Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.

Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

Zyxin contributes to coupling between cell junctions and contractile actomyosin networks during apical constriction.

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and ß-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions' centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved.

Introducing spatial availability, a singly-constrained measure of competitive accessibility.

Accessibility indicators are widely used in transportation, urban and healthcare planning, among many other applications. These measures are weighted sums of reachable opportunities from a given origin, conditional on the cost of movement, and are estimates of the potential for spatial interaction. Over time, various proposals have been forwarded to improve their interpretability: one of those methodological additions have been the introduction of competition. In this paper we focus on competition, but first demonstrate how a widely used measure of accessibility with congestion fails to properly match the opportunity-seeking population. We then propose an alternative formulation of accessibility with competition, a measure we call spatial availability. This measure relies on proportional allocation balancing factors (friction of distance and population competition) that are equivalent to imposing a single constraint on conventional gravity-based accessibility. In other words, the proportional allocation of opportunities results in a spatially available opportunities value which is assigned to each origin that, when all origin values are summed, equals the total number of opportunities in the region. We also demonstrate how Two-Stage Floating Catchment Area (2SFCA) methods are equivalent to spatial availability and can be reconceptualized as singly-constrained accessibility. To illustrate the application of spatial availability and compare it to other relevant measures, we use data from the 2016 Transportation Tomorrow Survey of the Greater Golden Horseshoe area in southern Ontario, Canada. Spatial availability is an important contribution since it clarifies the interpretation of accessibility with competition and paves the way for future applications in equity analysis (e.g., spatial mismatch, opportunity benchmarking, policy intervention scenario analysis).

Public transport access to drug treatment before and during COVID-19: Implications for the opioid epidemic.

Public transport disruptions caused by the COVID-19 pandemic had wide-ranging impacts on the ability of individuals to access health care. Individuals with opioid use disorder represent an especially vulnerable population due to the necessity of frequent, supervised doses of opioid agonists. Focused on Toronto, a major Canadian city suffering from the opioid epidemic, this analysis uses novel realistic routing methodologies to quantify how travel times to individuals\220 nearest clinics changed due to public transport disruptions from 2019 to 2020. Individuals seeking opioid agonist treatment face very constrained windows of access due to the need to manage work and other essential activities. We find that thousands of households in the most materially and socially deprived neighbourhoods crossed 30 and 20-minute travel time thresholds to their nearest clinic. As even small changes to travel times can lead to missed appointments and heighten the chances of overdose and death, understanding the distribution of those most impacted can help inform future policy measures to ensure adequate access to care.

Changes in accessibility to emergency and community food services during COVID-19 and implications for low income populations in Hamilton, Ontario.

In this paper we analyze the changes in accessibility to emergency and community food services before and during the COVID-19 pandemic in the City of Hamilton, Ontario. Many of these food services are the last line of support for households facing food insecurity; as such, their relevance cannot be ignored in the midst of the economic upheaval caused by the pandemic. Our analysis is based on the application of balanced floating catchment areas and concentrates on households with lower incomes (

Demand and level of service inflation in Floating Catchment Area (FCA) methods.

Floating Catchment Area (FCA) methods are a popular tool to investigate accessibility to public facilities, in particular health care services. FCA approaches are attractive because, unlike other accessibility measures, they take into account the potential for congestion of facilities. This is done by 1) considering the population within the catchment area of a facility to calculate a variable that measures level of service, and then 2) aggregating the level of service by population centers subject to catchment area constraints. In this paper we discuss an effect of FCA approaches, an artifact that we term demand and level of service inflation. These artifacts are present in previous implementations of FCA methods. We argue that inflation makes interpretation of estimates of accessibility difficult, which has possible deleterious consequences for decision making. Next, we propose a simple and intuitive approach to proportionally allocate demandand and level of service in FCA calculations. The approach is based on a standardization of the impedance matrix, similar to approaches popular in the spatial statistics and econometrics literature. The result is a more intiuitive measure of accessibility that 1) provides a local version of the provider-to-population ratio; and 2) preserves the level of demand and the level of supply in a system. We illustrate the relevant issues with some examples, and then empirically by means of a case study of accessibility to family physicians in the Hamilton Census Metropolitan Area (CMA), in Ontario, Canada. Results indicate that demand and supply inflation/deflation affect the interpretation of accessibility analysis using existing FCA methods, and that the proposed adjustment can lead to more intuitive results.

The health impacts of weekday traffic: A health risk assessment of PM2.5 emissions during congested periods.

Little work has accounted for congestion, using data that reflects driving patterns, traffic volume, and speed, to examine the association between traffic emissions and human health. In this study, we performed a health risk assessment of PM2.5 emissions during congestion periods in the Greater Toronto and Hamilton Area (GTHA), Canada. Specifically, we used a micro-level approach that combines the Stochastic User Equilibrium Traffic Assignment Algorithm with a MOVES emission model to estimate emissions considering congestion conditions. Subsequently, we applied a concentration-response function to estimate PM2.5-related mortality, and the associated health costs. Our results suggest that traffic congestion has a substantial impact on human health and the economy in the GTHA, especially at the most congested period (7:00am). Considering daily mortality, our results showed an impact of 206 (boundary test 95%: 116; 297) and 119 (boundary test 95%: 67; 171) deaths per year (all-cause and cardiovascular mortality, respectively). The economic impact from daily mortality is approximately $1.3 billion (boundary test 95%: 0.8; 1.9), and $778 million (boundary test 95%: 478; 981), for all-cause and cardiovascular mortality, respectively. Our study can guide reliable projections of transportation and air pollution levels, improving the capability of the medical community to prepare for future trends.

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation.

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here we identify a myosin light-chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endoderm precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, ß-catenin, and cadherin become highly enriched at the apical junctions of apically constricting cells and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis.

Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette.

A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.

Triggering a cell shape change by exploiting preexisting actomyosin contractions.

Apical constriction changes cell shapes, driving critical morphogenetic events, including gastrulation in diverse organisms and neural tube closure in vertebrates. Apical constriction is thought to be triggered by contraction of apical actomyosin networks. We found that apical actomyosin contractions began before cell shape changes in both Caenorhabitis elegans and Drosophila. In C. elegans, actomyosin networks were initially dynamic, contracting and generating cortical tension without substantial shrinking of apical surfaces. Apical cell-cell contact zones and actomyosin only later moved increasingly in concert, with no detectable change in actomyosin dynamics or cortical tension. Thus, apical constriction appears to be triggered not by a change in cortical tension, but by dynamic linking of apical cell-cell contact zones to an already contractile apical cortex.

Asymmetric cell division: a new way to divide unequally.

It has long been known that cells can divide unequally by shifting the mitotic spindle to one side. Two recent reports identify an alternative way to generate daughter cells of different sizes.