Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of MET intracellular kinase domain in two fusion protein backgrounds: wild type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase αC helix, pointing to differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a ß5 motif that acts as a structural pivot for kinase domain activation in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

The Burden of Radiation Exposure During Transcatheter Closure of Atrial Septal Defect.

Radiation reduction in the pediatric cardiac catheterization laboratory is well-suited for targeted quality improvement (QI) interventions. Transcatheter atrial septal defect (ASD) closure was chosen for this QI project based on a homogenous procedural population and inter-operator variability in radiation usage, with the aim to reduce radiation exposure during ASD device closure by 50% over 1 year. The aim for this project was defined and a Key Driver Diagram (KDD) was created with three domains for change: modification of procedural practice, reporting and monitoring/feedback, and team engagement. All patients undergoing attempted transcatheter ASD closure were considered for inclusion. The primary outcome, % reduction in median radiation dose (DAP/Kg), was determined through comparison with a historical cohort. Additional radiation metrics, procedural characteristics, and adverse events (AE) were compared to the historical cohort. Radiation exposure (DAP/kg) was reduced by 55% with a median dose reduction from 26 (15, 61) in a historical cohort to 12 (6, 22) in the intervention population (p <0.001). Fluoroscopy time and cine acquisition utilization significantly decreased. Procedure time, procedural success (defined as successful delivery of the device) and AE did not increase in the QI cohort. Successful practice changes included standardized procedural strategies to limit fluoroscopy and cine acquisition, improved fluoroscopic practice, engagement of the multidisciplinary team, and feedback with data reporting by electronic and in-person reminders. In conclusion, application of QI methodologies such as KDD with engagement of a multidisciplinary team can effectively reduce radiation in the pediatric catheterization laboratory.

Active site labeling of fatty acid and polyketide acyl-carrier protein transacylases.

Metabolic engineering of fatty acids and polyketides remains challenging due to unresolved protein-protein interactions that are essential to synthase activity. While several chemical probes have been developed to capture and visualize protein interfaces in these systems, acyl carrier protein (ACP) transacylase (AT) domains remain elusive. Herein, we combine a mutational strategy with fluorescent probe design to expedite the study of AT domains from fatty acid and polyketide synthases. We describe the design and evaluation of inhibitor-inspired and substrate-mimetic reporters containing sulfonyl fluoride and ß-lactone warheads. Moreover, specific active-site labeling occurs by optimizing pH, time, and probe concentration, and selective labeling is achieved in the presence of inhibitors of competing domains. These findings provide a panel of AT-targeting probes and set the stage for future combinatorial biosynthetic and drug discovery initiatives.

Taxon-specific aerosolization of bacteria and viruses in an experimental ocean-atmosphere mesocosm.

Ocean-derived, airborne microbes play important roles in Earth's climate system and human health, yet little is known about factors controlling their transfer from the ocean to the atmosphere. Here, we study microbiomes of isolated sea spray aerosol (SSA) collected in a unique ocean-atmosphere facility and demonstrate taxon-specific aerosolization of bacteria and viruses. These trends are conserved within taxonomic orders and classes, and temporal variation in aerosolization is similarly shared by related taxa. We observe enhanced transfer into SSA of Actinobacteria, certain Gammaproteobacteria, and lipid-enveloped viruses; conversely, Flavobacteriia, some Alphaproteobacteria, and Caudovirales are generally under-represented in SSA. Viruses do not transfer to SSA as efficiently as bacteria. The enrichment of mycolic acid-coated Corynebacteriales and lipid-enveloped viruses (inferred from genomic comparisons) suggests that hydrophobic properties increase transport to the sea surface and SSA. Our results identify taxa relevant to atmospheric processes and a framework to further elucidate aerosolization mechanisms influencing microbial and viral transport pathways.

Sea Spray Aerosol Structure and Composition Using Cryogenic Transmission Electron Microscopy.

