Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity.

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria.

Mutations in FBXO7 have been discovered to be associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerisation domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts to facilitate SCFFbxo7-mediated ubiquitination of MiD49. The L250P mutation reduces the SCFFbxo7 ligase-mediated ubiquitination of a subset of its known substrates. Although MiD49/51 expression was reduced in patient cells, there was no effect on the mitochondrial network. However, patient cells show reduced levels of mitochondrial function and mitophagy, higher levels of ROS and are less viable under stress. Our study demonstrates that Fbxo7 and PI31 regulate proteasomes and mitochondria and reveals a new function for PI31 in enhancing the SCFFbxo7 E3 ubiquitin ligase activity.

The effect of power stretchers on occupational injury rates in an urban emergency medical services system.

BACKGROUND: To examine occupational injury rates in a dual-response emergency medical services (EMS) system before and after implementation of a power-lift stretcher system. METHODS: The seasonally-adjusted occupational injury rate was estimated relative to medical call volume (per 1000 calls) and workers (per 100 FTEs) from 2009 to 2019, and stratified by severity (lost-time, healthcare only), role (EMS, FIRE) and type (patient-handling). Power-lift stretchers were adopted between 2013 and 2015. Preinjury versus postinjury rates were compared using binomial tests. Interrupted time series (ITS) analysis was used to estimate the trend and change in injuries related to patient-handling, with occupational illnesses serving as control. RESULTS: Binomial tests revealed varied results, with reductions in the injury rate per 1000 calls (-14.0%) and increases in the rate per 100 FTEs (+14.1%); rates also differed by EMS role and injury severity. ITS analysis demonstrated substantial reductions in patient-handling injuries following implementation of power-lift stretchers, both in the injury rate per 1000 calls (-50.4%) and per 100 FTEs (-46.6%), specifically among individuals deployed on the ambulance. Injury rates were slightly elevated during the winter months (+0.8 per 100 FTEs) and lower during spring (-0.5 per 100 FTEs). CONCLUSIONS: These results support the implementation of power-lift stretchers for injury prevention in EMS systems and demonstrate advantages of ITS analysis when data span long preintervention and postintervention periods.

Into the fold: advances in understanding aPKC membrane dynamics.

Atypical protein kinase Cs (aPKCs) are part of the PKC family of protein kinases and are atypical because they don't respond to the canonical PKC activators diacylglycerol (DAG) and Ca2+. They are central to the organization of polarized cells and are deregulated in several cancers. aPKC recruitment to the plasma membrane compartment is crucial to their encounter with substrates associated with polarizing functions. However, in contrast with other PKCs, the mechanism by which atypical PKCs are recruited there has remained elusive until recently. Here, we bring aPKC into the fold, summarizing recent reports on the direct recruitment of aPKC to membranes, providing insight into seemingly discrepant findings and integrating them with existing literature.

Database quality assessment in research in paramedicine: a scoping review.

BACKGROUND: Research in paramedicine faces challenges in developing research capacity, including access to high-quality data. A variety of unique factors in the paramedic work environment influence data quality. In other fields of healthcare, data quality assessment (DQA) frameworks provide common methods of quality assessment as well as standards of transparent reporting. No similar DQA frameworks exist for paramedicine, and practices related to DQA are sporadically reported. This scoping review aims to describe the range, extent, and nature of DQA practices within research in paramedicine. METHODS: This review followed a registered and published protocol. In consultation with a professional librarian, a search strategy was developed and applied to MEDLINE (National Library of Medicine), EMBASE (Elsevier), Scopus (Elsevier), and CINAHL (EBSCO) to identify studies published from 2011 through 2021 that assess paramedic data quality as a stated goal. Studies that reported quantitative results of DQA using data that relate primarily to the paramedic practice environment were included. Protocols, commentaries, and similar study types were excluded. Title/abstract screening was conducted by two reviewers; full-text screening was conducted by two, with a third participating to resolve disagreements. Data were extracted using a piloted data-charting form. RESULTS: Searching yielded 10,105 unique articles. After title and abstract screening, 199 remained for full-text review; 97 were included in the analysis. Included studies varied widely in many characteristics. Majorities were conducted in the United States (51%), assessed data containing between 100 and 9,999 records (61%), or assessed one of three topic areas: data, trauma, or out-of-hospital cardiac arrest (61%). All data-quality domains assessed could be grouped under 5 summary domains: completeness, linkage, accuracy, reliability, and representativeness. CONCLUSIONS: There are few common standards in terms of variables, domains, methods, or quality thresholds for DQA in paramedic research. Terminology used to describe quality domains varied among included studies and frequently overlapped. The included studies showed no evidence of assessing some domains and emerging topics seen in other areas of healthcare. Research in paramedicine would benefit from a standardized framework for DQA that allows for local variation while establishing common methods, terminology, and reporting standards.

