Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M.

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

It Is Surprisingly Difficult to Measure Income Segregation.

Recent studies have shown that U.S. Census- and American Community Survey (ACS)-based estimates of income segregation are subject to upward finite sampling bias (Logan et al. 2018; Logan et al. 2020; Reardon et al. 2018). We identify two additional sources of bias that are larger and opposite in sign to finite sampling bias: measurement error-induced attenuation bias and temporal pooling bias. The combination of these three sources of bias make it unclear how income segregation has trended. We formalize the three types of bias, providing a method to correct them simultaneously using public data from the decennial census and ACS from 1990 to 2015-2019. We use these methods to produce bias-corrected estimates of income segregation in the United States from 1990 to 2019. We find that (1) segregation is on the order of 50% greater than previously believed; (2) the increase from 2000 to the 2005-2009 period was much greater than indicated by previous estimates; and (3) segregation has declined since 2005-2009. Correcting these biases requires good estimates of the reliability of self-reported income and of the year-to-year volatility in neighborhood mean incomes.

Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems.

Engineered microbes can be used for producing value-added chemicals from renewable feedstocks, relieving the dependency on nonrenewable resources such as petroleum. These microbes often are composed of synthetic metabolic pathways; however, one major problem in establishing a synthetic pathway is the challenge of precisely controlling competing metabolic routes, some of which could be crucial for fitness and survival. While traditional gene deletion and/or coarse overexpression approaches do not provide precise regulation, cis-repressors (CRs) are RNA-based regulatory elements that can control the production levels of a particular protein in a tunable manner. Here, we describe a protocol for a generally applicable fluorescence-activated cell sorting technique used to isolate eight subpopulations of CRs from a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of 15 individual CRs with a broad range of protein production profiles. Using these new CRs, we demonstrated a change in production levels of a fluorescent reporter by over two orders of magnitude and further showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used four CRs to tune the production of the enzyme PpsA, involved in pyruvate to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning PpsA translation levels increased the overall titer of muconate. Therefore, CRs provide an approach to precisely tune protein levels in metabolic pathways and will be an important tool for other metabolic engineering efforts.

School racial segregation and long-term cardiovascular health among Black adults in the US: A quasi-experimental study.

BACKGROUND: Cardiovascular 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 FINDINGS: We 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. CONCLUSIONS: School 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.

School Racial Segregation and the Health of Black Children.

OBJECTIVES: Few 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. METHODS: We 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. RESULTS: In 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. CONCLUSIONS: School 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.

Phosphorylation and acetylation of mitochondrial transcription factor A promote transcription processivity without compromising initiation or DNA compaction.

Mitochondrial transcription factor A (TFAM) plays important roles in mitochondrial DNA compaction, transcription initiation, and in the regulation of processes like transcription and replication processivity. It is possible that TFAM is locally regulated within the mitochondrial matrix via such mechanisms as phosphorylation by protein kinase A and nonenzymatic acetylation by acetyl-CoA. Here, we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirmed using EMSAs that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM and provide an in-depth analysis of acetylated sites on TFAM. We show that both modifications of TFAM increase the processivity of mitochondrial RNA polymerase during transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification retains its full activity in transcription initiation. We conclude that TFAM phosphorylation by protein kinase A and nonenzymatic acetylation by acetyl-CoA are unlikely to occur at the mitochondrial DNA and that modified free TFAM retains its vital functionalities like compaction and transcription initiation while enhancing transcription processivity.

Increases in Income-Related Disparities in Early Elementary School Obesity, 1998-2014.