The composition and surface properties of atmospheric aerosol particles largely control their impact on climate by affecting their ability to uptake water, react heterogeneously, and nucleate ice in clouds. However, in the vacuum of a conventional electron microscope, the native surface and internal structure often undergo physicochemical rearrangement resulting in surfaces that are quite different from their atmospheric configurations. Herein, we report the development of cryogenic transmission electron microscopy where laboratory generated sea spray aerosol particles are flash frozen in their native state with iterative and controlled thermal and/or pressure exposures and then probed by electron microscopy. This unique approach allows for the detection of not only mixed salts, but also soft materials including whole hydrated bacteria, diatoms, virus particles, marine vesicles, as well as gel networks within hydrated salt droplets-all of which will have distinct biological, chemical, and physical processes. We anticipate this method will open up a new avenue of analysis for aerosol particles, not only for ocean-derived aerosols, but for those produced from other sources where there is interest in the transfer of organic or biological species from the biosphere to the atmosphere.

Deficit-lesion correlations in syntactic comprehension in aphasia.

The effects of lesions on syntactic comprehension were studied in thirty-one people with aphasia (PWA). Participants were tested for the ability to parse and interpret four types of syntactic structures and elements - passives, object extracted relative clauses, reflexives and pronouns - in three tasks - object manipulation, sentence picture matching with full sentence presentation and sentence picture matching with self-paced listening presentation. Accuracy, end-of-sentence RT and self-paced listening times for each word were measured. MR scans were obtained and analyzed for total lesion volume and for lesion size in 48 cortical areas. Lesion size in several areas of the left hemisphere was related to accuracy in particular sentence types in particular tasks and to self-paced listening times for critical words in particular sentence types. The results support a model of brain organization that includes areas that are specialized for the combination of particular syntactic and interpretive operations and the use of the meanings produced by those operations to accomplish task-related operations.

Mechanisms underlying syntactic comprehension deficits in vascular aphasia: new evidence from self-paced listening.

Sixty-one people with aphasia (pwa) and 41 matched controls were tested for the ability to understand sentences that required the ability to process particular syntactic elements and assign particular syntactic structures. Participants paced themselves word-by-word through 20 examples of 11 spoken sentence types and indicated which of two pictures corresponded to the meaning of each sentence. Sentences were developed in pairs such that comprehension of the experimental version of a pair required an aspect of syntactic processing not required in the corresponding baseline sentence. The need for the syntactic operations required only in the experimental version was triggered at a "critical word" in the experimental sentence. Listening times for critical words in experimental sentences were compared to those for corresponding words in the corresponding baseline sentences. The results were consistent with several models of syntactic comprehension deficits in pwa: resource reduction, slowed lexical and/or syntactic processing, abnormal susceptibility to interference from thematic roles generated non-syntactically. They suggest that a previously unidentified disturbance limiting the duration of parsing and interpretation may lead to these deficits, and that this mechanism may lead to structure-specific deficits in pwa. The results thus point to more than one mechanism underlying syntactic comprehension disorders both across and within pwa.

Working memory and the revision of syntactic and discourse ambiguities.

Two hundred participants, 50 in each of 4 age ranges (19-29 years, 30-49 years, 50-69 years, 70-90 years) were tested for short-term working memory, speed of processing, and online processing of 3 types of sentences in which an initially assigned syntactic structure and/or semantic interpretation had to be revised. Self-paced reading times were longer for the segments that signaled the need for revision; there also were interactions of age and sentence type and speed of processing and sentence type, but not of working memory and sentence type on reading times for these segments. The results provide evidence that working memory does not support the processes that revise the structure and interpretation of sentences and discourse.

Dissociations and associations of performance in syntactic comprehension in aphasia and their implications for the nature of aphasic deficits.

Sixty-one pwa were tested on syntactic comprehension in three tasks: sentence-picture matching, sentence-picture matching with auditory moving window presentation, and object manipulation. There were significant correlations of performances on sentences across tasks. First factors on which all sentence types loaded in unrotated factor analyses accounted for most of the variance in each task. Dissociations in performance between sentence types that differed minimally in their syntactic structures were not consistent across tasks. These results replicate previous results with smaller samples and provide important validation of basic aspects of aphasic performance in this area of language processing. They point to the role of a reduction in processing resources and of the interaction of task demands and parsing and interpretive abilities in the genesis of patient performance.

Short-term memory, working memory, and syntactic comprehension in aphasia.

