Lactate Elicits ER-Mitochondrial Mg2+ Dynamics to Integrate Cellular Metabolism.

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.

Membraneless channels sieve cations in ammonia-oxidizing marine archaea.

Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.

Rampant asexual reproduction and limited dispersal in a mangrove population of the coral Porites divaricata.

Corals are critical to marine biodiversity. Reproduction and dispersal are key to their resilience, but rarely quantified in nature. Exploiting a unique system-a fully censused, longitudinally characterized, semi-isolated population inhabiting mangroves-we used 2bRAD sequencing to demonstrate that rampant asexual reproduction most likely via parthenogenesis and limited dispersal enable the persistence of a natural population of thin-finger coral (Porites divaricata). Unlike previous studies on coral dispersal, knowledge of colony age and location enabled us to identify plausible parent-offspring relationships within multiple clonal lineages and develop tightly constrained estimates of larval dispersal; the best-fitting model indicates dispersal is largely limited to a few metres from parent colonies. Our results explain why this species is adept at colonizing mangroves but suggest limited genetic diversity in mangrove populations and limited connectivity between mangroves and nearby reefs. As P. divaricata is gonochoristic, and parthenogenesis would be restricted to females (whereas fragmentation, which is presumably common in reef and seagrass habitats, is not), mangrove populations likely exhibit skewed sex ratios. These findings suggest that coral reproductive diversity can lead to distinctly different demographic outcomes in different habitats. Thus, coral conservation will require the protection of the entire coral habitat mosaic, and not just reefs.

Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide.

Lipopolysaccharide (LPS) transport to the outer membrane (OM) is a crucial step in the biogenesis of microbial surface defenses. Although many features of the translocation mechanism have been elucidated, molecular details of LPS insertion via the LPS transport (Lpt) OM protein LptDE remain elusive. Here, we integrate native MS with hydrogen-deuterium exchange MS and molecular dynamics simulations to investigate the influence of substrate and peptide binding on the conformational dynamics of LptDE. Our data reveal that LPS induces opening of the LptD ß-taco domain, coupled with conformational changes on ß-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilizes the ß-taco, thereby preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both the ß-barrel and ß-taco domains of LptD, and suggest a means for developing antimicrobial therapeutics targeting this essential process in Gram-negative ESKAPE pathogens.

Domestic violence assault during the first year of the COVID-19 pandemic: a longitudinal community study.

BACKGROUND: The consequences of the COVID-19 pandemic have been far-reaching, disproportionately impacting vulnerable populations. Of particular concern is the impact on individuals experiencing domestic violence (DV), an urgent public health issue. There have been numerous reports of pandemic-related surges in DV, and it has been speculated that prolonged periods of state-mandated isolation may be the source of these surges. The current study utilized publicly available records to examine fluctuations in DV coinciding with COVID-19 lockdown restrictions in a diverse metropolitan county. METHODS: Data were extracted from local police blotters and mapping engines in Orange County, California (United States), documenting police-reported DV assault. All incidents were coded for time to examine the time course of DV among other types of assault, allowing for a longitudinal view of incidents over a 66-week window. Changepoint analyses were used to determine whether and when DV assaults changed when mapped with coinciding tightening or loosening of restrictions county-wide. Piecewise regression analyses evaluated whether any detected fluctuations were statistically meaningful. RESULTS: In Santa Ana, rates saw a small but significant spike in the week following the first major lockdown in March 2020 (b = .04, SE = .02, t = 2.37, p = .01), remaining stable at this higher level thereafter (b = -.003, SE = .003, t = -1.29, p = .20). In Anaheim, no meaningful change in DV assault rates was observed at any time interval. CONCLUSION: Results suggest that surges in DV vary between communities and that systemic issues may set the stage for the surge of an already endemic problem.

Mechanisms of E. coli chemotaxis signaling pathways visualized using cryoET and computational approaches.

Chemotaxis signaling pathways enable bacteria to sense and respond to their chemical environment and, in some species, are critical for lifestyle processes such as biofilm formation and pathogenesis. The signal transduction underlying chemotaxis behavior is mediated by large, highly ordered protein complexes known as chemosensory arrays. For nearly two decades, cryo-electron tomography (cryoET) has been used to image chemosensory arrays, providing an increasingly detailed understanding of their structure and function. In this mini-review, we provide an overview of the use of cryoET to study chemosensory arrays, including imaging strategies, key results, and outstanding questions. We further discuss the application of molecular modeling and simulation techniques to complement structure determination efforts and provide insight into signaling mechanisms. We close the review with a brief outlook, highlighting promising future directions for the field.