OBJECTIVE: Research shows that population-level rates of obesity, which rose dramatically from the 1970s through the mid-2000s, have since plateaued or even started to decline. However, overall improvements may mask differences in trends for different subgroups. For instance, obesity rates have continued to climb among low-income adolescents, leading to growing income-related gaps in obesity. By comparison, we know little about whether income-related disparities have also changed among elementary school children. To address this gap, we examined two cohorts of the Early Childhood Longitudinal Study - Kindergarten cohort, which followed children entering school in 1998 and 2010. We hypothesized that income-related disparities in obesity have also grown larger over time among young children. METHODS: We used data from nationally representative samples of children who entered kindergarten in 1998 and 2010. We documented rates of overweight and obesity from kindergarten through third grade, examined how rates differed for children from high- and low-income families, and tested whether income-related disparities changed over time. RESULTS: Rates of overweight and obesity were 2 to 5 percentage points higher in the later cohort, and overall increases masked substantial variation by income. Specifically, these increases were driven by children in lower-income households, resulting in substantially larger income-related disparities in overweight and obesity in the later cohort. CONCLUSIONS: As we hypothesized, income-related disparities in young children's obesity grew between 1998 and 2014. This suggests that efforts to curb increasing rates of obesity may have been more successful for higher-income families. We discuss potential mechanisms that may account for increasing disparities.

Long-Term Outcome of a Brief Intervention to Address Adolescent Drug Abuse in a School Setting.

The present study used data from a randomized controlled trial on brief interventions with adolescents to identify distinct longitudinal patterns of substance use and identify predictors, as well as outcomes associated with those use patterns. Data were originally collected for the purpose of evaluating two brief intervention conditions with adolescents who had been identified in a school setting as abusing alcohol or other drugs (total sample, N = 315). Adolescents were randomly assigned to a two-session adolescent only brief intervention (BI-A), a two-session adolescent- plus an additional parent session (BI-AP), or an assessment only control session (CON). We located 74 participants to assess them at approximately 3.5 years post-intervention. Three distinct cluster patterns were identified, including a low decreasing, moderate increasing, and high decreasing pattern of use. The low decreasing cluster was associated with the BI-A condition, mono-substance use, and comorbid anxiety symptoms at baseline. The moderate increasing cluster was associated with the BI-AP condition, polysubstance use, and comorbid conduct disorder symptoms at baseline. No variables were found to be predictive of membership within the high decreasing cluster. There were also no differences found between clusters on adjustment outcomes in young adulthood. Overall findings from this study support the long-term efficacy of a brief intervention, without parent involvement, for adolescents experiencing mild to moderate substance abuse problems. Findings also highlight the importance of early intervention and the tailoring of interventions to meet the unique needs of adolescents.

Educational Opportunity in Early and Middle Childhood: Using Full Population Administrative Data to Study Variation by Place and Age.

I use standardized test scores from roughly forty-five million students to describe the temporal structure of educational opportunity in more than eleven thousand school districts in the United States. Variation among school districts is considerable in both average third-grade scores and test score growth rates. The two measures are uncorrelated, indicating that the characteristics of communities that provide high levels of early childhood educational opportunity are not the same as those that provide high opportunities for growth from third to eighth grade. This suggests that the role of schools in shaping educational opportunity varies across school districts. Variation among districts in the two temporal opportunity dimensions implies that strategies to improve educational opportunity may need to target different age groups in different places.

From Census Tracts to Local Environments: An Egocentric Approach to Neighborhood Racial Change.

Most quantitative studies of neighborhood racial change rely on census tracts as the unit of analysis. However, tracts are insensitive to variation in the geographic scale of the phenomenon under investigation and to proximity among a focal tract's residents and those in nearby territory. Tracts may also align poorly with residents' perceptions of their own neighborhood and with the spatial reach of their daily activities. To address these limitations, we propose that changes in racial structure (i.e., in overall diversity and group-specific proportions) be examined within multiple egocentric neighborhoods, a series of nested local environments surrounding each individual that approximate meaningful domains of experience. Our egocentric approach applies GIS procedures to census block data, using race-specific population densities to redistribute block counts of whites, blacks, Hispanics, and Asians across 50-meter by 50-meter cells. For each cell, we then compute the proximity-adjusted racial composition of four different-sized local environments based on the weighted average racial group counts in adjacent cells. The value of this approach is illustrated with 1990-2000 data from a previous study of 40 large metropolitan areas. We document exposure to increasing neighborhood racial diversity during the decade, although the magnitude of this increase in diversity-and of shifts in the particular races to which one is exposed-differs by local environment size and racial group membership. Changes in diversity exposure at the neighborhood level also depend on how diverse the metro area as a whole has become.

