Type 2 diabetes mellitus (T2DM) continues to disproportionately affect African Americans, significantly impacting morbidity and mortality. Research suggests that addressing barriers that stem from socioeconomic circumstances, systemic inequalities, biological factors, and cultural factors may positively influence biometric indicators of health and diabetes control.The aim of this study was to evaluate a diabetes shared medical appointment (SMA) model program that has been culturally tailored to address the unique social determinants of health barriers faced by an inner city African American population in Norfolk, Virginia.A pilot study using a within-group pretest–posttest design was conducted. Information was collected from self-report surveys that included the Modified Michigan Diabetes Knowledge Test, the Diabetes Self-Efficacy Scale, and researcher generated surveys before and after a single-session three-hour SMA program.The program increased perceived diabetes self-care confidence and perceived overall diabetes knowledge levels. Increases in knowledge scores were seen but not statistically significant. Participants reported high levels of satisfaction with the program model.Findings indicate that this model is an effective and engaging method of improving self-care ability and diabetes disease management knowledge among African Americans. Addressing unique circumstances and barriers experienced by this population may be more effective than usual traditional care approaches. The SMA model is designed to allow more time for patient education regarding disease prevention and management using a multidisciplinary interprofessional approach which may encourage the development of self-care skills 3 nonprofit organization that offers a unique culturally tailored diabetes SMA model program specifically adapted to for low-income, underserved, and/or medically vulnerable African Americans living in Norfolk, Virginia. The program is aimed at increasing health education and awareness using an interactive group model approach. Participants initially report to the affiliated clinic, Primary Care Specialists, where they have a one-on-one visit with a nurse practitioner (NP) or medical doctor (MD). At that time, participants have a diabetes-focused exam and are given the opportunity to discuss individual treatment plans, any concerns, and ask personal questions. Participants are provided with printouts of their most recent diabetes-related laboratory information and biometric data before being escorted to the Healthy Living Center, located in the same building.The three-hour group education portion of the SMA takes place in a classroom and simulated kitchen setting with 8–12 participants. Instructors are community-based African American medical professionals from the local Primary Care Specialists clinic who are intimately familiar with the social determinants of health barriers among Norfolk’s inner-city African American community. Curriculum design was informed by these barriers which include a lack of access to affordable healthy food options, low health literacy, and difficulty effectively integrating diet and healthy lifestyle recommendations. The team of instructors includes a MD, NPs, and a pharmacist. The core curriculum follows the Association of Diabetes Care and Education Specialists (ADCES) 7 Self-Care Behaviors education format which incorporates the following elements: healthy coping; healthy eating; being active; diabetes monitoring; medication adherence; problem-solving; and reducing health risks  included African Americans aged 33 to 83 years with a confirmed diagnosis of T2DM Table . Individet al., Information was collected from self-report surveys administered to participants before and after the single-session culturally tailored diabetes SMA model program. The Diabetes Self-Efficacy Scale (DSES) was used to evaluate pre- and post-intervention differences in perceived self-care ability. It is comprised of eight questions that are answered using a Likert scale designed to measure self-efficacy for common diabetes-related behavioral and medical management issues. The Modified Michigan Diabetes Knowledge Test (MMDKT) was used to evaluate pre- and post-knowledge levels of the T2DM disease process and management. The modified version used in this study is written in a true/false/don’t know format  (Table P = 0.020).The mean total DSES score before the intervention was 54.65 points (SD = 16.816), and the mean total score after the intervention was 68.53 SD = 10.255) (Table 55 (TableP = 0.001).Although there was an increase in mean total MMDKT scores after the intervention, the increase was not statistically significant Table . HoweverParticipants used a Likert scale to rate satisfaction with the culturally tailored diabetes SMA model program on a scale of 1  to 10 (extremely satisfied) upon completing the program. The mean satisfaction score was 9.82 .Innovative diabetes interventions are necessary to respond to the growing disparities in diabetes prevalence and outcomes among minority groups in the United States. The ongoing deep-rooted racial disparities seen in diabetes management and outcomes indicate that the current approach to diabetes care is not adequately tailored to meet the needs of the African American population. Designing diabetes education and self-management content and activities to better meet the requirements of specific patient populations may strengthen patients’ ability to assimilate self-care recommendations into their daily lives. Interactive culturally tailored models, such as this one, that are also led by African American clinicians, may be more influential in empowering African American individuals to set and achieve personal health goals by instilling confidence in self-care abilities. The SMA program examined here can be used as a model for adapting traditional diabetes care to develop engaging