Isolation-by-distance and genetic parentage analysis provide similar larval dispersal estimates.

Larval exchange among marine populations is a vital driver of population dynamics and has the potential to inform conservation actions, but accurately measuring dispersal remains challenging. Consequently, empirical dispersal kernels have been measured for only a few marine species. Here, we obtained indirect dispersal estimates using an isolation-by-distance (IBD) model in the coral reef fish Elacatinus lori and assessed the accuracy of these estimates by comparing them to direct measurements of dispersal from genetic parentage analysis. Specifically, drawing on the IBD slope and effective population density, we indirectly estimated sigma (σ), the spread of a dispersal distribution. While the spread of the directly measured distribution was σ = 3.93 km (95% CI: 3.29-4.71 km), the spread of the IBD distribution was σ = 4.10 km (95% CI: 3.23-5.03) and σ = 2.90 km (95% CI: 2.26-3.59), assuming a random or monogamous mating system, respectively. Parameterizing Laplace dispersal kernels with these values of σ yielded patterns that were remarkably similar to a kernel fit to the direct parentage data. We also found that, like many marine fishes, E. lori has a large effective population size. However, uncertainty in effective size did not ultimately have a strong effect on the IBD-based dispersal estimates. Taken together, these findings illustrate that accurate dispersal estimates can be produced by indirect IBD methods and suggest that this more feasible approach to estimating dispersal may be broadly applicable to the study of marine larval dispersal.

Incorporating putatively neutral and adaptive genomic data into marine conservation planning.

The availability of genomic data for an increasing number of species makes it possible to incorporate evolutionary processes into conservation plans. Recent studies show how genetic data can inform spatial conservation prioritization (SCP), but they focus on metrics of diversity and distinctness derived primarily from neutral genetic data sets. Identifying adaptive genetic markers can provide important information regarding the capacity for populations to adapt to environmental change. Yet, the effect of including metrics based on adaptive genomic data into SCP in comparison to more widely used neutral genetic metrics has not been explored. We used existing genomic data on a commercially exploited species, the giant California sea cucumber (Parastichopus californicus), to perform SCP for the coastal region of British Columbia (BC), Canada. Using a RAD-seq data set for 717 P. californicus individuals across 24 sampling locations, we identified putatively adaptive (i.e., candidate) single nucleotide polymorphisms (SNPs) based on genotype-environment associations with seafloor temperature. We calculated various metrics for both neutral and candidate SNPs and compared SCP outcomes with independent metrics and combinations of metrics. Priority areas varied depending on whether neutral or candidate SNPs were used and on the specific metric used. For example, targeting sites with a high frequency of warm-temperature-associated alleles to support persistence under future warming prioritized areas in the southern coastal region. In contrast, targeting sites with high expected heterozygosity at candidate loci to support persistence under future environmental uncertainty prioritized areas in the north. When combining metrics, all scenarios generated intermediate solutions, protecting sites that span latitudinal and thermal gradients. Our results demonstrate that distinguishing between neutral and adaptive markers can affect conservation solutions and emphasize the importance of defining objectives when choosing among various genomic metrics for SCP.

Incorporación de Datos Genómicos Putativamente Neutros y Adaptativos dentro de la Planeación de la Conservación Marina Resumen La disponibilidad de los datos genómicos para un número creciente de especies posibilita la incorporación de los procesos evolutivos dentro de los planes de conservación. Los estudios recientes muestran cómo los datos genéticos pueden informar a la priorización de la conservación espacial (PCE) pero tienden a enfocarse más en las medidas de la diversidad y la distinción derivadas principalmente de los conjuntos de datos genéticos neutrales. La identificación de los marcadores genéticos adaptativos puede proporcionar información importante con respecto a la capacidad de las poblaciones para adaptarse al cambio ambiental. Aun así, no se ha explorado el efecto de la inclusión de las medidas basadas en los datos genéticos adaptativos dentro de la PCE y cómo se comparan con las medidas genéticas neutrales de uso más amplio. Usamos datos genómicos existentes sobre una especie de explotación comercial, el pepino de mar gigante de California (Parastichopus californicus), para realizar la PCE para la región costera de la Columbia Británica (BC) en Canadá. Usamos un conjunto de datos RAD-seq para 717 individuos de la especie P. californicus en 24 localidades de muestreo para identificar los polimorfismos de un solo nucleótido (PSNs) putativamente adaptativos (es decir, candidatos) con base en las asociaciones genotipo-ambiente manifestadas con la temperatura del fondo marino. Calculamos varias medidas para los PSNs neutrales y los PSNs candidatos y comparamos los resultados de la PCE con medidas independientes y con combinaciones de medidas. Las áreas prioritarias variaron dependiendo de si se usaron los SNP neutrales o los candidatos y de la medida específica que se utilizó. Por ejemplo, enfocarse en sitios con una frecuencia alta de alelos asociados con agua cálida para fortalecer la persistencia frente al futuro calentamiento prioriza las áreas en la región del sur de la costa. Al contrario, enfocarse en sitios con una alta heterocigosidad esperada en los loci de los candidatos para fortalecer la persistencia frente a la incertidumbre ambiental prioriza las áreas en la parte norte de la costa. Cuando combinamos las medidas, todos los escenarios generaron soluciones intermedias, protegiendo así los sitios que abarcan gradientes latitudinales y gradientes térmicos. Nuestros resultados demuestran que la distinción entre los marcadores neutrales y los adaptativos puede afectar las soluciones de conservación y también enfatizan la importancia de la definición de los objetivos cuando se elige entre varias medidas genómicas para la PCE.

