Massively parallel single-cell sequencing of diverse microbial populations.

Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.

Controlling organization and forces in active matter through optically defined boundaries.

Living systems are capable of locomotion, reconfiguration and replication. To perform these tasks, cells spatiotemporally coordinate the interactions of force-generating, 'active' molecules that create and manipulate non-equilibrium structures and force fields of up to millimetre length scales1-3. Experimental active-matter systems of biological or synthetic molecules are capable of spontaneously organizing into structures4,5 and generating global flows6-9. However, these experimental systems lack the spatiotemporal control found in cells, limiting their utility for studying non-equilibrium phenomena and bioinspired engineering. Here we uncover non-equilibrium phenomena and principles of boundary-mediated control by optically modulating structures and fluid flow in an engineered system of active biomolecules. Our system consists of purified microtubules and light-activatable motor proteins that crosslink and organize the microtubules into distinct structures upon illumination. We develop basic operations-defined as sets of light patterns-to create, move and merge the microtubule structures. By combining these operations, we create microtubule networks that span several hundred micrometres in length and contract at speeds up to an order of magnitude higher than the speed of an individual motor protein. We manipulate these contractile networks to generate and sculpt persistent fluid flows. The principles of boundary-mediated control that we uncover may be used to study emergent cellular structures and forces and to develop programmable active-matter devices.

Patients with substance use disorders receiving continued care in skilled nursing facilities following hospitalization.

Background: As hospitals in the US face pressures to reduce lengths of stay, healthcare systems are increasingly utilizing skilled nursing facilities (SNFs) to continue treating patients stable enough to leave the hospital, but not to return home. Substance use disorder (SUD) can complicate care of patients transferred to SNFs. The objective of this paper is to understand SNF experiences for this population of patients with comorbid SUD transferred to SNFs and examine care experiences in these facilities. Methods: This secondary mixed-methods analysis focuses on SNF experiences from a clinical trial of patient navigation services for medically-hospitalized adults with comorbid opioid, cocaine, and/or alcohol use disorder. This study compared baseline assessments and medical record review for participants (N = 400) with vs. without SNF transfer, and analyzed semi-structured qualitative interviews with a subsample of 15 participants purposively selected based on their transfer to a SNF. Results: Over 1 in 4 participants had a planned discharged to a SNF (26.8% sub-acute, 3.3% acute). Compared to participants with other types of discharge, participants discharged to a SNF had longer initial hospitalizations (4.9 vs. 11.8 days, p < 0.001), and were more likely to be White (38.6 vs. 50.8%; p = 0.02), female (38.9 vs. 52.5%; p = 0.01), have opioid use disorder (75.7 vs. 85.0%, p = 0.03), and be hospitalized for infection (43.6 vs. 58.3%; p = 0.007), and less likely to have worked prior to hospitalization (24.3 vs. 12.5%; p = 0.006). Qualitative narratives identified several themes from the SNF experience, including opioid analgesic dosing issues, challenges to the use of opioid agonist treatment of OUD, illicit opioid dealing/use, and limited access to addiction recovery support services during and following the SNF stay. Conclusions: SNFs are a common disposition for patients in need of subacute services following hospitalization but may be ill-equipped to properly manage patients in need of new or continuing SUD treatment.

Organizational Acceptability of Implementing SBIRT for Adolescents in Primary Care.

INTRODUCTION: Adolescent illicit drug, tobacco, and alcohol use can result in sudden and long-term negative health consequences. Primary care environments present the optimal opportunity for screening and brief interventions that target prevention and curtailing use. Screening, Brief Intervention, and Referral to Treatment (SBIRT) is a service delivery method that could potentially be well-integrated into primary care settings and used to serve a high volume of adolescents. Methods: This qualitative analysis of clinic staff interviews (N = 20), collected during a large cluster-randomized trial to implement two models of adolescent SBIRT, examined barriers and facilitating factors to overall acceptability of SBIRT. This study was conducted in a large, urban Federally Qualified Health Center (FQHC) at 7 sites throughout Baltimore City, Maryland, USA. Participants from each clinic included a range of various roles and responsibilities including: medical assistants (n = 3), nurses (n = 3), primary care providers (n = 4), behavioral health counselors (n = 4), and administrators (n = 6). Results: Results indicate both barriers and facilitating factors for acceptability of SBIRT in terms of (1) universal screening, (2) provider time demands, (3) behavioral health collaboration, and (4) behavioral health caseloads. Discussion: Universal screening was acceptable to participants across organizational roles, but brief interventions and referrals to treatment were found substantially less acceptable.

Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling.

Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4(-/-)) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4(-/-) mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.

Cervical cancer screening and follow-up in 4 geographically diverse US health care systems, 1998 through 2007.

BACKGROUND: Cervical cancer screening and follow-up guidelines have changed considerably in recent years, but to the authors' knowledge few published reports exist to estimate the impact of these changes in community-based settings. The authors examined the patterns and results of cervical cancer testing and follow-up over a decade in 4 geographically diverse US health care systems to inform the future evaluation of changes resulting from increased uptake of the human papillomavirus (HPV) vaccination. METHODS: The authors studied women aged 21 to 65 years who were members of one of these health systems at any time between 1998 and 2007. Data were collected and standardized across sites, based on receipt of Papanicolaou (Pap) and HPV tests, HPV vaccination, cervical biopsies, and treatment of cervical dysplasia. Annual rates (per 1000 person-years) of Pap testing, HPV testing, and cervical biopsy and treatment procedures were calculated. Screening intervals and trends in the results of screening Pap tests and cervical biopsies also were examined. RESULTS: Pap testing rates decreased (from 483 per 1000 person-years in 2000 to 412 per 1000 person-years in 2007) and HPV testing rates increased over the study period. Screening frequency varied across health care systems, and many women continued to receive annual testing. All 4 sites moved to less frequent screening over the study period without marked changes in the overall use of cervical biopsy or treatment. CONCLUSIONS: Despite differences over time and across health plans in rates of cervical cancer testing and follow-up cervical procedures, the authors found no notable differences in Pap test results, diagnostic or treatment procedure rates, or pathological outcomes. This finding suggests that the longer screening intervals did not lead to more procedures or more cancer diagnoses.

Specialty use among patients with treated hypertension in a patient-centered medical home.

BACKGROUND: Little is known about how delivery of primary care in the patient-centered medical home (PCMH) influences outpatient specialty care use. OBJECTIVE: To describe changes in outpatient specialty use among patients with treated hypertension during and after PCMH practice transformation. DESIGN: One-group, 48-month interrupted time series across baseline, PCMH implementation and post-implementation periods. PATIENTS: Adults aged 18-85 years with treated hypertension. INTERVENTION: System-wide PCMH redesign implemented across 26 clinics in an integrated health care delivery system, beginning in January 2009. MAIN MEASURES: Resource Utilization Band variables from the Adjusted Clinical Groups case mix software characterized overall morbidity burden (low, medium, high). Negative binomial regression models described adjusted annual differences in total specialty care visits. Poisson regression models described adjusted annual differences in any use (yes/no) of selected medical and surgical specialties. KEY RESULTS: Compared to baseline, the study population averaged 7% fewer adjusted specialty visits during implementation (P < 0.001) and 4% fewer adjusted specialty visits in the first post-implementation year (P = 0.02). Patients were 12% less likely to have any cardiology visits during implementation and 13% less likely during the first post-implementation year (P < 0.001). In interaction analysis, patients with low morbidity had at least 27% fewer specialty visits during each of 3 years following baseline (P < 0.001); medium morbidity patients had 9% fewer specialty visits during implementation (P < 0.001) and 5% fewer specialty visits during the first post-implementation year (P = 0.007); high morbidity patients had 3% (P = 0.05) and 5% (P = 0.009) higher specialty use during the first and second post-implementation years, respectively. CONCLUSIONS: Results suggest that more comprehensive primary care in this PCMH redesign enabled primary care teams to deliver more hypertension care, and that many needs of low morbidity patients were within the scope of primary care practice. New approaches to care coordination between primary care teams and specialists should prioritize high morbidity, clinically complex patients.

Changes in office visit use associated with electronic messaging and telephone encounters among patients with diabetes in the PCMH.

PURPOSE: Telephone- and Internet-based communication are increasingly common in primary care, yet there is uncertainty about how these forms of communication affect demand for in-person office visits. We assessed whether use of copay-free secure messaging and telephone encounters was associated with office visit use in a population with diabetes. METHODS: We used an interrupted time series design with a patient-quarter unit of analysis. Secondary data from 2008-2011 spanned 3 periods before, during, and after a patient-centered medical home (PCMH) redesign in an integrated health care delivery system. We used linear regression models to estimate proportional changes in the use of primary care office visits associated with proportional increases in secure messaging and telephone encounters. RESULTS: The study included 18,486 adults with diabetes. The mean quarterly number of primary care contacts increased by 28% between the pre-PCMH baseline and the postimplementation periods, largely driven by increased secure messaging; quarterly office visit use declined by 8%. In adjusted regression analysis, 10% increases in secure message threads and telephone encounters were associated with increases of 1.25% (95% CI, 1.21%-1.29%) and 2.74% (95% CI, 2.70%-2.77%) in office visits, respectively. In an interaction model, proportional increases in secure messaging and telephone encounters remained associated with increased office visit use for all study periods and patient subpopulations (P<.001). CONCLUSIONS: Before and after a medical home redesign, proportional increases in secure messaging and telephone encounters were associated with additional primary care office visits for individuals with diabetes. Our findings provide evidence on how new forms of patient-clinician communication may affect demand for office visits.

