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Difference-in-differences (DiD) is a powerful, quasi-experimental research design widely used in longitudinal policy evaluations with health outcomes. However, DiD designs face several challenges to ensuring reliable causal inference, such as when policy settings are more complex. Recent economics literature has revealed that DiD estimators may exhibit bias when heterogeneous treatment effects, a common consequence of staggered policy implementation, are present. To deepen our understanding of these advancements in epidemiology, in this methodologic primer, we start by presenting an overview of DiD methods. We then summarize fundamental problems associated with DiD designs with heterogeneous treatment effects and provide guidance on recently proposed heterogeneity-robust DiD estimators, which are increasingly being implemented by epidemiologists. We also extend the discussion to violations of the parallel trends assumption, which has received less attention. Last, we present results from a simulation study that compares the performance of several DiD estimators under different scenarios to enhance understanding and application of these methods.

School racial segregation is increasingly recognized as a threat to US public health: rising segregation in recent decades has been linked to a range of poor health outcomes for Black Americans. Key theorized mediators of these harms remain underexamined, including experiences of interpersonal and institutional racism driving increased stress, and peers health behaviors influencing students own. Using cross-sectional survey data on a national sample of adolescents, we investigated associations between school segregation and these two potential mediating pathways, operationalized as adolescents perceptions of prejudice from fellow students and the health behaviors of their peers (drinking and smoking). We further investigated whether associations were modified by individual race/ethnicity and school racial composition. Pooling across all schools and students, higher levels of school segregation were associated with decreased perceptions of peer prejudice (OR 0.54, 95% CI = 0.34-0.86), but not with peers health behaviors. However, this masked important differences by respondents race/ethnicity and school racial/ethnic composition. In predominantly White schools, school segregation was not associated with Black students perceptions of peers prejudice, but higher levels of segregation were associated with increased rates of peers drinking and smoking. In predominantly non-White schools, in contrast-where most Black students are educated-higher levels of school segregation were not associated with perceived peer prejudice nor unhealthier peer behaviors for Black students (in fact, peers health behaviors improved). And across both school types, higher levels of district segregation were associated with lower odds of reporting peer prejudice among non-Black students of color. Our findings suggest that the paths between school segregation and poor health depend on the type of school children attend in segregated districts. In schools predominantly serving students of color, structural factors upheld by school segregation-i.e., material, educational, disciplinary, or economic disadvantage-likely dominate over peer behaviors as the primary drivers of segregations health harms.

Labyrinthulomycetes are a group of ubiquitous and diverse unicellular Stramenopiles and have long been known for their vital role in ocean carbon cycling. However, their ecological function from the perspective of organic matter degradation remains poorly understood. This study reports high-quality genomes of two newly isolated Labyrinthulomycetes strains, namely, Botryochytrium sp. strain S-28 and Oblongichytrium sp. strain S-429, and provides molecular analysis of their ecological functions using comparative genomics and a biochemical assay. Our results suggest that Labyrinthulomycetes may occupy multiple ecological niches in marine ecosystems because of the significant differences in gene function among different genera. Certain strains could degrade wheat bran independently by secreting cellulase. The key glycoside hydrolase families (GH1, GH5, and GH9) related to cellulase and the functional domains of carbohydrate-active enzymes (CAZymes) were more enriched in their genomes. This group can actively participate in marine biochemical cycles as decomposers. In contrast, other strains that could not produce cellulase may thrive as "leftover scavengers" and act as a source of nutrients to the higher-trophic-level plankton. In addition, our findings emphasize the dual roles of endoglucanase, acting as both exo- and endoglucanases, in the process of cellulose degradation. Using genomic, biochemical, and phylogenetic analyses, our study provides a broader insight into the nutritional patterns and ecological functions of Labyrinthulomycetes. IMPORTANCE Unicellular heterotrophic eukaryotes are an important component of marine ecosystems. However, their ecological functions and modes of nutrition remain largely unknown. Our current understanding of marine microbial ecology is incomplete without integrating these heterotrophic microeukaryotes into the food web models. This study focuses on the unicellular fungus-like protists Labyrinthulomycetes and provides two high-quality genomes of cellulase-producing Labyrinthulomycetes. Our study uncovers the basis of their cellulase production by deciphering the results of genomic, biochemical, and phylogenetic analyses. This study instigates a further investigation of the molecular mechanism of organic matter utilization by Labyrinthulomycetes in the world's oceans.

