Coordinated DNA polymerization by Polγ and the region of LonP1 regulated proteolysis.

The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.

Structure-specific roles for PolG2-DNA complexes in maintenance and replication of mitochondrial DNA.

The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma (Pol γ) enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2-DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol γ subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease.

Structural insight and characterization of human Twinkle helicase in mitochondrial disease.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

The biological embedding of social adversity: How adolescent housing insecurity impacts inflammation over time.

BACKGROUND & PURPOSE: Adolescent housing insecurity is a dynamic form of social adversity that impacts child health outcomes worldwide. However, the means by which adolescent housing insecurity may become biologically embedded to influence health outcomes over the life course remain unclear. Therefore, we aimed to utilize life course perspectives and advanced causal inference methods to evaluate the potential for inflammation to contribute to the biological embedding of adolescent housing insecurity. MATERIALS AND METHODS: Using prospective data from the Great Smoky Mountains Study, we investigated the relationship between adolescent housing insecurity and whole-blood spot samples assayed for C-reactive protein (CRP). Adolescent housing insecurity was created based on annual measures of frequent residential moves, reduced standard of living, forced separation from the home, and foster care. Annual measures of CRP ranged from 0.001 mg/L to 13.6 mg/L (median = 0.427 mg/L) and were log10 transformed to account for positively skewed values. We used g-estimation of structural nested mean models to estimate a series of conditional average causal effects of adolescent housing insecurity on CRP levels from ages 11 to 16 years and interpreted the results within life course frameworks of accumulation, recency, and sensitive periods. PRINCIPAL RESULTS: Of the 1,334 participants, 427 [44.3 %] were female. Based on the conditional average causal effect, one exposure to adolescent housing insecurity from ages 11 to 16 years led to a 6.4 % (95 % CI = 0.69 - 12.4) increase in later CRP levels. Exposure at 14 years of age led to a 27.9 % increase in CRP levels at age 15 (95 % CI = 6.5 - 53.5). Recent exposures to adolescent housing insecurity (<3 years) suggested stronger associations with CRP levels than distant exposures (>3 years), but limited statistical power prevented causal conclusions regarding recency effects at the risk of a Type II Error. MAJOR CONCLUSIONS: These findings highlight inflammation-as indicated by increased CRP levels-as one potential mechanism for the biological embedding of adolescent housing insecurity. The results also suggest that adolescent housing insecurity-particularly recent, repeated, and mid-adolescent exposures-may increase the risk of poor health outcomes and should be considered a key intervention target.

Methylome-wide association study of anxiety disorders.

Anxiety Disorders (ANX) such as panic disorder, generalized anxiety disorder, and phobias, are highly prevalent conditions that are moderately heritable. Evidence suggests that DNA methylation may play a role, as it is involved in critical adaptations to changing environments. Applying an enrichment-based sequencing approach covering nearly 28 million autosomal CpG sites, we conducted a methylome-wide association study (MWAS) of lifetime ANX in 1132 participants (618 cases/514 controls) from the Netherlands Study of Depression and Anxiety. Using epigenomic deconvolution, we performed MWAS for the main cell types in blood: granulocytes, T-cells, B-cells and monocytes. Cell-type specific analyses identified 280 and 82 methylome-wide significant associations (q-value < 0.1) in monocytes and granulocytes, respectively. Our top finding in monocytes was located in ZNF823 on chromosome 19 (p = 1.38 × 10-10) previously associated with schizophrenia. We observed significant overlap (p < 1 × 10-06) with the same direction of effect in monocytes (210 sites), T-cells (135 sites), and B-cells (727 sites) between this Discovery MWAS signal and a comparable replication dataset from the Great Smoky Mountains Study (N = 433). Overlapping Discovery-Replication MWAS signal was enriched for findings from published GWAS of ANX, major depression, and post-traumatic stress disorder. In monocytes, two specific sites in the FZR1 gene showed significant replication after Bonferroni correction with an additional 15 nominally replicated sites in monocytes and 4 in T-cells. FZR1 regulates neurogenesis in the hippocampus, and its knockout leads to impairments in associative fear memory and long-term potentiation in mice. In the largest and most extensive methylome-wide study of ANX, we identified replicable methylation sites located in genes of potential relevance for brain mechanisms of psychiatric conditions.