patient-centered programs that better serve culturally or ethnically diverse populations. This pilot study supports that this culturally tailored diabetes SMA model is a highly acceptable method of addressing diabetes care needs in African Americans, while significantly enhancing self-care confidence. While statistical significance in improved knowledge levels as measured by the MMDKT scores was not demonstrated, findings suggested both actual and perceived noteworthy knowledge acquisition from the SMA program which could prove to be clinically significant in terms of improved disease outcomes.COVID-19 gathering restrictions significantly limited the number of patients that could enroll in the SMA or participate in the study, resulting in a small sample size (N = 37). Additionally, despite efforts to minimize effects, low literacy levels in the sample may have impacted the ability of some participants to fully understand the questions and provide accurate answers on the self-report surveys. Future studies with a larger sample and additional data collection methods may strengthen findings.We have outlined an innovative program that considers the multifactorial interplay of social determinants of health, institutional racism, biological differences, and widespread socioeconomic disparities unique to African Americans that shows improvement in perceived self-efficacy levels and self-rated knowledge levels, while simultaneously having high levels of participant satisfaction. The findings from this pilot study support future efforts to further explore the effectiveness of this culturally tailored model for improving diabetes knowledge and self-management ability among minority and underserved populations.


The formulation of sustainable fish feeds based on plant ingredients supplemented by alternative ingredients to plant  and genetic selection of fish for plant-based diets were tested on rainbow trout in two separate experiments. Plant-based diets and corresponding diets supplemented with an ingredient mix: insect, micro-algae and yeast in Experiment A, and insect and yeast in Experiment B were compared to commercial-like diets. In experiment A, the mix-supplemented diet was successful in compensating the altered growth performance of fish fed their respective plant-based diet compared to those fed the commercial diet, by restoring feed conversion. In experiment B, the selected line demonstrated improved growth performances of fish fed mix-supplemented and plant-based diets compared to the non-selected line. Metabolomics demonstrated a plasma compositional stability in fish fed mix-supplemented and basal plant-based diets comprising an amino acid accumulation and a glucose depletion, compared to those fed commercial diets. The selected line fed mix-supplemented and commercial diets showed changes in inositol, ethanol and methanol compared to the non-selected line, suggesting an involvement of microbiota. Changes in plasma glycine-betaine content in fish fed the mix-supplemented diet suggest the ability of the selected line to adapt to alternative ingredients. Plant ingredients make up at least 90% of the fish feed used for many fish species, including carnivorous ones such as the rainbow trout3. However, sustainable fish feeds free of marine ingredients are not yet sufficiently performant to guarantee a fair economic income to farmers. Fish feeds can be improved by selecting high-quality plant proteins, or by including alternative ingredients of plants ingredients and fish products, that meet the nutritional requirements of fish, increase farming profitability, and support environmental preservation and consumer expectations. A substantial research effort has been conducted to assess the efficacy of a wide range of alternative ingredients to plant-based feedstuffs, from single-cell ingredients to animal by-products.Combined with responsible fishing policies, the increasing demand for aquatic products challenges the development of sustainable and efficient fish feed for aquaculture, which currently provides about 50% of the aquatic products consumed7. Some of these microorganisms also contribute to restore the digestive and microbiota functions altered by plant-based diets5. On one hand, the production of bacteria and yeast has been optimised and can be implemented at a large scale. On the other hand, most of the micro-algae productions are still at the pilot scale on a few taxa collected from wild stock8. Alternative ingredients based on animal by-products tend to mimic natural fish food. This is the case, for example, of terrestrial insects10, whose larvae are beginning to be produced at a pilot scale and even at an industrial scale. These ingredients contain substantial protein levels and are well adapted to fish amino acid requirements5. They also contain specific functional compounds relevant for feed acceptability, gut integrity preservation, and metabolism. The incorporation of most of these alternative ingredients in fish feed has only been tested on individual ingredients; however, combining them could better fulfil fish nutritional requirements through their complementarity.Single-cell ingredients, such as micro-algae and yeast used as supplements in plant-based fish feeds, satisfy protein and lipid requirements of carnivorous fish12. Although farmed fish are likely to be subjected to natural selection, as commercial feeds now contain high levels of plant-based feedstuff13, continuous selection over generations has refined selected lines that outperform these non-selected lines when fed plant-based diets17.Genetically selecting fish is a complementary strategy to adapt fish to these feeds free of fish ingredients. This strategy is based on selecting fish that display the highest performances when fed plant-based diets over several generations. There is indeed a genetic basis for the adaptation to plant-based diets that has been