Pathogen-induced biosynthetic pathways encode defense-related molecules in bread wheat.

Wheat is a widely grown food crop that suffers major yield losses due to attack by pests and pathogens. A better understanding of biotic stress responses in wheat is thus of major importance. The recently assembled bread wheat genome coupled with extensive transcriptomic resources provides unprecedented new opportunities to investigate responses to pathogen challenge. Here, we analyze gene coexpression networks to identify modules showing consistent induction in response to pathogen exposure. Within the top pathogen-induced modules, we identify multiple clusters of physically adjacent genes that correspond to six pathogen-induced biosynthetic pathways that share a common regulatory network. Functional analysis reveals that these pathways, all of which are encoded by biosynthetic gene clusters, produce various different classes of compounds­namely, flavonoids, diterpenes, and triterpenes, including the defense-related compound ellarinacin. Through comparative genomics, we also identify associations with the known rice phytoalexins momilactones, as well as with a defense-related gene cluster in the grass model plant Brachypodium distachyon. Our results significantly advance the understanding of chemical defenses in wheat and open up avenues for enhancing disease resistance in this agriculturally important crop. They also exemplify the power of transcriptional networks to discover the biosynthesis of chemical defenses in plants with large, complex genomes.

Healthcare cost burden of acute chest pain presentations.

BACKGROUND: This study aimed to estimate the direct healthcare cost burden of acute chest pain attendances presenting to ambulance in Victoria, Australia, and to identify key cost drivers especially among low-risk patients. METHODS: State-wide population-based cohort study of consecutive adult patients attended by ambulance for acute chest pain with individual linkage to emergency and hospital admission data in Victoria, Australia (1 January 2015-30 June 2019). Direct healthcare costs, adjusted for inflation to 2020-2021 ($A), were estimated for each component of care using a casemix funding method. RESULTS: From 241 627 ambulance attendances for chest pain during the study period, mean chest pain episode cost was $6284, and total annual costs were estimated at $337.4 million ($68 per capita per annum). Total annual costs increased across the period ($310.5 million in 2015 vs $384.5 million in 2019), while mean episode costs remained stable. Cardiovascular conditions (25% of presentations) were the most expensive (mean $11 523, total annual $148.7 million), while a non-specific pain diagnosis (49% of presentations) was the least expensive (mean $3836, total annual $93.4 million). Patients classified as being at low risk of myocardial infarction, mortality or hospital admission (Early Chest pain Admission, Myocardial infarction, and Mortality (ECAMM) score) represented 31%-57% of the cohort, with total annual costs estimated at $60.6 million-$135.4 million, depending on the score cut-off used. CONCLUSIONS: Total annual costs for acute chest pain presentations are increasing, and a significant proportion of the cost burden relates to low-risk patients and non-specific pain. These data highlight the need to improve the cost-efficiency of chest pain care pathways.

Impact of prehospital opioid dose on angiographic and clinical outcomes in acute coronary syndromes.

BACKGROUND: An adverse interaction whereby opioids impair and delay the gastrointestinal absorption of oral P2Y12 inhibitors has been established, however the clinical significance of this in acute coronary syndrome (ACS) is uncertain. We sought to characterise the relationship between prehospital opioid dose and clinical outcomes in patients with ACS. METHODS: Patients given opioid treatment by emergency medical services (EMS) with ACS who underwent percutaneous coronary intervention (PCI) between 1 January 2014 and 31 December 2018 were included in this retrospective cohort analysis using data linkage between the Ambulance Victoria, Victorian Cardiac Outcomes Registry and Melbourne Interventional Group databases. Patients with cardiogenic shock, out-of-hospital cardiac arrest and fibrinolysis were excluded. The primary end point was the risk-adjusted odds of 30-day major adverse cardiac events (MACE) between patients who received opioids and those that did not. RESULTS: 10 531 patients were included in the primary analysis. There was no significant difference in 30-day MACE between patients receiving opioids and those who did not after adjusting for key patient and clinical factors. Among patients with ST-elevation myocardial infarction (STEMI), there were significantly more patients with thrombolysis in myocardial infarction (TIMI) 0 or 1 flow pre-PCI in a subset of patients with high opioid dose versus no opioids (56% vs 25%, p<0.001). This remained significant after adjusting for known confounders with a higher predicted probability of TIMI 0/1 flow in the high versus no opioid groups (33% vs 11%, p<0.001). CONCLUSIONS: Opioid use was not associated with 30-day MACE. There were higher rates of TIMI 0/1 flow pre-PCI in patients with STEMI prescribed opioids. Future prospective research is required to verify these findings and investigate alternative analgesia for ischaemic chest pain.

