A Bacterial Effector Mimics a Host HSP90 Client to Undermine Immunity.

The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity.

Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

A domain of all trades: The enzymatic versatility of the NiRAN domain.

The SARS-CoV-2 NiRAN domain is essential for viral replication. Despite adopting a pseudokinase fold, it catalyzes three distinct biochemical reactions from a single active site. In this issue of Molecular Cell, Small et al.1 elucidate the structural intricacies of the NiRAN domain shedding light on the factors that underlie its remarkable versatility.

A Single Kinase Generates the Majority of the Secreted Phosphoproteome.

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Continuous symmetry breaking in a two-dimensional Rydberg array.

Spontaneous symmetry breaking underlies much of our classification of phases of matter and their associated transitions1-3. The nature of the underlying symmetry being broken determines many of the qualitative properties of the phase; this is illustrated by the case of discrete versus continuous symmetry breaking. Indeed, in contrast to the discrete case, the breaking of a continuous symmetry leads to the emergence of gapless Goldstone modes controlling, for instance, the thermodynamic stability of the ordered phase4,5. Here, we realize a two-dimensional dipolar XY model that shows a continuous spin-rotational symmetry using a programmable Rydberg quantum simulator. We demonstrate the adiabatic preparation of correlated low-temperature states of both the XY ferromagnet and the XY antiferromagnet. In the ferromagnetic case, we characterize the presence of a long-range XY order, a feature prohibited in the absence of long-range dipolar interaction. Our exploration of the many-body physics of XY interactions complements recent works using the Rydberg-blockade mechanism to realize Ising-type interactions showing discrete spin rotation symmetry6-9.

Structural and mechanistic basis for protein glutamylation by the kinase fold.

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active-site Glu in SidE, resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

The mechanism of RNA capping by SARS-CoV-2.

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology.

High-Dimensional Single-Cell Mass Cytometry Demonstrates Differential Platelet Functional Phenotypes in Infants With Congenital Heart Disease.

BACKGROUND: Congenital heart disease (CHD) is a group of complex heart defects associated with hematologic abnormalities, including increased risk of thrombotic and bleeding events. Past studies have observed evidence of platelet hyperreactivity, while other studies showed decreased platelet activation in patients with CHD. The goal of this study was to develop a mass spectrometry approach to characterize single platelets in infants with CHD and identify potential etiology for such discrepant results. METHODS: We enrolled 19 infants with CHD along with 21 non-CHD controls at Yale New Haven Children's Heart Center. A single-cell high-dimensional mass cytometry method was developed to quantitatively interrogate platelet surface markers in whole blood. Additionally, plasma cytokine analysis was performed through a multiplexed panel of 52 vascular and inflammatory markers to assess for platelet releasates. RESULTS: We found that infants with CHD had significant differences in platelet activation and functional markers by mass cytometry compared with non-CHD controls. Based on cell surface markers, we classified the platelets into 8 subpopulations (P0 to P7). Distinct subpopulations of platelets (P1, P4, and P5) exhibiting decreased aggregatory phenotype but altered secretory phenotypes were also identified and found to be more abundant in the blood of infants with CHD. Electron microscopy identified increased proportion of hypogranular platelets in CHD. Moreover, cytokine analysis demonstrated an overall increase in plasma cytokines and biomarkers in CHD, including IL (interleukin)-6, IL-8, IL-27, RANTES, and VWF (von Willebrand factor), which are expressed in platelet granules and can be released upon activation. CONCLUSIONS: We developed a robust mass cytometry approach to identify platelet phenotypic heterogeneity. Infants with CHD had alterations in distinct subpopulations of platelets with overall reduced aggregatory phenotype and secretory dysfunction. These findings suggest that platelets in infants with CHD may be exhausted due to persistent stimulation and may explain both bleeding and thrombotic vascular complications associated with CHD.

Implementation, enrollment, and engagement in an opt-out telehealth pharmacist-assisted tobacco treatment program for patients seen in oncology outpatient clinics.

