Transcription templated assembly of the nucleolus in the C. elegans embryo.

The nucleolus is a multicomponent structure made of RNA and proteins that serves as the site of ribosome biogenesis within the nucleus. It has been extensively studied as a prototype of a biomolecular condensate whose assembly is driven by phase separation. While the steady-state size of the nucleolus is quantitatively accounted for by the thermodynamics of phase separation, we show that experimental measurements of the assembly dynamics are inconsistent with a simple model of a phase-separating system relaxing to its equilibrium state. Instead, we show that the dynamics are well described by a model in which the transcription of ribosomal RNA actively drives nucleolar assembly. We find that our model of active transcription-templated assembly quantitatively accounts for the rapid kinetics observed in early embryos at different developmental stages, and for different RNAi perturbations of embryo size. Our model predicts a scaling of the time to assembly with the volume of the nucleus to the one-third power, which is confirmed by experimental data. Our study highlights the role of active processes such as transcription in controlling the placement and timing of assembly of membraneless organelles.

The shared decision-making tool for caregivers of children with prenatal opioid exposure: A pilot and acceptability study.

This pilot and acceptability study sought to report provider acceptability and developmental concerns expressed by caregivers of children with prenatal opioid exposure using the Shared Decision-Making Tool (SDMT), an instrument created by study authors. Data were collected from five health care and early intervention providers and 83 caregivers from a medical clinic and early intervention service center. Descriptive statistics were used to identify frequency, mean level, and prioritization of developmental concerns using the SDMT, and to summarize provider acceptability about integrating the SDMT into their workflow. Communication was the most frequently cited concern in four consecutive age categories, followed by Inattention/impulsivity and Problem behavior. All providers "strongly agreed" or "agreed" with all statements on the provider feedback survey, except two instances. Results of this study support the SDMT as a potential tool to help engage caregivers and providers of children with prenatal opioid exposure in the shared decision-making process by standardizing communication related to areas of developmental concern and caregivers' priority needs. Findings from this pilot study will inform modifications to the SDMT and administration instructions before our next study, which will examine psychometric properties and caregiver acceptability of the scale.

Arabidopsis callus grafts: a system to study symplasmic intercellular macromolecular transport and gene silencing spread.

In the present study, we present callus grafting, comprising a method for reproducibly generating tissue chimeras from callus cultures of Arabidopsis thaliana. In this way, callus cultures of different genetic backgrounds may be co-cultivated such that cell-to-cell connectivity is achieved as a chimeric tissue is formed. To track intercellular connectivity and transport between non-clonal callus cells, we used transgenic lines expressing fluorescently tagged mobile and non-mobile fusion constructs. Using fluorescently-labelled reporter lines that label plasmodesmata, we show that secondary complex plasmodesmata are present at the cell walls of connected cells. We use this system to investigate cell-to-cell transport across the callus graft junction and show that different proteins and RNAs are mobile between non-clonal callus cells. Finally, we take advantage of the callus culture system to probe intercellular connectivity of grafted leaf and root calli and the effect of different light regimes of cell-to-cell transport. Taking advantage of the ability of callus to be cultivated in the complete absence of light, we show that the rate of silencing spread is significantly decreased in chimeric calli cultivated in total darkness. We propose that callus grafting is a fast and reliable method for analysing the capacity of a macromolecule to be exchanged between cells independent of the vasculature.

Phase Separation in Biology and Disease; Current Perspectives and Open Questions.

In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ∼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.

Let's phase it: viruses are master architects of biomolecular condensates.

Viruses compartmentalize their replication and assembly machinery to both evade detection and concentrate the viral proteins and nucleic acids necessary for genome replication and virion production. Accumulating evidence suggests that diverse RNA and DNA viruses form replication organelles and nucleocapsid assembly sites using phase separation. In general, the biogenesis of these compartments is regulated by two types of viral protein, collectively known as antiterminators and nucleocapsid proteins, respectively. Herein, we discuss how RNA viruses establish replication organelles and nucleocapsid assembly sites, and the evidence that these compartments form through phase separation. While this review focuses on RNA viruses, accumulating evidence suggests that all viruses rely on phase separation and form biomolecular condensates important for completing the infectious cycle.

Single-Molecule Tracking of RNA Polymerase In and Out of Condensates in Live Bacterial Cells.

Biomolecular condensates, first discovered in eukaryotic cells, were recently found in bacteria. The small size of these organisms presents unique challenges for identifying and characterizing condensates. Here, we describe a single-molecule approach for studying biomolecular condensates in live bacterial cells. Specifically, we outline a protocol to quantify the mobility of RNA polymerase in E. coli using HILO (highly inclined and laminated optical sheet) illumination with the photoconvertible fluorophore mMaple3. Our analysis classifies the trajectories of individual molecules by their local density, enabling a comparison of molecular mobilities between different subcellular compartments.