Variation in Health Care Processes, Quality and Outcomes According to Day and Time of Chest Pain Presentation via Ambulance.

BACKGROUND: Previous studies examining temporal variations in cardiovascular care have largely been limited to assessing weekend and after-hours effects. We aimed to determine whether more complex temporal variation patterns might exist in chest pain care. METHODS: This was a population-based study of consecutive adult patients attended by emergency medical services (EMS) for non-traumatic chest pain without ST elevation in Victoria, Australia between 1 January 2015 and 30 June 2019. Multivariable models were used to assess whether time of day and week stratified into 168 hourly time periods was associated with care processes and outcomes. RESULTS: There were 196,365 EMS chest pain attendances; mean age 62.4 years (standard deviation [SD] 18.3) and 51% females. Presentations demonstrated a diurnal pattern, a Monday-Sunday gradient (Monday peak) and a reverse weekend effect (lower rates on weekends). Five temporal patterns were observed for care quality and process measures, including a diurnal pattern (longer emergency department [ED] length of stay), an after-hours pattern (lower angiography or transfer for myocardial infarction, pre-hospital aspirin administration), a weekend effect (shorter ED clinician review, shorter EMS off-load time), an afternoon/evening peak period pattern (longer ED clinician review, longer EMS off-load time) and a Monday-Sunday gradient (ED clinician review, EMS offload time). Risk of 30-day mortality was associated with weekend presentation (Odds ratio [OR] 1.15, p=0.001) and morning presentation (OR 1.17, p<0.001) while risk of 30-day EMS reattendance was associated with peak period (OR 1.16, p<0.001) and weekend presentation (OR 1.07, p<0.001). CONCLUSIONS: Chest pain care demonstrates complex temporal variation beyond the already established weekend and after-hours effect. Such relationships should be considered during resource allocation and quality improvement programs to improve care across all days and times of the week.

Sex differences in prehospital analgesia in patients presenting with acute coronary syndromes and their association with clinical outcomes.

OBJECTIVES: This study examined if sex differences in prehospital pain scores, opioid administration, and clinical outcomes exist in acute coronary syndrome (ACS) patients. BACKGROUND: Sex differences persist in ACS presentation, management, and outcomes. The impact of sex differences on prehospital pain management of ACS with opioids is unknown. METHODS: Patients presenting with ACS via ambulance (2014-2018) that underwent percutaneous coronary intervention (PCI) were prospectively collected via the Victorian Cardiac Outcomes Registry and Melbourne Interventional Group, linked to the Ambulance Victoria database. The primary outcome was 30-day major adverse cardiac events (MACE). Secondary outcomes were descriptive analyses of prehospital pain score, intravenous morphine equivalent analgesic dosing, plus predictors of MACE and thrombolysis in myocardial infarction (TIMI) 0-1 flow pre-PCI. RESULTS: A total of 10,547 patients were included (female: 2775 [26%]). Opioids were administered to 1585 (57%) females, 5068 (65%) males (p < 0.001). Adjusted 30-day MACE was similar between opioid groups in both sexes (female: odds ratio [OR]: 1.21, confidence interval [CI] 0.82-1.79, p = 0.34; male: OR: 0.89, CI: 0.68-1.16, p = 0.40). Median pain score at presentation was 6 (interquartile range [IQR]: 4, 8) for both sexes. Median opioid dose was 2.5 mg (IQR: 0, 10) in females and 5 mg (IQR: 0, 10) in males (p < 0.001), with similar pain relief achieved. Adjusted rates of TIMI 0-1 pre-PCI were higher in patients administered opioids (female: OR 2.9, CI: 2.07-4.07, p < 0.001; male: OR: 2.67, CI: 2.19-3.25, p < 0.001). CONCLUSIONS: Female patients undergoing PCI received less opioid analgesia, but no sex differences in prehospital pain scores were seen. Opioid administration was associated with impaired antegrade flow in the culprit artery in both sexes, but not short-term MACE. Trials evaluating nonopioid analgesics in ACS are needed.

