Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Liquid-liquid phase separation underpins the formation of replication factories in rotaviruses.

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.

Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation.

Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility in silico. To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2K294E). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2K294E versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2K294E formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2K294E factories had delayed fusion kinetics. Moreover, NSP2K294E and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2WT. Finally, NSP2K294E exhibited defects in its capacity to induce LLPS droplet formation in vitro when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. IMPORTANCE Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.

Phase separation and viral factories: unveiling the physical processes supporting RNA packaging in dsRNA viruses.

Understanding of the physicochemical properties and functions of biomolecular condensates has rapidly advanced over the past decade. More recently, many RNA viruses have been shown to form cytoplasmic replication factories, or viroplasms, via phase separation of their components, akin to numerous cellular membraneless organelles. Notably, diverse viruses from the Reoviridae family containing 10-12 segmented double-stranded RNA genomes induce the formation of viroplasms in infected cells. Little is known about the inner workings of these membraneless cytoplasmic inclusions and how they may support stoichiometric RNA assembly in viruses with segmented RNA genomes, raising questions about the roles of phase separation in coordinating viral genome packaging. Here, we discuss how the molecular composition of viroplasms determines their properties, highlighting the interplay between RNA structure, RNA remodelling, and condensate self-organisation. Advancements in RNA structural probing and theoretical modelling of condensates can reveal the mechanisms through which these ribonucleoprotein complexes support the selective enrichment and stoichiometric assembly of distinct viral RNAs.

Structural basis of rotavirus RNA chaperone displacement and RNA annealing.

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

Pubertal Timing Across Asian American, Native Hawaiian, and Pacific Islander Subgroups.

Importance: Earlier puberty is associated with adverse health outcomes, such as mental health issues in adolescence and cardiometabolic diseases in adulthood. Despite rapid growth of the Asian American, Native Hawaiian, and Pacific Islander populations in the US, limited research exists on their pubertal timing, potentially masking health disparities. Objective: To examine pubertal timing among Asian American, Native Hawaiian, and Pacific Islander children and adolescents by disaggregating ethnic subgroups. Design, Setting, and Participants: This retrospective cohort study included Asian American, Native Hawaiian, and Pacific Islander youths aged 5 to 18 years assessed for pubertal development at Kaiser Permanente Northern California, a large, integrated health care delivery system. Follow-up occurred from March 2005, through December 31, 2019. Data were analyzed in October 2023. Exposure: Race and ethnicity, categorized into 11 ethnic subgroups: Asian Indian, Chinese, Filipino, Japanese, Korean, Native Hawaiian and Pacific Islander, Other South Asian, Other Southeast Asian, Vietnamese, multiethnic, and multiracial. Main Outcomes and Measures: Pubertal timing was determined using physician-assessed sexual maturity ratings (SMRs). Outcomes included the median age at transition from SMR 1 (prepubertal) to SMR 2 or higher (pubertal) for onset of genital development (gonadarche) in boys, breast development (thelarche) in girls, and pubic hair development (pubarche) in both boys and girls. Results: In this cohort of 107 325 Asian American, Native Hawaiian, and Pacific Islander children and adolescents (54.61% boys; 12.96% Asian Indian, 22.24% Chinese, 26.46% Filipino, 1.80% Japanese, 1.66% Korean, 1.96% Native Hawaiian and Pacific Islander, 0.86% Other South Asian, 3.26% Other Southeast Asian, 5.99% Vietnamese, 0.74% multiethnic, and 22.05% multiracial), the overall median ages for girls' pubarche and thelarche were 10.98 years (95% CI, 10.96-11.01 years) and 10.13 years (95% CI, 10.11-10.15 years), respectively. For boys' pubarche and gonadarche, median ages were 12.08 years (95% CI, 12.06-12.10 years) and 11.54 years (95% CI, 11.52-11.56 years), respectively. Differences between subgroups with earliest and latest median age at onset were 14 months for girls' pubarche, 8 months for thelarche, 8 months for boys' pubarche, and 4 months for gonadarche. In general, Asian Indian, Native Hawaiian and Pacific Islander, and Other South Asian subgroups had the earliest ages at onset across pubertal markers, while East Asian youths exhibited the latest onset. Restricting to those with healthy body mass index did not substantially change the findings. Conclusions and Relevance: In this cohort study of Asian American, Native Hawaiian, and Pacific Islander children and adolescents, pubertal timing varied considerably across ethnic subgroups. Further investigation is warranted to assess whether these differences contribute to observed health disparities in adulthood, such as type 2 diabetes and cardiovascular diseases.