demonstrated in various fish species19 allows us to create a comprehensive view of the metabolic syndrome of fish fed plant-based diets, previously described through transcriptomics and proteomics. These more recent approaches demonstrate the combined alteration of protein, lipid and carbohydrate metabolisms induced by plant-based diets in specific pathways23, including in selected lines26. Metabolomic approaches link the disruption of energy and protein metabolisms to the asynchronous absorption of essential nutrients in fish fed plant-based diets27, which can be restored by supplementing fish feeds with alternative ingredients30. Essential liver functions, such as phospholipid synthesis and one-carbon metabolism, are also affected, as demonstrated for instance by the status of betaine and related metabolites33. Moreover, specific metabolites  account for protective adaptations to preserve cell vital functions such as osmolality and buffering capacity34. All these changes constitute the metabolic scope of fish fed plant-based diets. Genetic selection for adaptation to a plant-based diet can compensate for digestive alterations36 and the related protein metabolic disturbances17 induced by plant-based diets. Further investigations using metabolomics and genomic approaches have shown other beneficial outcomes of this selection on various metabolic pathways35. However, a coherent metabolic scheme has not yet emerged, and this could be partly related to the existence of the different lines selected for adaptation to plant-based diets36. These dietary and/or selection-induced metabolic changes are informative on the mechanisms that lead to modifications in the performances of fish fed plant-based diets. However, the metabolic consequences of combining supplementation with a mix of alternative ingredients to plant-based ingredients with trout line selection are still unknown.The development of metabolomic approaches to fish nutrition37 and the existence of selected lines that have adapted to plant-based diets36.The purpose of this study was to test the relevance of using a mix of alternative ingredients, namely yeast, micro-algae and insects, in selected and non-selected trout lines, in order to restore fish performance and metabolism that have been altered by a plant-based diet. In a first step, two mix-supplemented diets were provided to a non-selected line and compared to their respective non-supplemented plant-based diets that differed in the level of protein purification of plant ingredients. These four diets were then compared to a commercial diet composed of marine ingredients. In a second step, the mix-supplemented diets, fine-tuned from the previous ones, were tested on a line selected for a plant-based diet as well as on a control non-selected line. We used rainbow trout, one of the first fish species reared in Europe, as a model species for salmonids. This species benefits from solid background data on long-term plant-based diet feeding throughout the life cycle of the fishThe growth performances and plasma metabolome were analysed in rainbow trout fed either plant-based diets (PB) or corresponding plant-based diets supplemented with a mix of alternative ingredients, insect (I), spirulina (S) and yeast (Y), compared to commercial-like diets comprising marine ingredients (COM) in two separate experiments Fig. . In a fiP < 0.05) than that of the fish fed the PB1 diet. Fish fed the other diets showed intermediate responses. The specific growth rate was not significantly different (ANOVA) between diets although the same pattern of differences in final body weight could be observed as the one noted between the ISY2 and PB1 diets. The voluntary feed intake and the feed conversion ratio were significantly higher in fish fed the PB1 diet than in fish fed the PB2 and ISY1 diets (P < 0.05), and the latter were significantly higher (P < 0.05) than in fish fed the commercial diet COM-A.The growth performances and the somatic indices are presented in Table P<0.05) than those of fish fed the plant-based diet (PB3) but they were significantly lower than those of fish fed the COM-B diet when compared within each of the fish line. The feed conversion ratio of fish fed the IY3 supplemented diet was significantly higher than that of the COM-B but not significantly different from the PB3 diets within each of the fish line. When comparing the selected and the non-selected lines, we observed that the final body weight, the specific growth rate, and the voluntary feed intake were significantly higher in selected line than in non-selected line for fish fed IY3 and PB3 diets but such difference was not observed in COM-B diet. The feed conversion ratio of the selected line were significantly higher than that of the non selected line whatever the diet. However, we noted a highly significant interaction between line and diet in terms of final body weight (P = 7.10–4), specific growth rate (P = 8.10–3) and voluntary feed intake (P = 5.10–7). The selected line of fish fed the IY3 diet had a significantly higher final body weight and a significantly higher feed intake than the non-selected line fed the same diet, but such a difference was not observed in the fish that were fed the COM-B diet. The feed conversion ratio was significantly higher in fish of the selected line fed the supplemented plant-based diet IY3 than in those fed the two other diets while in non selected line the feed conversion ratio of the fish fed the COM-B diet was significantly higher than those fed the two other diets.In Experiment B Table , the finIn Experiment A, a PCA showed that the plasma NMR profiles of fish fed the PB1 and PB2 plant-based and the ISY1 and ISY2 supplemented plant-based diets were slightly discriminated compared to the fish fed the COM-A diet  compared to the corresponding plant-based diets  profile using volcano plots showed that there were very few discriminating