Transcription elongation mechanisms of RNA polymerases I, II, and III and their therapeutic implications.

Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.

Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.

The small-molecule BMH-21 directly inhibits transcription elongation and DNA occupancy of RNA polymerase I in vivo and in vitro.

Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small-molecule BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use.

Defining the divergent enzymatic properties of RNA polymerases I and II.

Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult because studies are often conducted under disparate experimental conditions and techniques. Here, we directly compare and reveal functional differences between Saccharomyces cerevisiae Pols I and II using a series of quantitative in vitro transcription assays. We find that Pol I single-nucleotide and multinucleotide addition rate constants are faster than those of Pol II. Pol I elongation complexes are less stable than Pol II elongation complexes, and Pol I is more error prone than Pol II. Collectively, these data show that the enzymatic properties of the Pols have diverged over the course of evolution, optimizing these enzymes for their unique cellular responsibilities.

Human Melioidosis Caused by Novel Transmission of Burkholderia pseudomallei from Freshwater Home Aquarium, United States1.

Nearly all cases of melioidosis in the continental United States are related to international travel to areas to which Burkholderia pseudomallei, the bacterium that causes melioidosis, is endemic. We report the diagnosis and clinical course of melioidosis in a patient from the United States who had no international travel history and the public health investigation to determine the source of exposure. We tested environmental samples collected from the patient's home for B. pseudomallei by PCR and culture. Whole-genome sequencing was conducted on PCR-positive environmental samples, and results were compared with sequences from the patient's clinical specimen. Three PCR-positive environmental samples, all collected from a freshwater home aquarium that had contained imported tropical fish, were a genetic match to the clinical isolate from the patient. This finding suggests a novel route of exposure and a potential for importation of B. pseudomallei, a select agent, into the United States from disease-endemic areas.

The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells.

Fatty acid uptake and metabolism are often dysregulated in cancer cells. Fatty acid activation is a critical step that allows these biomolecules to enter cellular metabolic pathways such as mitochondrial ß-oxidation for ATP generation or the lipogenic routes that generate bioactive lipids such as the inositol phospholipids. Fatty acid activation by the addition of coenzyme A is catalysed by a family of enzymes called the acyl CoA synthetase ligases (ACSL). Furthermore, enhanced expression of particular ACSL isoforms, such as ACSL4, is a feature of some more aggressive cancers and may contribute to the oncogenic phenotype. This study focuses on ACSL3 and ACSL4, closely related structural homologues that preferentially activate palmitate and arachidonate fatty acids, respectively. In this study, immunohistochemical screening of multiple soft tissue tumour arrays revealed that ACSL3 and ACSL4 were highly, but differentially, expressed in a subset of leiomyosarcomas, fibrosarcomas and rhabdomyosarcomas, with consistent cytoplasmic and granular stainings of tumour cells. The intracellular localisations of endogenously expressed ACSL3 and ACSL4 were further investigated by detailed subcellular fractionation analyses of HT1080 fibrosarcoma and MCF-7 breast cancer cells. ACSL3 distribution closely overlapped with proteins involved in trafficking from the trans-Golgi network and endosomes. In contrast, the ACSL4 localisation pattern more closely followed that of calnexin which is an  endoplasmic reticulum resident chaperone. Confocal immunofluorescence imaging of MCF-7 cells confirmed the intracellular localisations of both enzymes. These observations reveal new information regarding the compartmentation of fatty acid metabolism in cancer cells.

Caregiver-reported religious beliefs and complementary and alternative medicine use among children admitted to an epilepsy monitoring unit.