Seeing Biomolecular Condensates Through the Lens of Viruses.

Phase separation of viral biopolymers is a key factor in the formation of cytoplasmic viral inclusions, known as sites of virus replication and assembly. This review describes the mechanisms and factors that affect phase separation in viral replication and identifies potential areas for future research. Drawing inspiration from studies on cellular RNA-rich condensates, we compare the hierarchical coassembly of ribosomal RNAs and proteins in the nucleolus to the coordinated coassembly of viral RNAs and proteins taking place within viral factories in viruses containing segmented RNA genomes. We highlight the common characteristics of biomolecular condensates in viral replication and how this new understanding is reshaping our views of virus assembly mechanisms. Such studies have the potential to uncover unexplored antiviral strategies targeting these phase-separated states.

Structural Racism and Adolescent Mental Health Disparities in Northern California.

Importance: Understanding how structural racism is associated with adolescent mental health is critical to advance health equity. Objective: To assess associations between neighborhood privilege, measured by the Index of Concentration at the Extremes (ICE) and adolescent depressive symptoms, suicidality, and related racial and ethnic disparities. Design, Setting, and Participants: This was a retrospective cohort study using electronic health records of adolescents aged 12 to 16 years who attended well-teen visits between 2017 and 2021. Kaiser Permanente Northern California is an integrated health care delivery system serving 4.6 million members. The cohort included 34 252 individuals born singleton at an affiliated facility from January 1, 2005, to December 31, 2009, and who had completed at least 1 mental health screener during a well-teen visit by November 23, 2021. Exposures: American Community Survey 2016 to 2021 5-year estimates were used to calculate ICE scores for adolescents' residential census tract at ages 10 to 11. Three ICE measures were used as proxies of structural racism: racial privilege (ICE-race and ethnicity; hereinafter ICE-race), economic privilege (ICE-income), and combined economic and racial privilege (ICE-income plus race and ethnicity; herinafter ICE-income plus race). ICE scores were categorized into quintiles based on California statewide distributions. Main Outcomes and Measures: Depressive symptoms and suicidality were assessed through self-report screeners during well-teen visits. Depressive symptoms were considered to be present if patients had a score on the Patient Health Questionnaire-2 of 3 or higher (the tool uses a Likert scale to determine the frequency [0 = not at all; 3 = nearly every day] that they had depressed mood and lack of pleasure in usual activities in the past 2 weeks; responses were summed and dichotomized). Results: Analyses included 34 252 adolescents (12-16 years of age; mean [SD] age, 13.7 [0.8] years; 17 557 [51.3%] male, 7284 [21.3%] Asian or Pacific Islander, 2587 [7.6%] Black], 9061 [26.5%] Hispanic, 75 [0.2%] American Indian or Indigenous, 12 176 [35.5%] White, and 3069 [9%] other or unknown). Risks of depressive symptoms and suicidality generally increased with each level of declining neighborhood privilege. Adjusted risk ratios comparing adolescents from neighborhoods with the least to most racial and economic privilege were 1.37 (95% CI, 1.20-1.55) for depressive symptoms and 1.59 (95% CI, 1.23-2.05) for suicidality. Racial disparities between Black and White youth and Hispanic and White youth decreased after adjusting for each ICE measure, and became nonsignificant in models adjusting for ICE-race and ICE-income plus race. Conclusions and Relevance: In this cohort study, lower neighborhood privilege was associated with greater risks of adolescent depressive symptoms and suicidality. Furthermore, adjusting for neighborhood privilege reduced mental health disparities affecting Black and Hispanic adolescents. These findings suggest that efforts to promote equity in adolescent mental health should extend beyond the clinical setting and consider the inequitable neighborhood contexts that are shaped by structural racism.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus.

Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.

Maternal Depression and Antidepressant Use During Pregnancy and Associations with Depressive Symptoms and Suicidality in Adolescent Children.

Purpose: Children of mothers with prenatal depression have elevated risk for depression later in life. Pregnant women are hesitant to use antidepressants due to fear of adverse fetal effects. To inform prevention, this study examined associations between maternal prenatal depression and antidepressant use, and adolescent depressive symptoms and suicidality. Patients and Methods: Prospective data from 74,695 mother-adolescent dyads from the Kaiser Permanente Northern California integrated healthcare delivery system were used. Three prenatal exposure groups were examined: maternal depression and antidepressants (Med); depression and no antidepressants (No-Med); neither depression nor antidepressants (NDNM). Adolescent depressive symptoms (Patient Health Questionnaire-2 score ≥3) and suicidality were assessed for 12- to 18-year-olds. Associations were analyzed using mixed effects logistic regression, adjusted for confounders. Results: Maternal prenatal depression was associated with higher odds of adolescent depressive symptoms (Med odds ratio [OR]: 1.50, 95% confidence interval [CI]: 1.23-1.84; No-Med OR: 1.59, CI: 1.34-1.88) and suicidality (Med OR: 2.36, CI: 1.67-3.34; No-Med OR: 1.54, CI: 1.10-2.14) compared to no prenatal depression (NDNM). Adolescents exposed to prenatal depression and antidepressants were not at greater odds of depressive symptoms (Med OR: 0.95, CI: 0.74-1.21) compared to those not exposed to antidepressants (No-Med). However, they showed non-significant but greater odds of suicidality (Med OR: 1.54, CI: 0.99-2.39). Conclusion: Our findings suggest that maternal prenatal depression is associated with adolescent depressive symptoms and suicidality, and that exposure to antidepressants in utero does not increase risk of depressive symptoms, specifically. While not statistically significant, the increased odds of suicidality among adolescents exposed to antidepressants suggest a possible association; however, further investigation is needed. After replication, the findings of this study may inform shared clinical decision-making when considering options regarding antidepressant use for the treatment of maternal prenatal depression.

Neighborhood Racial and Economic Privilege and Timing of Pubertal Onset in Girls.

PURPOSE: Early puberty is associated with adverse health outcomes over the life course, and Black and Hispanic girls experience puberty earlier than girls of other racial/ethnic backgrounds. Neighborhood racial and economic privilege may contribute to these disparities by conferring differential exposure to mechanisms (e.g., stress, obesity, endocrine disruptors) underlying early puberty. We examined associations between neighborhood privilege, measured by the Index of Concentration at the Extremes (ICE), and age at pubic hair onset (pubarche) and breast development onset (thelarche) in a large multiethnic cohort. METHODS: A cohort of 46,299 girls born 2005-2011 at Kaiser Permanente Northern California medical facilities were followed until 2021. Pubertal development was assessed routinely by pediatricians using the Sexual Maturity Rating scale. ICE quintiles for race/ethnicity, income, and income + race/ethnicity were calculated using American Community Survey 2010 5-year estimates and linked to census tract at birth. We fit multilevel Weibull regression models accommodating left, right, and interval censoring for all analyses. RESULTS: ICE measures were monotonically associated with pubertal onset, with the strongest associations observed for ICE-race/ethnicity. Adjusting for maternal education, age at delivery, and parity, girls from the least versus most privileged ICE-race/ethnicity quintiles were at increased risk for earlier pubarche (hazard ratio: 1.30, 95% confidence interval: 1.21, 1.38) and thelarche (hazard ratio: 1.45, 95% confidence interval: 1.36, 1.54). These associations remained significant after adjusting for girls' race/ethnicity and childhood body mass index. Additionally, adjustment for ICE partially attenuated Black-White and Hispanic-White disparities in pubertal onset. DISCUSSION: Neighborhood privilege may contribute to pubertal timing and related disparities.