compounds  as it was shown that rearing conditions can modulate the response of fish to plant-based diets44. This highlights the crucial importance of repeated tests to ensure robust progress in the development of sustainable fish feed.This work aimed at testing the current key elements for improved growth performances of fish fed plant-based diets. This comprised the relevant choice of plant-based ingredients, the supplementation with a mix of alternative ingredients  and the genetic selection for fish adaptation to plant-based diet. The results presented here clearly demonstrated that each of these elements contributed to improve growth performances compared to fish fed a control plant-based diet. However, the underlying mechanisms for improved growth performances related either to formulation of plant-based diet or to genetic selection were different. Full plant-based diets usually negatively affect the feed intake of numerous fish species due to low acceptability of plant-based ingredients30. It is, however, not possible to draw conclusions on the additive or synergic effect of the ingredients tested as the inclusion level of each of these ingredients was rather low (5%).Concerning the mix-supplemented diets, the growth performances of fish fed the three diets tested  tended to reach that of fish fed commercial-like diets, demonstrating the beneficial effect of combining alternative ingredients that have already been tested separately26. A higher voluntary feed intake has also been observed in existing selected trout lines adapted to plant-based diets compared to non-selected lines45. Interestingly, we observed a highly significant interaction between selection effect and diet effect for the feed intake and a less significant interaction for growth rate. This interaction was demonstrated by the significantly higher feed intake of the selected line fed the mix-supplemented diet and the plant-based diet compared to the non-selected line, and no differences in the feed intake between lines of fish fed the commercial diet. Finally, the growth performances of fish in the selected line, fed the mix-supplemented diets, were significantly improved compared to the referent plant-based diet but did not reached that of the non-selected line fed a commercial-like diet knowing that only half of the genetic effect was tested in our experiment. This indicates however that the selected line has both the ability to adapt to a full plant-based diet and the ability to adapt to alternative ingredients.Genetically selecting fish for higher growth when fed a plant-based diet increased voluntary food intake more than final body weight, resulting in reduced feed efficiency. This confirms the genetic background for feed-acceptability of plant-based diets observed in salmonidsThe improved zootechnical performances of fish fed mix-supplemented diets and that of fish of the selected lines constitutes an interesting background to the analysis of the underlying metabolic processes involved in restoring fish performances by using plasma metabolomics. The plasma was collected after the post-prandial absorption of feed nutrients to study fish metabolome rather than food metabolome. We demonstrated that the fish metabolome is remarkably stable with full plant-based diets.31 induced by gut intolerance to plant ingredients. In the two present experiments, we confirmed that essential amino acids circulating in the plasma were maintained at high levels in fish fed plant-based diets compared to those fed commercial-like reference diets that include fish ingredients. This delay could be related to amino acid unbalance which in turn could amplify their effects on feed intake43. Furthermore, the plant-based diet also induced a lower circulating glucose compared to the commercial-like reference diet. The commercial-like diet also contained plant-based ingredients rich in complex carbohydrates that could induce a high circulating level of glucose in salmonids47. The asynchronous supply of energy substrates and essential amino acids results in a typical metabolic scope of fish fed plant-based diets compared to commercial-like diets. Our results clearly demonstrate that the plasma metabolomes of fish fed all plant-based diets were maintained within this metabolic scope regardless of the improvement tested, either by diet formulation or by supplementation with specific sustainable ingredients.The metabolome of fish fed a plant-based diet compared to that of fish fed a marine diet is classically characterised by a delay in the absorption of essential amino acids29. The few observed changes were contingent on the plant-based diet tested. Histidine was reduced by the supplementation with a mix of insect, yeast and spirulina ingredients compared to the PB1 diet in line with changes in growth performances. The plasma level of histidine might reflect the preservation of vital cell functions such as osmolality and buffering capacity48. These functions could be affected by gut integrity and immunity alterations induced by plant-based diets40 and could be restored with alternative ingredients supplementation. These known alterations might have been reduced with the other two plant-based diets -namely PB2 and PB3- by the use of whole plant ingredients rather than processed or purified ones. In Experiment B, glycine-betaine  relative intensity was increased by the supplementation of a mix of insect and yeast ingredients of the third plant-based diet, PB3 in line with changes in growth performances. Levels of choline, glycine-betaine and their derived metabolites  are regularly found to be affected in fish fed plant-based diets49. Glycine-betaine is involved in a methyl-donor function50, in the synthesis of phospholipids and fatty acids51 and in cell osmotic protection52. The observed difference in glycine-betaine could thus be related to the restoration of one of these functions by