Unraveling hierarchical genetic structure in a marine metapopulation: A comparison of three high-throughput genotyping approaches.

Marine metapopulations often exhibit subtle population structure that can be difficult to detect. Given recent advances in high-throughput sequencing, an emerging question is whether various genotyping approaches, in concert with improved sampling designs, will substantially improve our understanding of genetic structure in the sea. To address this question, we explored hierarchical patterns of structure in the coral reef fish Elacatinus lori using a high-resolution approach with respect to both genetic and geographic sampling. Previously, we identified three putative E. lori populations within Belize using traditional genetic markers and sparse geographic sampling: barrier reef and Turneffe Atoll; Glover's Atoll; and Lighthouse Atoll. Here, we systematically sampled individuals at ~10 km intervals throughout these reefs (1,129 individuals from 35 sites) and sequenced all individuals at three sets of markers: 2,418 SNPs; 89 microsatellites; and 57 nonrepetitive nuclear loci. At broad spatial scales, the markers were consistent with each other and with previous findings. At finer spatial scales, there was new evidence of genetic substructure, but our three marker sets differed slightly in their ability to detect these patterns. Specifically, we found subtle structure between the barrier reef and Turneffe Atoll, with SNPs resolving this pattern most effectively. We also documented isolation by distance within the barrier reef. Sensitivity analyses revealed that the number of loci (and alleles) had a strong effect on the detection of structure for all three marker sets, particularly at small spatial scales. Taken together, these results illustrate empirically that high-throughput genotyping data can elucidate subtle genetic structure at previously-undetected scales in a dispersive marine fish.

The Evolution of Marine Larval Dispersal Kernels in Spatially Structured Habitats: Analytical Models, Individual-Based Simulations, and Comparisons with Empirical Estimates.

Understanding the causes of larval dispersal is a major goal of marine ecology, yet most research focuses on proximate causes. Here we ask how ultimate, evolutionary causes affect dispersal. Building on Hamilton and May's classic 1977 article "Dispersal in Stable Habitats," we develop analytic and simulation models for the evolution of dispersal kernels in spatially structured habitats. First, we investigate dispersal in a world without edges and find that most offspring disperse as far as possible, opposite the pattern of empirical data. Adding edges to our model world leads to nearly all offspring dispersing short distances, again a mismatch with empirical data. Adding resource heterogeneity improves our results: most offspring disperse short distances with some dispersing longer distances. Finally, we simulate dispersal evolution in a real seascape in Belize and find that the simulated dispersal kernel and an empirical dispersal kernel from that seascape both have the same shape, with a high level of short-distance dispersal and a low level of long-distance dispersal. The novel contributions of this work are to provide a spatially explicit analytic extension of Hamilton and May's 1977 work, to demonstrate that our spatially explicit simulations and analytic models provide equivalent results, and to use simulation approaches to investigate the evolution of dispersal kernel shape in spatially complex habitats. Our model could be modified in various ways to investigate dispersal evolution in other species and seascapes, providing new insights into patterns of marine larval dispersal.

Mechanism of Acid-Triggered Cargo Release from Lipid Bilayer-Coated Mesoporous Silica Particles.