Commentary: Integrative, multi-level explanatory models are needed to understand recent trends in sex, gender, and internalizing conditions, reflections on Keyes and Platt (2023).

Keyes' and Platt's (The Journal of Child Psychology and Psychiatry, 2023) review provides much-needed systematic evidence about why internalizing symptoms have increased and it clarifies the role of novel risk factors. The findings highlight that multiple factors at multiple levels are responsible for this phenomenon, many with small effects, within a complex interplay that is rarely well captured. As new insights emerge across disciplines, an important step is to renew efforts to integrate them to understand how internalizing symptoms develop for different people.

Taking John Schulenberg's "long view" on successful transitions to adulthood: Associations with adult substance use.

Can positive transitions into young adulthood at age 25 prevent problematic substance use at age 31, even in the context of childhood adverse family environments, conduct problems, and adolescent substance use? We lean on John Schulenberg's developmental framework to examine this question, focusing on the potential young adult milestones of high school and college graduation, employment, residential independence, romantic partnership, and parenthood. Data came from a prospective-longitudinal multi-method study with N = 1199 participants who were first assessed at age 5 years old and followed to age 31. An accumulation of positive transitions in young adulthood (age 25) was associated with lower likelihood of age 31 problematic cannabis use. The protective effect for problematic cannabis use remained even when adjusting for childhood adverse family environments and was primarily driven by successful college graduation and/or home ownership. The accumulation of positive transitions protected individuals at modest to somewhat elevated risk due to childhood adverse family environments from experiencing age 31 cannabis use problems. However, for other individuals with very high numbers of conduct problems, or with high levels of adolescent substance use, the protective effects of accumulated positive transitions to young adulthood were less strong or nonexistent. Moreover, individuals who completed college or obtained full-time employment by 25 were more likely to report problematic age 31 alcohol use. These findings highlight the central tenets of John Schulenberg's developmental framework, including the examination of ontogenetic continuity and discontinuity, the interplay of developmentally distal and proximal effects, and the identification of developmental protective factors that may sway people toward or away from substance use.

Using Wearable Digital Devices to Screen Children for Mental Health Conditions: Ethical Promises and Challenges.

In response to a burgeoning pediatric mental health epidemic, recent guidelines have instructed pediatricians to regularly screen their patients for mental health disorders with consistency and standardization. Yet, gold-standard screening surveys to evaluate mental health problems in children typically rely solely on reports given by caregivers, who tend to unintentionally under-report, and in some cases over-report, child symptomology. Digital phenotype screening tools (DPSTs), currently being developed in research settings, may help overcome reporting bias by providing objective measures of physiology and behavior to supplement child mental health screening. Prior to their implementation in pediatric practice, however, the ethical dimensions of DPSTs should be explored. Herein, we consider some promises and challenges of DPSTs under three broad categories: accuracy and bias, privacy, and accessibility and implementation. We find that DPSTs have demonstrated accuracy, may eliminate concerns regarding under- and over-reporting, and may be more accessible than gold-standard surveys. However, we also find that if DPSTs are not responsibly developed and deployed, they may be biased, raise privacy concerns, and be cost-prohibitive. To counteract these potential shortcomings, we identify ways to support the responsible and ethical development of DPSTs for clinical practice to improve mental health screening in children.

A Pilot Randomized Controlled Trial of the Family Assessment and Feedback Intervention (FAFI): Effects on Mental Health Literacy and Attitudinal Engagement with Health Supports and Services.