Control of atypical PKCι membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface.

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently, it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudosubstrate region and the C1 domain as direct membrane interaction modules; however, their relative importance and coupling are unknown. We combined molecular modeling and functional assays to show that the regulatory module of aPKCι, comprising the PB1 pseudosubstrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial ß-strand (beta-strand linker). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a hitherto unknown regulatory mechanism of aPKCι membrane binding and release during cell polarization.

Patterns of change in prehospital spinal motion restriction: A retrospective database review.

BACKGROUND: Acute management of trauma patients with potential spine injuries has evolved from uniform spinal immobilization (SI) to spinal motion restriction (SMR). Little research exists describing how these changes have been implemented. This study aims to describe and analyze the practice of SMR in one emergency medical services (EMS) agency over the time frame of SMR adoption. METHODS: This was a retrospective database review of electronic patient care reports from 2009 to 2020. The effects of key practice changes (revised documentation and a collar-only treatment option) were analyzed in an interrupted time series using the rate of SI/SMR as the primary outcome. Secondary outcomes included patient age, sex, acuity, mechanism of injury, treatment provided, cervical collar size, and positioning. These were assessed for changes from year to year by Poisson regression. Associations between patient and treatment characteristics were investigated with binomial logistic regression. RESULTS: There were 25,747 instances of SI/SMR included. Among all patients, the median age was 40 (interquartile range 24-56), 58% (14,970) were male, and 20% (5062) were high-acuity. The rate of SI/SMR declined from 31.2 to 12.7 treatments per 100 trauma calls per month. The proportion of high-acuity patients increased by 9.6% per year on average (95% CI 8.7%-10.0%). When first available, collar-only treatment was provided to 47% of patients, rising by 6.3% per year (95% CI 3.2%-9.5%) to 60% in 2020. Collar-only treatment (compared to board-and-collar) was more likely to be applied to low-acuity patients (as compared to high): odds ratio 3.01 (95% CI 2.64-3.43). CONCLUSIONS: This study shows decreasing SI/SMR treatment and changing patient and practice characteristics. These patterns of care cannot be attributed solely to formal protocol changes. Similar patterns and their possible explanations should be investigated elsewhere.

Characterising methamphetamine use to inform health and social policies in Manitoba, Canada: a protocol for a retrospective cohort study using linked administrative data.

INTRODUCTION: Rising use of methamphetamine is causing significant public health concern in Canada. The biological and behavioural effects of methamphetamine range from wakefulness, vigour and euphoria to adverse physical health outcomes like myocardial infarction, haemorrhagic stroke, arrhythmia and seizure. It can also cause severe psychological complications such as psychosis. National survey data point to increasing rates of methamphetamine use, as well as increasing ease of access and serious methamphetamine-related harms. There is an urgent need for evidence to address knowledge gaps, provide direction to harm reduction and treatment efforts and inform health and social policies for people using methamphetamine. This protocol describes a study that aims to address this need for evidence. METHODS: The study will use linked, whole population, de-identified administrative data from the Manitoba Population Research Data Repository. The cohort will include individuals in the city of Winnipeg, Manitoba, who came into contact with the health system for reasons related to methamphetamine use from 2013 to 2021 and a comparison group matched on age, sex and geography. We will describe the cohort's sociodemographic characteristics, calculate incidence and prevalence of mental disorders associated with methamphetamine use and examine rates of health and social service use. We will evaluate the use of olanzapine pharmacotherapy in reducing adverse emergency department outcomes. In partnership with Indigenous co-investigators, outcomes will be stratified by First Nations and Métis identity. ETHICS AND DISSEMINATION: The study was approved by the University of Manitoba Health Research Ethics Board, and access datasets have been granted by all data providers. We also received approval from the First Nations Health and Social Secretariat of Manitoba's Health Information Research Governance Committee and the Manitoba Métis Federation. Dissemination will be guided by an 'Evidence 2 Action' group of public rightsholders, service providers and knowledge users who will ensure that the analyses address the critical issues.