Complementary and alternative medicine (CAM) includes a wide range of practices and products that are generally outside the use of conventional medicine as practiced in Western cultures. Use of CAM in persons with epilepsy is high, even compared to individuals with other chronic health conditions. In this study, we surveyed caregivers of children admitted to a regional epilepsy monitoring unit (EMU) in the southeast United States to assess CAM use among patients (N=225). Thirteen percent of respondents indicated current use of CAM by their child, 16% reported past use, and 43% reported interest in future use, most commonly in marijuana as a potential treatment (23%). Over 25% of respondents expressed interest in CAM use related to side effects of anti-epileptic medications. Regarding prayer as a form of CAM, a large majority of respondents in this sample identified as Christian and actively prayed for their child's illness, revealing a high prevalence of spiritual practices in this population. Eighty-one percent of respondents reported that they had not discussed CAM use with their doctor. Discussing CAM use with a health care provider was significantly related to past CAM use (p<.02), but not current use or willingness to try CAM in the future (p>.05). These results have important implications for future practice and support increased communication and patient education, as many anti-epileptic medications interact with certain herbs and supplements, posing a potential health risk and treatment barrier in this population.

Scale-up of collaborative TB/HIV activities in Guyana.

OBJECTIVE: To assess scale-up of recommended tuberculosis (TB)/HIV activities in Guyana and to identify specific strategies for further expansion. METHODS: Medical records and clinic registers were reviewed at nine TB clinics and 10 HIV clinics. At TB clinics, data were collected on HIV testing and antiretroviral therapy (ART) for patients with TB/HIV; at HIV clinics, data were collected on intensified case finding (ICF), tuberculin skin test (TST) results, and provision of isoniazid preventive therapy (IPT). RESULTS: At TB clinics, among 461 patients newly diagnosed with TB, 419 (90.9%) had a known HIV status and 121 (28.9%) were HIV-infected. Among the 63 patients with TB/HIV, 33 (52.4%) received ART. Among the 45 patients with TB/HIV for whom dates of HIV diagnosis were available, 38 (84.4%) individuals knew their HIV status prior to TB diagnosis. At HIV clinics, among 127 patients eligible to receive a TST, 87 (68.5%) received a TST, 66 (75.9%) had a TST result, seven (10.6%) had a newly positive result, two had a previously positive result, and six of nine patients with positive results (66.7%) received IPT. ICF could not be assessed because of incomplete or discrepant documentation. CONCLUSIONS: An in-depth evaluation of TB/HIV activities successfully identified areas of success and remaining challenges. At TB clinics, HIV testing rates are high; further scale-up of ART for persons with TB/HIV is needed. At HIV clinics, use of TST to focus IPT is a feasible and efficient strategy; improving rates of annual TST screening will allow for further expansion of IPT.

Effects of intrauterine substance and postnatal violence exposure on aggression in children.

During the cocaine epidemic of the 1980s and early 1990s, many expressed fears that children with intrauterine cocaine exposure (IUCE) would grow up to be unusually violent. The present study examines the relationship of caregiver reports of school-age children's aggressive behavior with IUCE and postnatal exposure to violence. Respondents were 140 low-income, primarily African American children, ages 8-11, and each child's current primary caregiver from a longitudinal study evaluating potential long term sequelae of IUCE. Multiple regression analyses were used to investigate the independent and interactive effects of level of IUCE (None (n = 69), Lighter (n = 47), Heavier (n = 24)) and exposure to violence (Violence Exposure Scale for Children-Revised) on aggressive behavior (Child Behavior Checklist), while also controlling for other intrauterine substance exposures and additional contextual factors. Children's self-reported exposure to violence was significantly positively associated with caregivers' reports of aggressive behavior (ß = 2.17, P = .05), as was concurrent caregiver's psychiatric distress (ß = .15, P = .003). However, neither IUCE nor its interaction with exposure to violence showed a significant association with aggressive behavior. Findings suggest the importance of postnatal social environment rather than IUCE in predicting aggressive behavior in childhood.

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release.

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF) IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3' end was fastest when the 3' terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.

Protocol for monitoring and analyzing single nucleotide incorporation by S. cerevisiae RNA polymerases.

Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel. Finally, we detail how to quantify the gel images. For complete details on the use and execution of this protocol, please refer to Appling et al. (2015).1.

Associations of household unmet basic needs and health outcomes among very low birth weight children.