Sixty-one people with aphasia were tested on 10 tests of short-term memory (STM) and for the ability to use syntactic structure to determine the meanings of 11 types of sentences in three tasks-object manipulation, picture matching, and picture matching with self-paced listening. Multilevel models showed relationships between measures of the ability to retain and manipulate item and order information in STM and accuracy and reaction time (RT), and a greater relationship between these STM measures and accuracy and RT for several more complex sentence types in individual tasks. There were no effects of measures of STM that reflect the use of phonological codes or rehearsal on comprehension. There was only one effect of STM measures on self-paced listening times. There were double dissociations between performance on STM and individual comprehension tasks, indicating that normal STM is not necessary to perform normally on these tasks. The results are most easily related to the view that STM plays a facilitatory role in supporting the use of the products of the comprehension process to accomplish operations related to tasks.

Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel.

Microalgae are a promising feedstock for biodiesel and other liquid fuels due to their fast growth rate, high lipid yields, and ability to grow in a broad range of environments. However, many microalgae achieve maximal lipid yields only under stress conditions hindering growth and providing compositions not ideal for biofuel applications. Metabolic engineering of algal fatty acid biosynthesis promises to create strains capable of economically producing fungible and sustainable biofuels. The algal fatty acid biosynthetic pathway has been deduced by homology to bacterial and plant systems, and much of our understanding is gleaned from basic studies in these systems. However, successful engineering of lipid metabolism in algae will necessitate a thorough characterization of the algal fatty acid synthase (FAS) including protein-protein interactions and regulation. This review describes recent efforts to engineer fatty acid biosynthesis toward optimizing microalgae as a biodiesel feedstock.

Effects of age, speed of processing, and working memory on comprehension of sentences with relative clauses.

Two hundred participants, 50 in each of four age ranges (19-29, 30-49, 50-69, 70-90) were tested for working memory, speed of processing, and the processing of sentences with relative clauses. In Experiment 1, participants read four sentence types (cleft subject, cleft object, subject-subject, subject-object) in a word-by-word, non-cumulative, self-paced reading task and made speeded plausibility judgments about them. In Experiment 2, participants read two types of sentences, one of which contained a doubly center embedded relative clause. Older participants' comprehension was less accurate and there was age-related slowing of online processing times in all but the simplest sentences, which increased in syntactically complex sentences in Experiment 1. This pattern suggests an age-related decrease in the efficiency of parsing and interpretation. Slower speed of processing and lower working memory were associated with longer online processing times only in Experiment 2, suggesting that task-related operations are related to general speed of processing and working memory. Lower working memory was not associated with longer reading times in more complex sentences, consistent with the view that general working memory is not critically involved in online syntactic processing. Longer online processing at the most demanding point in the most demanding sentence was associated with better comprehension, indicating that it reflects effective processing under some certain circumstances. However, the poorer comprehension performance of older individuals indicates that their slower online processing reflects inefficient processing even at these points.

Rasch models of aphasic performance on syntactic comprehension tests.

Responses of 42 people with aphasia to 11 sentence types in enactment and sentence-picture matching tasks were characterized using Rasch models that varied in the inclusion of the factors of task, sentence type, and patient group. The best fitting models required the factors of task and patient group but not sentence type. The results provide evidence that aphasic syntactic comprehension is best accounted for by models that include different estimates of patient ability in different tasks and different difficulty of all sentences in different groups of patients, but that do not include different estimates of patient ability for different types of sentences.

A study of syntactic processing in aphasia II: neurological aspects.

This paper presents the results of a study of the effects of left hemisphere strokes on syntactically-based comprehension in aphasic patients. We studied 42 patients with aphasia secondary to left hemisphere strokes and 25 control subjects for the ability to assign and interpret three syntactic structures (passives, object extracted relative clauses, and reflexive pronouns) in enactment, sentence-picture matching and grammaticality judgment tasks. We measured accuracy, RT and self-paced listening times in SPM and GJ. We obtained magnetic resonance (MR) and 5-deoxyglucose positron emission tomography (FDG PET) data on 31 patients and 12 controls. The percent of selected regions of interest (ROIs) that was lesioned on MR and the mean normalized PET counts per voxel in ROIs were calculated. In regression analyses, lesion measures in both perisylvian and non-perisylvian ROIs predicted performance. Patients who performed at similar levels behaviorally had lesions of very different sizes, and patients with equivalent lesion sizes varied greatly in their level of performance. The data are consistent with a model in which the neural tissue that is responsible for the operations underlying sentence comprehension and syntactic processing is localized in different neural regions in different individuals.