Proper nutrition is critical for maternal and neonatal health. In January 2017, the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) in San Francisco, California, began providing an additional $40 per month in fruit and vegetable (F&V) benefits to pregnant clients with the goal of improving food security and nutrition-related outcomes. We evaluated whether pregnant women on WIC who received this additional F&V benefit exhibited better perinatal and birth outcomes compared with those who received standard WIC benefits. We used 2010-2019 birth certificate data from the National Center for Health Statistics. The intervention group consisted of WIC participants living in San Francisco (SF) County (intervention county) and whose first trimester started after January 2017. We used a quasi-experimental synthetic control method to compare trends between the intervention and control groups (a weighted sample of other California counties that did not distribute additional F&V benefits). Outcomes included low birth weight, preterm birth, small-for-gestational-age, gestational diabetes, and gestational weight gain. No significant differences in maternal and neonatal outcomes among WIC recipients in SF and synthetic control group were observed after the F&V benefits were distributed. Prior studies have shown that additional F&V benefits have positive effects on maternal and infant outcomes, indicating that F&V vouchers are a promising strategy for supporting equitable health outcomes. Our null results suggest that more rigorous research is needed to determine their optimal dose and duration, especially in high-cost-of-living areas, and to examine more upstream and structural interventions.

Residential racial segregation is a key manifestation of anti-Black structural racism, thought to be a fundamental cause of poor health; evidence has shown that it yields neighborhood disinvestment, institutional discrimination, and targeting of unhealthy products like tobacco and alcohol. Yet research on the long-term impacts of childhood exposure to residential racial segregation is limited. Here, we analyzed data on 1823 Black participants in the Panel Study of Income Dynamics, estimating associations between childhood segregation trajectories and young adult health. Black young adults who consistently lived in high-segregation neighborhoods throughout childhood experienced unhealthier smoking and drinking behaviors and higher odds of obesity compared to other trajectory groups, including children who moved into or out of high-segregation neighborhoods. Results were robust to controls for neighborhood and family poverty. Findings underscore that for Black children who grow up in segregated neighborhoods, the roots of structurally-determined health inequities are established early in life.

Residential racial segregation in the U.S. has been hypothesized as a fundamental cause driving health disparities across racial groups. Potential mechanisms include economic and social marginalization, subsequent constrained opportunities, and high stress. Yet evidence on residential segregation's association with health among Black and White children-particularly longitudinally-is sparse. This study aims to address this gap. We used data from the Panel Study of Income Dynamics (PSID), a national longitudinal study of U.S. households, analyzing information on 1,251 Black and 1,427 White children who participated in the Child Development Supplement (CDS) at least twice (survey waves 1997, 2002, 2007, 2014). We fit individual fixed-effects models to estimate the within-person association of neighborhood-level residential segregation, measured with local Getis-Ord G* statistics, with three outcomes (general health, weight status, and behavioral problems). We examined heterogeneous effects by age and sex. We also examined associations between health and childhood segregation trajectories, i.e., the pattern of children's residential segregation exposures from birth through when their health outcomes were measured, providing additional insight on dynamic experiences of segregation. In fixed effects models, among Black children, higher segregation was associated with worse self-rated health, especially for Black children who were older (aged 11-17 years). In trajectory models, among White children, moving out of highly segregated neighborhoods was associated with a lower probability of poor self-rated health, while moving into those neighborhoods or back and forth between neighborhood types were both associated with increased behavioral problems. Our findings highlight the importance of early-life residential segregation in shaping persistent racial health disparities, as well as the costs of segregation for all children living in highly segregated neighborhoods.

BackgroundCardiovascular disease (CVD) disproportionately affects Black adults in the United States. This is increasingly acknowledged to be due to inequitable distribution of health-promoting resources. One potential contributor is inequities in educational opportunities, although it is unclear what aspects of education are most salient. School racial segregation may affect cardiovascular health by increasing stress, constraining socioeconomic opportunities, and altering health behaviors. We investigated the association between school segregation and Black adults' CVD risk.Methods and findingsWe leveraged a natural experiment created by quasi-random (i.e., arbitrary) timing of local court decisions since 1991 that released school districts from court-ordered desegregation. We used the Panel Study of Income Dynamics (PSID) (1991 to 2017), linked with district-level school segregation measures and desegregation court order status. The sample included 1,053 Black participants who ever resided in school districts that were under a court desegregation order in 1991. The exposure was mean school segregation during observed schooling years. Outcomes included several adult CVD risk factors and outcomes. We fitted standard ordinary least squares (OLS) multivariable linear regression models, then conducted instrumental variables (IV) analysis, using the proportion of schooling years spent in districts that had been released from court-ordered desegregation as an instrument. We adjusted for individual- and district-level preexposure confounders, birth year, and state fixed effects. In standard linear models, school segregation was associated with a lower probability of good self-rated health (-0.05 percentage points per SD of the segregation index; 95% CI: -0.08, -0.03; p < 0.001) and a higher probability of binge drinking (0.04 percentage points; 95% CI: 0.002, 0.07; p = 0.04) and heart disease (0.01 percentage points; 95% CI: 0.002, 0.15; p = 0.007). IV analyses also found that school segregation was associated with a lower probability of good self-rated health (-0.09 percentage points; 95% CI: -0.17, -0.02, p = 0.02) and a higher probability of binge drinking (0.17 percentage points; 95% CI: 0.04, 0.30, p = 0.008). For IV estimates, only binge drinking was robust to adjustments for multiple hypothesis testing. Limitations included self-reported outcomes and potential residual confounding and exposure misclassification.ConclusionsSchool segregation exposure in childhood may have longstanding impacts on Black adults' cardiovascular health. Future research should replicate these analyses in larger samples and explore potential mechanisms. Given the recent rise in school segregation, this study has implications for policies and programs to address racial inequities in CVD.