the ingredients supplied. Alternatively, glycine-betaine is a specific ingredient of yeast culture substrate and thus its accumulation in plasma could also be directly related to the supplementation of a plant-based diet with a yeast extract.There were very few changes in plasma metabolomes induced by supplementation with a mix of alternative ingredients. Moreover, the metabolic effects of each of the ingredients tested are difficult to detect at such low inclusion level of 5%, and they are more discernible in tissues, such as muscle and liver, than in plasma53. However, the ability to use plant-based diets is a complex trait which integrates low palatability of diets54, gut susceptibility to anti-nutritional factors56, alteration in liver function57, microbiota modulation related to complex carbohydrates59, and metabolic pathway adaptation to an unbalanced supply of essential nutrients such as fatty acids and amino acids61. In terms of functionality, it has been established that selected lines17 were able to compensate for the delay in amino acid absorption generally observed in salmonids fed plant-based feedstuffs61. We did not observe such a difference in amino acid accumulation between the selected and non-selected lines, but this might be explained by either the plasma sampling that took place later after the last meal than in other studies17 or by the differences between the metabolic characteristics of the selected lines36.The plasma metabolome was faintly but specifically affected by the selection of fish for adaptation to plant-based diets. It was surprisingly similar in the selected line and in the non-selected line, both in fish fed a commercial-like diet (COM-B) and in fish fed a mix-supplemented diet (IY3). The lines selected for a given trait usually exhibit specific differences in the related metabolic pathways, for instance, a line selected for lipid content41. This suggests, however, that selected lines could adapt their lipid metabolism when fed plant-based diets although in itself this could not contribute to improved growth performances. Inositol is also highlighted in the plasma metabolome of a rainbow trout selected line compared to a non-selected one35. The changes in inositol are especially interesting as it is a precursor of phospholipids, and in its phosphorylated state, it induces various biological functions including insulin resistance, osmoregulation and antioxidant responses63. One less speculative explanation for the lowering of the inositol status in the selected line is that it could simply be evidence of the redesign of essential phosphatidyl-inositol in different tissues, including the brain. This could be complemented by a better oral acceptability of plant-based diets mediated by inositol64 and thus could lead to a better adaptation of the fish to plant-based diets by increasing their feed intake. The lines were also differentiated in both COM and IY3 diets by the status of ethanol and methanol, 48 hours after the last meal. This could be related to a microbiota adaptation to plant-based diets as it has been established that the lines of rainbow trout selected for adaptability to plant-based diets showed higher gut and microbiota tolerance to plant ingredients45. In addition, the microbiota could produce ethanol from carbohydrates65 and this production could vary with the two types of diets tested (COM-B and IY3) depending on the composition of the microbiota and the nature of the carbohydrates. The analysis of the digesta metabolome a few hours after the last meal in a selected line of rainbow trout fed a plant-based diet reveals differences in numerous metabolites of the carbohydrate metabolism35. Thus, the specific contribution of microbiota end-product substrates to fish metabolism needs to be further investigated by comparative analysis of post-prandial time course in digesta and plasma metabolomes. The circulating ethanol and methanol in plasma could also be of external origin due to manufacturing residues of the ingredients and the diet, but this cannot account for a line effect. Alternatively, it could be an end product of fish energy metabolism, but endogenous ethanol is only produced in a few species of fish enduring extreme anaerobic conditions66.There were few but interesting discriminations in the plasma metabolome between lines of trout either non-selected or selected for their adaptation to plant-based diets. Some of these discriminations were both observed in fish fed mix-supplemented diet and commercial-like diet whereas others were observed mainly in fish fed a mix-supplemented diet. It is interesting to further explore these differences related to complementary metabolic pathways. The lines were differentiated in both the COM-B and IY3 diets by inositol and circulating lipids related to unsaturated fatty acid and lipoproteins. The changes in plasma circulating lipids are usually related to differences in fatty acid composition of the plant-based diet16. The other differences observed for glycine betaine and related compounds in the plasma metabolome of the selected line fed a supplemented plant-based diet could be associated with yeast supplementation28. This demonstrates for the first time that selected lines can not only adapt their lipid and energy metabolism to the better utilisation of plant-based diets, but also that they could adapt their metabolism to alternative ingredients complementing the plant-based diets.Line-specific differences related within fish fed the mix-supplemented diet IY3 were observed for plasma glucose and glycine-betaine that were higher in the selected line than in the non-selected one. This was associated with an increased feed intake together with a decreased feed efficiency. The higher glucose in the selected line fed a mix-supplemented diet mimics the accumulation of glucose observed in fish fed COM diets, as previously discussed. Together with