The influence of ambulance offload time on 30-day risks of death and re-presentation for patients with chest pain.

OBJECTIVE: To assess whether ambulance offload time influences the risks of death or ambulance re-attendance within 30 days of initial emergency department (ED) presentations by adults with non-traumatic chest pain. DESIGN, SETTING: Population-based observational cohort study of consecutive presentations by adults with non-traumatic chest pain transported by ambulance to Victorian EDs, 1 January 2015 - 30 June 2019. PARTICIPANTS: Adults (18 years or older) with non-traumatic chest pain, excluding patients with ST elevation myocardial infarction (pre-hospital electrocardiography) and those who were transferred between hospitals or not transported to hospital (eg, cardiac arrest or death prior to transport). MAIN OUTCOME MEASURES: Primary outcome: 30-day all-cause mortality (Victorian Death Index data). SECONDARY OUTCOME: Transport by ambulance with chest pain to ED within 30 days of initial ED presentation. RESULTS: We included 213 544 people with chest pain transported by ambulance to EDs (mean age, 62 [SD, 18] years; 109 027 women [51%]). The median offload time increased from 21 (IQR, 15-30) minutes in 2015 to 24 (IQR, 17-37) minutes during the first half of 2019. Three offload time tertiles were defined to include approximately equal patient numbers: tertile 1 (0-17 minutes), tertile 2 (18-28 minutes), and tertile 3 (more than 28 minutes). In multivariable models, 30-day risk of death was greater for patients in tertile 3 than those in tertile 1 (adjusted rates, 1.57% v 1.29%; adjusted risk difference, 0.28 [95% CI, 0.16-0.42] percentage points), as was that of a second ambulance attendance with chest pain (adjusted rates, 9.03% v 8.15%; adjusted risk difference, 0.87 [95% CI, 0.57-1.18] percentage points). CONCLUSIONS: Longer ambulance offload times are associated with greater 30-day risks of death and ambulance re-attendance for people presenting to EDs with chest pain. Improving the speed of ambulance-to-ED transfers is urgently required.

Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production.

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3ß-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3ß-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

A noncanonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat.

Plants produce an array of natural products with important ecological functions. These compounds are often decorated with oligosaccharide groups that influence bioactivity, but the biosynthesis of such sugar chains is not well understood. Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits, as exemplified by avenacins, antimicrobial defense compounds produced by oats. Avenacins have a branched trisaccharide moiety consisting of l-arabinose linked to 2 d-glucose molecules that is critical for antifungal activity. Plant natural product glycosylation is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). We previously characterized the arabinosyltransferase that initiates the avenacin sugar chain; however, the enzymes that add the 2 remaining d-glucose molecules have remained elusive. Here we characterize the enzymes that catalyze these last 2 glucosylation steps. AsUGT91G16 is a classical cytosolic UGT that adds a 1,2-linked d-glucose molecule to l-arabinose. Unexpectedly, the enzyme that adds the final 1,4-linked d-glucose (AsTG1) is not a UGT, but rather a sugar transferase belonging to Glycosyl Hydrolase family 1 (GH1). Unlike classical UGTs, AsTG1 is vacuolar. Analysis of oat mutants reveals that AsTG1 corresponds to Sad3, a previously uncharacterized locus shown by mutation to be required for avenacin biosynthesis. AsTG1 and AsUGT91G16 form part of the avenacin biosynthetic gene cluster. Our demonstration that a vacuolar transglucosidase family member plays a critical role in triterpene biosynthesis highlights the importance of considering other classes of carbohydrate-active enzymes in addition to UGTs as candidates when elucidating pathways for the biosynthesis of glycosylated natural products in plants.

Emergency medicine patient wait time multivariable prediction models: a multicentre derivation and validation study.

