Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells.

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

RATIONALE: Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. METHODS: Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. RESULTS: We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 µL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. CONCLUSIONS: GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.

An ecological view of executive function in young children: Variation in and predictors of executive function skills over one school year.

For many years, researchers studied executive functions (EFs) in the laboratory with a focus on understanding an individual child's development and brain processes in a controlled environment. Building on this foundational research, there is a growing interest in EFs in the context of a child's dynamic, social world, and the contextual and compositional factors influencing EF development. This paper provides a descriptive view of EFs in 1112 K-3 children from six schools in Phoenix, AZ, USA. The study's goals were to examine (1) variation in EF scores between and within schools and classrooms, (2) predictors of variation in children's spring EF scores, and (3) individual and compositional predictors of children's spring EF scores. Our findings indicate greater variation in children's EF within schools than between, with very little or no variation arising from differences between schools. Though we observed greater variation within classrooms than between them, a notable amount of variance in children's spring EF scores appears to arise from differences between classrooms. Classroom-level variables, including a fall leave-out classroom mean (without the students' own score) and the number of children in the top or bottom grade-level quartiles in each classroom, were significant predictors of variation in spring EF scores as well as in fall to spring changes in EF. In some cases, the classroom variables were stronger predictors than individual fall scores. Findings suggest that understanding variation and cultivating growth in EF skills requires intervention, measurement, and analytic approaches that extend beyond the individual to include compositional features of the classroom environment. RESEARCH HIGHLIGHTS: Variation in children's EF scores (n = 1112 children) was greater within schools (n = 6 schools) than between, with very little or no variance arising from differences between schools. While variance was greater within classrooms than between (n = 67 classrooms), a notable amount of variance in children's spring EF scores appears to arise from differences between classrooms. Classroom-level variables (e.g., leave-out mean, number of children in the top or bottom grade-level quartiles in each classroom) were significant predictors of variation and of changes in spring EF outcomes. In some cases, the classroom variables were stronger predictors of spring EF than individual fall scores.

An observational approach for exploring variability in young children's regulation-related skills within classroom contexts.

Prior work has conceptualized children's executive function and self-regulation skills as relatively stable across short periods of time. Grounded in long-standing contextual theories of human development, this study introduces a new observational tool for measuring children's regulatory skills across different naturally occurring situations within early childhood classrooms. Using 460 observations of 91 children (M age = 5.54 years) in 16 socio-demographically diverse Prekindergarten and Kindergarten classrooms, we found that this tool-the Regulation-Related Skills Measure (RRSM)-reliably captured observed dimensions of young children's attention control and inhibitory control, but failed to appropriately represent more "internal" regulatory processes (e.g., working memory). Associations between the RRSM and other measures of children's executive function and self-regulation (i.e., direct assessments, adult reports) were low to moderate (r = 0.03 to 0.44), suggesting these tools are likely to be complementary in that they provide overlapping but ultimately distinct information regarding children's regulatory performance. Finally, results suggested substantial within-child variation in regulatory behaviors across different situations within the classroom, with the same children demonstrating consistently stronger attention control and inhibitory control during transitions than during either teacher- or student-directed activities. These findings underscore the situationally-dependent nature of children's self-regulatory performance, with implications for both theory and practice. Future research is needed to replicate these findings in more diverse, representative samples of children.

An ecological perspective on early educator well-being at the start of the COVID-19 pandemic.

Early educator well-being is increasingly understood as a critical ingredient of high-quality early education and care. The COVID-19 pandemic has threatened educator well-being by exacerbating existing stressors and introducing novel stressors to all aspects of early educators' lives, and early educators have had differential access to resources to cope with these new circumstances. Using survey data collected between April and June 2020 with a sample of 666 early educators in community-based center, family child care, Head Start, and public school prekindergarten programs across Massachusetts, we document the pandemic's initial influence on educators' sense of well-being. Adopting an ecological perspective, we consider educator-, program-, and community-level factors that may be associated with reported changes in well-being. Most educators indicated that their mental and financial well-being had been affected. These changes were not systematically associated with most contextual factors, although there was clear evidence of variability in reported impacts by provider type. These findings underscore the need to support educator well-being, as well as to create policy solutions that meet the heterogeneous needs of this essential workforce.