Patient and provider medication preferences affect treatment outcomes among adolescents and young adults with opioid use disorder.

BACKGROUND: The opioid epidemic in the United States has not spared youth or young adults, as evidenced by a six-fold increase in opioid use disorder (OUD) diagnoses in the last two decades. Given this dramatic rise, a call for greater uptake and accessibility of medications for opioid use disorder (MOUDs) among youth and young adults has ensued, resulting in an increasing number of MOUD treatment pathways for this vulnerable population. METHODS: This secondary data analysis seeks to characterize patient and provider preferences for MOUD treatment pathways, and test for associations between baseline MOUD treatment preferences and opioid use and treatment adherence outcomes. Participants included 288 youth and young adults (age 15-21 years), recruited from a residential treatment program in Maryland. The study assessed patient preferences at baseline (n = 253) and provider preferences at patient treatment discharge (n = 224). Mixed-effects negative binomial regression models were conducted for opioid use outcomes, and logistic regressions were conducted for treatment adherence outcomes. RESULTS: Results indicate that congruence of treatment with patients' (Incidence Rate Ratio [IRR] = 0.65) and providers' (IRR = 0.66) preferences was significantly associated with reduced self-reported days of opioid use in the past 90 days, but only for patients receiving extended-release naltrexone (XR-NTX). Results also indicated that patients were less likely to switch medication treatment pathways (e.g., from XR-NTX to buprenorphine, or vice versa) during follow-up if they received their preferred treatment at baseline, a finding which held true for both XR-NTX (Odds Ratio [OR] = 0.32) and buprenorphine (OR = 0.22). CONCLUSIONS: Receipt of MOUD congruent with patient and provider preferences was associated with reduced opioid use and greater treatment adherence in this sample of youth and young adults with OUD.

Telemetry without collars: performance of fur- and ear-mounted satellite tags for evaluating the movement and behaviour of polar bears.

The study of animal movement provides insights into underlying ecological processes and informs analyses of behaviour and resource use, which have implications for species management and conservation. The tools used to study animal movement have evolved over the past decades, allowing for data collection from a variety of species, including those living in remote environments. Satellite-linked radio and GPS collars have been used to study polar bear (Ursus maritimus) ecology and movements throughout the circumpolar Arctic for over 50 years. However, due to morphology and growth constraints, only adult female polar bears can be reliably collared. Collars have proven to be safe, but there has been opposition to their use, resulting in a deficiency in data across much of the species' range. To bolster knowledge of movement characteristics and behaviours for polar bears other than adult females, while also providing an alternative to collars, we tested the use of fur- and ear-mounted telemetry tags that can be affixed to polar bears of any sex and age. We tested three fur tag designs (SeaTrkr, tribrush and pentagon tags), which we affixed to 15 adult and 1 subadult male polar bears along the coast of Hudson Bay during August-September 2021-2022. Fur tags were compared with ear tags deployed on 42 subadult and adult male polar bears captured on the coast or the sea ice between 2016 and 2022. We used data from the tags to quantify the amount of time subadult and adult males spent resting versus traveling while on land. Our results show the three fur tag designs remained functional for shorter mean durations (SeaTrkr = 58 days; tribrush = 47 days; pentagon = 22 days) than ear tags (121 days), but positional error estimates were comparable among the Argos-equipped tags. The GPS/Iridium-equipped SeaTrkr fur tags provided higher resolution and more frequent location data. Combined, the tags provided sufficient data to model different behavioural states. Furthermore, as hypothesized, subadult and adult male polar bears spent the majority of their time resting while on land, increasing time spent traveling as temperatures cooled. Fur tags show promise as a short-term means of collecting movement data from free-ranging polar bears. Supplementary Information: The online version contains supplementary material available at 10.1186/s40317-024-00373-2.

Spreading a medical home redesign: effects on emergency department use and hospital admissions.