OBJECTIVE: Patients, families and community members would like emergency department wait time visibility. This would improve patient journeys through emergency medicine. The study objective was to derive, internally and externally validate machine learning models to predict emergency patient wait times that are applicable to a wide variety of emergency departments. METHODS: Twelve emergency departments provided 3 years of retrospective administrative data from Australia (2017-2019). Descriptive and exploratory analyses were undertaken on the datasets. Statistical and machine learning models were developed to predict wait times at each site and were internally and externally validated. Model performance was tested on COVID-19 period data (January to June 2020). RESULTS: There were 1 930 609 patient episodes analysed and median site wait times varied from 24 to 54 min. Individual site model prediction median absolute errors varied from±22.6 min (95% CI 22.4 to 22.9) to ±44.0 min (95% CI 43.4 to 44.4). Global model prediction median absolute errors varied from ±33.9 min (95% CI 33.4 to 34.0) to ±43.8 min (95% CI 43.7 to 43.9). Random forest and linear regression models performed the best, rolling average models underestimated wait times. Important variables were triage category, last-k patient average wait time and arrival time. Wait time prediction models are not transferable across hospitals. Models performed well during the COVID-19 lockdown period. CONCLUSIONS: Electronic emergency demographic and flow information can be used to approximate emergency patient wait times. A general model is less accurate if applied without site-specific factors.

Effect of Lower vs Higher Oxygen Saturation Targets on Survival to Hospital Discharge Among Patients Resuscitated After Out-of-Hospital Cardiac Arrest: The EXACT Randomized Clinical Trial.

Importance: The administration of a high fraction of oxygen following return of spontaneous circulation in out-of-hospital cardiac arrest may increase reperfusion brain injury. Objective: To determine whether targeting a lower oxygen saturation in the early phase of postresuscitation care for out-of-hospital cardiac arrest improves survival at hospital discharge. Design, Setting, and Participants: This multicenter, parallel-group, randomized clinical trial included unconscious adults with return of spontaneous circulation and a peripheral oxygen saturation (Spo2) of at least 95% while receiving 100% oxygen. The trial was conducted in 2 emergency medical services and 15 hospitals in Victoria and South Australia, Australia, between December 11, 2017, and August 11, 2020, with data collection from ambulance and hospital medical records (final follow-up date, August 25, 2021). The trial enrolled 428 of a planned 1416 patients. Interventions: Patients were randomized by paramedics to receive oxygen titration to achieve an oxygen saturation of either 90% to 94% (intervention; n = 216) or 98% to 100% (standard care; n = 212) until arrival in the intensive care unit. Main Outcomes and Measures: The primary outcome was survival to hospital discharge. There were 9 secondary outcomes collected, including hypoxic episodes (Spo2 <90%) and prespecified serious adverse events, which included hypoxia with rearrest. Results: The trial was stopped early due to the COVID-19 pandemic. Of the 428 patients who were randomized, 425 were included in the primary analysis (median age, 65.5 years; 100 [23.5%] women) and all completed the trial. Overall, 82 of 214 patients (38.3%) in the intervention group survived to hospital discharge compared with 101 of 211 (47.9%) in the standard care group (difference, -9.6% [95% CI, -18.9% to -0.2%]; unadjusted odds ratio, 0.68 [95% CI, 0.46-1.00]; P = .05). Of the 9 prespecified secondary outcomes collected during hospital stay, 8 showed no significant difference. A hypoxic episode prior to intensive care was observed in 31.3% (n = 67) of participants in the intervention group and 16.1% (n = 34) in the standard care group (difference, 15.2% [95% CI, 7.2%-23.1%]; OR, 2.37 [95% CI, 1.49-3.79]; P < .001). Conclusions and Relevance: Among patients achieving return of spontaneous circulation after out-of-hospital cardiac arrest, targeting an oxygen saturation of 90% to 94%, compared with 98% to 100%, until admission to the intensive care unit did not significantly improve survival to hospital discharge. Although the trial is limited by early termination due to the COVID-19 pandemic, the findings do not support use of an oxygen saturation target of 90% to 94% in the out-of-hospital setting after resuscitation from cardiac arrest. Trial Registration: ClinicalTrials.gov Identifier: NCT03138005.

