SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.

Loss of transient receptor potential channel 5 causes obesity and postpartum depression.

Hypothalamic neural circuits regulate instinctive behaviors such as food seeking, the fight/flight response, socialization, and maternal care. Here, we identified microdeletions on chromosome Xq23 disrupting the brain-expressed transient receptor potential (TRP) channel 5 (TRPC5). This family of channels detects sensory stimuli and converts them into electrical signals interpretable by the brain. Male TRPC5 deletion carriers exhibited food seeking, obesity, anxiety, and autism, which were recapitulated in knockin male mice harboring a human loss-of-function TRPC5 mutation. Women carrying TRPC5 deletions had severe postpartum depression. As mothers, female knockin mice exhibited anhedonia and depression-like behavior with impaired care of offspring. Deletion of Trpc5 from oxytocin neurons in the hypothalamic paraventricular nucleus caused obesity in both sexes and postpartum depressive behavior in females, while Trpc5 overexpression in oxytocin neurons in knock-in mice reversed these phenotypes. We demonstrate that TRPC5 plays a pivotal role in mediating innate human behaviors fundamental to survival, including food seeking and maternal care.

Identification, Characterization, and Heritability of Murine Metastable Epialleles: Implications for Non-genetic Inheritance.

Generally repressed by epigenetic mechanisms, retrotransposons represent around 40% of the murine genome. At the Agouti viable yellow (Avy) locus, an endogenous retrovirus (ERV) of the intracisternal A particle (IAP) class retrotransposed upstream of the agouti coat-color locus, providing an alternative promoter that is variably DNA methylated in genetically identical individuals. This results in variable expressivity of coat color that is inherited transgenerationally. Here, a systematic genome-wide screen identifies multiple C57BL/6J murine IAPs with Avy epigenetic properties. Each exhibits a stable methylation state within an individual but varies between individuals. Only in rare instances do they act as promoters controlling adjacent gene expression. Their methylation state is locus-specific within an individual, and their flanking regions are enriched for CTCF. Variably methylated IAPs are reprogrammed after fertilization and re-established as variable loci in the next generation, indicating reconstruction of metastable epigenetic states and challenging the generalizability of non-genetic inheritance at these regions.

Including environmental and climatic considerations for sustainable coral reef restoration.

Coral reefs provide ecosystem benefits to millions of people but are threatened by rapid environmental change and ever-increasing human pressures. Restoration is becoming a priority strategy for coral reef conservation, yet implementation remains challenging and it is becoming increasingly apparent that indirect conservation and restoration approaches will not ensure the long-term sustainability of coral reefs. The important role of environmental conditions in restoration practice are currently undervalued, carrying substantial implications for restoration success. Giving paramount importance to environmental conditions, particularly during the pre-restoration planning phase, has the potential to bring about considerable improvements in coral reef restoration and innovation. This Essay argues that restoration risk may be reduced by adopting an environmentally aware perspective that gives historical, contemporary, and future context to restoration decisions. Such an approach will open up new restoration opportunities with improved sustainability that have the capacity to dynamically respond to environmental trajectories.

Efficacy of T-cell assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis.

ABSTRACT: Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK)-cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Assessment of Fc receptor-triggered NK-cell and T-cell receptor (TCR)-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH than routine K562 cell-based assays, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis after K562 target cell stimulation displayed a higher interindividual variability, in part explained by differences in NK-cell differentiation or large functional reductions after shipment. We thus recommend combined analysis of TCR-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis.

A glossary of plant cell structures: Current insights and future questions.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

Food is Medicine for HIV: Improved health and hospitalizations in the Changing Health through Food Support (CHEFS-HIV) pragmatic randomized trial.

