Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules.

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.

Extracellular fluid viscosity enhances cell migration and cancer dissemination.

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

The impact of JUUL market entry on cigarette sales: evidence from a major chain retailer in Canada.

BACKGROUND: Electronic nicotine delivery systems (ENDS), such as the JUUL system, are nicotine products for adults who currently smoke cigarettes but are looking for an alternative to combustible cigarettes. Sales of ENDS products were legislatively acknowledged and authorized federally in Canada with the Royal Assent of the Tobacco and Vaping Products Act in 2018. METHODS: With the unique dataset from a major chain retailer in Canada, we evaluated the impacts of JUUL market entry on cigarette sales across Canada from January 2017 to August 2019 using two-way fixed effects panel regression models by leveraging on the entry time variation at the city level. We conducted various robustness checks and a permutation test to validate our results. RESULTS: Our estimates suggested that JUUL market entry was, on average, significantly correlated with a 1.65% per-month decrease in cigarette sales during the initial months, and with a potentially larger impact on urban areas. Our results were robust across various specifications and tests. These findings implied that JUUL and combustible cigarettes act as economic substitutes during the study time period in Canada. CONCLUSIONS: These results suggested that local availability of ENDS products, such as JUUL, has the potential to reduce local cigarette consumption.

Estimating the Effects of Non-Pharmaceutical Interventions and Population Mobility on Daily COVID-19 Cases: Evidence from Ontario.

This study uses coronavirus disease 2019 (COVID-19) case counts and Google mobility data for 12 of Ontario's largest Public Health Units from Spring 2020 until the end of January 2021 to evaluate the effects of non-pharmaceutical interventions (NPIs; policy restrictions on business operations and social gatherings) and population mobility on daily cases. Instrumental variables (IV) estimation is used to account for potential simultaneity bias, because both daily COVID-19 cases and NPIs are dependent on lagged case numbers. IV estimates based on differences in lag lengths to infer causal estimates imply that the implementation of stricter NPIs and indoor mask mandates are associated with reductions in COVID-19 cases. Moreover, estimates based on Google mobility data suggest that increases in workplace attendance are correlated with higher case counts. Finally, from October 2020 to January 2021, daily Ontario forecasts from Box-Jenkins time-series models are more accurate than official forecasts and forecasts from a susceptible-infected-removed epidemiology model.

Cette étude cherche à évaluer les effets des interventions non pharmaceutiques (INPs; restrictions sur les activités commerciales et rassemblements sociaux) et de la mobilité de la population sur le nombre de cas d'infection par jour, en utilisant les nombres de cas d'infection par la maladie à coronavirus 2019 (COVID-19) et les données de mobilité de Google pour 12 des plus grands Bureaux de Santé publique de l'Ontario entre le printemps 2020 et la fin janvier 2021. La méthode des variables instrumentales (VI) permet de rendre compte d'un biais potentiel de simultanéité puisque les taux quotidiens de COVID-19 et les INPs dépendent, tous les deux, du nombre de cas décalés. Les estimations par les VI basées sur les différences de durée des décalages d'ajustement pour inférer des estimations causales impliquent que de plus strictes INPs et le port obligatoire du masque dans les endroits fermés sont associés à une réduction de cas d'infection. Par ailleurs, Les estimations basées sur les données de mobilité de Google montrent que la présence accrue sur le lieu du travail est corrélée avec un plus grand nombre de cas d'infection. Finalement, d'octobre 2020 à Janvier 2021, les prévisions faites à partir de modèles de Box-Jenkins en série chronologique s'avèrent plus précises que les prévisions officielles et que celles utilisant le modèle épidémiologique susceptible ­ infecté ­ retiré.

An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling.

Cell-to-cell communication via the Notch pathway is mediated between the membrane-bound Notch receptor and either of its canonical membrane-bound ligands Delta or Serrate. Notch ligands mediate receptor transactivation between cells and also mediate receptor cis-inhibition when Notch and ligand are co-expressed on the same cell. We demonstrate in Drosophila that removal of any of the EGF-like repeats (ELRs) 4, 5 or 6 results in a Serrate molecule capable of transactivating Notch but exhibiting little or no Notch cis-inhibition capacity. These forms of Serrate require Epsin (Liquid facets) to transduce a signal, suggesting that ELR 4-6-deficient ligands still require endocytosis for Notch activation. We also demonstrate that ELRs 4-6 are responsible for the dominant-negative effects of Serrate ligand forms that lack the intracellular domain and are therefore incapable of endocytosis in the ligand-expressing cell. We find that ELRs 4-6 of Serrate are conserved across species but do not appear to be conserved in Delta homologs.

Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy.

The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.

Notch signaling at a glance.

Cell-cell interactions define a quintessential aspect of multicellular development. Metazoan morphogenesis depends on a handful of fundamental, conserved cellular interaction mechanisms, one of which is defined by the Notch signaling pathway. Signals transmitted through the Notch surface receptor have a unique developmental role: Notch signaling links the fate of one cell with that of a cellular neighbor through physical interactions between the Notch receptor and the membrane-bound ligands that are expressed in an apposing cell. The developmental outcome of Notch signals is strictly dependent on the cellular context and can influence differentiation, proliferation and apoptotic cell fates. The Notch pathway is conserved across species (Artavanis-Tsakonas et al., 1999; Bray, 2006; Kopan and Ilagan, 2009). In humans, Notch malfunction has been associated with a diverse range of diseases linked to changes in cell fate and cell proliferation including cancer (Louvi and Artavanis-Tsakonas, 2012). In this Cell Science at a Glance article and the accompanying poster we summarize the molecular biology of Notch signaling, its role in development and its relevance to disease.

Prospective self-gated nonenhanced magnetic resonance angiography of the peripheral arteries.

Most nonenhanced MRA techniques for evaluating peripheral artery disease (PAD) require cardiac synchronization through physiological gating. Electrocardiographic gating is the most popular method for cardiac synchronization; however, it is subject to interference from switching magnetic field gradients and radiofrequency pulses. A method is described for self-gated nonenhanced MRA that does not require the use of electrocardiographic gating. Imaging was prospectively triggered by detecting the acceleration of blood flow during systole with a reference-less phase contrast navigator. The technique was implemented for nonsubtractive nonenhanced MRA using quiescent-interval single-shot MRA. The lower extremity peripheral arteries of eight healthy subjects were imaged using electrocardiographic-, pulse-, and self-gated quiescent-interval single-shot. Self-gated quiescent-interval single-shot triggered with 99% accuracy. There were no significant differences in relative contrast, contrast-to-noise ratio, or image quality between self-gated and electrocardiographic-gated quiescent-interval single-shot MRA (P > 0.05). Image quality with pulse gating was inferior.

Conserved genes act as modifiers of invertebrate SMN loss of function defects.

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

Do more stringent policies reduce daily COVID-19 case counts? Evidence from Canadian provinces.

The enactment of COVID-19 policies in Canada falls under provincial jurisdiction. This study exploits time-series variation across four Canadian provinces to evaluate the effects of stricter COVID-19 policies on daily case counts. Employing data from this time-period allows an evaluation of the efficacy of policies independent of vaccine impacts. While both OLS and IV results offer evidence that more stringent Non-Pharmaceutical Interventions (NPIs) can reduce daily case counts within a short time-period, IV estimates are larger in magnitude. Hence, studies that fail to control for simultaneity bias might produce confounded estimates of the efficacy of NPIs. However, IV estimates should be treated as correlations given the possibility of other unobserved determinants of COVID-19 spread and mismeasurement of daily cases. With respect to specific policies, mandatory mask usage in indoor spaces and restrictions on business operations are significantly associated with lower daily cases. We also test the efficacy of different forecasting models. Our results suggest that Gradient Boosted Regression Trees (GBRT) and Seasonal Autoregressive-Integrated Moving Average (SARIMA) models produce more accurate short-run forecasts relative to Vector Auto Regressive (VAR), and Susceptible-Infected-Removed (SIR) epidemiology models. Forecasts from SIR models are also inferior to results from basic OLS regressions. However, predictions from models that are unable to correct for endogeneity bias should be treated with caution.

Forecasting daily COVID-19 cases with gradient boosted regression trees and other methods: evidence from U.S. cities.