PURPOSE: The patient-centered medical home (PCMH) is being rapidly deployed in many settings to strengthen US primary care, improve quality, and control costs; however, evidence supporting this transformation is still lacking. We describe the Group Health experience in attempting to replicate the effects on health care use seen in a PCMH prototype clinic via a systemwide spread using Lean as the change strategy. METHODS: We used an interrupted time series analysis with a patient-month unit of analysis over a 4-year period that included baseline, implementation, and stabilization periods for 412,943 patients. To account for secular trends across these periods, we compared changes in use of face-to-face primary care visits, emergency department visits, and inpatient admissions with those of a nonequivalent comparison group of patients served by community network practices. RESULTS: After accounting for secular trends among network patients, patients empaneled to the PCMH clinics had 5.1% and 6.7% declines in primary care office visits in early and later stabilization years, respectively, after the implementation year. This trend was accompanied by a 123% increase in the use of secure electronic message threads and a 20% increase in telephone encounters. Declines were also seen in emergency department visits at 1 and 2 years (13.7% and 18.5%) compared with what would be expected based on secular trends in network practices. No statistically significant changes were found for hospital admissions. CONCLUSIONS: The Group Health experience shows it is possible to reduce emergency department use with PCMH transformation across a diverse set of clinics using a clear change strategy (Lean) and sufficient resources and supports.

Development of a diagnostic test set to assess agreement in breast pathology: practical application of the Guidelines for Reporting Reliability and Agreement Studies (GRRAS).

BACKGROUND: Diagnostic test sets are a valuable research tool that contributes importantly to the validity and reliability of studies that assess agreement in breast pathology. In order to fully understand the strengths and weaknesses of any agreement and reliability study, however, the methods should be fully reported. In this paper we provide a step-by-step description of the methods used to create four complex test sets for a study of diagnostic agreement among pathologists interpreting breast biopsy specimens. We use the newly developed Guidelines for Reporting Reliability and Agreement Studies (GRRAS) as a basis to report these methods. METHODS: Breast tissue biopsies were selected from the National Cancer Institute-funded Breast Cancer Surveillance Consortium sites. We used a random sampling stratified according to woman's age (40-49 vs. ≥50), parenchymal breast density (low vs. high) and interpretation of the original pathologist. A 3-member panel of expert breast pathologists first independently interpreted each case using five primary diagnostic categories (non-proliferative changes, proliferative changes without atypia, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive carcinoma). When the experts did not unanimously agree on a case diagnosis a modified Delphi method was used to determine the reference standard consensus diagnosis. The final test cases were stratified and randomly assigned into one of four unique test sets. CONCLUSIONS: We found GRRAS recommendations to be very useful in reporting diagnostic test set development and recommend inclusion of two additional criteria: 1) characterizing the study population and 2) describing the methods for reference diagnosis, when applicable.

The Microbiome-TIME Axis: A Host of Possibilities.

Cancer continues to be a significant source of mortality and morbidity worldwide despite progress in cancer prevention, early detection, and treatment. Fortunately, immunotherapy has been a breakthrough in the treatment of many cancers. However, the response to immunotherapy treatment and the experience of associated side effects varies significantly between patients. Recently, attention has been given to understanding the role of the tumor immune microenvironment (TIME) in the development, progression, and treatment response of cancer. A new understanding of the role of the microbiota in the modulation of the TIME has further complicated the story but also unlocked a new area of adjuvant therapeutic research. The complex balance of tumor-permissive and tumor-suppressive immune environments requires further elucidation in order to be harnessed as a therapeutic target. Because both the TIME and the microbiome show importance in these areas, we propose here the concept of the "microbiome-TIME axis" to review the current field of research and future directions.

A systematic review of typologies on aged care system components to facilitate complex comparisons.

Objectives: Typologies are frequently utilised in analyses of the quality, funding, and efficiency of aged care systems. This review aims to provide a comprehensive resource identifying and critiquing existing aged care typologies. Methods: Systematic search of MEDLINE, Econlit, Google Scholar, greylit.org and Open Grey databases from inception to July 2020, including typologies of national, regional or provider aged care systems. Article screening, data extraction, and quality appraisal were conducted in duplicate. Results: 14 aged care typologies were identified; five applied to residential care, two to home care and seven to mixed settings; eight examined national systems and seven regional or provider systems. Five typologies classifying national financing or home care services, provider financing of staff and services and quality of residential care were considered high quality. The schematic provided summarises the focus area and aids in typology selection. Discussion: The aged care typologies identified cover a wide range of areas and contexts of aged care provision. This schematic, summary and critique will aid researchers, providers, and aged care policy makers to examine their own setting, compare it to other approaches to aged care provision and assist in identifying alternatives and important considerations, when undertaking aged care reform.