OBJECTIVE: We examined associations of past year household hardships (housing, energy, food, and healthcare hardships) with postnatal growth, developmental risk, health status, and hospitalization among children 0-36 months born with very low birth weight (VLBW) and the extent that these relationships differed by receipt of child supplemental security income (SSI). STUDY DESIGN: We examined cross-sectional data from 695 families. Growth was measured as weight-for-age z-score change. Developmental risk was defined as ≥1 concerns on the "Parents' Evaluation of Developmental Status" screening tool. Child health status was categorized as excellent/good vs. fair/poor. Hospitalizations excluded birth hospitalizations. RESULTS: Compared to children with no household hardships, odds of developmental risk were greater with 1 hardship (aOR 2.0 [1.26, 3.17]) and ≥2 hardships (aOR) 1.85 [1.18, 2.91], and odds of fair/poor child health (aOR) 1.59 [1.02, 2.49] and hospitalizations (aOR) 1.49 [1.00, 2.20] were greater among children with ≥2 hardships. In stratified analysis, associations of hardships and developmental risk were present for households with no child SSI and absent for households with child SSI. CONCLUSION: Household hardships were associated with developmental risk, fair/poor health status, and hospitalizations among VLBW children. Child SSI may be protective against developmental risk among children living in households with hardships.

Uncovering the mechanisms of transcription elongation by eukaryotic RNA polymerases I, II, and III.

Eukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical in vitro transcription assays for direct comparison of their elongation rates, elongation complex (EC) stabilities, and fidelities. Pol I is the fastest, most likely to misincorporate, forms the least stable EC, and is most sensitive to alterations in reaction buffers. Pol II is the slowest of the Pols, forms the most stable EC, and negligibly misincorporated an incorrect nucleotide. The enzymatic properties of Pol III were intermediate between Pols I and II in all assays examined. These results reveal unique enzymatic characteristics of the Pols that provide new insights into their evolutionary divergence.

RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21.

Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols' elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.

US Housing insecurity and the health of very young children.

OBJECTIVES: We investigated the association between housing insecurity and the health of very young children. METHODS: Between 1998 and 2007, we interviewed 22,069 low-income caregivers with children younger than 3 years who were seen in 7 US urban medical centers. We assessed food insecurity, child health status, developmental risk, weight, and housing insecurity for each child's household. Our indicators for housing insecurity were crowding (> 2 people/bedroom or>1 family/residence) and multiple moves (≥ 2 moves within the previous year). RESULTS: After adjusting for covariates, crowding was associated with household food insecurity compared with the securely housed (adjusted odds ratio [AOR] = 1.30; 95% confidence interval [CI] = 1.18, 1.43), as were multiple moves (AOR = 1.91; 95% CI = 1.59, 2.28). Crowding was also associated with child food insecurity (AOR = 1.47; 95% CI = 1.34, 1.63), and so were multiple moves (AOR = 2.56; 95% CI = 2.13, 3.08). Multiple moves were associated with fair or poor child health (AOR = 1.48; 95% CI =1.25, 1.76), developmental risk (AOR 1.71; 95% CI = 1.33, 2.21), and lower weight-for-age z scores (-0.082 vs -0.013; P= .02). CONCLUSIONS: Housing insecurity is associated with poor health, lower weight, and developmental risk among young children. Policies that decrease housing insecurity can promote the health of young children and should be a priority.

Food insecurity and risk of poor health among US-born children of immigrants.

OBJECTIVES: We investigated the risk of household food insecurity and reported fair or poor health among very young children who were US citizens and whose mothers were immigrants compared with those whose mothers had been born in the United States. METHODS: Data were obtained from 19,275 mothers (7216 of whom were immigrants) who were interviewed in hospital-based settings between 1998 and 2005 as part of the Children's Sentinel Nutrition Assessment Program. We examined whether food insecurity mediated the association between immigrant status and child health in relation to length of stay in the United States. RESULTS: The risk of fair or poor health was higher among children of recent immigrants than among children of US-born mothers (odds ratio [OR] = 1.26; 95% confidence interval [CI] = 1.02, 1.55; P < .03). Immigrant households were at higher risk of food insecurity than were households with US-born mothers. Newly arrived immigrants were at the highest risk of food insecurity (OR = 2.45; 95% CI = 2.16, 2.77; P < .001). Overall, household food insecurity increased the risk of fair or poor child health (OR = 1.74; 95% CI = 1.57, 1.93; P < .001) and mediated the association between immigrant status and poor child health. CONCLUSIONS: Children of immigrant mothers are at increased risk of fair or poor health and household food insecurity. Policy interventions addressing food insecurity in immigrant households may promote child health.