Oligonucleotide-Recognizing Topoisomerase Inhibitors (OTIs): Precision Gene Editors for Neurodegenerative Diseases?

Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson-Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.

Paramedic attitudes towards prehospital spinal care: a cross-sectional survey.

BACKGROUND: The optimal application of spinal motion restriction (SMR) in the prehospital setting continues to be debated. Few studies have examined how changing guidelines have been received and interpreted by emergency medical services (EMS) personnel. This study surveys paramedics' attitudes, observations, and self-reported practices around the treatment of potential spine injuries in the prehospital setting. METHODS: This was a cross-sectional survey of a North American EMS agency. After development and piloting, the final version of the survey contained four sections covering attitudes towards 1) general practice, 2) specific techniques, 3) assessment protocols, and 4) mechanisms of injury (MOI). Questions used Likert-scale, multiple-choice, yes/no, and free-text responses. Exploratory factor analysis (EFA) was used to identify latent constructs within responses, and factor scores were analyzed by ordinal logistic regression for associations with demographic characteristics (including qualification level, gender, and years of experience). MOI evaluations were assessed for inter-rater reliability (Fleiss' kappa). Inductive qualitative content analysis, following Elo & Kyngäs (2008), was used to examine free-text responses. RESULTS: Two hundred twenty responses were received (36% of staff). Raw results indicated that respondents felt that SMR was seen as less important than in the past, that they were treating fewer patients than previously, and that they follow protocol in most situations. The EFA identified two factors: one (Judging MOIs) captured paramedics' estimation that the presented MOI could potentially cause a spine injury, and another (Treatment Value) reflected respondents' composite view of the effectiveness, importance, and applicability of SMR. Respondents with advanced life support (ALS) qualification were more likely to be skeptical of the value of SMR compared to those at the basic life support (BLS) level (OR: 2.40, 95%CI: 1.21-4.76, p = 0.01). Overall, respondents showed fair agreement in the evaluation of MOIs (k = 0.31, 95%CI: 0.09-0.49). Content analysis identified tension expressed by respondents between SMR-as-directed and SMR-as-applied. CONCLUSION: Results of this survey show that EMS personnel are skeptical of many elements of SMR but use various strategies to balance protocol adherence with optimizing patient care. While identifying several areas for future research, these findings argue for incorporating provider feedback and judgement into future guideline revision.

Bimodal regulation of axonal transport by the GDNF-RET signalling axis in healthy and diseased motor neurons.

Deficits in axonal transport are one of the earliest pathological outcomes in several models of amyotrophic lateral sclerosis (ALS), including SOD1G93A mice. Evidence suggests that rescuing these deficits prevents disease progression, stops denervation, and extends survival. Kinase inhibitors have been previously identified as transport enhancers, and are being investigated as potential therapies for ALS. For example, inhibitors of p38 mitogen-activated protein kinase and insulin growth factor receptor 1 have been shown to rescue axonal transport deficits in vivo in symptomatic SOD1G93A mice. In this work, we investigated the impact of RET, the tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), as a modifier of axonal transport. We identified the fundamental interplay between RET signalling and axonal transport in both wild-type and SOD1G93A motor neurons in vitro. We demonstrated that blockade of RET signalling using pharmacological inhibitors and genetic knockdown enhances signalling endosome transport in wild-type motor neurons and uncovered a divergence in the response of primary motor neurons to GDNF compared with cell lines. Finally, we showed that inhibition of the GDNF-RET signalling axis rescues in vivo transport deficits in early symptomatic SOD1G93A mice, promoting RET as a potential therapeutic target in the treatment of ALS.

Database quality assessment in research in paramedicine: a scoping review protocol.