Introduction: There is a vast literature on the performance of different short-term forecasting models for country specific COVID-19 cases, but much less research with respect to city level cases. This paper employs daily case counts for 25 Metropolitan Statistical Areas (MSAs) in the U.S. to evaluate the efficacy of a variety of statistical forecasting models with respect to 7 and 28-day ahead predictions. Methods: This study employed Gradient Boosted Regression Trees (GBRT), Linear Mixed Effects (LME), Susceptible, Infectious, or Recovered (SIR), and Seasonal Autoregressive Integrated Moving Average (SARIMA) models to generate daily forecasts of COVID-19 cases from November 2020 to March 2021. Results: Consistent with other research that have employed Machine Learning (ML) based methods, we find that Median Absolute Percentage Error (MAPE) values for both 7-day ahead and 28-day ahead predictions from GBRTs are lower than corresponding values from SIR, Linear Mixed Effects (LME), and Seasonal Autoregressive Integrated Moving Average (SARIMA) specifications for the majority of MSAs during November-December 2020 and January 2021. GBRT and SARIMA models do not offer high-quality predictions for February 2021. However, SARIMA generated MAPE values for 28-day ahead predictions are slightly lower than corresponding GBRT estimates for March 2021. Discussion: The results of this research demonstrate that basic ML models can lead to relatively accurate forecasts at the local level, which is important for resource allocation decisions and epidemiological surveillance by policymakers.

Cytoskeletal activation of NHE1 regulates cell volume and DNA methylation.

The regulation of mammalian cell volume is crucial for maintaining key cellular processes. Cells can rapidly respond to osmotic and hydrostatic pressure imbalances during environmental challenges, generating fluxes of water and ions that alter volume within minutes. While the role of ion pump and leak in cell volume regulation has been well-established, the role of the actomyosin cytoskeleton and its substantial interplay with ion transporters are still unclear. In this work, we discover a system of cell volume regulation controlled by cytoskeletal activation of ion transporters. Under hypotonic shock, NIH-3T3 and MCF-10A display a 20% secondary volume increase (SVI) following the initial regulatory volume decrease. We show that SVI is initiated by Ca 2+ influx through stretch activated channel Piezo1 and subsequent actomyosin remodeling. Rather than contracting cells, actomyosin triggers cell swelling by activating Na + -H + exchanger 1 (NHE1) through their co-binding partner ezrin. Cytoskeletal activation of NHE1 can be similarly triggered by mechanical stretch and attenuated by soft substrates. This mechanism is absent in certain cancer cell lines such as HT1080 and MDA-MB-231, where volume regulation is dominated by intrinsic response of ion transporters. Moreover, cytoskeletal activation of NHE1 during SVI induces nuclear deformation, leading to DNA demethylation and a significant, immediate transcriptomic response in 3T3 cells, a phenomenon that is absent in HT1080 cells. Overall, our findings reveal the central role of Ca 2+ and actomyosin-mediated mechanosensation in the regulation of ion transport, cell volume, DNA methylation, and transcriptomics.

Identifying the Socioeconomic, Demographic, and Political Determinants of Social Mobility and Their Effects on COVID-19 Cases and Deaths: Evidence From US Counties.

Background: The spread of COVID-19 at the local level is significantly impacted by population mobility. The U.S. has had extremely high per capita COVID-19 case and death rates. Efficient nonpharmaceutical interventions to control the spread of COVID-19 depend on our understanding of the determinants of public mobility. Objective: This study used publicly available Google data and machine learning to investigate population mobility across a sample of US counties. Statistical analysis was used to examine the socioeconomic, demographic, and political determinants of mobility and the corresponding patterns of per capita COVID-19 case and death rates. Methods: Daily Google population mobility data for 1085 US counties from March 1 to December 31, 2020, were clustered based on differences in mobility patterns using K-means clustering methods. Social mobility indicators (retail, grocery and pharmacy, workplace, and residence) were compared across clusters. Statistical differences in socioeconomic, demographic, and political variables between clusters were explored to identify determinants of mobility. Clusters were matched with daily per capita COVID-19 cases and deaths. Results: Our results grouped US counties into 4 Google mobility clusters. Clusters with more population mobility had a higher percentage of the population aged 65 years and over, a greater population share of Whites with less than high school and college education, a larger percentage of the population with less than a college education, a lower percentage of the population using public transit to work, and a smaller share of voters who voted for Clinton during the 2016 presidential election. Furthermore, clusters with greater population mobility experienced a sharp increase in per capita COVID-19 case and death rates from November to December 2020. Conclusions: Republican-leaning counties that are characterized by certain demographic characteristics had higher increases in social mobility and ultimately experienced a more significant incidence of COVID-19 during the latter part of 2020.

Discovery of Phospholipase D Inhibitors with Improved Drug-like Properties and Central Nervous System Penetrance.