Dissimulation strategies on standard neuropsychological tests: A qualitative investigation.

OBJECTIVE: Most previous studies in the area of malingering neuropsychological deficits have focused on the development and validation of measures to detect falsification of symptoms or suboptimal performance. The present study employs qualitative methods to investigate strategies employed by individuals attempting to feign cognitive impairment. This study aims to achieve a richer and more detailed understanding of the phenomenon of feigning. METHOD: Detailed semi-structured interviews were administered to 15 non-neurological individuals instructed to feign cognitive impairment on a standard neuropsychological test battery. The interviews, which considered the strategies used and the thinking underlying participants' choices, were subjected to Thematic Analysis. RESULTS: Thematic Analysis revealed three main organising themes. Participants described Using Strategies, on specific tests and generally across the battery, Having a Rationale for their decisions and spontaneously commented on their Experience of the Task. CONCLUSION: The findings of the present study reveal numerous potentially useful identifiers of feigning strategies, including many not previously reported. The resulting themes point to the development of more effective methods for detecting feigned cognitive impairments and could have a significant impact on the way that neuropsychological testing sessions are conducted.

The Influence of Conduct Problems and Callous-Unemotional Traits on Academic Development Among Youth.

The present study attempted to address developmental differences within the large group of youth with conduct problems through an examination of the relationship between callous-unemotional traits and academic outcomes in an effort to expand the field's understanding of heterogeneity in outcomes associated with behavior problems. Data were collected from a cohort of 3rd grade students (N = 942; 51 % female; 45.6 % Hispanic/Latino, 41.1 % Black/African American, 4.7 % Non-Hispanic White; mean age = 8.07 years) in eighteen public elementary schools, as well as their parents and teachers. Hierarchical linear modeling revealed that callous-unemotional traits were associated with lower quality student-teacher relationships and worse performance on standardized math and reading exams over and above the effects of conduct problems. These findings suggest that school-based interventions may be particularly effective in ameliorating some of the deficits noted within this subset of youth exhibiting conduct problems. This finding has important policy implications as the field of developmental science attempts to design and enrich programs that focus on improving social-emotional learning.

Measuring psychosocial environments using individual responses: an application of multilevel factor analysis to examining students in schools.

Interest in understanding how psychosocial environments shape youth outcomes has grown considerably. School environments are of particular interest to prevention scientists as many prevention interventions are school-based. Therefore, effective conceptualization and operationalization of the school environment is critical. This paper presents an illustration of an emerging analytic method called multilevel factor analysis (MLFA) that provides an alternative strategy to conceptualize, measure, and model environments. MLFA decomposes the total sample variance-covariance matrix for variables measured at the individual level into within-cluster (e.g., student level) and between-cluster (e.g., school level) matrices and simultaneously models potentially distinct latent factor structures at each level. Using data from 79,362 students from 126 schools in the National Longitudinal Study of Adolescent to Adult Health (formerly known as the National Longitudinal Study of Adolescent Health), we use MLFA to show how 20 items capturing student self-reported behaviors and emotions provide information about both students (within level) and their school environment (between level). We identified four latent factors at the within level: (1) school adjustment, (2) externalizing problems, (3) internalizing problems, and (4) self-esteem. Three factors were identified at the between level: (1) collective school adjustment, (2) psychosocial environment, and (3) collective self-esteem. The finding of different and substantively distinct latent factor structures at each level emphasizes the need for prevention theory and practice to separately consider and measure constructs at each level of analysis. The MLFA method can be applied to other nested relationships, such as youth in neighborhoods, and extended to a multilevel structural equation model to better understand associations between environments and individual outcomes and therefore how to best implement preventive interventions.

Social and Emotional Learning Services and Child Outcomes in Third Grade: Evidence from a Cohort of Head Start Participants.

