A multicentre case series of analytically confirmed gamma-hydroxybutyrate intoxications in Western Australian emergency departments: Pre-hospital circumstances, co-detections and clinical outcomes.

INTRODUCTION: Gamma-hydroxybutyrate (GHB) use is associated with high risk of accidental overdose. This study examined the pre-hospital circumstances, demographic characteristics and clinical outcomes of analytically confirmed GHB emergency department (ED) presentations in Western Australia (WA). METHODS: This case series was conducted across three WA EDs involved in the Emerging Drugs Network of Australia, from April 2020 to July 2022. Patient demographics, pre-hospital drug exposure circumstances and ED presentation and outcome characteristics were collected from ambulance and hospital medical records of GHB-confirmed cases. RESULTS: GHB was detected in 45 ED presentations. The median age was 34 years and 53.3% (n = 24) were female. Most patients arrived at the ED by ambulance (n = 37, 85.7%) and required immediate emergency care (Australasian Triage Score 1 or 2 = 97.8%). One-third of patients were admitted to intensive care (n = 14, 31.1%). Methylamphetamine was co-detected in 37 (82.2%) GHB-confirmed cases. Reduced conscious state was indicated by first recorded Glasgow Coma Scale of ≤8 (n = 29, 64.4%) and observations of patients becoming, or being found, 'unresponsive' and 'unconscious' in various pre-hospital settings (n = 28, 62.2%). 'Agitated' and/or 'erratic' mental state and behavioural observations were recorded in 20 (44.4%) cases. DISCUSSION AND CONCLUSIONS: Analytically verified data from ED presentations with acute toxicity provides an objective information source on drug use trends and emerging public health threats. In our study, patients presenting to WA EDs with GHB intoxication were acutely unwell, often requiring intensive care treatment. The unexpectedly high proportion of female GHB intoxications and methylamphetamine co-ingestion warrants further exploration.

Illicit drugs in the emergency department: Can we determine on clinical grounds if patients are intoxicated? Results from the Western Australian Illicit Substance Evaluation (WISE) study.

INTRODUCTION: Presentations related to illicit drugs are a feature of emergency department practice. Clinicians may form a belief that a patient is intoxicated with illicit drugs based on patient self-report, clinical features on presentation and the local prevalence of illicit drug use. But evidence of the accuracy of this assessment is lacking. The Western Australian Illicit Substance Evaluation (WISE) study enrolled patients believed by their treating clinician to be intoxicated with illicit drugs, and this analysis aims to evaluate the validity of this belief. METHODS: A blood sample was taken on patient arrival and details of patient history, examination and interventions were collected by clinical and research staff. Toxicological examination of biological samples used liquid chromatography-mass spectrometry techniques including Quadrupole Time of Flight screening and Triple Quadrupole targeted analyses. RESULTS: Of 632 study presentations, 518 had illicit drugs detected representing a positive predictive value of 0.82 (95% confidence interval 78.7, 84.9). Those with illicit drugs detected were significantly less likely to arrive by police transport (p = 0.010) or to have used alcohol (p < 0.001). They were significantly more likely to report illicit drug use (p < 0.001) and a much smaller proportion were admitted to a psychiatric ward (3.5% vs. 19.3%, p < 0.0001). Heart rate and systolic blood pressure were significantly higher in the illicit drug group (p = 0.004 and p = 0.003). DISCUSSION AND CONCLUSIONS: In this study, the positive predictive value of clinicians determining if their patient had taken illicit drugs was 0.82. Contemporaneous biochemical analysis in the clinical setting would increase this accuracy and inform patient care.

Analytically confirmed illicit and novel psychoactive drug use in Western Australian emergency departments: initial results from the Emerging Drugs Network of Australia (EDNA).