ObjectivesFew researchers have evaluated whether school racial segregation, a key manifestation of structural racism, affects child health, despite its potential impacts on school quality, social networks, and stress from discrimination. We investigated whether school racial segregation affects Black children's health and health behaviors.MethodsWe estimated the association of school segregation with child health, leveraging a natural experiment in which school districts in recent years experienced increased school segregation. School segregation was operationalized as the Black-White dissimilarity index. We used ordinary least squares models as well as quasi-experimental instrumental variables analysis, which can reduce bias from unobserved confounders. Data from the Child Development Supplement of the Panel Study of Income Dynamics (1997-2014, n = 1248 Black children) were linked with district-level school segregation measures. Multivariable regressions were adjusted for individual-, neighborhood-, and district-level covariates. We also performed subgroup analyses by child sex and age.ResultsIn instrumental variables models, a one standard deviation increase in school segregation was associated with increased behavioral problems (2.53 points on a 27-point scale; 95% CI, 0.26 to 4.80), probability of having ever drunk alcohol (0.23; 95% CI, 0.049 to 0.42), and drinking at least monthly (0.20; 95% CI, 0.053 to 0.35). School segregation was more strongly associated with drinking behaviors among girls.ConclusionsSchool segregation was associated with worse outcomes on several measures of well-being among Black children, which may contribute to health inequities across the life span. These results highlight the need to promote school racial integration and support Black youth attending segregated schools.

BackgroundThraustochytrids are unicellular fungal-like marine protists with ubiquitous existence in marine environments. They are well-known for their ability to produce high-valued omega-3 polyunsaturated fatty acids (ω-3-PUFAs) (e.g., docosahexaenoic acid (DHA)) and hydrolytic enzymes. Thraustochytrid biomass has been estimated to surpass that of bacterioplankton in both coastal and oceanic waters indicating they have an important role in microbial food-web. Nevertheless, the molecular pathway and regulatory network for PUFAs production and the molecular mechanisms underlying ecological functions of thraustochytrids remain largely unknown.ResultsThe genomes of two thraustochytrids strains (Mn4 and SW8) with ability to produce DHA were sequenced and assembled with a hybrid sequencing approach utilizing Illumina short paired-end reads and Pacific Biosciences long reads to generate a highly accurate genome assembly. Phylogenomic and comparative genomic analyses found that DHA-producing thraustochytrid strains were highly similar and possessed similar gene content. Analysis of the conventional fatty acid synthesis (FAS) and the polyketide synthase (PKS) systems for PUFAs production only detected incomplete and fragmentary pathways in the genome of these two strains. Surprisingly, secreted carbohydrate active enzymes (CAZymes) were found to be significantly depleted in the genomes of these 2 strains as compared to other sequenced relatives. Furthermore, these two strains possess an expanded gene repertoire for signal transduction and self-propelled movement, which could be important for their adaptations to dynamic marine environments.ConclusionsOur results demonstrate the possibility of a third PUFAs synthesis pathway besides previously described FAS and PKS pathways encoded in the genome of these two thraustochytrid strains. Moreover, lack of a complete set of hydrolytic enzymatic machinery for degrading plant-derived organic materials suggests that these two DHA-producing strains play an important role as a nutritional source rather than a nutrient-producer in marine microbial-food web. Results of this study suggest the existence of two types of saprobic thraustochytrids in the world's ocean. The first group, which does not produce cellulosic enzymes and live as 'left-over' scavenger of bacterioplankton, serves as a dietary source for the plankton of higher trophic levels and the other possesses capacity to live on detrital organic matters in the marine ecosystems.