A variety of universal school-based social and emotional learning (SEL) programs have been designed in the past decades to help children improve social-emotional and academic skills. Evidence on the effectiveness of SEL programs has been mixed in the literature. Using data from a longitudinal follow-up study of children (n = 414) originally enrolled in a clustered randomized controlled trial (RCT) when they were in Head Start, we examined whether universal SEL services in third grade were associated with the development of children from disadvantaged families. We took advantage of pairwise matching in the RCT design to compare children who had similar family background and preschool experiences but received different doses of SEL services in third grade. The results showed that the frequent (i.e., weekly to daily) exposure to SEL opportunities was associated with favorable social-emotional and academic development in third grade, including increased social skills, student-teacher relationship, and academic skills, as well as reduced impulsiveness.

Translating multilevel theory into multilevel research: challenges and opportunities for understanding the social determinants of psychiatric disorders.

INTRODUCTION: The observation that features of the social environment, including family, school, and neighborhood characteristics, are associated with individual-level outcomes has spurred the development of dozens of multilevel or ecological theoretical frameworks in epidemiology, public health, psychology, and sociology, among other disciplines. Despite the widespread use of such theories in etiological, intervention, and policy studies, challenges remain in bridging multilevel theory and empirical research. METHODS: This paper set out to synthesize these challenges and provide specific examples of methodological and analytical strategies researchers are using to gain a more nuanced understanding of the social determinants of psychiatric disorders, with a focus on children's mental health. To accomplish this goal, we begin by describing multilevel theories, defining their core elements, and discussing what these theories suggest is needed in empirical work. In the second part, we outline the main challenges researchers face in translating multilevel theory into research. These challenges are presented for each stage of the research process. In the third section, we describe two methods being used as alternatives to traditional multilevel modeling techniques to better bridge multilevel theory and multilevel research. These are (1) multilevel factor analysis and multilevel structural equation modeling; and (2) dynamic systems approaches. CONCLUSIONS: Through its review of multilevel theory, assessment of existing strategies, and examination of emerging methodologies, this paper offers a framework to evaluate and guide empirical studies on the social determinants of child psychiatric disorders as well as health across the life course.

Implications of different methods for specifying classroom composition of externalizing behavior and its relationship to social-emotional outcomes.

In this paper, we examine common methods for using individual-level data to represent classroom composition by examining exemplary studies that thoughtfully incorporate such measures. Building on these studies, and using data from the Chicago School Readiness Project (CSRP), this paper examines theoretical and analytical implications of a set of different transformations of individual ratings of child externalizing behaviors in order to examine and compare the influence of these representations of classroom composition on Kindergarten internalizing behaviors, social competence, and attention/impulsivity problems. Results indicate that each Kindergarten outcome is influenced by distinct aspects of classroom composition of externalizing behaviors. Kindergarten internalizing behaviors are positively associated with the proportion of children in the Head Start classroom who started with externalizing scores above the 75th percentile regardless of the average value of externalizing behaviors in the classroom. In contrast, Kindergarten social competence is predicted by three aspects of the classroom distribution of externalizing behaviors in the fall of Head Start-the classroom mean, standard deviation, and skew. Finally, Kindergarten attention/impulsivity problems were not associated with any aspect of classroom composition of externalizing behavior examined in this paper.

Prenatal exposure to the mineralocorticoid receptor antagonist spironolactone disrupts hippocampal area CA2 connectivity and alters behavior in mice.

In the brain, the hippocampus is enriched with mineralocorticoid receptors (MR; Nr3c2), a ligand-dependent transcription factor stimulated by the stress hormone corticosterone in rodents. Recently, we discovered that MR is required for the acquisition and maintenance of many features of mouse area CA2 neurons. Notably, we observed that immunofluorescence for the vesicular glutamate transporter 2 (vGluT2), likely representing afferents from the supramammillary nucleus (SuM), was disrupted in the embryonic, but not postnatal, MR knockout mouse CA2. To test whether pharmacological perturbation of MR activity in utero similarly disrupts CA2 connectivity, we implanted slow-release pellets containing the MR antagonist spironolactone in mouse dams during mid-gestation. After confirming that at least one likely active metabolite crossed from the dams' serum into the embryonic brains, we found that spironolactone treatment caused a significant reduction of CA2 axon fluorescence intensity in the CA1 stratum oriens, where CA2 axons preferentially project, and that vGluT2 staining was significantly decreased in both CA2 and dentate gyrus in spironolactone-treated animals. We also found that spironolactone-treated animals exhibited increased reactivity to novel objects, an effect similar to what is seen with embryonic or postnatal CA2-targeted MR knockout. However, we found no difference in preference for social novelty between the treatment groups. We infer these results to suggest that persistent or more severe disruptions in MR function may be required to interfere with this type of social behavior. These findings do indicate, though, that developmental disruption in MR signaling can have persistent effects on hippocampal circuitry and behavior.