Lipid bilayer-coated mesoporous silica nanoparticles are unique core-shell nanomaterials currently being developed as drug delivery vehicles. To improve cargo loading and biocirculation, the pore structure and surface chemistry of the particle have been modified and well characterized. However, an understanding of cargo release mechanisms from cellular uptake pathways remains largely unexplored. Here, we present a study of the release mechanism of lipid bilayer-coated silica particles induced by endosomal-like pH change from 7.4 to 5.0. We found that this relatively small pH change produces rapid deformation of the supported lipid bilayer that ultimately results in holes in the membrane. Using a combination of dye release studies, wide-field and confocal fluorescence microscopies, and surface area modeling analysis, we determined that small blister-like structures are formed, which lead to lateral membrane displacement and hole formation. Possible mechanisms for the blister formation, which include curvature effects and interfacial interactions, are discussed.

The formation of marine kin structure: effects of dispersal, larval cohesion, and variable reproductive success.

The spatial distribution of relatives has profound effects on kin interactions, inbreeding, and inclusive fitness. Yet, in the marine environment, the processes that generate patterns of kin structure remain understudied because larval dispersal on ocean currents was historically assumed to disrupt kin associations. Recent genetic evidence of co-occurring siblings challenges this assumption and raises the intriguing question of how siblings are found together after a (potentially) disruptive larval phase. Here, we develop individual-based models to explore how stochastic processes operating at the individual level affect expected kinship at equilibrium. Specifically, we predict how limited dispersal, sibling cohesion, and variability in reproductive success differentially affect patterns of kin structure. All three mechanisms increase mean kinship within populations, but their spatial effects are markedly different. We find that (1) when dispersal is limited, kinship declines monotonically as a function of the distance between individuals; (2) when siblings disperse cohesively, kinship increases within a site relative to between sites; and (3) when reproductive success varies, kinship increases equally at all distances. The differential effects of these processes therefore only become apparent when individuals are sampled at multiple spatial scales. Notably, our models suggest that aggregative larval behaviors, such as sibling cohesion, are not necessary to explain documented levels of relatedness within marine populations. Together, these findings establish a theoretical framework for disentangling the drivers of marine kin structure.

Deficits in striatal dopamine release in cannabis dependence.

Most drugs of abuse lead to a general blunting of dopamine release in the chronic phase of dependence, which contributes to poor outcome. To test whether cannabis dependence is associated with a similar dopaminergic deficit, we examined striatal and extrastriatal dopamine release in severely cannabis-dependent participants (CD), free of any comorbid conditions, including nicotine use. Eleven CD and 12 healthy controls (HC) completed two positron emission tomography scans with [11C]-(+)-PHNO, before and after oral administration of d-amphetamine. CD stayed inpatient for 5-7 days prior to the scans to standardize abstinence. Magnetic resonance spectroscopy (MRS) measures of glutamate in the striatum and hippocampus were obtained in the same subjects. Percent change in [11C]-(+)-PHNO-binding potential (ΔBPND) was compared between groups and correlations with MRS glutamate, subclinical psychopathological and neurocognitive parameters were examined. CD had significantly lower ΔBPND in the striatum (P=0.002, effect size (ES)=1.48), including the associative striatum (P=0.003, ES=1.39), sensorimotor striatum (P=0.003, ES=1.41) and the pallidus (P=0.012, ES=1.16). Lower dopamine release in the associative striatum correlated with inattention and negative symptoms in CD, and with poorer working memory and probabilistic category learning performance in both CD and HC. No relationships to MRS glutamate and amphetamine-induced subclinical positive symptoms were detected. In conclusion, this study provides evidence that severe cannabis dependence-without the confounds of any comorbidity-is associated with a deficit in striatal dopamine release. This deficit extends to other extrastriatal areas and predicts subclinical psychopathology.

Development and Initial Psychometrics for a Therapist Competence Instrument for CBT for Youth Anxiety.

Therapist competence is an important component of treatment integrity. This article reports on the development and initial psychometric assessment of the Cognitive-Behavioral Treatment for Anxiety in Youth Competence Scale (CBAY-C), an observational instrument designed to capture therapist limited-domain competence (i.e., competence in the delivery of core interventions and delivery methods found in a specific psychosocial treatment program) in the delivery of the core practice elements in individual cognitive-behavioral treatment (ICBT) for youth anxiety. Treatment sessions (N = 744) from 68 youth participants (M age = 10.60 years, SD = 2.03; 82.3% Caucasian; 52.9% male) of the same ICBT program for youth anxiety from (a) an efficacy study and (b) an effectiveness study were independently scored by 4 coders using observational instruments designed to assess therapist competence, treatment adherence, treatment differentiation, alliance, and client involvement. Interrater reliability-intraclass correlation coefficients (2,2)-for the item scores averaged 0.69 (SD = 0.11). The CBAY-C item, scale, and subscale (Skills, Exposure) scores showed evidence of validity via associations with observational instruments of treatment adherence to ICBT for youth anxiety, theory-based domains (cognitive-behavioral treatment, psychodynamic, family, client centered), alliance, and client involvement. Important to note, although the CBAY-C scale, subscale, and item scores did overlap with a corresponding observational treatment adherence instrument independently rated by coders, the degree of overlap was moderate, indicating that the CBAY-C assesses a distinct component of treatment integrity. Applications of the instrument and future research directions discussed include the measurement of treatment integrity and testing integrity-outcome relations.