INTRODUCTION: The paramedic practice environment presents unique challenges to data documentation and access, as well as linkage to other parts of the healthcare system. Variable or unknown data quality can influence the validity of research in paramedicine. A number of database quality assessment (DQA) frameworks have been developed and used to evaluate data quality in other areas of healthcare. The extent these or other DQA practices have been applied to paramedic research is not known. Accordingly, this scoping review aims to describe the range, extent and nature of DQA practices within research in paramedicine. METHODS AND ANALYSIS: This scoping review will follow established methods for the conduct (Johanna Briggs Institute; Arksey and O'Malley) and reporting (Preferred Reporting Items in Systematic Reviews and Meta-Analyses extension for scoping reviews) of scoping reviews. In consultation with a professional librarian, a search strategy was developed representing the applicable population, concept and context. This strategy will be applied to MEDLINE (National Library of Medicine), Embase (Elsevier), Scopus (Elsevier) and CINAHL (EBSCO) to identify studies published from 2011 through 2021 that assess paramedic data quality as a stated goal. Studies will be included if they report quantitative results of DQA using data that relate primarily to the paramedic practice environment. Protocols, commentaries, case studies, interviews, simulations and experimental data-processing techniques will be excluded. No restrictions will be placed on language. Study selection will be performed by two reviewers, with a third available to resolve conflicts. Data will be extracted from included studies using a data-charting form piloted and iteratively revised based on studies known to be relevant. Results will be summarised in a chart of study characteristics, DQA-specific outcomes and key findings. ETHICS AND DISSEMINATION: Ethical approval is not required. Results will be submitted to relevant conferences and peer-reviewed journals. TRIAL REGISTRATION: 10.17605/OSF.IO/Z287T.

Sex and Gender Equity in Prehospital Electrocardiogram Acquisition.

INTRODUCTION: Research in cardiac care has identified significant gender-based differences across many outcomes. Women with heart disease are less likely both to be diagnosed and to receive standard care. Gender-based disparities in the prehospital setting are under-researched, but they were found to exist within rates of 12-lead electrocardiogram (ECG) acquisition within one urban Emergency Medical Services (EMS) agency. STUDY OBJECTIVE: This study evaluates the quality improvement (QI) initiative that was implemented in that agency to raise overall rates of 12-lead ECG acquisition and reduce the gap in acquisition rates between men and women. METHODS: This QI project included two interventions: revised indications for 12-lead acquisition, and training that highlighted sex- and gender-based differences relevant to patient care. To evaluate this project, a retrospective database review identified all patient contacts that potentially involved cardiac assessment over 18 months. The primary outcome was the rate of 12-lead acquisition among patients with qualifying complaints. This was assessed by mean rates of acquisition in before and after periods, as well as segmented regression in an interrupted time series. Secondary outcomes included differences in rates of 12-lead acquisition, both overall and in individual complaint categories, each compared between men/women and before/after the interventions. RESULTS: Among patients with qualifying complaints, the mean rate of 12-lead acquisition in the lead-in period was 22.5% (95% CI, 21.8% - 23.2%) with no discernible trend. The protocol change and training were each associated with a significant absolute level increase in the acquisition rate: 2.09% (95% CI, 0.21% - 4.0%; P = .03) and 3.2% (95% CI, 1.18% - 5.22%; P = .003), respectively. When compared by gender and time period, women received fewer 12-leads than men overall, and more 12-leads were acquired after the interventions than before. There were also significant interactions between gender and period, both overall (2.8%; 95% CI, 1.9% - 3.6%; P < .0001) and in all complaint categories except falls and heart problems. CONCLUSION: This QI project resulted in an increase in 12-leads acquired. Pre-existing gaps in rates of acquisition between men and women were reduced but did not disappear. On-going research is examining the reasons behind these differences from the perspective of prehospital providers.

Discovery of N-Trisubstituted Pyrimidine Derivatives as Type I RET and RET Gatekeeper Mutant Inhibitors with a Novel Kinase Binding Pose.

Mutations of the rearranged during transfection (RET) kinase are frequently reported in cancer, which make it as an attractive therapeutic target. Herein, we discovered a series of N-trisubstituted pyrimidine derivatives as potent inhibitors for both wild-type (wt) RET and RETV804M, which is a resistant mutant for several FDA-approved inhibitors. The X-ray structure of a representative inhibitor with RET revealed that the compound binds in a unique pose that bifurcates beneath the P-loop and confirmed the compound as a type I inhibitor. Through the structure-activity relationship (SAR) study, compound 20 was identified as a lead compound, showing potent inhibition of both RET and RETV804M. Additionally, compound 20 displayed potent antiproliferative activity of CCDC6-RET-driven LC-2/ad cells. Analysis of RET phosphorylation indicated that biological activity was mediated by RET inhibition. Collectively, N-trisubstituted pyrimidine derivatives could serve as scaffolds for the discovery and development of potent inhibitors of type I RET and its gatekeeper mutant for the treatment of RET-driven cancers.

Feasibility of Direct Current stimulation through hair using a dry electrode: potential for transcranial Direct Current Stimulation (tDCS) application.