Craters on the melanoma surface facilitate tumor-immune interactions and demonstrate pathologic response to checkpoint blockade in humans.

Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8+ T cells in the tumor. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8+ T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response.

Unpacking the Black Box of the CSRP Intervention: The Mediating Roles of Teacher-child Relationship Quality and Self-regulation.

This study examines the theory of change of the Chicago School Readiness Project (CSRP), testing a sequence of theory-derived mediating mechanisms including the quality of teacher-child relationships and children's self-regulation. The CSRP is a multi-component teacher- and classroom-focused intervention, and its cluster-randomized efficacy trial was conducted in 35 Head Start-funded classrooms. A series of increasingly complex and conservative structural equation models indicate that the CSRP carries its effects on children's academic and behavioral outcomes through changes in teacher-child relationship quality and children's self-regulation.

Does a Preschool Social and Emotional Learning Intervention Pay Off for Classroom Instruction and Children's Behavior and Academic Skills? Evidence From the Foundations of Learning Project.

This article tests the hypothesis that children's learning environment will improve through a social and emotional learning (SEL) intervention that provides preschool teachers with new skills to manage children's disruptive behavior by reporting results from the Foundations of Learning (FOL) Demonstration, a place-randomized, experimental evaluation conducted by MDRC. Research Findings: Findings demonstrate that the FOL intervention improved teachers' ability to address children's behavior problems and to provide a positive emotional climate in their classrooms. Importantly, the FOL intervention also improved the number of minutes of instructional time, although the quality of teachers' instruction was not improved. Finally, FOL benefited children's observed behavior in classrooms, with lower levels of conflictual interactions and, at the trend level, higher levels of engagement in classrooms activities, relative to similar students randomly assigned to control classrooms. Practice or Policy: This study is one of an emerging body of research on the efficacy of SEL programs for preschool children living in poverty. Understanding the value-added of these programs (e.g., in increased instructional time and increased classroom engagement) as well as their limitations (e.g., in teachers' instructional quality and children's academic skills) will help us design the next set of more effective interventions for low-income children.

TEACHING THROUGH INTERACTIONS: Testing a Developmental Framework of Teacher Effectiveness in over 4,000 Classrooms.

Validating frameworks for understanding classroom processes that contribute to student learning and development is important to advance the scientific study of teaching. This article presents one such framework, Teaching through Interactions, which posits that teacher-student interactions are a central driver for student learning and organizes teacher-student interactions into three major domains. Results provide evidence that across 4,341 preschool to elementary classrooms (1) teacher-student classroom interactions comprise distinct emotional, organizational, and instructional domains; (2) the three-domain latent structure is a better fit to observational data than alternative one- and two-domain models of teacher-student classroom interactions; and (3) the three-domain structure is the best-fitting model across multiple data sets.

Exploring early educator burnout and process quality in a statewide sample.

Building on literature linking educators' psychosocial well-being to early education and care quality, this study analyzed early educators' (N = 648) reports of burnout across a range of group-based care types in one state and examined the relation of burnout to setting quality. Confirmatory factor analysis showed the burnout measure, a self-reported emotional exhaustion scale, had a one-factor structure and adequate internal consistency among educators working in a range of early education and care settings. Measured by the scale, educators on average reported infrequent feelings of burnout. There were small but statistically significant differences in burnout scores by setting type, with Head Start educators on average reporting modestly more frequent burnout symptoms than educators in community-based centers (ß=0.29,b = 0.30, SE = 0.13, p = 0.014) or family childcare settings (ß=0.57,b = 0.60, SE = 0.14, p < 0.001). Only one significant association was observed between educators' self-reported burnout scores and setting quality after accounting for educator and setting characteristics: a negative association with child involvement (ß =  - 0.09,b = -0.04, SE = 0.02, p = 0.03). These findings contribute to the field's understanding of burnout as a component of educator well-being and provide initial insight about targeting supports to improve educator well-being.