Mobile Stroke Units Facilitate Prehospital Management of Intracerebral Hemorrhage.

Background and Purpose: Mobile stroke units (MSUs) improve reperfusion therapy times in acute ischemic stroke (AIS). However, prehospital management options for intracerebral hemorrhage (ICH) are less established. We describe the initial Melbourne MSU experience in ICH. Methods: Consecutive patients with ICH and AIS treated by the Melbourne MSU were included. We describe demographics, proportions of patients receiving specific therapies, and bypass to comprehensive/neurosurgical centers. We also compare operational time metrics between patients with MSU-ICH and MSU-AIS. Results: During a 2-year period, the Melbourne MSU managed 49 patients with ICH, mean (SD) age 74 (12) years, median (interquartile range) National Institutes of Health Stroke Scale 17 (12­20). Intravenous antihypertensives were the commonest treatment provided (46.9%). Bypass of a primary center to a comprehensive center with neurosurgical expertise occurred in 32.7% of patients with MSU-ICH compared with 20.5% of patients with MSU-AIS. Compared with patients with MSU-AIS, patients with MSU-ICH had faster onset-to-emergency-call, and onset-to-scene-arrival times at the median and 75th percentiles. Conclusions: MSUs can facilitate ultra-early ICH diagnosis, management, and triage.

Utility of Severity-Based Prehospital Triage for Endovascular Thrombectomy: ACT-FAST Validation Study.

BACKGROUND AND PURPOSE: Severity-based assessment tools may assist in prehospital triage of patients to comprehensive stroke centers (CSCs) for endovascular thrombectomy (EVT), but criticisms regarding diagnostic inaccuracy have not been adequately addressed. This study aimed to quantify the benefits and disadvantages of severity-based triage in a large real-world paramedic validation of the Ambulance Clinical Triage for Acute Stroke Treatment (ACT-FAST) algorithm. METHODS: Ambulance Victoria paramedics assessed the prehospital ACT-FAST algorithm in patients with suspected stroke from November 2017 to July 2019 following an 8-minute training video. All patients were transported to the nearest stroke center as per current guidelines. ACT-FAST diagnostic accuracy was compared with hospital imaging for the presence of large vessel occlusion (LVO) and need for CSC-level care (LVO, intracranial hemorrhage, and tumor). Patient-level time saving to EVT was modeled using a validated Google Maps algorithm. Disadvantages of CSC bypass examined potential thrombolysis delays in non-LVO infarcts, proportion of patients with false-negative EVT, and CSC overburdening. RESULTS: Of 517 prehospital assessments, 168/517 (32.5%) were ACT-FAST positive and 132/517 (25.5%) had LVO. ACT-FAST sensitivity and specificity for LVO was 75.8% and 81.8%, respectively. Positive predictive value was 58.8% for LVO and 80.0% when intracranial hemorrhage and tumor (CSC-level care) were included. Within the metropolitan region, 29/55 (52.7%) of ACT-FAST-positive patients requiring EVT underwent a secondary interhospital transfer. Prehospital bypass with avoidance of secondary transfers was modeled to save 52 minutes (95% CI, 40.0-61.5) to EVT commencement. ACT-FAST was false-positive in 8 patients receiving thrombolysis (8.1% of 99 non-LVO infarcts) and false-negative in 4 patients with EVT requiring secondary transfer (5.4% of 74 EVT cases). CSC bypass was estimated to over-triage 1.1 patients-per-CSC-per-week in our region. CONCLUSIONS: The overall benefits of an ACT-FAST algorithm bypass strategy in expediting EVT and avoiding secondary transfers are estimated to substantially outweigh the disadvantages of potentially delayed thrombolysis and over-triage, with only a small proportion of EVT patients missed.

Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity.