INTRODUCTION: The burden of acute illicit drug use in Australia is largely unknown. Establishing a prospective drug surveillance system in emergency departments using analytical confirmation may facilitate the early identification of emerging drugs. We describe demographic data and acute toxicity patterns, stratified by analytical confirmation of illicit drugs and novel psychoactive substances, to emergency departments in Western Australia. METHODS: Patients presenting with severe and/or unusual clinical features consistent with recreational drug toxicity were identified across five Western Australian emergency departments participating in the Emerging Drugs Network of Australia between April 2020 and December 2021. Demographic and toxicology patterns in patients with and without analytically confirmed illicit drugs/novel psychoactive substances from blood samples were collected during the emergency department presentation. RESULTS: The cohort included 434 severe and/or unusual toxicology presentations; median age 33 years (first and third quartiles 25-40 years), 268 (61.8%) males. Any substance (illicit, novel psychoactive substance, pharmaceutical) was detected in 405 (93.3%) presentations. Illicit drugs/novel psychoactive substances were detected in 257 (59.2%) presentations, including 73 (28.3%) with more than one confirmed illicit drug/novel psychoactive substance. Frequent illicit drugs identified were metamfetamine (n = 201, 77.9%) and gamma-hydroxybutyrate (n = 30, 11.6%). Forty-eight novel psychoactive substances were detected within 43 (16.7%) presentations. Novel benzodiazepines were most frequently detected (n = 29, 60.4%). Frequent pharmaceuticals detected included diazepam (n = 100, 26.1%) and clonazepam (n = 40, 10.4%). One hundred and fifty-five (35.7%) presentations were discharged home and 56 (12.9%) were admitted to intensive care. Presentations with detected illicit drugs/novel psychoactive substances had a lower median intensive care length of stay compared to presentations without detected illicit drugs/novel psychoactive substances (32.6 h versus 50.8 h respectively, P < 0.001). CONCLUSIONS: Integration of clinical and analytic data in patients with severe and/or unusual toxicology presentations via the Emerging Drugs Network of Australia provides insight into illicit drug/novel psychoactive substance use responsible for acute harm across Western Australian emergency departments.

'Trial by fire': An online survey exploring confidence of junior doctors in managing toxicology presentations to the emergency department.

INTRODUCTION: The often unknown nature of acute drug intoxication, especially with illicit drugs and emerging novel psychoactive substances, can present a significant challenge for emergency clinicians. Less experienced clinicians are particularly vulnerable to the diagnostic dilemmas of complex toxicology emergencies. We sought to better understand the confidence of junior doctors in assessing and managing toxicological emergencies across two emergency departments in Perth, Australia. METHODS: An online survey was conducted between August 2020 and February 2021. Self-rated confidence was measured on a five-point Likert Scale across 10 statements. Two open-ended questions were included to capture perceived barriers and facilitators impacting clinical confidence. Quantitative data were analysed using descriptive methods and Fisher's exact test. Free-text responses were analysed using content analysis. RESULTS: A total of 104 surveys were completed (19.2% interns, 40.4% resident medical officers and 40.4% registrars). Self-rated confidence varied across statements and by staff type. The lowest confidence rating was for managing a patient who had overdosed from an unknown substance (31.7%) and the highest rating for referring a patient to psychiatry following deliberate self-poisoning (86.6%). Confidence increased with greater clinical experience for all statements. Qualitative analysis revealed perceptions of clinical preparedness, complexity of patients and a safe and supportive culture as key factors impacting confidence. DISCUSSION AND CONCLUSIONS: Overcoming perceived deficits in knowledge and clinical experience were key to building confidence. Our findings highlight the need for improved access to toxicology-specific curricula and training, and strategies to ensure adequate supervision from senior clinicians.

The Emerging Drugs Network of Australia: A toxicosurveillance system of illicit and emerging drugs in the emergency department.