BACKGROUND: Policy support for "Food is Medicine"-medically tailored meals or groceries to improve health-is rapidly growing. No randomized trials have heretofore investigated the benefits of medically tailored food programs for people living with HIV (PLHIV). METHODS: The CHEFS-HIV pragmatic randomized trial included PLHIV who were clients of Project Open Hand (POH), a San Francisco-based nonprofit food organization. The intervention arm (n = 93) received comprehensive medically tailored meals, groceries, and nutritional education. Control participants (n = 98) received less intensive (POH "standard of care") food services. Health, nutrition, and behavioral outcomes were assessed at baseline and 6 months later. Primary outcomes measured were viral non-suppression and health related quality of life. Mixed models estimated treatment effects as differences-in-differences between arms. RESULTS: The intervention arm had lower odds of hospitalization (odds ratio [OR] = 0.11), food insecurity (OR = 0.23), depressive symptoms (OR = 0.32), antiretroviral therapy adherence <90% (OR = 0.18), and unprotected sex (OR = 0.18), and less fatty food consumption (ß= -0.170 servings/day) over 6 months, compared to the control arm. There was no difference between study arms in viral non-suppression and health-related quality of life over 6 months. CONCLUSIONS: A "Food-is-Medicine" intervention reduced hospitalizations and improved mental and physical health among PLHIV, despite no impact on viral suppression. CLINICAL TRIALS REGISTRATION: NCT03191253.

Comparing Methods for Detection and Quantification of Plasmodesmal Callose in Nicotiana benthamiana Leaves During Defense Responses.

Callose, a ß-(1,3)-d-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD or, conversely, by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing likely intercellular trafficking activity during plant immunity. Despite the popularity of this metric, there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying plasmodesmal callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescence microscopy to measure callose deposition in fixed tissue. Manual or semiautomated workflows for image analysis were also compared and found to produce similar results, although the semiautomated workflow produced a wider distribution of data points. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts.

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.

Early menopause and hormone therapy as determinants for lung health outcomes: a secondary analysis using the PLCO trial.

RATIONALE: Early natural menopause (early-M; <45 years of age) increases the risk of lung morbidities and mortalities in smokers. However, it is largely unknown whether early-M due to surgery demonstrates similar effects and whether menopausal hormone therapy (MHT) is protective against lung diseases. OBJECTIVES: To assess the associations of early-M and MHT with lung morbidities and mortalities using the prospective Prostate, Lung, Colorectal and Ovarian (PLCO) trial. METHODS: We estimated the risk among 69 706 postmenopausal women in the PLCO trial, stratified by menopausal types and smoking status. RESULTS: Early-M was associated with an increased risk of most lung disease and mortality outcomes in ever smokers with the highest risk seen for respiratory mortality (HR 1.98, 95% CI 1.34 to 2.92) in those with bilateral oophorectomy (BO). Early-M was positively associated with chronic bronchitis, and all-cause, non-cancer and respiratory mortality in never smokers with natural menopause or BO, with the highest risk seen for BO- respiratory mortality (HR 1.91, 95% CI 1.16 to 3.12). Ever MHT was associated with reduced all-cause, non-cancer and cardiovascular mortality across menopause types regardless of smoking status and was additionally associated with reduced risk of non-ovarian cancer, lung cancer (LC) and respiratory mortality in ever smokers. Among smokers, ever MHT use was associated with a reduction in HR for all-cause, non-cancer and cardiovascular mortality in a duration-dependent manner. CONCLUSIONS: Smokers with early-M should be targeted for smoking cessation and LC screening regardless of menopause types. MHT users had a lower likelihood of dying from LC and respiratory diseases in ever smokers.

Airway epithelial cell response to RSV is mostly impaired in goblet and multiciliated cells in asthma.

BACKGROUND: In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most-but not all-studies. Moreover, the molecular mechanisms causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood. METHODS: Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (n=8) and healthy donors (n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After 3 days, cells were processed for single-cell RNA sequencing. RESULTS: A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed. CONCLUSION: Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.

Roles of ROS and redox in regulating cell-to-cell communication: Spotlight on viral modulation of redox for local spread.

Reactive oxygen species (ROS) are important signalling molecules that influence many aspects of plant biology. One way in which ROS influence plant growth and development is by modifying intercellular trafficking through plasmodesmata (PD). Viruses have evolved to use PD for their local cell-to-cell spread between plant cells, so it is therefore not surprising that they have found ways to modulate ROS and redox signalling to optimise PD function for their benefit. This review examines how intracellular signalling via ROS and redox pathways regulate intercellular trafficking via PD during development and stress. The relationship between viruses and ROS-redox systems, and the strategies viruses employ to control PD function by interfering with ROS-redox in plants is also discussed.