Academic Performance of Subsequent Schools and Impacts of Early Interventions: Evidence from a Randomized Controlled Trial in Head Start Settings.

The role of subsequent school contexts in the long-term effects of early childhood interventions has received increasing attention, but has been understudied in the literature. Using data from the Chicago School Readiness Project (CSRP), a cluster-randomized controlled trial conducted in Head Start programs, we investigate whether the intervention had differential effects on academic and behavioral outcomes in kindergarten if children attended high- or low-performing schools subsequent to the preschool intervention year. To address the issue of selection bias, we adopt an innovative method, principal score matching, and control for a set of child, mother, and classroom covariates. We find that exposure to the CSRP intervention in the Head Start year had significant effects on academic and behavioral outcomes in kindergarten for children who subsequently attended high-performing schools, but no significant effects on children attending low-performing schools. Policy implications of the findings are discussed.

Executive function, self-regulation skills, behaviors, and socioeconomic status in early childhood.

BACKGROUND AND OBJECTIVES: Prior research has established steep socioeconomic status (SES) disparities in children's cognitive skills at kindergarten entry. Yet, few studies have had comprehensive, multi-informant data to examine SES-related differences in foundational social and emotional skills and executive function. The objective of the current study is to systematically examine SES-related differences in young children's executive function (EF), self-regulation skills, and behaviors. METHODS: The current study analyzed data on 2,309 young children from the Early Learning Study at Harvard (ELS@H). Multi-method (direct-assessment and reports) and multi-informant (parents and early education and care educators) information on children's executive function, self-regulation skills, and internalizing, externalizing, and adaptive behaviors were used. A parametric framework employing Ordinary Least Squares (OLS) estimation was used to quantify the size of the SES-related differences in this set of children's foundational social-emotional skills. RESULTS: On average, there were differences of 0.24-0.45 SD for EF, 0.22-0.32 SD for self-regulation skills, and 0.27-0.54 SD for behaviors favoring children from the highest SES quartile of the distribution of SES relative to children from the lowest quartile. The SES-related differences were consistent across direct assessment, parent reports, and educator reports. Some differences were larger for older children relative to their younger counterparts. CONCLUSIONS: Findings indicate a need for comprehensive intervention efforts well before kindergarten entry aimed at closing early disparities in children's foundational social and emotional skills and executive function.

Changes in Children's Behavioral Health and Family Well-Being During the COVID-19 Pandemic.

OBJECTIVE: The coronavirus disease 2019 pandemic and associated public health measures have influenced all aspects of life for children and families. In this study, we examine changes in children's behavioral health and families' well-being at the start of the pandemic. METHOD: We used longitudinal data on 2880 children from 1 US state collected over 3 waves to compare family and child well-being before and after a state-wide stay-at-home advisory set in March 2020. We descriptively examined levels and changes in 4 child behavioral health outcomes (externalizing, internalizing, adaptive, and dysregulated behaviors) and 4 family well-being outcomes (parental mental health, parental stress, parent-child relationship conflict, and household chaos) across the preshutdown and postshutdown periods. Fixed effects regression models were used to predict within-child and within-family differences in preshutdown and postshutdown outcomes. RESULTS: Fixed effects analyses showed children's externalizing (0.09 points; 95% confidence interval [CI] 0.05-0.13), internalizing (0.04 points; 95% CI, 0.01-0.08), and dysregulated (0.11 points; 95% CI, 0.06-0.16) behaviors increased after the shutdown, whereas children's adaptive behaviors declined (-0.10 points; 95% CI, -0.15 to -0.05). Parental mental health issues (0.22 points; 95% CI, 0.17-0.27), parental stress (0.08 points; 95% CI, 0.03-0.12), parent-child relationship conflict (0.10 points; 95% CI, 0.04-0.16), and household chaos (0.10 points; 95% CI, 0.05-0.14) all increased relative to preshutdown levels. CONCLUSION: Many children experienced declines in behavioral health and many families experienced declines in well-being in the early months of the public health crisis, suggesting the need for family-focused and child-focused policies to mitigate these changes.