Phospholipase D (PLD) is a phospholipase enzyme responsible for hydrolyzing phosphatidylcholine into the lipid signaling molecule, phosphatidic acid, and choline. From a therapeutic perspective, PLD has been implicated in human cancer progression as well as a target for neurodegenerative diseases, including Alzheimer's. Moreover, knockdown of PLD rescues the ALS phenotype in multiple Drosophila models of ALS (amyotrophic lateral sclerosis) and displays modest motor benefits in an SOD1 ALS mouse model. To further validate whether inhibiting PLD is beneficial for the treatment of ALS, a brain penetrant small molecule inhibitor with suitable PK properties to test in an ALS animal model is needed. Using a combination of ligand-based drug discovery and structure-based design, a dual PLD1/PLD2 inhibitor was discovered that is single digit nanomolar in the Calu-1 cell assay and has suitable PK properties for in vivo studies. To capture the in vivo measurement of PLD inhibition, a transphosphatidylation pharmacodynamic LC-MS assay was developed, in which a dual PLD1/PLD2 inhibitor was found to reduce PLD activity by 15-20-fold.

Estimating the effects of cigarette taxes on birth outcomes.

Employing provincial data from 1979 to 2004 allows us to exploit the significant (45 percent to 60 percent) reduction in excise taxes in Eastern Canada enacted in February 1994 to estimate the impacts of cigarette taxes on birth outcomes. Empirical estimates suggest that an increase in cigarette taxes is significantly associated with lower infant mortalities. However, we also find some evidence of a counter-intuitive positive correlation between taxes and fetal deaths. Overall, conditional on methodology, we find increased lagged per capita health expenditures and the number of physicians to be significantly associated with improvements in birth outcomes.

Estimating the impacts of cigarette taxes on youth smoking participation, initiation, and persistence: empirical evidence from Canada.

In response to the widespread availability of illegal contraband, the federal and five provincial governments in Canada implemented a 40-60% reduction to cigarette excise taxes in February 1994. We exploit this unique and discrete policy shock by estimating the effects of cigarette taxes on youth smoking with data from the 1992-1996 Waterloo Smoking Prevention Program, 1991 General Social Survey, 1994 Youth Smoking Survey, 1996-1997 and 1998-1999 National population Health Surveys, and the 1999 Canadian Tobacco Use Monitoring Survey. Empirical estimates yield daily and occasional participation elasticities from -0.10 to -0.14, which is consistent with findings from recent U.S.-based research. A key contribution of this research is in the analysis of lower taxes on a panel of 591 youths from the Waterloo Smoking Prevention Program, who did not smoke in 1993, but 43% of whom confirm smoking participation following the tax reduction. Employing these data reveals elasticities from -0.2 to -0.5, which suggest that even significant and discrete changes in taxes might have limited impacts on the initiation and persistence of youth smoking.

Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target.

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a devastating neurodegenerative disorder lacking effective treatments. ALS pathology is linked to mutations in >20 different genes indicating a complex underlying genetic architecture that is effectively unknown. Here, in an attempt to identify genes and pathways for potential therapeutic intervention and explore the genetic circuitry underlying Drosophila models of ALS, we carry out two independent genome-wide screens for modifiers of degenerative phenotypes associated with the expression of transgenic constructs carrying familial ALS-causing alleles of FUS (hFUSR521C) and TDP-43 (hTDP-43M337V). We uncover a complex array of genes affecting either or both of the two strains, and investigate their activities in additional ALS models. Our studies indicate the pathway that governs phospholipase D activity as a major modifier of ALS-related phenotypes, a notion supported by data we generated in mice and others collected in humans.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.

Distinct types of glial cells populate the Drosophila antenna.

BACKGROUND: The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix-loop-helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna. RESULTS: We have used different P(Gal4) lines to drive Green Fluorescent Protein (GFP) in distinct populations of cells within the Drosophila antenna. Mz317::GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional approximately 30 glial cells detected by GH146::GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317-glia and GH146-glia respectively. In the adult, processes of GH146-glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317-glia form a peripheral layer. Ablation of GH146-glia does not result in any significant effects on the patterning of the olfactory receptor axons. CONCLUSION: We have demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146-glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca.

Programmed cell death and context dependent activation of the EGF pathway regulate gliogenesis in the Drosophila olfactory system.

In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? We found that ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase transiently, and antagonism of Epidermal Growth Factor (EGF) pathway signaling compromises their development. We suggest that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death.