States as Sites of Educational (In)Equality: State Contexts and the Socioeconomic Achievement Gradient.

Socioeconomic achievement gaps have long been a central focus of educational research. However, not much is known about how (and why) between-district gaps vary among states, even though states are a primary organizational level in the decentralized education system in the United States. Using data from the Stanford Education Data Archive (SEDA), this study describes state-level socioeconomic achievement gradients and the growth of these gradients from Grades 3 to 8. We also examine state-level correlates of the gradients and their growth, including school system funding equity, preschool enrollment patterns, the distribution of teachers, income inequality, and segregation. We find that socioeconomic gradients and their growth rates vary considerably among states, and that between-district income segregation is positively associated with the socioeconomic achievement gradient.

Has Income Segregation Really Increased? Bias and Bias Correction in Sample-Based Segregation Estimates.

Several recent studies have concluded that residential segregation by income in the United States has increased in the decades since 1970, including a significant increase after 2000. Income segregation measures, however, are biased upward when based on sample data. This is a potential concern because the sampling rate of the American Community Survey (ACS)-from which post-2000 income segregation estimates are constructed-was lower than that of the earlier decennial censuses. Thus, the apparent increase in income segregation post-2000 may simply reflect larger upward bias in the estimates from the ACS, and the estimated trend may therefore be inaccurate. In this study, we first derive formulas describing the approximate sampling bias in two measures of segregation. Next, using Monte Carlo simulations, we show that the bias-corrected estimators eliminate virtually all of the bias in segregation estimates in most cases of practical interest, although the correction fails to eliminate bias in some cases when the population is unevenly distributed among geographic units and the average within-unit samples are very small. We then use the bias-corrected estimators to produce unbiased estimates of the trends in income segregation over the last four decades in large U.S. metropolitan areas. Using these corrected estimates, we replicate the central analyses in four prior studies on income segregation. We find that the primary conclusions from these studies remain unchanged, although the true increase in income segregation among families after 2000 was only half as large as that reported in earlier work. Despite this revision, our replications confirm that income segregation has increased sharply in recent decades among families with children and that income inequality is a strong and consistent predictor of income segregation.

Patterns of literacy among U.S. students.

How well do U.S. students read? In this article, Sean Reardon, Rachel Valentino, and Kenneth Shores rely on studies using data from national and international literacy assessments to answer this question. In part, the answer depends on the specific literacy skills assessed. The authors show that almost all U.S. students can "read" by third grade, if reading is defined as proficiency in basic procedural word-reading skills. But reading for comprehension--integrating background knowledge and contextual information to make sense of a text--requires a set of knowledge-based competencies in addition to word-reading skills. By the standards used in various large-scale literacy assessments, only about a third of U.S. students in middle school possess the knowledge-based competencies to "read" in this more comprehensive sense. This low level of literacy proficiency does not appear to be a result of declining performance over time. Literacy skills of nine-year-olds in the United States have increased modestly over the past forty years, while the skills of thirteen- and seventeen-year-olds have remained relatively flat. Literacy skills vary considerably among students, however. For example, the literacy skills of roughly 10 percent of seventeen-year-olds are at the level of the typical nine-year-old. This variation is patterned in part by race, ethnicity, and socioeconomic background. Black and Hispanic students enter high school with average literacy skills three years behind those of white and Asian students; students from low-income families enter high school with average literacy skills five years behind those of high-income students. These are gaps that no amount of remedial instruction in high school is likely to eliminate. And while the racial and ethnic disparities are smaller than they were forty to fifty years ago, socioeconomic disparities in literacy skills are growing. Nor is the low level of literacy skills particularly a U.S. phenomenon. On international comparisons, American students perform modestly above average compared with those in other developed countries (and well above average among a larger set of countries). Moreover, there is no evidence that U.S. students lose ground relative to those in other countries during the middle school years. Thus, although literacy skills in the United States are lower than needed to meet the demands of modern society, the same is true in most other developed countries.

