in-hospital and out-of-hospital data for all stroke and TBI patients attended by Ambulance Victoria, Australia. Logistic regression predicted the survival for ischemic and haemorrhagic strokes as well as TBI. The constituents of RSI, such a medications, intubation success and time intervals were analysed against survival using interactions to asses if RSI impacts survival differently for strokes compared to TBI. RESULTS: This analysis found significant interactions in the RSI-only group for age, number of intubation attempts, atropine, fentanyl, pulse rate and perhaps scene time and time- to-RSI. Such interactions imply that RSI impact survival differently for TBI versus strokes. Additionally, no significant difference in survival for TBI was found, with a - 0.7% lesser survival for RSI compared to no-RSI; OR 0.86 (95% CI 0.67 to 1.11; p = 0.25). Survival for haemorrhagic stroke was - 14.1% less for RSI versus no-RSI; OR 0.44 (95% CI 0.33 to 0.58; p = 0.01) and was - 4.3%; OR 0.67 (95% CI 0.49 to 0.91; p = 0.01) lesser for ischemic strokes. CONCLUSIONS: Rapid sequence intubation and related factors interact with stroke and TBI, which suggests that RSI effects stroke survival in a different way from TBI. If RSI impact survival differently for strokes compared to TBI, then perhaps the TBI evidence cannot be used for stroke RSI.

Low-Scaling Algorithm for Nudged Elastic Band Calculations Using a Surrogate Machine Learning Model.

We present the incorporation of a surrogate Gaussian process regression (GPR) atomistic model to greatly accelerate the rate of convergence of classical nudged elastic band (NEB) calculations. In our surrogate model approach, the cost of converging the elastic band no longer scales with the number of moving images on the path. This provides a far more efficient and robust transition state search. In contrast to a conventional NEB calculation, the algorithm presented here eliminates any need for manipulating the number of images to obtain a converged result. This is achieved by inventing a new convergence criteria that exploits the probabilistic nature of the GPR to use uncertainty estimates of all images in combination with the force in the saddle point in the target model potential. Our method is an order of magnitude faster in terms of function evaluations than the conventional NEB method with no accuracy loss for the converged energy barrier values.

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

This review covers the current knowledge of the cytochrome P450 enzymes (CYPs) of the human pathogen Mycobacterium tuberculosis (Mtb) and their endogenous redox partners, focusing on their biological function, expression, regulation, involvement in antibiotic resistance, and suitability for exploitation as antitubercular targets. The Mtb genome encodes twenty  CYPs and nine associated redox partners required for CYP catalytic activity. Transposon insertion mutagenesis studies have established the (conditional) essentiality of several of these enzymes for in vitro growth and host infection. Biochemical characterization of a handful of Mtb CYPs has revealed that they have specific physiological functions in bacterial virulence and persistence in the host. Analysis of the transcriptional response of Mtb CYPs and redox partners to external insults and to first-line antibiotics used to treat tuberculosis showed a diverse expression landscape, suggesting for some enzymes a potential role in drug resistance. Combining the knowledge about the physiological roles and expression profiles indicates that, at least five Mtb CYPs, CYP121A1, CYP125A1, CYP139A1, CYP142A1, and CYP143A1, as well as two ferredoxins, FdxA and FdxC, can be considered promising novel therapeutic targets.

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants.

Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.

The association of paramedic rapid sequence intubation and survival in out-of-hospital stroke.

INTRODUCTION: Ambulance transport of patients with stroke is common, with rapid sequence intubation (RSI) to secure the airway used regularly. Randomised controlled trial evidence exists to support the use of RSI in traumatic brain injuries (TBIs), but it is not clear whether the RSI evidence from TBI can be applied to the patient with stroke. To this end, we analysed a retrospective stroke dataset to compare survival of patients with RSI compared with patients that did not receive RSI. METHODS: This study was a retrospective analysis of 10 years of in-hospital and out-of-hospital data for all patients with stroke attended by Ambulance Victoria, in Victoria Australia. Generalised boosted logistic regression was used to predict propensity scores, with initial vital signs, age and demographic variables as well as measures of illness severity and comorbidity included in the prediction model. This analysis employed a 1:1 nearest-neighbour matching which was applied to generate a dataset from which we calculated the OR of survival to hospital discharge of patients receiving RSI versus no-RSI. The sensitivity of these results to unmeasured confounding was assessed with deterministic sensitivity analysis. RESULTS: The propensity score-matched cohort showed a decreased survival for RSI in strokes with an OR 0.61 (95% CI 0.45 to 0.82; p=0.001) when compared with no-RSI. A subgroup analysis showed no significant survival difference for ischaemic strokes: OR 0.66 (95% CI 40 to 1.07; p=0.09). The survival for haemorrhagic stroke was OR 0.60 (95% CI 0.41 to 0.90; p=0.01) lesser for RSI. Results were likely robust to unmeasured confounding and missing data. CONCLUSIONS: Our retrospective analysis shows a decrease in survival when RSI is utilised by paramedics for stroke. Since RSI is commonly used for strokes, controlled trial evidence to support this practice is urgently needed.

Effects of the Introduction of Intranasal Fentanyl on Reduction of Pain Severity Score in Children: An Interrupted Time-Series Analysis.

OBJECTIVES: Children are at risk of inadequate analgesia due to paramedics' inexperience in assessing children and challenges in administering analgesics when the patient is distressed and uncooperative. This study reports on the outcome of a change to practice guidelines that added intranasal fentanyl and intramuscular morphine within a large statewide ambulance service. METHODS: This retrospective study included patients younger than 15 years treated by paramedics between January 2008 and December 2011. The primary outcome of interest was the proportion of patients having a 2/10 or greater reduction in pain severity score using an 11-point Verbal Numeric Rating Scale before and after the intervention. Segmented regression analysis was used to estimate the effect of the intervention over time. A multiple regression model calculated odds ratios with 95% confidence intervals. RESULTS: A total of 92,378 children were transported by paramedics during the study period, with 9833 cases included in the analysis. The median age was 11 years; 61.6% were male. Before the intervention, 88.1% (n = 3114) of children receiving analgesia had a reduction of pain severity of 2 or more points, with 94.2% (n = 5933) achieving this benchmark after intervention (P < 0.0001). The odds of a reduction in pain of 2 or more points increased by 1.01 per month immediately before the intervention and 2.33 after intervention (<0.0001). CONCLUSIONS: This large study of a system-wide clinical practice guideline change has demonstrated a significant improvement in the outcome of interest. However, a proportion of children with moderate to severe pain did not receive analgesia.