Glycosylation of small molecules is critical for numerous biological processes in plants, including hormone homeostasis, neutralization of xenobiotics, and synthesis and storage of specialized metabolites. Glycosylation of plant natural products is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits such as disease resistance and flavor and have numerous pharmaceutical applications. Most characterized plant natural product UGTs are glucosyltransferases, and little is known about enzymes that add other sugars. Here we report the discovery and characterization of AsAAT1 (UGT99D1), which is required for biosynthesis of the antifungal saponin avenacin A-1 in oat (Avena strigosa). This enzyme adds l-Ara to the triterpene scaffold at the C-3 position, a modification critical for disease resistance. The only previously reported plant natural product arabinosyltransferase is a flavonoid arabinosyltransferase from Arabidopsis (Arabidopsis thaliana). We show that AsAAT1 has high specificity for UDP-ß-l-arabinopyranose, identify two amino acids required for sugar donor specificity, and through targeted mutagenesis convert AsAAT1 into a glucosyltransferase. We further identify a second arabinosyltransferase potentially implicated in the biosynthesis of saponins that determine bitterness in soybean (Glycine max). Our investigations suggest independent evolution of UDP-Ara sugar donor specificity in arabinosyltransferases in monocots and eudicots.

Predicting Ambulance Patient Wait Times: A Multicenter Derivation and Validation Study.

STUDY OBJECTIVE: To derive and internally and externally validate machine-learning models to predict emergency ambulance patient door-to-off-stretcher wait times that are applicable to a wide variety of emergency departments. METHODS: Nine emergency departments provided 3 years (2017 to 2019) of retrospective administrative data from Australia. Descriptive and exploratory analyses were undertaken on the datasets. Statistical and machine-learning models were developed to predict wait times at each site and were internally and externally validated. RESULTS: There were 421,894 episodes analyzed, and median site off-load times varied from 13 (interquartile range [IQR], 9 to 20) to 29 (IQR, 16 to 48) minutes. The global site prediction model median absolute errors were 11.7 minutes (95% confidence interval [CI], 11.7 to 11.8) using linear regression and 12.8 minutes (95% CI, 12.7 to 12.9) using elastic net. The individual site model prediction median absolute errors varied from the most accurate at 6.3 minutes (95% CI, 6.2 to 6.4) to the least accurate at 16.1 minutes (95% CI, 15.8 to 16.3). The model technique performance was the same for linear regression, random forests, elastic net, and rolling average. The important variables were the last k-patient average waits, triage category, and patient age. The global model performed at the lower end of the accuracy range compared with models for the individual sites but was within tolerable limits. CONCLUSION: Electronic emergency demographic and flow information can be used to estimate emergency ambulance patient off-stretcher times. Models can be built with reasonable accuracy for multiple hospitals using a small number of point-of-care variables.

The Impact of the COVID-19 Pandemic on Demand for Emergency Ambulances in Victoria, Australia.

Objective: Relatively little has been reported about the impact of COVID-19 restrictions on emergency ambulance services. We describe the influence of the COVID-19 pandemic on the emergency ambulance system in Victoria, Australia.Methods: We performed an interrupted time series analysis of consecutive calls for ambulance from January 2018 to February 2021, including two waves of COVID-19. The COVID-19 lockdown period included seven months of stay-at-home restrictions (16/03/2020-18/10/2020). Nineteen weeks of post-lockdown data were included (19/10/2020-28/02/2021).Results: In total, 2,356,326 consecutive calls were included. COVID-19 lockdown was associated with an absolute reduction of 64,991 calls (almost 2,100 calls/week). According to time series analysis, lockdown was associated with a 12.6% reduction in weekly calls (IRR = 0.874 [95% CI 0.811, 0.941]), however no change in long-term trend (IRR = 1.000 [95% CI 0.996, 1.003]). During lockdown, the long-term trend of attendances to patients with suspected acute coronary syndromes (ACS, IRR = 1.006 [95% CI 1.004, 1.009]) and mental health-related issues (IRR = 1.005 [95% CI 1.002, 1.008]) increased. After lockdown, the call volume was 5.6% below pre-COVID-19 predictions (IRR = 0.944 [95% CI 0.909, 0.980]), however attendances for suspected ACS were higher than predicted (IRR = 1.069 [95% CI 1.009, 1.132]). Ambulance response times deteriorated, and total case times were longer than prior to the pandemic, driven predominantly by extended hospital transfer times.Conclusion: The COVID-19 pandemic had a dramatic impact on the emergency ambulance system. Despite lower call volumes post-lockdown than predicted, we observed deteriorating ambulance response times, extended case times and hospital delays. The pattern of attendance to patients with suspected ACS potentially highlights the collateral burden of delaying treatment for urgent conditions.