Biomolecular condensate phase diagrams with a combinatorial microdroplet platform.

The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.

Explaining the Black-White Disparity in Preterm Birth: A Consensus Statement From a Multi-Disciplinary Scientific Work Group Convened by the March of Dimes.

In 2017-2019, the March of Dimes convened a workgroup with biomedical, clinical, and epidemiologic expertise to review knowledge of the causes of the persistent Black-White disparity in preterm birth (PTB). Multiple databases were searched to identify hypothesized causes examined in peer-reviewed literature, 33 hypothesized causes were reviewed for whether they plausibly affect PTB and either occur more/less frequently and/or have a larger/smaller effect size among Black women vs. White women. While definitive proof is lacking for most potential causes, most are biologically plausible. No single downstream or midstream factor explains the disparity or its social patterning, however, many likely play limited roles, e.g., while genetic factors likely contribute to PTB, they explain at most a small fraction of the disparity. Research links most hypothesized midstream causes, including socioeconomic factors and stress, with the disparity through their influence on the hypothesized downstream factors. Socioeconomic factors alone cannot explain the disparity's social patterning. Chronic stress could affect PTB through neuroendocrine and immune mechanisms leading to inflammation and immune dysfunction, stress could alter a woman's microbiota, immune response to infection, chronic disease risks, and behaviors, and trigger epigenetic changes influencing PTB risk. As an upstream factor, racism in multiple forms has repeatedly been linked with the plausible midstream/downstream factors, including socioeconomic disadvantage, stress, and toxic exposures. Racism is the only factor identified that directly or indirectly could explain the racial disparities in the plausible midstream/downstream causes and the observed social patterning. Historical and contemporary systemic racism can explain the racial disparities in socioeconomic opportunities that differentially expose African Americans to lifelong financial stress and associated health-harming conditions. Segregation places Black women in stressful surroundings and exposes them to environmental hazards. Race-based discriminatory treatment is a pervasive stressor for Black women of all socioeconomic levels, considering both incidents and the constant vigilance needed to prepare oneself for potential incidents. Racism is a highly plausible, major upstream contributor to the Black-White disparity in PTB through multiple pathways and biological mechanisms. While much is unknown, existing knowledge and core values (equity, justice) support addressing racism in efforts to eliminate the racial disparity in PTB.

Fundamental social causes of inequalities in colorectal cancer mortality: A study of behavioral and medical mechanisms.

BACKGROUND: Fundamental cause theory posits that social conditions strongly influence the risk of health risks. This study identifies risk mechanisms that social conditions associated with socioeconomic status (SES) and race/ethnicity shape in the production of colorectal cancer (CRC) mortality. METHODS: Two large datasets in the United States examining behavioral and medical preventive factors (N = 4.63-million people) were merged with population-level mortality data observing 761,100 CRC deaths among 3.31-billion person-years of observation to examine trends in CRC mortality from 1999-2012. Analyses examined the changing role of medical preventions and health behaviors in catalyzing SES and racial/ethnic inequalities in CRC mortality. RESULTS: Lower SES as well as Black, Hispanic, Asian/Pacific Islander, and Native American race/ethnicity were associated with decreased access to age-appropriate screening and/or increased prevalence of behavioral risk factors. Analyses further revealed that SES and racial/ethnic inequalities were partially determined by differences in engagement in two preventive factors: use of colonoscopy, and participation in physical activity. DISCUSSION: Social inequalities were not completely determined by behavioral risk factors. Nevertheless, a more equitable distribution of preventive medicines has the potential to reduce both the risk of, and social inequalities in, CRC mortality.