OBJECTIVE: To describe the workflow, reach, cost, and self-reported quit rates for an opt-out tobacco treatment program (TTP) for patients seen in 43 oncology outpatient clinics. METHODS: Between May 25, 2021, and December 31, 2022, adult patients (≥18 years) visiting clinics affiliated with the Medical University of South Carolina Hollings Cancer Center were screened for smoking status. Those currently smoking were referred to a telehealth pharmacy-assisted TTP. An attempt was made to contact referred patients by phone. Patients reached were offered free smoking cessation counseling and a 2-week starter kit of nicotine replacement medication. A random sample of 420 patients enrolled in the TTP were selected to participate in a telephone survey to assess smoking status 4 to 12 months after enrollment. RESULTS: During the reference period 35,756 patients were screened and 9.3% were identified as currently smoking. Among the 3319 patients referred to the TTP at least once, 2393 (72.1%) were reached by phone, of whom 426 (12.8%) were ineligible for treatment, 458 (13.8%) opted out of treatment, and 1509 (45.5%) received treatment. More than 90% of TTP enrollees smoked daily, with an average of 13.1 cigarettes per day. Follow-up surveys were completed on 167 of 420 patients, of whom 23.4% to 33.5% reported not smoking; if all nonresponders to the survey are counted as smoking, the range of quit rates is 9.3% to 13.3%. CONCLUSION: The findings demonstrate the feasibility of reaching and delivering smoking cessation treatments to patients from a diverse set of geographically dispersed oncology clinics.

Breast lymphedema following breast-conserving treatment for breast cancer: current status and future directions.

PURPOSE: To examine the current evidence on breast lymphedema (BL) diagnosis and treatment after breast-conserving surgery, identify gaps in the literature, and propose future research directions. METHODS: A comprehensive literature review was conducted using Ovid, PubMed, and Cochrane, including studies published between 2000 and 2023. References were reviewed manually for eligible studies. Inclusion criteria were as follows: patients who underwent breast conserving treatment (surgery ± radiation) for breast cancer, goals of the paper included analyzing or reviewing BL measurement with ultrasound or tissue dielectric constant, or BL treatment. Twenty-seven manuscripts were included in the review. RESULTS: There is variation in incidence, time course, and risk factors for BL. Risk factors for BL included breast size, primary and axillary surgery extent, radiation, and chemotherapy but require further investigation. Diagnostic methods for BL currently rely on patient report and lack standardized criteria. Tissue dielectric constant (TDC) and ultrasound (US) emerged as promising ambulatory BL assessment tools; however, diagnostic thresholds and validation studies with ICG lymphography are needed to establish clinical utility. The evidence base for treatment of BL is weak, lacking high-quality studies. CONCLUSION: The natural history of BL is not well defined. TDC and US show promise as ambulatory assessment tools for BL; however, further validation with lymphatic imaging is required. BL treatment is not established in the literature. Longitudinal, prospective studies including pre-radiation measurements and validating with lymphatic imaging are required. These data will inform screening, diagnostic criteria, and evidence-based treatment parameters for patients with BL after breast-conserving surgery and radiation.

Neutrality in plant-herbivore interactions.

Understanding the distribution of herbivore damage among leaves and individual plants is a central goal of plant-herbivore biology. Commonly observed unequal patterns of herbivore damage have conventionally been attributed to the heterogeneity in plant quality or herbivore behaviour or distribution. Meanwhile, the potential role of stochastic processes in structuring plant-herbivore interactions has been overlooked. Here, we show that based on simple first principle expectations from metabolic theory, random sampling of different sizes of herbivores from a regional pool is sufficient to explain patterns of variation in herbivore damage. This is despite making the neutral assumption that herbivory is caused by randomly feeding herbivores on identical and passive plants. We then compared its predictions against 765 datasets of herbivory on 496 species across 116° of latitude from the Herbivory Variability Network. Using only one free parameter, the estimated attack rate, our neutral model approximates the observed frequency distribution of herbivore damage among plants and especially among leaves very well. Our results suggest that neutral stochastic processes play a large and underappreciated role in natural variation in herbivory and may explain the low predictability of herbivory patterns. We argue that such prominence warrants its consideration as a powerful force in plant-herbivore interactions.

Parental Perspectives From the Survey of Sleep Quality in the PICU Validation Study on Environmental Factors Causing Sleep Disruption in Critically Ill Children.

OBJECTIVES: Sleep promotion bundles being tested in PICUs use elements adapted from adult bundles. As children may react differently than adults in ICU environments, this study investigated what parents report disrupted the sleep of their child in a PICU. DESIGN: Secondary analysis of a multicenter validation study of the Survey of Sleep quality in the PICU. SETTING: Four Northeastern U.S. PICUs, one hospital-based pediatric sleep laboratory. PATIENTS: Parents sleeping at the bedside of a child in the PICU or hospital-based sleep laboratory. INTERVENTIONS: Anonymous one-time survey eliciting parts of hospital or ICU environments that have been described as disruptive to sleep in validated adult ICU and pediatric inpatient questionnaires. MEASUREMENTS AND MAIN RESULTS: Level of sleep disruption was scored by Likert scale, with higher scores indicating more disruption. Age, demographics, baseline sleep, and PICU exposures were used to describe causes of sleep disruption in a PICU. Of 152 PICU parents, 71% of their children's sleep was disrupted significantly by at least one aspect of being in the PICU. The most prevalent were "being in pain or uncomfortable because they are sick" (38%), "not sleeping at home" (30%), "alarms on machines" (28%), and "not sleeping on their home schedule" (26%). Only 5% were disrupted by excessive nocturnal light exposure. Overall sleep disruption was not different across four PICUs or in those receiving sedation. The validation study control group, healthy children undergoing polysomnography, had less sleep disruption than those in a PICU despite sleeping in a hospital-based sleep laboratory. CONCLUSIONS: There are multiple aspects of critical care environments that affect the sleep of children, which are different from that of adults, such as disruption to home schedules. Future interventional sleep promotion bundles should include sedated children and could be applicable in multicenter settings.