Melbourne Mobile Stroke Unit and Reperfusion Therapy: Greater Clinical Impact of Thrombectomy Than Thrombolysis.

Background and Purpose- Mobile stroke units (MSUs) are increasingly used worldwide to provide prehospital triage and treatment. The benefits of MSUs in giving earlier thrombolysis have been well established, but the impacts of MSUs on endovascular thrombectomy (EVT) and effect on disability avoidance are largely unknown. We aimed to determine the clinical impact and disability reduction for reperfusion therapies in the first operational year of the Melbourne MSU. Methods- Treatment time metrics for MSU patients receiving reperfusion therapy were compared with control patients presenting to metropolitan Melbourne stroke units via standard ambulance within MSU operating hours. The primary outcome was median time difference in first ambulance dispatch to treatment modeled using quantile regression analysis. Time savings were subsequently converted to disability-adjusted life years avoided using published estimates. Results- In the first 365-day operation of the Melbourne MSU, prehospital thrombolysis was administered to 100 patients (mean age, 73.8 years; 62% men). The median time savings per MSU patient, compared with the control cohort, was 26 minutes (P<0.001) for dispatch to hospital arrival and 15 minutes (P<0.001) for hospital arrival to thrombolysis. The calculated overall time saving from dispatch to thrombolysis was 42.5 minutes (95% CI, 36.0-49.0). In the same period, 41 MSU patients received EVT (mean age, 76 years; 61% men) with median dispatch-to-treatment time saving of 51 minutes ([95% CI, 30.1-71.9], P<0.001). This included a median time saving of 17 minutes ([95% CI, 7.6-26.4], P=0.001) for EVT hospital arrival to arterial puncture for MSU patients. Estimated median disability-adjusted life years saved through earlier provision of reperfusion therapies were 20.9 for thrombolysis and 24.6 for EVT. Conclusions- The Melbourne MSU substantially reduced time to reperfusion therapies, with the greatest estimated disability avoidance driven by the more powerful impact of earlier EVT. These findings highlight the benefits of prehospital notification and direct triage to EVT centers with facilitated workflow on arrival by the MSU.

Drivers of metabolic diversification: how dynamic genomic neighbourhoods generate new biosynthetic pathways in the Brassicaceae.

Plants produce an array of specialized metabolites with important ecological functions. The mechanisms underpinning the evolution of new biosynthetic pathways are not well-understood. Here, we exploit available genome sequence resources to investigate triterpene biosynthesis across the Brassicaceae. Oxidosqualene cyclases (OSCs) catalyze the first committed step in triterpene biosynthesis. Systematic analysis of 13 sequenced Brassicaceae genomes was performed to identify all OSC genes. The genome neighbourhoods (GNs) around a total of 163 OSC genes were investigated to identify Pfam domains significantly enriched in these regions. All-vs-all comparisons of OSC neighbourhoods and phylogenomic analysis were used to investigate the sequence similarity and evolutionary relationships of the numerous candidate triterpene biosynthetic gene clusters (BGCs) observed. Functional analysis of three representative BGCs was carried out and their triterpene pathway products were elucidated. Our results indicate that plant genomes are remarkably plastic, and that dynamic GNs generate new biosynthetic pathways in different Brassicaceae lineages by shuffling the genes encoding a core palette of triterpene-diversifying enzymes, presumably in response to strong environmental selection pressure. These results illuminate a genomic basis for diversification of plant-specialized metabolism through natural combinatorics of enzyme families, which can be mimicked using synthetic biology to engineer diverse bioactive molecules.

The protosteryl and dammarenyl cation dichotomy in polycyclic triterpene biosynthesis revisited: has this 'rule' finally been broken?