Highly dynamic evolution of the chemosensory system driven by gene gain and loss across subterranean beetles.

Chemical cues in subterranean habitats differ highly from those on the surface due to the contrasting environmental conditions, such as absolute darkness, high humidity or food scarcity. Subterranean animals underwent changes to their sensory systems to facilitate the perception of essential stimuli for underground lifestyles. Despite representing unique systems to understand biological adaptation, the genomic basis of chemosensation across cave-dwelling species remains unexplored from a macroevolutionary perspective. Here, we explore the evolution of chemoreception in three beetle tribes that underwent at least six independent transitions to the underground, through a phylogenomics spyglass. Our findings suggest that the chemosensory gene repertoire varies dramatically between species. Overall, no parallel changes in the net rate of evolution of chemosensory gene families were detected prior, during, or after the habitat shift among subterranean lineages. Contrarily, we found evidence of lineage-specific changes within surface and subterranean lineages. However, our results reveal key duplications and losses shared between some of the lineages transitioning to the underground, including the loss of sugar receptors and gene duplications of the highly conserved ionotropic receptors IR25a and IR8a, involved in thermal and humidity sensing among other olfactory roles in insects. These duplications were detected both in independent subterranean lineages and their surface relatives, suggesting parallel evolution of these genes across lineages giving rise to cave-dwelling species. Overall, our results shed light on the genomic basis of chemoreception in subterranean beetles and contribute to our understanding of the genomic underpinnings of adaptation to the subterranean lifestyle at a macroevolutionary scale.

New insights into plasmodesmata: complex 'protoplasmic connecting threads'.

Intercellular communication in plants, as in other multicellular organisms, allows cells in tissues to coordinate their responses for development and in response to environmental stimuli. Much of this communication is facilitated by plasmodesmata (PD), consisting of membranes and cytoplasm, that connect adjacent cells to each other. PD have long been viewed as passive conduits for the movement of a variety of metabolites and molecular cargoes, but this perception has been changing over the last two decades or so. Research from the last few years has revealed the importance of PD as signaling hubs and as crucial players in hormone signaling. The adoption of advanced biochemical approaches, molecular tools and high-resolution imaging modalities have led to several recent breakthroughs in our understanding of the roles of PD, revealing the structural and regulatory complexity of these 'protoplasmic connecting threads'. We highlight several of these findings that we think well illustrate the current understanding of PD as functioning at the nexus of plant physiology, development, and acclimation to the environment.

Impact of continuous testosterone exposure on reproductive physiology, activity, and pain-related behavior in young adult female rats.

Testosterone may reduce pain in cisgender women and transgender men. Rodents can provide a useful model for investigating physiological effects of hormone therapy. To this end, continuous-release testosterone or blank (placebo) capsules were implanted s.c. into young adult female rats, and three weeks later rats were either ovariectomized or sham-ovariectomized. Testosterone treatment that mimicked previously reported endogenous levels in males eliminated estrous cycling and decreased uterine weight. Testosterone also significantly increased body weight and suppressed the increases in daily wheel running observed in placebo controls over time. Subsequent ovariectomy or sham-ovariectomy decreased wheel running in all groups, but testosterone-treated rats recovered significantly more quickly than did placebo-treated rats. Neither testosterone nor ovariectomy significantly altered hindpaw mechanical threshold. Two weeks after sham/ovariectomy surgery, injection of Complete Freund Adjuvant (CFA) into one hindpaw reduced wheel running and mechanical threshold in all groups; running significantly decreased from the first to second day after CFA in testosterone- but not in placebo-treated rats. Morphine 1.0 but not 3.2 mg/kg increased CFA-suppressed wheel running similarly in all groups, whereas both doses of morphine increased CFA-suppressed mechanical threshold. These data suggest that weeks-long testosterone treatment with or without ovariectomy may provide a useful physiological model of testosterone therapy as used in human gender transition. Although testosterone administered at levels similar to those in gonadally intact males tended to hasten female rats' recovery from surgery, it did not decrease maximal pain-related behaviors after surgery or hindpaw inflammatory insult, nor did it alter opioid antinociception.

Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.

Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.

The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors.

Most vertebrate RNA viruses show pervasive suppression of CpG and UpA dinucleotides, closely resembling the dinucleotide composition of host cell transcriptomes. In contrast, CpG suppression is absent in both invertebrate mRNA and RNA viruses that exclusively infect arthropods. Arthropod-borne (arbo) viruses are transmitted between vertebrate hosts by invertebrate vectors and thus encounter potentially conflicting evolutionary pressures in the different cytoplasmic environments. Using a newly developed Zika virus (ZIKV) model, we have investigated how demands for CpG suppression in vertebrate cells can be reconciled with potentially quite different compositional requirements in invertebrates and how this affects ZIKV replication and transmission. Mutant viruses with synonymously elevated CpG or UpA dinucleotide frequencies showed attenuated replication in vertebrate cell lines, which was rescued by knockout of the zinc-finger antiviral protein (ZAP). Conversely, in mosquito cells, ZIKV mutants with elevated CpG dinucleotide frequencies showed substantially enhanced replication compared to wild type. Host-driven effects on virus replication attenuation and enhancement were even more apparent in mouse and mosquito models. Infections with CpG- or UpA-high ZIKV mutants in mice did not cause typical ZIKV-induced tissue damage and completely protected mice during subsequent challenge with wild-type virus, which demonstrates their potential as live-attenuated vaccines. In contrast, the CpG-high mutants displayed enhanced replication in Aedes aegypti mosquitoes and a larger proportion of mosquitoes carried infectious virus in their saliva. These findings show that mosquito cells are also capable of discriminating RNA based on dinucleotide composition. However, the evolutionary pressure on the CpG dinucleotides of viral genomes in arthropod vectors directly opposes the pressure present in vertebrate host cells, which provides evidence that an adaptive compromise is required for arbovirus transmission. This suggests that the genome composition of arbo flaviviruses is crucial to maintain the balance between high-level replication in the vertebrate host and persistent replication in the mosquito vector.

U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

Implementing group care in Dutch and Surinamese maternity and child care services: the vital importance of addressing outer context barriers.

BACKGROUND: By addressing physical and psychosocial needs, group care (GC) improves health-related behaviours, peer support, parent-provider interactions and may improve birth outcomes. Hence, global implementation of GC is encouraged. Context analyses prior to implementation are vital to elucidate which local factors may support or hinder implementation. METHODS: Contextual analyses conducted in the Netherlands and Suriname were compared to identify the factors relevant to the implementability of GC as perceived by healthcare professionals (HCPs). 32 semi-structured interviews were conducted with Dutch and Surinamese healthcare professionals. Audio recordings were transcribed verbatim and coded using the Framework approach. The Consolidated Framework for Implementation Research guided the development of the interview guide and of the coding tree. RESULTS: Outer setting: Concerns regarding funding surfaced in both countries. Due to limited health insurance coverage, additional fees would limit accessibility in Suriname. In the Netherlands, midwives dreaded lower revenue due to reimbursement policies that favour one-on-one care. Inner setting: Appropriate space for GC was absent in one Dutch and three Surinamese facilities. Role division regarding GC implementation was clearer in the Netherlands than in Suriname. INNOVATION: HCPs from both countries expected increased social support, health knowledge among women, and continuity of care(r). Individuals/innovation deliverers: Self-efficacy and motivation emerged as intertwined determinants to GC implementation in both countries. Individuals/innovation recipients: Competing demands can potentially lower acceptability of GC in both countries. While Dutch HCPs prioritised an open dialogue with mothers, Surinamese HCPs encouraged the inclusion of partners. PROCESS: Campaigns to raise awareness of GC were proposed. Language barriers were a concern for Dutch but not for Surinamese HCPs. CONCLUSIONS: While the most striking differences between both countries were found in the outer setting, they trickle down and affect all layers of context. Ultimately, at a later stage, the process evaluation will show if those outer setting barriers we identified prior to implementation actually hindered GC implementation. Changes to the health care systems would ensure sustained implementation in both countries, and this conclusion feeds into a more general discussion: how to proceed when contextual analyses reveal barriers that cannot be addressed with the time and resources available.