OBJECTIVE: The unprecedented rise in synthetic drugs, many containing unknown toxic agents, has made timely analytical diagnosis more difficult, and has reduced the confidence of clinicians providing ED management to this population of patients. This has also impacted the quality of evidence informing harm reduction responses. The Emerging Drugs Network of Australia (EDNA) brings together emergency physicians, toxicologists and forensic laboratories to establish a standardised ED toxicosurveillance system in Australia. METHODS: Blood analysis of intoxicated patients will be conducted by forensic laboratories to enable precise identification of the substances causing acute toxicity. This will be linked with clinical data collected at the time of ED presentation to enable analysis of the clinical effects and outcomes associated with different illicit and emerging drugs. Toxicological and clinical data collected across sentinel sites will align with a nationally endorsed minimum dataset. RESULTS: EDNA's collaborative network will establish a national system of surveillance and reporting of illicit and emerging drugs causing acute toxicity. Standardisation of data collection recorded in a national clinical registry will provide more robust data on epidemiology and associated harms. This will facilitate the translation of clinical and toxicological evidence into timely, appropriate harm reduction and policy. CONCLUSION: Our work represents a collaborative response to calls for more sophisticated data on emerging drug trends in Australia. EDNA will improve coordination between clinicians and analytical services by way of its standardised approach to surveillance and reporting.

In†Vivo Imaging of the Tumor-Associated Enzyme NCEH1 with a Covalent PET Probe.

Herein, we report the development of an 18 F-labeled, activity-based small-molecule probe targeting the cancer-associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile 18 F radionuclide incorporation required for PET imaging. The resulting molecule, [18 F]JW199, labels active NCEH1 in live cells at nanomolar concentrations and greater than 1000-fold selectivity relative to other serine hydrolases. [18 F]JW199 displays rapid, NCEH1-dependent accumulation in mouse tissues. Finally, we demonstrate that [18 F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple-negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis.

Consumer involvement in emergency medicine research: Lessons from engaging sepsis survivors.

Collaboration with consumers is an emerging focus for medical researchers worldwide. Public involvement is increasingly encouraged, and in some cases stipulated by funding bodies, in order to secure financial support. While consumer involvement could be viewed as another hurdle in the funding application process, it can add immense value to research outcomes. However, given the diverse and transient nature of our consumer group, how can we develop meaningful public engagement in emergency medicine research?

Photoproximity Profiling of Protein-Protein Interactions in Cells.

We report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction- and light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled unbiased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of PhotoPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD, and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic subcellular and protein interaction "social network" of a redox-sensitive protein in cells with high temporal resolution.

An early warning system for emerging drugs of concern in the emergency department: Protocol for the Western Australian Illicit Substance Evaluation (WISE) study.

OBJECTIVE: An ever-increasing number of novel psychoactive substances are being detected worldwide. These emerging drugs have been demonstrated to cause toxicity in clusters, and deaths have been reported. We urgently need to learn more about their effects. We report the protocol for the Western Australian Illicit Substance Evaluation (WISE) study, a research project investigating illicit drug use in the ED. METHODS: Patients can be enrolled if the treating clinician strongly suspects they are currently intoxicated with a stimulant, hallucinogenic or cannabinoid drug; and an i.v. cannula or blood tests are required for routine clinical care. Patients are enrolled under a waiver of consent. A single additional blood tube is collected, de-identified and frozen on site. A temporary link between patient identification number and study identification number is retained for up to 10 business days post-hospital discharge to allow for clinical data collection, before this is destroyed and the patients become permanently de-identified. Samples are transported for external liquid chromatography-mass spectrometry analysis in batches once de-identified. RESULTS: The key outcome will be identification of any psychoactive drugs present in the blood sample, together with their respective concentration. This will be linked to the clinical effects, as well as being compared with the substance the patient believed they had taken. CONCLUSION: We consider the novel approach outlined forms a template for an early warning system for emerging drugs of concern, while also providing vital and comprehensive information on current drugs of abuse, their clinical effects and their impact on the health system.

Restricted fluid resuscitation in suspected sepsis associated hypotension (REFRESH): a pilot randomised controlled trial.