Income inequality and income segregation.

This article investigates how the growth in income inequality from 1970 to 2000 affected patterns of income segregation along three dimensions: the spatial segregation of poverty and affluence, race-specific patterns of income segregation, and the geographic scale of income segregation. The evidence reveals a robust relationship between income inequality and income segregation, an effect that is larger for black families than for white families. In addition, income inequality affects income segregation primarily through its effect on the large-scale spatial segregation of affluence rather than by affecting the spatial segregation of poverty or by altering small-scale patterns of income segregation.

Meta-analysis of supplemental treatment for depressive and anxiety disorders in patients being treated for alcohol dependence.

Approximately half of those receiving treatment for an alcohol use disorder (AUD) also suffer with an anxiety or depressive (internalizing) disorder. Because all internalizing disorders mark a poor alcohol treatment outcome, it seems reasonable to supplement AUD treatment with a psychiatric intervention when these disorders co-occur with AUD. However, this conclusion may be faulty given that the various possible interrelationships between AUD and internalizing disorders do not uniformly imply a high therapeutic yield from this approach. Unfortunately, the studies conducted to date have been too few and too small to resolve this important clinical issue with confidence. Therefore, we used a meta-analytic method to synthesize the effects from published randomized controlled trials examining the impact of supplementing AUD treatment with a psychiatric treatment for co-occurring internalizing disorder (N = 15). We found a pooled effect size (d) of .32 for internalizing outcomes and .22 for a composite of alcohol outcomes; however, the alcohol outcomes effect sizes were greater than this for some specific outcome domains. Subgroups that differed in terms of internalizing outcomes included treatment type (medication vs. cognitive behavioral therapy) and treatment focus (anxiety vs. depression). There was also a trend for the studies with better internalizing disorder outcomes to have better alcohol outcomes. These results indicate that clinical outcomes (both psychiatric and alcohol-related) could be somewhat improved by supplementing AUD treatment with psychiatric treatment for co-occurring internalizing disorder.

Race and space in the 1990s: changes in the geographic scale of racial residential segregation, 1990-2000.

We use newly developed methods of measuring spatial segregation across a range of spatial scales to assess changes in racial residential segregation patterns in the 100 largest U.S. metropolitan areas from 1990 to 2000. Our results point to three notable trends in segregation from 1990 to 2000: (1) Hispanic-white and Asian-white segregation levels increased at both micro- and macro-scales; (2) black-white segregation declined at a micro-scale, but was unchanged at a macro-scale; and (3) for all three racial groups and for almost all metropolitan areas, macro-scale segregation accounted for more of the total metropolitan area segregation in 2000 than in 1990. Our examination of the variation in these trends among the metropolitan areas suggests that Hispanic-white and Asian-white segregation changes have been driven largely by increases in macro-scale segregation resulting from the rapid growth of the Hispanic and Asian populations in central cities. The changes in black-white segregation, in contrast, appear to be driven by the continuation of a 30-year trend in declining micro-segregation, coupled with persistent and largely stable patterns of macro-segregation.

The geographic scale of metropolitan racial segregation.