A study of syntactic processing in aphasia I: behavioral (psycholinguistic) aspects.

This paper presents the results of a study of syntactically based comprehension in aphasic patients. We studied 42 patients with aphasia secondary to left hemisphere strokes and 25 control participants. We measured off-line, end-of-sentence, performance (accuracy and reaction time) in two tasks that require comprehension--enactment and sentence-picture matching--and in grammaticality judgment, with whole sentence auditory presentation. We also used sentence-picture matching and grammaticality judgment as tasks in two self-paced listening studies with the same patients to measure on-line performance. In each task and presentation format, we presented sentences that tested the ability to assign and interpret three structural contrasts chosen to examine different basic syntactic operations: actives and passives, subject and object extracted relative clauses, and reflexive pronouns and matched sentences without these elements. We examined these behavioral data to determine patterns of impairment in individual patients and in groups of patients, using correlational analyses, factor analyses, and analyses of variance. The results showed that almost no individual patients had stable deficits referable to the ability to interpret individual syntactic structures, that a variety of structural features contributed to sentence processing complexity both on-line and off-line, that correct responses were associated with normal on-line and errors with abnormal performance, and that the major determinant of performance is a factor that affected performance on all sentence types. The results indicate that the major cause of aphasic impairments of syntactically based comprehension are intermittent reductions in the processing capacity available for syntactic, interpretive, and task-related operations.

Emergency department procedural sedation and analgesia: A Canadian Community Effectiveness and Safety Study (ACCESS).

OBJECTIVES: To determine the effectiveness and safety of procedural sedation and analgesia (PSA) in a Canadian community emergency department (ED) staffed primarily by family physicians and to assess the role of capnometry monitoring in PSA. METHODS: One hundred and sixty (160) consecutive procedural sedation cases were reviewed from the ED of a rural hospital in Huntsville, Ont. The ED is mainly staffed by family physicians who have received in-house training in PSA. Safety and effectiveness measures were extrapolated from a standardized PSA form by a blinded research assistant. RESULTS: The mean age of the patient population was 33.6 years (standard deviation = 23.6). Fifty-four percent of the patients were male, and 33% of the cases were pediatric. PSA medications included propofol (84%), fentanyl (51%) and midazolam (15%), and the procedural success rate was 95.6%. The adverse event (AE) rate was 18% and included apnea (10%), inadequate sedation (3%), bradycardia (2%), desaturation (1%), hypotension (1%) and bag-valve-mask use (1%). In those aged > or = 65 years there was a greater incidence of apnea. There were no episodes of emesis and there were no intubations. A modified jaw thrust manoeuvre was used in 23% of the cases. I the 64% of cases where capnometry was used, there was no association between its use and any AE measures. CONCLUSION: Procedural sedation was safe and effective in our environment. Capnometry recording did not appear to alter outcomes, although the data are incomplete.

Domestic violence. Incidence and prevalence in a northern emergency department.

OBJECTIVE: To examine the incidence and prevalence of domestic violence (DV) against women presenting to emergency departments. DESIGN: Prospective cohort study to determine health status and exposure to DV. SETTING: Hospital emergency department in urban northern Canada. PARTICIPANTS: Random sample of women older than 16 presenting to the emergency department for any reason. MAIN OUTCOME MEASURES: Demographic variables, exposure to DV. RESULTS: Of 1800 potential subjects, 577 (32%) did not fit inclusion criteria. Of the remaining 1223, 983 (80%) agreed to participate. Mean age was 41, 135 of participants (14%) were aboriginal, and 546 (56%) were married. Overall, 725 (74%) had current partners. Incidence of DV resulting in emergency department presentation on the day of assessment was 2%. Of women with partners, 66 (9%) had previously been threatened or injured by those partners. Lifetime prevalence of DV was 51%; physical DV was experienced by 40%. One-year prevalence was 26%. CONCLUSION: Incidence of DV was lower than expected; prevalence of DV was high.