New Homogeneous Spatial Areas Identified Using Case-Crossover Spatial Lag Grid Differences between Aerosol Optical Depth-PM2.5 and Respiratory-Cardiovascular Emergency Department Visits and Hospitalizations.

Optimal use of Hierarchical Bayesian Model (HBM)-assembled aerosol optical depth (AOD)-PM2.5 fused surfaces in epidemiologic studies requires homogeneous temporal and spatial fused surfaces. No analytical method is available to evaluate spatial heterogeneity. The temporal case-crossover design was modified to assess the spatial association between four experimental AOD-PM2.5 fused surfaces and four respiratory-cardiovascular hospital events in 12 km2 grids. The maximum number of adjacent lag grids with significant odds ratios (ORs) identified homogeneous spatial areas (HOSAs). The largest HOSA included five grids (lag grids 04; 720 km2) and the smallest HOSA contained two grids (lag grids 01; 288 km2). Emergency department asthma and inpatient asthma, myocardial infarction, and heart failure ORs were significantly higher in rural grids without air monitors than in urban grids with air monitors at lag grids 0, 1, and 01. Rural grids had higher AOD-PM2.5 concentration levels, population density, and poverty percentages than urban grids. Warm season ORs were significantly higher than cold season ORs for all health outcomes at lag grids 0, 1, 01, and 04. The possibility of elevated fine and ultrafine PM and other demographic and environmental risk factors synergistically contributing to elevated respiratory-cardiovascular chronic diseases in persons residing in rural areas was discussed.

SARS-CoV-2 seroprevalence in healthcare workers and risk factors.

BACKGROUND: Exposure of healthcare workers (HCW) to SARS-CoV-2 is a public health concern. Not only are HCWs particularly exposed to SARS-CoV-2, but their contamination can also weaken the healthcare system. METHODS: We analyzed exposure of French University Hospital HCWs to SARS-CoV-2 through history of positive RT-PCR test and SARS-CoV-2 seroprevalence. Potential risk factors, such as age, BMI, having children or not, working in a COVID-19 unit, or smoking were explored. RESULTS: From May to June 2020, among the 8960 employees of the University Hospital of Nancy, a serological test was performed in 4696 HCWs. The average (SD) age was 40.4 (11.4) years, and the sample included 3926 women (83.6%). Of the 4696 HCWs, 1050 were smokers (22.4%). Among them, 2231 HCWs had a history of COVID-19 symptoms and/or flu-like syndrome (47.5%) and 238 were seropositive (5.1%). Neither gender, sex, BMI, nor having children were associated with a history of positive RT-PCR test or seropositive status. Previous work in a COVID-19 unit was associated with a history of positive RT-PCR test (p = 0.045), but not with seroprevalence (p = 0.215). As expected, history of COVID-19 clinical manifestations was more frequent in HCWs with positive serology than in HCWs with negative serology (adjusted OR = 1.9, 95%CI [1.4-2.5], p < 0.001). Less expected, smoking was associated with a reduced risk of seropositivity among HCWs (adjusted OR = 0.6, 95%CI [0.4-0.9], p = 0.019). CONCLUSION: HCW are patently exposed to SARS-CoV-2. Care to COVID-19 patients was not associated with a higher SARS-CoV-2 seroprevalence. Smoking appears here associated to a lower seroprevalence.

Breastfeeding Initiation and Continuation Among Women with Substance and Tobacco Use During Pregnancy: Findings from the Pregnancy Risk Assessment Monitoring System 2016-2018.

Background and Aims: Substance and tobacco use is associated with poor maternal and child health outcomes. Although these have each been linked to lower breastfeeding rates when examined separately, studies have yet to examine how the combination of tobacco and other substance use influences breastfeeding initiation and continuation. The aim of this study was to examine how the combination of smoking tobacco and use of illicit substances influences the odds of breastfeeding initiation and continuation. Materials and Methods: This retrospective cohort study (n = 15,634) used survey data from the 2016-2018 Centers for Disease Control and Prevention (CDC) Pregnancy Risk Assessment Monitoring System from eight US states to examine the association of tobacco and illicit substance use with breastfeeding initiation and continuation (≥6 weeks). The odds of breastfeeding initiation and continuation for individuals with and without prenatal tobacco and illicit substance use, adjusting for maternal and infant characteristics, were estimated using weighted, multivariable logistic regression models. Results: The combination of prenatal tobacco and illicit substance use was associated with a 42% reduction in the odds of initiating breastfeeding (adjusted odds ratio [aOR] 0.58 [95% confidence interval, CI 0.39-0.87]) and a 39% reduction in the odds of breastfeeding for at least 6 weeks (aOR 0.61 [95% CI 0.41-0.92]) when compared with those without tobacco and substance use. Conclusion: The odds of breastfeeding initiation and continuation are significantly lower among individuals with both prenatal tobacco and illicit substance use. Future studies are needed to identify barriers to breastfeeding within this population, to inform patient-centered interventions aimed at overcoming these barriers.