This article addresses an aspect of racial residential segregation that has been largely ignored in prior work: the issue of geographic scale. In some metropolitan areas, racial groups are segregated over large regions, with predominately white regions, predominately black regions, and so on, whereas in other areas, the separation of racial groups occurs over much shorter distances. Here we develop an approach-featuring the segregation profile and the corresponding macro/micro segregation ratio-that offers a scale-sensitive alternative to standard methodological practice for describing segregation. Using this approach, we measure and describe the geographic scale of racial segregation in the 40 largest U.S. metropolitan areas in 2000. We find considerable heterogeneity in the geographic scale of segregation patterns across both metropolitan areas and racial groups, a heterogeneity that is not evident using conventional "aspatial" segregation measures. Moreover, because the geographic scale of segregation is only modestly correlated with the level of segregation in our sample, we argue that geographic scale represents a distinct dimension of residential segregation. We conclude with a brief discussion of the implications of our findings for investigating the patterns, causes, and consequences of residential segregation at different geographic scales.

Beyond the Census Tract: Patterns and Determinants of Racial Segregation at Multiple Geographic Scales.

The census tract-based residential segregation literature rests on problematic assumptions about geographic scale and proximity. We pursue a new tract-free approach that combines explicitly spatial concepts and methods to examine racial segregation across egocentric local environments of varying size. Using 2000 census data for the 100 largest U.S. metropolitan areas, we compute a spatially modified version of the information theory index H to describe patterns of black-white, Hispanic-white, Asian-white, and multi-group segregation at different scales. The metropolitan structural characteristics that best distinguish micro-segregation from macro-segregation for each group combination are identified, and their effects are decomposed into portions due to racial variation occurring over short and long distances. A comparison of our results to those from tract-based analyses confirms the value of the new approach.

Retail tobacco outlet density and youth cigarette smoking: a propensity-modeling approach.

OBJECTIVES: We examined whether retail tobacco outlet density was related to youth cigarette smoking after control for a diverse range of neighborhood characteristics. METHODS: Data were gathered from 2116 respondents (aged 11 to 23 years) residing in 178 census tracts in Chicago, Ill. Propensity score stratification methods for continuous exposures were used to adjust for potentially confounding neighborhood characteristics, thus strengthening causal inferences. RESULTS: Retail tobacco outlets were disproportionately located in neighborhoods characterized by social and economic disadvantage. In a model that excluded neighborhood confounders, a marginally significant effect was found. Youths in areas at the highest 75th percentile in retail tobacco outlet density were 13% more likely (odds ratio [OR]=1.13; 95% confidence interval [CI]=0.99, 1.28) to have smoked in the past month compared with those living at the lowest 25th percentile. However, the relation became stronger and significant (OR=0.21; 95% CI=1.04, 1.41) after introduction of tract-level confounders and was statistically significant in the propensity score-adjusted model (OR = 1.20; 95% CI = 1.001, 1.44). Results did not differ significantly between minors and those legally permitted to smoke. CONCLUSIONS: Reductions in retail tobacco outlet density may reduce rates of youth smoking.

Differences in onset and persistence of substance abuse and dependence among whites, blacks, and Hispanics.

OBJECTIVE: This article describes patterns of onset, persistence, and cessation of substance abuse among whites, blacks, and Hispanics that are masked in cross-sectional prevalence data. METHODS: The authors analyzed longitudinal data from a sample of 1,004 white, black, and Hispanic respondents from Chicago to investigate processes of onset, persistence, and cessation of substance abuse and dependence for two age cohorts, 15 and 18 at baseline and 17 and 20 at follow-up. RESULTS: The data show few racial or ethnic differences in the prevalence of alcohol and marijuana abuse and dependence at age 15. Rates of onset of alcohol abuse and dependence among whites between ages 15 and 17 were significantly higher than for blacks and Hispanics, and the rates of onset of marijuana abuse and dependence among blacks between ages 18 and 20 were significantly higher than for whites and Hispanics of the same age group. There were few significant differences among the three groups in the persistence rates of abuse and dependence. CONCLUSION: By age 20 the rates of marijuana abuse and dependence are significantly higher among blacks than among whites and Hispanics.