Conventional transcranial direct current stimulation (tDCS) protocols typically deliver 2 mA for 20-30 minutes. The most common administration uses a wet electrode approach which dries out in ~60 minutes at room temperature. This restricts its application to limited duration electrode-scalp contact use cases unless additional conductive media (saline, gel, or paste) is re-applied. This problem is further compounded by the subject's hair which not only presents administration challenges (interferes with electrode attachment and adhesion) but also acts as a conduit of current flow into the scalp resulting in current hotspots. This non-uniform current injection results in increased skin sensation. The aim of this study was to determine suitability of a commercially available hydrogel for DC delivery through hair. Experiments involved both non-clinical testing on an agar block and clinical testing on subjects' forearms. Electrodes were positioned on the posterior side of the forearm that has hair for the clinical testing. Typical dose as used in tDCS was delivered and pain scores were collected. Results indicate suitable current delivery performance and all subjects tolerated delivery with pain scores ranging between 0-6. Our study paves the way for future testing on the scalp for tDCS application.Clinical Relevance-This study demonstrates the possibility of delivering tDCS through hair via dry electrodes. Specific use cases that cannot use a traditional wet electrode approach stand to benefit from the results of our work.

A genetically-encoded crosslinker screen identifies SERBP1 as a PKCε substrate influencing translation and cell division.

The PKCε-regulated genome protective pathway provides transformed cells a failsafe to successfully complete mitosis. Despite the necessary role for Aurora B in this programme, it is unclear whether its requirement is sufficient or if other PKCε cell cycle targets are involved. To address this, we developed a trapping strategy using UV-photocrosslinkable amino acids encoded in the PKCε kinase domain. The validation of the mRNA binding protein SERBP1 as a PKCε substrate revealed a series of mitotic events controlled by the catalytic form of PKCε. PKCε represses protein translation, altering SERBP1 binding to the 40 S ribosomal subunit and promoting the assembly of ribonucleoprotein granules containing SERBP1, termed M-bodies. Independent of Aurora B, SERBP1 is shown to be necessary for chromosome segregation and successful cell division, correlating with M-body formation. This requirement for SERBP1 demonstrates that Aurora B acts in concert with translational regulation in the PKCε-controlled pathway exerting genome protection.

Emergency care with lay responders in underserved populations: a systematic review.

OBJECTIVE: To assess the individual and community health effects of task shifting for emergency care in low-resource settings and underserved populations worldwide. METHODS: We systematically searched 13 databases and additional grey literature for studies published between 1984 and 2019. Eligible studies involved emergency care training for laypeople in underserved or low-resource populations, and any quantitative assessment of effects on the health of individuals or communities. We conducted duplicate assessments of study eligibility, data abstraction and quality. We synthesized findings in narrative and tabular format. FINDINGS: Of 19 308 papers retrieved, 34 studies met the inclusion criteria from low- and middle-income countries (21 studies) and underserved populations in high-income countries (13 studies). Targeted emergency conditions included trauma, burns, cardiac arrest, opioid poisoning, malaria, paediatric communicable diseases and malnutrition. Trainees included the general public, non-health-care professionals, volunteers and close contacts of at-risk populations, all trained through in-class, peer and multimodal education and public awareness campaigns. Important clinical and policy outcomes included improvements in community capacity to manage emergencies (14 studies), patient outcomes (13 studies) and community health (seven studies). While substantial effects were observed for programmes to address paediatric malaria, trauma and opioid poisoning, most studies reported modest effect sizes and two reported null results. Most studies were of weak (24 studies) or moderate quality (nine studies). CONCLUSION: First aid education and task shifting to laypeople for emergency care may reduce patient morbidity and mortality and build community capacity to manage health emergencies for a variety of emergency conditions in underserved and low-resource settings.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.

Activated neutrophil fluorescent imaging technique for human lungs.

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Identification of neutrophil activation in real time in the lungs of patients may permit biological stratification of patients in otherwise heterogenous cohorts typically defined by clinical criteria. No methods for identifying neutrophil activation in real time in the lungs of patients currently exist. We developed a bespoke molecular imaging probe targeting three characteristic signatures of neutrophil activation: pinocytosis, phagosomal alkalinisation, and human neutrophil elastase (HNE) activity. The probe functioned as designed in vitro and ex vivo. We evaluated optical endomicroscopy imaging of neutrophil activity using the probe in real-time at the bedside of healthy volunteers, patients with bronchiectasis, and critically unwell mechanically ventilated patients. We detected a range of imaging responses in vivo reflecting heterogeneity of condition and severity. We corroborated optical signal was due to probe function and neutrophil activation.