Substance Use and Utilization of Prenatal and Postpartum Care.

OBJECTIVES: Prenatal and postpartum care for women with substance use is important due to increased risk of poor health outcomes. The influence of substance use on perinatal care utilization is not well characterized, especially postpartum care. The objective of this study was to examine the effect of substance use during pregnancy on prenatal and postpartum care utilization in a nationally representative sample and to identify maternal characteristics associated with inadequate prenatal and postpartum care among women with substance use. METHODS: Pregnancy Risk Assessment Monitoring System data (2016-2018) from 8 states were used for this study. Logistic regression models adjusted for complex survey weights and confounder variables were used to estimate the odds of not receiving adequate prenatal care and postpartum care. Weighted Rao-Scott chi-square tests were used to examine maternal characteristics associated with care utilization among women who reported substance use during pregnancy. RESULTS: The study included 15,131 women, with 5.3% who reported illicit substance use during pregnancy. In multivariable models, substance use was associated with an increase in the odds of not receiving adequate prenatal care (OR 1.69, CI 1.32, 2.17) and not receiving postpartum care (OR: 1.47, CI 1.10, 1.95). Among women who reported substance use, depression and smoking status were associated with not receiving adequate prenatal or postpartum care. CONCLUSIONS: Substance use during pregnancy is independently associated with disparities in prenatal and postpartum care access. Future studies are needed to identify how barriers lead to care inequalities and importantly, to identify strategies to improve care utilization.

Pricing indirect emissions accelerates low-carbon transition of US light vehicle sector.

Large-scale electric vehicle adoption can greatly reduce emissions from vehicle tailpipes. However, analysts have cautioned that it can come with increased indirect emissions from electricity and battery production that are not commonly regulated by transport policies. We combine integrated energy modeling and life cycle assessment to compare optimal policy scenarios that price emissions at the tailpipe only, versus both tailpipe and indirect emissions. Surprisingly, scenarios that also price indirect emissions exhibit higher, rather than reduced, sales of electric vehicles, while yielding lower cumulative tailpipe and indirect emissions. Expected technological change ensures that emissions from electricity and battery production are more than offset by reduced emissions of gasoline production. Given continued decarbonization of electricity supply, results show that a large-scale adoption of electric vehicles is able to reduce CO2 emissions through more channels than previously expected. Further, carbon pricing of stationary sources will also favor electric vehicles.

Effectiveness of Interdisciplinary Leadership Training for Early Career Professionals in the Field of Developmental Disabilities.

INTRODUCTION: The purpose of this paper is to describe the curriculum and self-reported outcomes on measures of interdisciplinary leadership skills for work within the field of developmental disabilities from trainees in one interprofessional training program. METHODS: The paper highlights one program's curriculum and strategy for capturing self-report survey measures from trainees in cohorts from 2014-2018 (n = 86) on two surveys (Interdisciplinary Attitudes and Skills and Leadership Self-Evaluation Form) and three time points across the training year: before training (T1), mid-year (T2), and after training (T3). RESULTS: Data from 86 trainees are reported including demographics (nearly 80% white, 92% female), non-descriptive statistics due to non-normative samples, and tertiles demonstrating changes between time points. Significant differences between medians are reported between T1-T3 specifically related to utilizing interdisciplinary skills and gains in leadership competencies. Specific utilization of skills was reported to be 'Greatly' attributable to the LEND program related to sharing ideas and asking for help across disciplines. DISCUSSION: Trainees' self-report from before training to after training indicates an increase in competence and utilization of interdisciplinary skills to be expected from participation in the curriculum. Self-report measures are.

Brief Report: Typical Visual Updating in Autism.

Individuals with Autism Spectrum Disorder (ASD) can struggle with visual updating. In a previous picture morphing study (Burnett and Jellema 2012) adults with ASD recognized the second picture significantly later when seeing one picture gradually changing into another. The aim of the current study was to test whether this previously reported perceptual atypicality may be due to general perceptual deficits. We therefore employed a modified picture morphing task. Against expectations, people with ASD showed typical performance in the task and no general perceptual deficits in relation to the picture morphing paradigm. Our results suggest that reported difficulties with visual updating in ASD may be due to temporal task restrictions and do not reflect a genuine problem with visual updating.

Dissecting the complexity of biomolecular condensates.

Biomolecular condensates comprise a diverse and ubiquitous class of membraneless organelles. Condensate assembly is often described by liquid-liquid phase separation. While this process explains many key features, it cannot account for the compositional or architectural complexity that condensates display in cells. Recent work has begun to dissect the rich network of intermolecular interactions that give rise to biomolecular condensates. Here, we review the latest results from theory, simulations and experiments, and discuss what they reveal about the structure-function relationship of condensates.