Polarization-sensitive optical coherence tomography and scleral collagen fiber orientation in osteogenesis imperfecta.

Osteogenesis imperfecta (OI), a rare genetic connective tissue disorder, primarily arises from pathogenic variants affecting the production or structure of collagen type I. In addition to skeletal fragility, individuals with OI may face an increased risk of developing ophthalmic diseases. This association is believed to stem from the widespread presence of collagen type I throughout various parts of the eye. However, the precise consequences of abnormal collagen type I on different ocular tissues remain unknown. Of particular significance is the sclera, where collagen type I is abundant and crucial for maintaining the structural integrity of the eye. Recent research on healthy individuals has uncovered a unique organizational pattern of collagen fibers within the sclera, characterized by fiber arrangement in both circular and radial layers around the optic nerve head. While the precise function of this organizational pattern remains unclear, it is hypothesized to play a role in providing mechanical support to the optic nerve. The objective of this study is to investigate the impact of abnormal collagen type I on the sclera by assessing the fiber organization near the optic nerve head in individuals with OI and comparing them to healthy individuals. Collagen fiber orientation of the sclera was measured using polarization-sensitive optical coherence tomography (PS-OCT), an extension of the conventional OCT that is sensitive to materials that exhibit birefringence (axial changes in light refraction). Birefringence was quantified and used as imaging contrast to extract collagen fiber orientation as well as the thickness of the radially oriented scleral layer. Three individuals with OI, exhibiting different degrees of disease severity, were assessed and analyzed, along with seventeen healthy individuals. Mean values obtained from individuals with OI were descriptively compared to those of the healthy participant group. PS-OCT revealed a similar orientation pattern of scleral collagen fibers around the optic nerve head between OI individuals and healthy individuals. However, two OI participants exhibited reduced mean birefringence of the radially oriented scleral layer compared to the healthy participant group (OI participant 1 oculus dexter et sinister (ODS): 0.34°/µm, OI participant 2: ODS 0.26°/µm, OI participant 3: OD: 0.29°/µm, OS: 0.28°/µm, healthy participants: ODS 0.38 ± 0.05°/µm). The radially oriented scleral layer was thinner in all OI participants although within ±2 standard deviations of the mean observed in healthy individuals (OI participant 1 OD: 101 µm, OS 104 µm, OI participant 2: OD 97 µm, OS 98 µm, OI participant 3: OD: 94 µm, OS 120 µm, healthy participants: OD 122.8 ± 13.6 µm, OS 120.8 ± 15.1 µm). These findings imply abnormalities in collagen organization or composition, underscoring the necessity for additional research to comprehend the ocular phenotype in OI.

An intervention to improve lumbar puncture rates for meningitis surveillance in children at four secondary health facilities in Malawi: A before/after analysis.

OBJECTIVES: A lumbar puncture (LP) procedure plays a key role in meningitis diagnosis. In Malawi and other sub-Saharan African countries, LP completion rates are sometimes poor, making meningitis surveillance challenging. Our objective was to measure LP rates following an intervention to improve these during a sentinel hospital meningitis surveillance exercise in Malawi. METHODS: We conducted a before/after intervention analysis among under-five children admitted to paediatric wards at four secondary health facilities in Malawi. We used local and World Health Organization (WHO) guidelines to determine indications for LP, as these are widely used in low- and middle-income countries (LMIC). The intervention comprised of refresher trainings for facility staff on LP indications and procedure, use of automated reminders to perform LP in real time in the wards, with an electronic data management system, and addition of surveillance-specific clinical officers to support existing health facility staff with performing LPs. Due to the low numbers in the before/after analysis, we also performed a during/after analysis to supplement the findings. RESULTS: A total of 13,375 under-five children were hospitalised over the 21 months window for this analysis. The LP rate was 10.4% (12/115) and 60.4% (32/53) in the before/after analysis, respectively, and 43.8% (441/1006) and 72.5% (424/599) in the supplemental during/after analysis, respectively. In our intervention-specific analysis among the three individual components, there were improvements in the LP rate by 48% (p < 0.001) following the introduction of surveillance-specific clinical officers, 10% (p < 0.001) following the introduction of automated reminders to perform an LP and 13% following refresher training. CONCLUSIONS: This analysis demonstrated a rise in LP rates following our intervention. This intervention package may be considered for planning future facility-based meningitis surveillances in similar low-resource settings.

