High-Throughput 3D Cellular Imaging

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 - July 30, 200

Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy

Trends and Insights from Transportation Congestion Pricing Policy Research: A Bibliometric Analysis

Toll-based congestion pricing (CP) policies are increasingly implemented globally for alleviating road traffic congestion. Several interconnected factors affecting or induced by CP implementation include air quality/emissions, travel time, and road user safety. We sought to examine and characterize research output and patterns across several domains (e.g., health, policy acceptability) surrounding toll-based CP policies, in order to identify where research has focused and where gaps exist. We conducted a structured review and identified 2333 relevant publications, using semi-supervised and machine learning strategies combined with manual review. Annual publication counts peaked in 2015 (n = 122). Themes identified from title and abstract terms included policy implementation characteristics, advanced transportation modeling methods and approaches, and public perception and acceptability. Authorship networks indicated a lack of interdisciplinary research. Country analyses identified the US, China, and the UK as the most frequently represented countries, and underrepresentation from low-income countries. Findings indicate that research focused on specific road user types (e.g., pedestrians) and safety impacts, and equity considerations were relatively sparse compared to other topics (e.g., policy economics, public perception). Additional research on these critical topics is necessary to ensure that such policies are designed to promote positive and equitable effects on road user health and safety

Defining Emergency Department Asthma Visits for Public Health Surveillance, North Carolina, 2008–2009

IntroductionWhen using emergency department (ED) data sets for public health surveillance, a standard approach is needed to define visits attributable to asthma. Asthma can be the first (primary) or a subsequent (2nd through 11th) diagnosis. Our study objective was to develop a definition of ED visits attributable to asthma for public health surveillance. We evaluated the effect of including visits with an asthma diagnosis in primary-only versus subsequent positions.MethodsThe study was a cross-sectional analysis of population-level ED surveillance data. Of the 114 North Carolina EDs eligible to participate in a statewide surveillance system in 2008–2009, we used data from the 111 (97%) that participated during those years. Included were all ED visits with an ICD-9-CM diagnosis code for asthma in any diagnosis position (1 through 11). We formed 11 strata based on the diagnosis position of asthma and described common chief complaint and primary diagnosis categories for each. Prevalence ratios compared each category’s proportion of visits that received either asthma- or cardiac-related procedure codes.ResultsRespiratory diagnoses were most common in records of ED visits in which asthma was the first or second diagnosis, while primary diagnoses of injury and heart disease were more common when asthma appeared in positions 3–11. Asthma-related chief complaints and procedures were most common when asthma was the first or second diagnosis, whereas cardiac procedures were more common in records with asthma in positions 3–11.ConclusionED visits should be defined as asthma-related when asthma is in the first or second diagnosis position

A novel Vision Zero leadership training model to support collaboration and strategic action planning

Introduction: While collaboration and cooperation are regarded as foundational to Vision Zero (VZ) and Safe Systems initiatives, there is little guidance on structuring VZ collaboration, conducting collaborative goal setting, and aligning tangible action across organizations. As part of a larger VZ mutual learning model, we developed a VZ Leadership Team Institute to support communities in collaborative VZ strategic planning and goal setting. The purpose of this paper is to describe the development and evaluation of the Institute, which can serve as a foundation for other initiatives seeking to move VZ planning and implementation forward in a collaborative, systems-aware manner.Methods: In June 2021, eight multi-disciplinary teams of 3–6 persons each (n = 42 participants) attended the Institute, representing leaders from communities of various sizes. Surveys were administered pre, immediately post, and 6 months following the Institute. We measured confidence in a range of skills (on a 5-point scale, 1: not confident to 5: very confident). Surveys also measured coalition collaboration pre-Institute and 6 months post-Institute (on a 4-point scale, 1: strongly disagree to 4: strongly agree).Results: The largest increases in confidence from pre- to immediately post-Institute were for collaboratively drafting objectives and actions for VZ goals (pre-mean: 2.6, SD: 0.9 to post-mean: 3.8, SD: 0.9); incorporating equity into goals (pre-mean: 2.8, SD: 1.0 to post-mean: 3.9, SD: 0.8); and knowing how to keep VZ planning and implementation efforts on track (pre-mean: 2.6, SD: 1.0 to post-mean: 3.7, SD: 0.7). For all measures, average confidence in skills decreased from immediately post-Institute to 6 months post-Institute, but remained greater than average scores pre-Institute. Several measures of coalition collaboration maintained high agreement across time, and mean agreement increased for reporting that the future direction of the coalition was clearly communicated to everyone (pre-mean: .6, SD: 0.8; 6 months post-mean: 3.1, SD: 0.4). However, average scores decreased for feeling like the coalition had adequate staffing (pre-mean: 3.0, SD: 0.6; 6 months post-mean: 2.3, SD: 0.5).Discussion: The Institute utilized innovative content, tools, and examples to support VZ coalitions’ collaborative and systems-aware planning and implementation processes. As communities work toward zero transportation deaths and serious injuries, providing effective support models to aid multidisciplinary planning and action around a Safe Systems approach will be important to accelerate progress toward a safer transportation system

A non-experimental study of oral anticoagulation therapy initiation before and after national patient safety goals

ObjectivesThe Joint Commission revised its National Patient Safety Goals (NPSGs) to include oral anticoagulation therapy (OAT) in 2008. We sought to examine the effect of including OAT in The Joint Commission's NPSGs on historically low rates of OAT initiation for individuals with incident atrial fibrillation (AF).SettingSoutheastern state in the USA.ParticipantsNorth Carolina State Health Plan claims data from 944 500 individuals enrolled between 1 January 2006 and 31 December 2010, supplemented with data from the Area Resource File and Online Survey, Certification and Reporting data network. We evaluated OAT initiation before and after the 2008 NPSGs revisions in a retrospective cohort new user design with an AF intervention group and two control groups: a positive control—patients estimated to be at very high risk of thromboembolism (mechanical heart valve and pulmonary embolism); and a negative control—patients with very low perceived risk of thromboembolism (paroxysmal AF). We developed multivariable models using a difference-in-difference parameterisation. Effects were estimated with generalised estimating equations.Primary outcome measureOAT initiation, a binary outcome defined as having a prescription drug claim for warfarin within 30 days of the index claim.ResultsOAT initiation was low (26.8%) for eligible individuals with incident AF in 2006–2008 but increased after NPSGs implementation (31.7%, p=0.022). OAT initiation was high but decreased in the positive control group (67.5% vs 62.0%, p=0.003). Multivariate analysis resulted in a relative 11% (95% CI (4% to 18%), p<0.01) increase in OAT initiation for incident AF patients.ConclusionsWe document a substantial increase in guideline concordant OAT initiation in incident AF after the establishment of NPSGs, suggesting that regulatory healthcare agency initiatives can influence clinical practice

ATP Binding by Monarch-1/NLRP12 Is Critical for Its Inhibitory Function

The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-κB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-κB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1

Population-based burden of COPD-related visits in the ED: Return ED visits, hospital admissions, and comorbidity risks

Little is known about the population-based burden of ED care for COPD

Strategic Planning to Reduce the Burden of Stroke Among Veterans: Using Simulation Modeling to Inform Decision Making

Reducing the burden of stroke is a priority for the Veterans Affairs (VA) Health System, reflected by the creation of the VA Stroke Quality Enhancement Research Initiative (QUERI). To inform the initiative's strategic planning, we estimated the relative population-level impact and efficiency of distinct approaches to improving stroke care in the United States Veteran population to inform policy and practice