This randomized controlled trial tested the Family Assessment and Feedback Intervention (FAFI), a new intervention to enhance family engagement with emotional and behavioral health services. The FAFI is a guided conversation with families about results of their multidimensional assessment that is set in the context of motivational enhancement. It differs from other assessment-with-feedback interventions by extending the focus of assessment beyond the target child to parents and the family environment, addressing parental emotional and behavioral problems and competencies, spanning a broad range of children's and parents' strengths and difficulties, and being generalizable to many settings and practitioners. Participants were 81 families in primary care pediatrics. The FAFI was associated with a significant increase in parental mental health literacy and with an increase in parental attitudinal engagement with health supports and services that closely approached statistical significance (p = .052), while controlling for children's age and gender and family socioeconomic status.

Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors.

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in kcat/Km in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.

Childhood loneliness as a specific risk factor for adult psychiatric disorders.

BACKGROUND: Loneliness is a major risk factor for both psychological disturbance and poor health outcomes in adults. This study aimed to assess whether childhood loneliness is associated with a long-term disruption in mental health that extends into adulthood. METHODS: This study is based on the longitudinal, community-representative Great Smoky Mountains Study of 1420 participants. Participants were assessed with the structured Child and Adolescent Psychiatric Assessment interview up to eight times in childhood (ages 9-16; 6674 observations; 1993-2000) for childhood loneliness, associated psychiatric comorbidities and childhood adversities. Participants were followed up four times in adulthood (ages 19, 21, 25, and 30; 4556 observations of 1334 participants; 1999-2015) with the structured Young Adult Psychiatric Assessment Interview for psychiatric anxiety, depression, and substance use outcomes. RESULTS: Both self and parent-reported childhood loneliness were associated with adult self-reported anxiety and depressive outcomes. The associations remained significant when childhood adversities and psychiatric comorbidities were accounted for. There was no evidence for an association of childhood loneliness with adult substance use disorders. More associations were found between childhood loneliness and adult psychiatric symptoms than with adult diagnostic status. CONCLUSION: Childhood loneliness is associated with anxiety and depressive disorders in young adults, suggesting that loneliness - even in childhood - might have long-term costs in terms of mental health. This study underscores the importance of intervening early to prevent loneliness and its sequelae over time.

Adult criminal outcomes of juvenile justice involvement.

BACKGROUND: The juvenile justice system in the USA adjudicates over seven hundred thousand youth in the USA annually with significant behavioral offenses. This study aimed to test the effect of juvenile justice involvement on adult criminal outcomes. METHODS: Analyses were based on a prospective, population-based study of 1420 children followed up to eight times during childhood (ages 9-16; 6674 observations) about juvenile justice involvement in the late 1990 and early 2000s. Participants were followed up years later to assess adult criminality, using self-report and official records. A propensity score (i.e. inverse probability) weighting approach was used that approximated an experimental design by balancing potentially confounding characteristics between children with v. without juvenile justice involvement. RESULTS: Between-groups differences on variables that elicit a juvenile justice referral (e.g. violence, property offenses, status offenses, and substance misuse) were attenuated after applying propensity-based inverse probability weights. Participants with a history of juvenile justice involvement were more likely to have later official and violent felony charges, and to self-report police contact and spending time in jail (ORs from 2.5 to 3.3). Residential juvenile justice involvement was associated with the highest risk of both, later official criminal records and self-reported criminality (ORs from 5.1 to 14.5). Sensitivity analyses suggest that our findings are likely robust to potential unobserved confounders. CONCLUSIONS: Juvenile justice involvement was associated with increased risk of adult criminality, with residential services associated with highest risk. Juvenile justice involvement may catalyze rather than deter from adult offending.

Cultural contributions to adults' self-rated mental health problems and strengths: 7 culture clusters, 28 societies, 16 906 adults.