A first-principles alternative to empirical solvent parameters.

The use of solvents is ubiquitous in chemistry. Empirical parameters, such as the Kamlet-Taft parameters and Gutmann donor/acceptor numbers, have long been used to predict and quantify the effects solvents have on chemical phenomena. Collectively however, such parameters are unsatisfactory, since each describes ultimately the same non-covalent solute-solvent and solute-solute interactions in completely disparate ways. Here we hypothesise that empirical solvent parameters are essentially proxy measures of the electrostatic terms that dominate solvent-solute interactions. On the basis of this hypothesis, we develop a new fundamental descriptor of these interactions, , and show that it is a self-consistent, probe-free, first principles alternative to established empirical solvent parameters.

Chronic respiratory symptoms following deployment-related occupational and environmental exposures among US veterans.

OBJECTIVES: Characterise inhalational exposures during deployment to Afghanistan and Southwest Asia and associations with postdeployment respiratory symptoms. METHODS: Participants (n=1960) in this cross-sectional study of US Veterans (Veterans Affairs Cooperative Study 'Service and Health Among Deployed Veterans') completed an interviewer-administered questionnaire regarding 32 deployment exposures, grouped a priori into six categories: burn pit smoke; other combustion sources; engine exhaust; mechanical and desert dusts; toxicants; and military job-related vapours gas, dusts or fumes (VGDF). Responses were scored ordinally (0, 1, 2) according to exposure frequency. Factor analysis supported item reduction and category consolidation yielding 28 exposure items in 5 categories. Generalised linear models with a logit link tested associations with symptoms (by respiratory health questionnaire) adjusting for other covariates. OR were scaled per 20-point score increment (normalised maximum=100). RESULTS: The cohort mean age was 40.7 years with a median deployment duration of 11.7 months. Heavy exposures to multiple inhalational exposures were commonly reported, including burn pit smoke (72.7%) and VGDF (72.0%). The prevalence of dyspnoea, chronic bronchitis and wheeze in the past 12 months was 7.3%, 8.2% and 15.6%, respectively. Burn pit smoke exposure was associated with dyspnoea (OR 1.22; 95% CI 1.06 to 1.47) and chronic bronchitis (OR 1.22; 95% CI 1.13 to 1.44). Exposure to VGDF was associated with dyspnoea (OR 1.29; 95% CI 1.14 to 1.58) and wheeze (OR 1.18; 95% CI 1.02 to 1.35). CONCLUSION: Exposures to burn pit smoke and military occupational VGDF during deployment were associated with an increased odds of chronic respiratory symptoms among US Veterans.

Family Rules and Routines During the Early Phases of Pediatric Cancer Treatment: Associations With Child Emotional and Behavioral Health.

OBJECTIVE: Consistent family rules and routines promote positive adaptation to stress and may be protective to child emotional and behavioral functioning. Few studies have quantified family engagement in these behaviors during pediatric cancer treatment or examined associations with child emotional and behavioral health. METHODS: In this cross-sectional observational study, 86 primary caregivers of youth ages 2-14 years (M = 7.9) with an initial diagnosis of cancer within 16 weeks reported on their frequency of engagement in family rules and routines (e.g., sleep, schoolwork, and meal routines) before their child's cancer diagnosis and their current frequency of engagement in the same routines. Caregivers also reported demographics, psychosocial distress, and child emotional and behavioral health outcomes. Analyses examined demographic and psychosocial factors associated with engagement in rules and routines during cancer treatment, and associations with child emotional and behavioral health. RESULTS: Families reported a lower frequency of engagement in rules and routines during cancer treatment, compared to before treatment (mean difference 0.8 SDs [95% confidence interval 0.7-1.1 SDs]). Caregiver factors associated with lower engagement in rules and routines during treatment included being married, having lower educational attainment, and higher levels of psychosocial distress. Families who engaged in higher levels of rules and routines during treatment reported fewer child externalizing and behavioral challenges. There was limited evidence of association between family rules and routines and child internalizing outcomes. CONCLUSIONS: Results found that engaging in family rules and routines during cancer treatment was associated with fewer child behavioral challenges during treatment. Future directions include longitudinal examinations of family rules, routines, and child emotional/behavioral outcomes to examine directional impact over time.