Beyond Equilibrium Phase Diagrams: Enzymatic Activity Shakes Up Bacterial Condensates.

Phase separation is a thermodynamic process, but cells are inherently out of equilibrium. Guilhas et al. (2020) identify an active process through which an ATP-dependent motor controls the number and position of biomolecular condensates in bacteria.

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation.

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

Nucleolar Organization and Functions in Health and Disease.

The nucleolus is a prominent, membraneless compartment found within the nucleus of eukaryotic cells. It forms around ribosomal RNA (rRNA) genes, where it coordinates the transcription, processing, and packaging of rRNA to produce ribosomal subunits. Recent efforts to characterize the biophysical properties of the nucleolus have transformed our understanding of the assembly and organization of this dynamic compartment. Indeed, soluble macromolecules condense from the nucleoplasm to form nucleoli through a process called liquid-liquid phase separation. Individual nucleolar components rapidly exchange with the nucleoplasm and separate within the nucleolus itself to form distinct subcompartments. In addition to its essential role in ribosome biogenesis, the nucleolus regulates many aspects of cell physiology, including genome organization, stress responses, senescence and lifespan. Consequently, the nucleolus is implicated in several human diseases, such as Hutchinson-Gilford progeria syndrome, Diamond-Blackfan anemia, and various forms of cancer. This Special Issue highlights new insights into the physical and molecular mechanisms that control the architecture and diverse functions of the nucleolus, and how they break down in disease.

Evaluation of Lanthanide-Doped Upconverting Nanoparticles for in Vitro and in Vivo Applications.

Because of their unique physicochemical properties, lanthanide-doped upconverting nanoparticles (Ln-UCNPs) have exceptional potential for biological applications. However, the use in biological systems is hampered by the limited understanding of their bionano interactions. Our multidisciplinary study has generated these insights through in-depth and quantitative analyses. The Ln-UCNPs examined here are spherical, monodisperse, and stable in aqueous environments. We show that Ln-UCNPs were associated with HeLa (cervical cancer) and LLC-PK1 (renal proximal tubule) cells and were nontoxic over a wide concentration range. Multiple biomarkers were assessed to monitor the cellular homeostasis in Ln-UCNP-treated cells. To this end, we evaluated the nuclear lamina, nucleoli, and nuclear transport factors. Single-cell analyses quantified the impact on Nrf2 and NF-κB, two transcription factors that control stress and immune responses. Moreover, we measured Ln-UCNP-induced changes in the abundance of molecular chaperones. Collectively, in vitro studies confirmed that Ln-UCNPs are nontoxic and trigger minor cellular stress responses. This lack of toxicity was verified in vivo, using the model organism Caenorhabditis elegans. The compatibility with biological systems prompted us to assess Ln-UCNPs as potential contrast agents for magnetic resonance imaging. We demonstrated that the Ln-UCNPs examined here were especially suitable as T2 contrast agents; they clearly outperformed the clinically used Gadovist. Taken together, our interdisciplinary work provides robust evidence for the nontoxicity of Ln-UCNPs. This sets the stage for the translation of Ln-UCNP for use in complex biological systems.

Contribution of Satellite-Derived Aerosol Optical Depth PM2.5 Bayesian Concentration Surfaces to Respiratory-Cardiovascular Chronic Disease Hospitalizations in Baltimore, Maryland.

The fine particulate matter baseline (PMB), which includes PM2.5 monitor readings fused with Community Multiscale Air Quality (CMAQ) model predictions, using the Hierarchical Bayesian Model (HBM), is less accurate in rural areas without monitors. To address this issue, an upgraded HBM was used to form four experimental aerosol optical depth (AOD)-PM2.5 concentration surfaces. A case-crossover design and conditional logistic regression evaluated the contribution of the AOD-PM2.5 surfaces and PMB to four respiratory-cardiovascular hospital events in all 99 12 km2 CMAQ grids, and in grids with and without ambient air monitors. For all four health outcomes, only two AOD-PM2.5 surfaces, one not kriged (PMC) and the other kriged (PMCK), had significantly higher Odds Ratios (ORs) on lag days 0, 1, and 01 than PMB in all grids, and in grids without monitors. In grids with monitors, emergency department (ED) asthma PMCK on lag days 0, 1 and 01 and inpatient (IP) heart failure (HF) PMCK ORs on lag days 01 were significantly higher than PMB ORs. Warm season ORs were significantly higher than cold season ORs. Independent confirmation of these results should include AOD-PM2.5 concentration surfaces with greater temporal-spatial resolution, now easily available from geostationary satellites, such as GOES-16 and GOES-17.