BACKGROUND: It is unknown how much variation in adult mental health problems is associated with differences between societal/cultural groups, over and above differences between individuals. METHODS: To test these relative contributions, a consortium of indigenous researchers collected Adult Self-Report (ASR) ratings from 16 906 18- to 59-year-olds in 28 societies that represented seven culture clusters identified in the Global Leadership and Organizational Behavioral Effectiveness study (e.g. Confucian, Anglo). The ASR is scored on 17 problem scales, plus a personal strengths scale. Hierarchical linear modeling estimated variance accounted for by individual differences (including measurement error), society, and culture cluster. Multi-level analyses of covariance tested age and gender effects. RESULTS: Across the 17 problem scales, the variance accounted for by individual differences ranged from 80.3% for DSM-oriented anxiety problems to 95.2% for DSM-oriented avoidant personality (mean = 90.7%); by society: 3.2% for DSM-oriented somatic problems to 8.0% for DSM-oriented anxiety problems (mean = 6.3%); and by culture cluster: 0.0% for DSM-oriented avoidant personality to 11.6% for DSM-oriented anxiety problems (mean = 3.0%). For strengths, individual differences accounted for 80.8% of variance, societal differences 10.5%, and cultural differences 8.7%. Age and gender had very small effects. CONCLUSIONS: Overall, adults' self-ratings of mental health problems and strengths were associated much more with individual differences than societal/cultural differences, although this varied across scales. These findings support cross-cultural use of standardized measures to assess mental health problems, but urge caution in assessment of personal strengths.

DNA methylation signatures of childhood trauma predict psychiatric disorders and other adverse outcomes 17 years after exposure.

Childhood trauma is robustly linked to a broad range of adverse outcomes with consequences persisting far into adulthood. We conducted a prospective longitudinal study to predict psychiatric disorders and other adverse outcomes from trauma-related methylation changes 16.9 years after trauma exposure in childhood. Methylation was assayed using a sequencing-based approach that provides near-complete coverage of all 28 million sites in the blood methylome. Methylation data involved 673 assays from 489 participants aged 13.6 years (SD = 1.9) with outcomes measures collected at age 30.4 (SD = 2.26). For a subset of 303 participants we also generated methylation data in adulthood. Trauma-related methylation risk scores (MRSs) significantly predicted adult depression, externalizing problems, nicotine dependence, alcohol use disorder, serious medical problems, social problems and poverty. The predictive power of the MRSs was higher than that of reported trauma and could not be explained by the reported trauma, correlations with demographic variables, or a continuity of the predicted health problems from childhood to adulthood. Rather than measuring the occurrence of traumatic events, the MRSs seemed to capture the subject-specific impact of trauma. The majority of predictive sites did not remain associated with the outcomes suggesting the signatures of trauma do not become biologically embedded in the blood methylome. Instead, the long-term effects of trauma therefore seemed more consistent with a developmental mechanism where the initial subject-specific impacts of trauma are magnified over time. The MRSs have the potential to be a novel clinical biomarker for the assessment of trauma-related health risks.

Intergenerational effects of the Fast Track intervention on the home environment: A randomized control trial.

BACKGROUND: Maladaptive family environments harm child development and are passed across generations. Childhood interventions may break this intergenerational cycle by improving the family environments children form as adults. The present study investigates this hypothesis by examining follow-up data collected 18 years after the end of the childhood Fast Track intervention designed to prevent externalizing problems. METHODS: We examined whether Fast Track assignment from grades 1 to 10 prevented the emergence of maladaptive family environments at age 34. A total of 400 (n = 206 in intervention condition, n = 194 controls) Fast Track participants who were parents at age 34 were surveyed about 11 aspects of their current family environment. The hypotheses and analytic plan were preregistered at https://osf.io/dz9t5 and the Fast Track trial was registered at clinicaltrials.gov (NCT01653535). RESULTS: Multiple group linear regression models revealed that mothers who participated in the Fast Track intervention as children had lower depression symptoms, alcohol problems, drug problems, corporal punishment use, and food insecurity compared to control group mothers. All effects were modest in magnitude. However, for these same mothers, the Fast Track intervention had no effect on cannabis problems, experiences of romantic partner violence, or maternal use of physical aggression or warmth with their children. Additionally, mothers in the Fast Track intervention group reported higher levels of family chaos than those in the control group, but this effect may be a byproduct of the higher number of children per household in the intervention group. No intervention effects were found for fathers who participated in the Fast Track intervention as children. CONCLUSIONS: Childhood assignment to Fast Track has some beneficial effects for girls, but not boys, on the family environments these individuals formed as adults 18 years later.

Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM.

The mitochondrial replisome replicates the 16.6 kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.

Pubertal timing moderates the same-day coupling between family hassles and negative affect in girls and boys.

This study examined the association between pubertal timing, daily affect, conduct problems, and the exposure to hassles across family, peer, and school contexts. Adolescents (M age = 12.27; 49.7% female; 62.6% White) completed ecological momentary assessments across 14 consecutive days (N = 388). Earlier maturing girls reported lower daily averages of positive affect compared to their same-sex, same-age peers. We did not find evidence for a relationship between pubertal timing and daily negative affect or conduct problems in girls, nor for daily negative and positive affect or conduct problems in boys. However, pubertal timing did moderate the day-level association between average negative affect and family hassles for both girls and boys. When experiencing more family hassles, earlier maturing girls reported greater negative affect relative to later maturing girls who experienced family hassles. In contrast, later maturing boys, relative to earlier maturing boys, reported higher levels of negative affect in the context of family hassles.

Early life adversity and psychopathology in preschoolers: mechanisms and moderators.

Developmental theories suggest that exposure to early life adversity (ELA) alters developing emotional response systems, predicting risk for psychopathology across the life span. The present study examines whether negative emotionality (NE), a trait-like measure of emotionality that develops during early childhood, mediates the association between ELA and psychopathology in a representative sample of 917 preschoolers (Mage = 3.84). Additionally, we explored whether cognitive control, which supports attentional focusing and inhibition and has been identified as a transdiagnostic protective factor, moderates the impact of heightened emotionality following adversity on psychopathology risk. We utilized parent report of adversity, psychopathology, and NE and parent report and task-based measures of cognitive control. Structural equation modeling of cross-sectional data revealed that NE partially mediated the link between ELA and psychopathology symptoms. Moreover, parent-reported cognitive control buffered this link such that the effect of ELA on psychopathology through NE was stronger in children with low versus high cognitive control. These results identify elevated NE as one mechanism linking ELA and psychopathology, specifically among children with poorer top-down control, informing our understanding of key risk and protective factors among adversity-exposed children.

The Vermont Family Based Approach in Primary Care Pediatrics: Effects on Children's and Parents' Emotional and Behavioral Problems and Parents' Health-Related Quality of Life.

This randomized controlled trial tested the Vermont Family Based Approach (VFBA) in primary care pediatrics. The VFBA is a model of healthcare delivery that shifts the focus from the individual to the family, emphasizes emotional and behavioral health, and uses evidence-based health promotion/prevention along with the treatment of emotional and behavioral problems. Participants were 81 families of 3-15-year-olds. For children, the VFBA was associated with greater reductions than the Control condition on the Child Behavior Checklist Emotionally Reactive, Withdrawn, Sleep Problems, Aggressive Behavior and Total Problems scales. For parents, the VFBA was associated with greater reductions than the Control condition on the Adult Self-Report Anxious/Depressed, Rule-Breaking Behavior, Internalizing Problems and Total Problems scales. The VFBA was also associated with greater improvement than the Control condition in the parents' health-related quality of life, as indicated by all scales of the Medical Outcomes Study Health Survey.

Day-to-day variation in adolescent food insecurity.

This study examined differences in both average and variability in daily adolescent food insecurity, by adolescents' levels of economic disadvantage and race/ethnicity. We used data from a 14-day ecological momentary assessment of 395 adolescents enrolled in public schools in North Carolina. Each evening, adolescents were asked questions about that day's food insecurity. Economically disadvantaged adolescents reported both higher average food insecurity and more day-to-day variability in food insecurity than non-economically disadvantaged adolescents. Controlling for economic disadvantage, Black adolescents also experienced both higher average food insecurity and more variability from day to day than White or Hispanic adolescents. For those receiving Supplemental Nutrition Assistance Program (SNAP) benefits, daily food insecurity was higher in the second half of the month after SNAP transfer than in the beginning of the month. Food insecurity among adolescents is not static but varies from day to day. This daily variation is greater for economically disadvantaged youth.