PURPOSE: To determine if a regimen of restricted fluids and early vasopressor compared to usual care is feasible for initial resuscitation of hypotension due to suspected sepsis. METHODS: A prospective, randomised, open-label, clinical trial of a restricted fluid resuscitation regimen in the first 6 h among patients in the emergency department (ED) with suspected sepsis and a systolic blood pressure under 100 mmHg, after minimum 1000 ml of IV fluid. Primary outcome was total fluid administered within 6 h post randomisation. RESULTS: There were 99 participants (50 restricted volume and 49 usual care) in the intention-to-treat analysis. Median volume from presentation to 6 h in the restricted volume group was 2387 ml [first to third quartile (Q1-Q3) 1750-2750 ml]; 30 ml/kg (Q1-Q3 32-39 ml/kg) vs. 3000 ml (Q1-Q3 2250-3900 ml); 43 ml/kg (Q1-Q3 35-50 ml/kg) in the usual care group (p < 0.001). Median duration of vasopressor support was 21 h (Q1-Q3 9-42 h) vs. 33 h (Q1-Q3 15-50 h), (p = 0.13) in the restricted volume and usual care groups, respectively. At 90-days, 4 of 48 (8%) in the restricted volume group and 3 of 47 (6%) in the usual care group had died. Protocol deviations occurred in 6/50 (12%) in restricted group and 11/49 (22%) in the usual care group, and serious adverse events in four cases (8%) in each group. CONCLUSIONS: A regimen of restricted fluids and early vasopressor in ED patients with suspected sepsis and hypotension appears feasible. Illness severity was moderate and mortality rates low. A future trial is necessary with recruitment of high-risk patients to determine effects on clinical outcomes in this setting.

CC-401 Promotes ß-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition.

Pharmacologic expansion of endogenous ß cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control ß-cell growth we screened ∼2400 bioactive compounds for rat ß-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat ß-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) ß-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat ß cells, which were broadly growth responsive to compound treatment, human ß-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3ß (GSK-3ß) or activin A receptor type II-like kinase/transforming growth factor-ß (ALK5/TGF-ß) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human ß-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced ß-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the ß-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control ß-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control ß-cell growth.

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging.

New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.

Patient perspectives on priorities for emergency medicine research: The PERSPEX study.

OBJECTIVES: To determine the priorities for emergency medicine research of patients currently in an ED and to compare their priorities with those of ACEM researchers. METHODS: A survey of current patients in the EDs of Royal Perth Hospital and Armadale Health Service. Patients gave their reason for presentation, suggested three important research priorities for emergency medicine and ranked their top 5 choices from a pre-specified list published by the ACEM researchers. Results were analysed using qualitative and quantitative research methods. RESULTS: A total of 430 patients completed the survey, of which 218 were men (50.7%), with median age 44 years (interquartile range [IQR] 30-61 years, range 18-92 years). The top 5 priorities suggested by patients were cardiology, trauma, ED processes, mental health and haematology/oncology. The top 5 patient rankings of the ACEM researcher list were resuscitation, trauma, cardiology, infectious diseases and paediatrics. Older age groups tended to rank cardiology high, while trauma and resuscitation were ranked high among all age groups. There was moderate agreement between patients and ACEM researchers (ρ = 0.51, P = 0.03). CONCLUSIONS: The top 5 emergency medicine research priorities nominated by patients in ED were cardiology, trauma, ED processes, mental health and haematology/oncology, although many 'system priorities' were identified as well. These priorities were generally consistent with ACEM researchers, but patients also suggested alternative directions for future research.

REstricted Fluid REsuscitation in Sepsis-associated Hypotension (REFRESH): study protocol for a pilot randomised controlled trial.