Covering: 1948 up to the end of 2018. The triterpene alcohols represent an important and diverse class of natural products. This diversity is believed to originate from the differential enzymatically controlled cyclisation of 2,3-oxidosqualene. It is now a well-established presumption that all naturally occurring tetra- and penta-cyclic triterpene alcohols can be rationalised by the resolution of one of two intermediary tetracyclic cations, termed the protosteryl and dammarenyl cations. Here, a discussion of typical key triterpene structures and their proposed derivation from either of these progenitors is followed by comparison with a recently reported novel pentacyclic triterpene orysatinol which appears to correspond to an unprecedented divergence from this dichotomous protosteryl/dammarenyl view of triterpene biogenesis. Not only does this discovery widen the potential scope of triterpene scaffolds that could exist in nature, it could call into question the reliability of stereochemical assignments of some existing triterpene structures that are supported by only limited spectroscopic evidence. The discovery of orysatinol provides direct experimental evidence to support considering more flexibility in the stereochemical interpretation of the biogenic isoprene rule.

Towards take-all control: a C-21ß oxidase required for acylation of triterpene defence compounds in oat.

Oats produce avenacins, antifungal triterpenes that are synthesized in the roots and provide protection against take-all and other soilborne diseases. Avenacins are acylated at the carbon-21 position of the triterpene scaffold, a modification critical for antifungal activity. We have previously characterized several steps in the avenacin pathway, including those required for acylation. However, transfer of the acyl group to the scaffold requires the C-21ß position to be oxidized first, by an as yet uncharacterized enzyme. We mined oat transcriptome data to identify candidate cytochrome P450 enzymes that may catalyse C-21ß oxidation. Candidates were screened for activity by transient expression in Nicotiana benthamiana. We identified a cytochrome P450 enzyme AsCYP72A475 as a triterpene C-21ß hydroxylase, and showed that expression of this enzyme together with early pathway steps yields C-21ß oxidized avenacin intermediates. We further demonstrate that AsCYP72A475 is synonymous with Sad6, a previously uncharacterized locus required for avenacin biosynthesis. sad6 mutants are compromised in avenacin acylation and have enhanced disease susceptibility. The discovery of AsCYP72A475 represents an important advance in the understanding of triterpene biosynthesis and paves the way for engineering the avenacin pathway into wheat and other cereals for control of take-all and other diseases.

Comparison of Magnetic Resonance Analysis of Myocardial Scarring With Biomarker Release Following S-T Elevation Myocardial Infarction.

BACKGROUND: Late gadolinium enhancement (LGE) with cardiac magnetic resonance (CMR) is commonly assumed to represent myocardial fibrosis; however, comparative human histological data are limited, and there is no consensus on the most accurate method for LGE quantitation. We evaluated the relationship between CMR assessment of regional fibrosis and infarct size assessment using serial biomarkers after ST elevation acute myocardial infarction (STEMI). METHODS: Ninety-three patients treated for STEMI (59±10 years, 86% male) underwent CMR 6 months after infarction. Infarct size was quantified by CMR-LGE using manual and range of semi-automated thresholds (range: 2-10 standard deviations [SD]) above reference myocardium and the full width-half maximum (FWHM) technique, and compared with the rise in serum biomarkers. The agreement between CMR and biomarker in the identification of large infarcts based on peak troponin (TnI) levels was also analysed. RESULTS: Quantification methods had a strong influence on the infarct size assessment with CMR-LGE. Significant correlations were observed between LGE and biomarkers across all of the signal intensity thresholds. Whilst there was a wide variation with respect to the estimation of total LGE size (from 6.8±7.7 to 32.1±11.3 grams), the variation in the correlation with peak troponin level was much smaller (r-values ranging from 0.670 to 0.876). There was good agreement between CMR-LGE and biomarker assessment of infarct size; the best agreement between CMR-LGE and large infarction using a threshold of 8SD for peak TnI>50ng/mL (Cohen's kappa (κ)=0.722), and a threshold of 4SD for peak TnI >95ng/mL (κ=0.761). CONCLUSIONS: The correlation between CMR-LGE quantification of infarct size and biomarker release following STEMI at a range of semi-automated thresholds was consistently strong, with good agreement between measures across a range of thresholds.