higher glutamine and alanine found in the selected line fed COM diets, this suggests that the selected line could better adapt its energy metabolism than the non-selected one. This confirms the specific metabolic adaptation of selected lines of rainbow trout fed plant-based dietsThis work explored for the first time three complementary strategies to adapt rainbow trout nutrition to sustainable fish feeds: alternative formulation of plant-based diets with raw feedstuffs, the supplementation of plant-based diets with a mix of alternative ingredients , and the adaptation of fish to plant-based diets by genetic selection. These different strategies were successful in improving the growth performances of fish fed plant-based diets, although not always up to the levels of fish fed a commercial-like diet. The study of the plasma metabolome revealed that the alternative formulation of a plant-based diet and its supplementation with alternative ingredients could help suppress the known alteration of fish metabolism due to gut alteration in different ways. This argues for a systematic analysis of the plasma metabolome in sustainable fish-feed development.https://doi.org/10.15454/GPYD-AM38 and PEIMA Sizun, France, https://doi.org/10.15454/1.5572329612068406E12. In the first experiment (Experiment A), rainbow trout were taken from an INRAE synthetic line. Synthetic line has been created in the 1980s through mixing of fish from different French farms populations, and a few from Denmark and USA to get a large genetic basis. It was then closed and maintained in our INRAE experimental facilities over generations with large number of breeders and without artificial selection to maintain genetic variability. The fish of this synthetic line were maintained and produced at the INRAE experimental hatchery . The fish of this synthetic line were transferred at the fry stage to the INRAE experimental facility at NUMEA Donzacq. In the second experiment (Experiment B), rainbow trout from an INRAE line, selected for its ability to survive and grow on a full plant-based diet for four generations15, was used to be compared with the INRAE synthetic non-selected line. These lines were maintained and reared at the INRAE experimental hatchery of PEIMA . The mating design is presented in Fig. selected). The other group was fertilised with individually frozen sperm from 31 males in the synthetic line (non-selected). The comparison between the selected line and the non-selected line thus corresponds to the half-response of the expected genetic selection since it only relates to the male pathway.Two experiments were conducted in two different INRAE experimental facilities, namely, NUMEA Donzacq, France, Tenebrio molitor larvae protein hydrolysate, by Spirulina solutions (www.spirulinasolutions.fr) for dried biomass of spirulina Arthrospira platensis cultivated in autotrophic conditions, and by Lesaffre Industry  for yeast Saccharomyces cerevisiae cell wall extract .The diet designs used in each of the two experiments are summarised in Fig. In Experiment B, three diets were formulated and produced by Le Gouessant : (a) a commercial-like diet (COM-B), (b) a plant-based diet (PB3) similar to the low-cost diet of the first experiment and (c) the same plant-based diet supplemented with 5% insect hydrolysate extract and 5% yeast wall extract (IY3) Table .The comparative formulations of the diets used in Experiments A and B are presented Table Fish care and sampling were undertaken in accordance with the European Directive 2010/63/EU and French legislation for animal welfare used for scientific purpose . Growth experiments, as well as fish manipulation and fish euthanasia were performed in accordance with the guidelines reviewed and the study was approved by ethical committees Ethical Committee Aquitaine Fish and Birds CEEA – 073 and Finisterian Ethical Committee in Animal Experiments CEEA - 074 (CEFEA) . The experimental facilities and each of the scientists and staff involved in experimental manipulation and euthanasia had their own agreements (A40-2281 and B64-10-003 respectively for the NUMEA facility and N° D29–277‑02 and A64-495-1 for the PEIMA facility). This study was carried out in compliance with the ARRIVE 2.0 guidelines. The fish were anaesthetised with benzocaine (10 mg/L) in Experiment A and with tricaine methylsulfonateMS-222(0.05 g/L) in Experiment B. The fish were then euthanised by a sharp blow to the head and bled in water by sectioning their gill arches.P = 0.96).The fish were reared in a flow-through system at natural photoperiod and constant oxygen (> 9.0 mg/L) at a temperature of 17 °C ± 1 °C. Fish were fed twice daily manually to satiation until first unfed pellets appeared, for 64 days and corresponding feed distribution was recorded. The whole tank biomass was weighed every 3 weeks after starving the fish for 24 hours. Total feed distribution and tank biomass were recorded during the entire experiment.In Experiment A, juvenile fish  were randomly distributed among 15 tanks (n = 30 fish per 100-L tank), representing each of the five diets in triplicates. The initial body weights were not significantly different between the tanks . The fish were reared in a flow-through system at natural photoperiod, constant oxygen (7.5 to 9.5 mg/L) and ambient temperature (12 °C to 18 °C). Fish were fed automatically to satiation twice daily for 85 days and the unfed pellets were collected and recorded to adjust the next day feed distribution. This latter feeding method used was more adapted to the size of the tank used. The whole tank biomass was weighed every 3 weeks after starving the fish for 24 hours. Total feed distribution and tank biomass were recorded during the entire experiment.In Experiment B, juvenile fish, half of which were taken from the non-selected line  the other half of which were taken from the selected line , were distributed among 18 tanks (n= 350 fish per 1800-L tank), representing each of the three diets in each of the two lines in triplicates. The initial body weights were not significantly different between the tanks within each fish line (Two way ANOVA F=1.30 Voluntary feed intake (amount of feed distributed per day), specific growth rate  – Ln)/number of days)) and feed conversion ratio (feed distributed/body weight gain) in each tank were calculated.Nine fish per treatment for Experiment A and 15 fish per treatment for Experiment B were sampled 48 hours after the last meal. In Experiment B, the sampling design was very heavy as it comprised not only blood sampling for metabolomics but also a comprehensive assessment of phenotypical and quality traits together with tissue sampling for biochemical analysis. It was not possible to include all the treatments. Thus the sampling was focused on one hand on the three diets only in the selected line and on the other hand on the mix-supplemented diet IY3 and the reference diet COM-B in both the selected line and the non-selected line, according to a Latin-square design. The fish were anaesthetised with benzocaine (10 mg/L) in Experiment A and with tricaine methylsulfonate MS-222 (0.05 g/L) in Experiment B. The blood was collected, centrifuged and the plasma was collected, snap-frozen in liquid nitrogen, and stored at − 24°C until the end of the sampling procedure, then stored at – 80 °C until analysis. The fish were then euthanised by a blunt blow on the head and bled in water by sectioning their gill arches.28. Briefly, plasma samples were first individually thawed, then diluted with D2O, the diluted plasma was then transferred to a 5-mm NMR tube for NMR acquisition, which was performed immediately after each sample preparation.Plasma samples were prepared as described previously1H-NMR acquisitions (1D 1H-NMR) were undertaken at the INRAE Metabolome platform (https://doi.org/10.15454/1.5572412770331912E12) using parameters previously described28. Briefly, for plasma, one-dimensional Carr-Purcell-Meiboom-Gill (CPMG) 1H-NMR spectra with water presaturation and a 90° pulse angle were acquired. In addition, a simple water presaturation pulse sequence (zgpr) was used to analyse circulating lipoproteins and the two corresponding datasets were combined as previously described67.One-dimensional 68. The spectra regions stemming from intelligent bucketing were named according to the centre of the corresponding region (e.g. B4.2670 for a bucket centred at 4.2670 ppm). If they met the criterion of a signal-to-noise ratio greater than 3, their area was integrated and standardised using constant sum normalisation (CSN). The assignments in the 1D 1H-NMR spectra were based on previous studies28, an in-house database, a public database  and the ChenomX NMR Suite library 8.3 . When annotated, each integrated spectral region was also named after the metabolite it corresponded to. A set of 37 annotated compounds (Table The free induction decays and spectra were processed using the NMRProcFlow tool (nmrprocflow.org)ds Table  was conshttp://www.biostatflow.org)69. Tank effect on fish initial body weight was tested using one-way ANOVA (P < 0.05). Dietary and/or line effects on fish performances were tested via variance analyses  to summarise most of the variance induced at tank level (N = 3). Normal distribution of the dependant variables was confirmed using the Shapiro-Wilk test (P > 0.05) at tank level. Multiple comparison Tukey’s tests were implemented to test for differences (P < 0.05) when ANOVA was significant.Univariate analyses were performed using the BioStatFlow tool based on R scripts 69. Principal component analyses (PCA) were used to assess the main sources of variance on metabolite profiles. These PCA were performed on fish fed the five diets in Experiment A, on fish from the selected line fed the three diets in Experiment B and on fish from both selected and non-selected lines fed the COM-B and IY3 diets in Experiment B. Partial least-squares discriminant analyses (OPLS-DA) were also performed to discriminate the differences in plasma metabolome related to fish line effect in fish fed the COM-B and IY3 diets.Plasma datasets of metabolomic profiles were mean-centred and scaled to unit variance prior to multivariate analyses using the BioStatFlow tool . A 1.2 log fold change threshold between means and a P-adjusted value < 0.05 were considered significant.The effects of the relative intensities of mix-supplemented diets on metabolites were compared to their respective plant-based diets on a volcano plot. Differences in individual metabolites were tested using the Wilcoxon test with P > 0.05 for 24 out of 37 annotated spectra regions) and, when necessary, the metabolite relative intensity was log2-transformed to correct the distribution (13 out of 37 annotated spectra regions). A Wilcoxon test was performed to assess pair-pair differences when ANOVA was significant.Two-way ANOVAs were performed on 37 annotated spectra regions of plasma Table  of the sSupplementary Information.