BACKGROUND: Guidelines recommend an initial intravenous (IV) fluid bolus of 30 ml/kg isotonic crystalloid for patients with sepsis and hypotension. However, there is a lack of evidence from clinical trials to support this. Accumulating observational data suggest harm associated with the injudicious use of fluids in sepsis. There is currently equipoise regarding liberal or restricted fluid-volume resuscitation as first-line treatment for sepsis-related hypotension. A randomised trial comparing these two approaches is, therefore, justified. METHODS/DESIGN: The REstricted Fluid REsuscitation in Sepsis-associated Hypotension trial (REFRESH) is a multicentre, open-label, randomised, phase II clinical feasibility trial. Participants will be patients presenting to the emergency departments of Australian metropolitan hospitals with suspected sepsis and a systolic blood pressure of < 100 mmHg, persisting after a 1000-ml fluid bolus with isotonic crystalloid. Participants will be randomised to either a second 1000-ml fluid bolus (standard care) or maintenance rate fluid only, with the early commencement of a vasopressor infusion to maintain a mean arterial pressure of > 65 mmHg, if required (restricted fluid). All will receive further protocolised fluid boluses (500 ml or 250 ml, respectively), if required during the 6-h study period. The primary outcome measure is total volume administered in the first 6 h. Secondary outcomes include fluid volume at 24 h, organ support 'free days' to day 28, 90-day mortality, and a range of feasibility and process-of-care measures. Participants will also undergo serial measurement, over the first 24 h, of biomarkers of inflammation, endothelial cell activation and glycocalyx degradation for comparison between the groups. DISCUSSION: This is the first randomised trial examining fluid volume for initial resuscitation in septic shock in an industrialised country. A pragmatic, open-label design will establish the feasibility of undertaking a large, international, multicentre trial with sufficient power to assess clinical outcomes. The embedded biomarker study aims to provide mechanistic plausibility for a larger trial by defining the effects of fluid volume on markers of systemic inflammation and the vascular endothelium. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Registry, ID: ACTRN12616000006448. Registered on 12 January 2016.

Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.

Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

Brominated Luciferins Are Versatile Bioluminescent Probes.

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

Design and Synthesis of an Alkynyl Luciferin Analogue for Bioluminescence Imaging.

Herein, the synthesis and characterization of an alkyne-modified luciferin is reported. This bioluminescent probe was accessed using C-H activation methodology and was found to be stable in solution and capable of light production with firefly luciferase. The luciferin analogue was also cell permeant and emitted more redshifted light than d-luciferin, the native luciferase substrate. Based on these features, the alkynyl luciferin will be useful for a variety of imaging applications.

A "Caged" Luciferin for Imaging Cell-Cell Contacts.

Cell-cell interactions underlie fundamental biological processes but remain difficult to visualize over long times and large distances in tissues and live organisms. Bioluminescence imaging with luciferase-luciferin pairs is sufficiently sensitive to image cells in vivo but lacks the spatial resolution to identify cellular locations and interactions. To repurpose this technology for visualizing cellular networks, we developed a "caged" luciferin that produces light only when cells are in close contact. This molecule comprises a nitroaromatic core that can be selectively reduced ("uncaged") by one cell type, liberating a luciferin that can be selectively consumed by neighboring, luciferase-expressing cells. When the two cell types are in contact, robust light emission is observed. This imaging strategy will enable the noninvasive visualization of cell-cell interactions relevant to organismal biology.

Rapid and scalable assembly of firefly luciferase substrates.

Bioluminescence imaging with luciferase-luciferin pairs is a popular method for visualizing biological processes in vivo. Unfortunately, most luciferins are difficult to access and remain prohibitively expensive for some imaging applications. Here we report cost-effective and efficient syntheses of d-luciferin and 6'-aminoluciferin, two widely used bioluminescent substrates. Our approach employs inexpensive anilines and Appel's salt to generate the luciferin cores in a single pot. Additionally, the syntheses are scalable and can provide multi-gram quantities of both substrates. The streamlined production and improved accessibility of luciferin reagents will bolster in vivo imaging efforts.