Spatio-temporal patterning of extensile active stresses in microtubule-based active fluids.

Microtubule-based active fluids exhibit turbulent-like autonomous flows, which are driven by the molecular motor powered motion of filamentous constituents. Controlling active stresses in space and time is an essential prerequisite for controlling the intrinsically chaotic dynamics of extensile active fluids. We design single-headed kinesin molecular motors that exhibit optically enhanced clustering and thus enable precise and repeatable spatial and temporal control of extensile active stresses. Such motors enable rapid, reversible switching between flowing and quiescent states. In turn, spatio-temporal patterning of the active stress controls the evolution of the ubiquitous bend instability of extensile active fluids and determines its critical length dependence. Combining optically controlled clusters with conventional kinesin motors enables one-time switching from contractile to extensile active stresses. These results open a path towards real-time control of the autonomous flows generated by active fluids.

Single-cell analysis of multiple invertible promoters reveals differential inversion rates as a strong determinant of bacterial population heterogeneity.

Population heterogeneity can promote bacterial fitness in response to unpredictable environmental conditions. A major mechanism of phenotypic variability in the human gut symbiont Bacteroides spp. involves the inversion of promoters that drive the expression of capsular polysaccharides, which determine the architecture of the cell surface. High-throughput single-cell sequencing reveals substantial population heterogeneity generated through combinatorial promoter inversion regulated by a broadly conserved serine recombinase. Exploiting control over population diversification, we show that populations with different initial compositions converge to a similar composition over time. Combining our data with stochastic computational modeling, we demonstrate that the differential rates of promoter inversion are a major mechanism shaping population dynamics. More broadly, our approach could be used to interrogate single-cell combinatorial phase variable states of diverse microbes including bacterial pathogens.

Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies.

Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi-one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.

Programming bacteria for multiplexed DNA detection.

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.

Ray Optics for Gliders.

Control of self-propelled particles is central to the development of many microrobotic technologies, from dynamically reconfigurable materials to advanced lab-on-a-chip systems. However, there are few physical principles by which particle trajectories can be specified and can be used to generate a wide range of behaviors. Within the field of ray optics, a single principle for controlling the trajectory of light─Snell's law─yields an intuitive framework for engineering a broad range of devices, from microscopes to cameras and telescopes. Here we show that the motion of self-propelled particles gliding across a resistance discontinuity is governed by a variant of Snell's law, and develop a corresponding ray optics for gliders. Just as the ratio of refractive indexes sets the path of a light ray, the ratio of resistance coefficients is shown to determine the trajectories of gliders. The magnitude of refraction depends on the glider's shape, in particular its aspect ratio, which serves as an analogue to the wavelength of light. This enables the demixing of a polymorphic, many-shaped, beam of gliders into distinct monomorphic, single-shaped, beams through a friction prism. In turn, beams of monomorphic gliders can be focused by spherical and gradient friction lenses. Alternatively, the critical angle for total internal reflection can be used to create shape-selective glider traps. Overall our work suggests that furthering the analogy between light and microscopic gliders may be used for sorting, concentrating, and analyzing self-propelled particles.

Examining the Effectiveness of the FaCES Adolescent SBIRT Intervention.

PURPOSE: The Facilitating Change for Excellence in SBIRT (FaCES) is a service package for adolescent primary care that was developed based on best practices and evidence, but was empirically untested. The aim of this study is to compare the FaCES intervention to treatment as usual (TAU) for rural adolescent primary care patients. METHODS: In this modified cluster-randomized stepped wedge design, providers who completed at least 20 adolescent TAU visits received training in the FaCES package in random order. Adolescent patients (N = 1,226) waiting for appointments were continuously recruited into the study and completed a baseline assessment before their scheduled appointment and an on-line 3-month follow-up. Participants received either FaCES or TAU, depending on whether their provider had been trained in FaCES. Due to COVID-19 disruptions, only 14 of the 29 providers were trained before study recruitment activities ceased. RESULTS: More than 80% of the sample indicated no prior use of tobacco, alcohol, or marijuana at study entry. The Arm × Time interaction failed to reach significance for the substance use outcomes considered. In the FaCES condition, the group with no prior use had an increased probability of substance use at 3-month follow-up, while the group reporting prior use had a decreased probability of use at follow-up. Participants who reported no use at baseline had an increased probability of use at follow-up, whether they received the FaCES intervention or TAU. DISCUSSION: This study was unable to demonstrate the effectiveness of FaCES. Findings suggest some natural movement in substance use risk over time.