Patterns, causes, and consequences of marine larval dispersal.

Quantifying the probability of larval exchange among marine populations is key to predicting local population dynamics and optimizing networks of marine protected areas. The pattern of connectivity among populations can be described by the measurement of a dispersal kernel. However, a statistically robust, empirical dispersal kernel has been lacking for any marine species. Here, we use genetic parentage analysis to quantify a dispersal kernel for the reef fish Elacatinus lori, demonstrating that dispersal declines exponentially with distance. The spatial scale of dispersal is an order of magnitude less than previous estimates-the median dispersal distance is just 1.7 km and no dispersal events exceed 16.4 km despite intensive sampling out to 30 km from source. Overlaid on this strong pattern is subtle spatial variation, but neither pelagic larval duration nor direction is associated with the probability of successful dispersal. Given the strong relationship between distance and dispersal, we show that distance-driven logistic models have strong power to predict dispersal probabilities. Moreover, connectivity matrices generated from these models are congruent with empirical estimates of spatial genetic structure, suggesting that the pattern of dispersal we uncovered reflects long-term patterns of gene flow. These results challenge assumptions regarding the spatial scale and presumed predictors of marine population connectivity. We conclude that if marine reserve networks aim to connect whole communities of fishes and conserve biodiversity broadly, then reserves that are close in space (<10 km) will accommodate those members of the community that are short-distance dispersers.

CheY's acetylation sites responsible for generating clockwise flagellar rotation in Escherichia coli.

Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K91 repression is manifested in the conformational dynamics of the ß4α4 loop, shifted toward an active state upon mutation. Removal of ß4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination, we further suggest that K109 acetylation is actively involved in generating clockwise rotation.

Sustained Implementation of Cognitive-Behavioral Therapy for Youth Anxiety and Depression: Long-term Effects of Structured Training and Consultation on Therapist Practice in the Field.

Identifying factors that promote sustained implementation of evidence-based treatments (EBTs) after therapists receive training is critical for professional psychology. To address the field's minimal knowledge in this area, we interviewed community-based therapists (N = 23) who had completed intensive training in cognitive behavioral therapy (CBT) for either anxiety or depression as part of a randomized effectiveness trial (Southam-Gerow et al., 2010; Weisz et al., 2009). Therapists were interviewed three to five years after completion of the initial trial, representing one of the longest-term follow-ups of therapist practices after training. Therapists viewed each protocol and their individual CBT strategies as effective and appropriate for the majority of their current anxiety and depression caseloads. However, therapists used parts of each protocol much more frequently than the protocol as a whole (i.e., 78.5% used parts of the Coping Cat, and 7.5% used the whole protocol; 58.6% used parts of the PASCET, and 20% used the whole protocol). Therapists reported using problem-solving the most and exposure exercises the least for current anxious cases; they used cognitive restructuring the most and homework the least for current depression cases. Interventions that were more difficult to implement in usual care settings were less likely to be sustained. Future efforts should evaluate the characteristics and structure of EBTs that are most acceptable to therapists and should investigate which kinds of ongoing learning supports will maintain therapist skills in and continued use of EBTs.

Acting Locally and Globally: Dissemination and Implementation Around the World and Next Door.

Murray et al. (this issue) present a fascinating account of their international dissemination and implementation (D&I) research focused on training therapists in Thailand and Iraq to provide a modular treatment approach called Common Elements Treatment Approach to youth. In this commentary, we use Murray et al. as a springboard to discuss a few general conclusions about the current direction of D&I research. Specifically, we reflect on current D&I models, highlighting their ecological focus and their emphasis on stakeholder involvement. Next, we discuss the central importance of implementation supports such as treatment programs, training approaches, assessment and outcome monitoring tools, and organizational interventions. We conclude with a consideration of how D&I work that aims to adapt implementation supports for local needs represent a key path to our goal of sustainability.