Pregnant women in treatment for opioid use disorder: Material hardships and psychosocial factors.

BACKGROUND: While pregnant women in treatment for opioid use disorder (OUD) face considerable challenges, common material hardships- food insecurity and housing instability, known to negatively impact maternal-child health, have been inadequately researched within this population. This study describes food/housing hardships and evaluates associations with key psychosocial factors. METHODS: A single-site prospective study, 100 3rd trimester women receiving prenatal care and medication-assisted treatment for OUD were interviewed, including screening for food/housing hardships, depressive symptoms, intimate partner vulnerability; and self-reported post-traumatic stress disorder (PTSD) history. We developed a three-level categorization combining food/housing screening outcomes: 1) "both insecure"; 2) "either secure"; and 3) "both secure". Bivariate analyses and linear path analyses evaluated associations among psychosocial variables using "both secure" as the referent group. RESULTS: Of 100 women, 56% reported food insecurity; 61% housing instability; 42% "both insecure"; 33% "either insecure"; 25% "both secure". In unadjusted food/housing groups "either insecure" and "both insecure" reported significantly greater depressive symptoms; "both insecure" additionally reported significantly greater intimate partner vulnerability. Path analyses adjusted for PTSD and compared with "both secure" (adjusted mean = 6.2): "either insecure" had greater depressive symptom scores (adjusted means = 9.8, p = .01) while "both insecure" had greater depressive scores (adjusted means 10.5, p = .002). In addition, "both insecure" had a clinically important 5.7 point greater intimate partner vulnerability score. There were no significant interactions between food/housing and PTSD. CONCLUSIONS: Even in women receiving prenatal care and treatment for OUD, food/housing material hardships and associated psychosocial factors are of major concern, requiring screening and remediation.

Could prenatal food insecurity influence neonatal abstinence syndrome severity?

BACKGROUND AND AIMS: In general populations, prenatal food insecurity negatively affects maternal and infant health. Our aim was to estimate and test the association between prenatal food insecurity and neonatal abstinence syndrome (NAS) severity. DESIGN/SETTING: Single-site prospective cohort design. Women receiving opioid agonist treatment with methadone or buprenorphine were interviewed (including demographics and food insecurity) during the third trimester at the combined obstetric/opioid use disorder treatment clinic at Boston Medical Center (BMC) in Boston, MA, USA, a large urban safety-net hospital. During postnatal hospitalization, infants were assessed and treated per hospital NAS protocol. Maternal clinic and infant hospitalization data were abstracted from medical records. PARTICIPANTS: Women (n = 75; aged ≥ 18 years; fluent English; singleton pregnancy; intending to deliver at BMC and maintain parental custody) receiving care in the specialized clinic were study eligible (2013-15). Women who delivered infants < 36 weeks gestational age or required prolonged newborn intensive care unit stay were excluded from analyses. PRIMARY MEASUREMENTS: Predictors: validated two-question Hunger Vital Sign™ food insecurity screener; outcomes: extent of NAS pharmacological treatment and length of hospital stay (LOS) for NAS. FINDINGS: Of the mother-infant dyads, 61 (81%) infants were treated pharmacologically for NAS. Mean hospital LOS was 19.9 (standard deviation = 9.4) days. Maternal food insecurity (n = 43, 57.3%) was associated with infant NAS pharmacological treatment in logistic regression analyses individually adjusted for prenatal: maternal depression [adjusted odds ratios (aOR) = 3.69 (95% confidence intervals (CI) = 1.02-13.43, P = 0.05)] and methadone agonist treatment [aOR = 4.17 (95% CI = 1.05-16.50, P = 0.04)]. Associations of food insecurity and LOS were inconclusive regardless of covariate control (P > 0.05). CONCLUSION: Among women receiving opioid agonist treatment, prenatal food insecurity appears to be associated with increased risk for neonatal abstinence syndrome pharmacological treatment.