The scale of data produced during the SARS-CoV-2 pandemic has been unprecedented, with more than 13 million sequences shared publicly at the time of writing. This wealth of sequence data provides important context for interpreting local outbreaks. However, placing sequences of interest into national and international context is difficult given the size of the global dataset. Often outbreak investigations and genomic surveillance efforts require running similar analyses again and again on the latest dataset and producing reports. We developed civet (cluster investigation and virus epidemiology tool) to aid these routine analyses and facilitate virus outbreak investigation and surveillance. Civet can place sequences of interest in the local context of background diversity, resolving the query into different ’catchments’ and presenting the phylogenetic results alongside metadata in an interactive, distributable report. Civet can be used on a fine scale for clinical outbreak investigation, for local surveillance and cluster discovery, and to routinely summarise the virus diversity circulating on a national level. Civet reports have helped researchers and public health bodies feedback genomic information in the appropriate context within a timeframe that is useful for public health. The timely sharing of genomic data during the SARS-CoV-2 pandemic has enabled large-scale national and international surveillance efforts around the world. On a finer scale, pathogen genomics can supplement infection prevention and control efforts in clinical settings, as well as aid in outbreak investigations in community settings –4. HowevAnalysing or interpreting genomic information alone without relevant epidemiological information can be misleading and lead to incorrect conclusions due to the incomplete nature of the data. The relatively low mutation rate of SARS-CoV-2, frequent occurrence of convergent mutations (homoplasies), and prevalence of incomplete genome sequences make it critical to integrate epidemiological information alongside the genomic data to provide the most accurate picture and extract the most value from any given dataset. This includes temporal and spatial information, but may also include outbreak-specific data such as profession, ward, clinical metadata, or the background of viral lineages actively circulating in the community. Outbreak investigations often require bespoke reports that present information in a transparent and accessible manner. The data presented must be easily interpretable by health care providers and teams involved in infection control, the majority of whom are not accustomed to incorporating this type of data into their decision making processes.https://hgwdev.gi.ucsc.edu/cgi-bin/hgPhyloPlace) [The virus genomics community has developed a number of tools for analysing and visualising virus genomic data on the order of magnitude of this pandemic. HgPhyloPlace uses UShER to rapidly place sequences of interest into a global SARS-CoV-2 phylogeny (loPlace) . Tree viloPlace) , MicroreloPlace)  and DendloPlace)  can effiloPlace) . HoweverloPlace) , 11. FurloPlace) . The advloPlace) –have beeWe developed civet (Cluster Investigation and Virus Epidemiology Tool) to address this challenge of integrating metadata while condensing huge quantities of genomic data, and thereby aid SARS-CoV-2 outbreak investigations and surveillance efforts. Civet enables robust phylogenetic analysis to be performed, dynamically querying a large background dataset and generating interactive reports integrating both epidemiological metadata and genomic analysis. Both Public Health Scotland and Public Health England have routinely used civet to inform local outbreaks of SARS-CoV-2 and a number of studies have already been published that have used civet as tool for genomic epidemiology –17.Civet is a Python-based tool with an embedded analysis pipeline implemented in Snakemake . Civet oAll the scenarios presented as example cases for civet use are synthetic examples inspired by actual data and details have been entirely anonymised and/or altered. The first case study is a constructed outbreak loosely based on a hospital outbreak, but details have been masked. The second case study uses only publically available data from COG-UK. The UK studies were done as part of surveillance for COVID-19 infections under the auspices of Section 251 of the National Health Service Act 2006 or Regulation 3 of The Health Service (Control of Patient Information) Regulations 2002, or both. They therefore did not require individual patient consent or ethical approval. The COVID-19 Genomics UK Consortium study protocol was approved by the Public Health England Research Ethics Governance Group (reference number R&D NR0195). The third case study in Trinidad and Tobago is wholly based on public data.https://github.com/COG-UK/datapipe) although a similarly centralised set up could be applied elsewhere. The COG-UK Datapipe filters the input data to only include sequences of high quality with more than 90% genome completeness and removes any non-UK outliers with a genetic distance from the root beyond four standard deviations from the mean of the dataset in each epiweek. The sequences are aligned against a reference genome sequence  and problematic sites such as the untranslated regions (UTRs) are masked out. Any background dataset used with civet should strive to be of the highest quality possible as catchment finding and phylogenetic inference will be sensitive to poor quality data. Civet can generate the background alignment, metadata file and a SNP summary file from an unaligned fasta sequence file, such as a download sequence file from GISAID [To run civet, the user must minimally provide a sequence alignment and metadata file representing the background diversity of the pathogen of interest. Users on CLIMB-COVID have this data provided by the COG-UK Datapipe  allows the user to specify mutations of interest and this can include deletions as well as SNPs. For catchment finding, deletions are ignored in a pairwise manner, as is common practice in many phylogenetic methods. Similarly, within iqtree deletions are classified as unknown and so do not influence the tree structure in the final report.Query identifiers are matched with the alignment in the background data, and the set of fasta sequence records is compiled from queries in both alignment files. Identical sequences are collapsed to a single unique sequence . Collapsgenome.ucsc.edu/cgi-bin/hgPhyloPlace).The gofasta method updown topranking has parameters that can deal with some missing data. By default and within civet, any sites that are different between the reference genome and the query genome are catalogued and for every pairwise comparison there is 10% ambiguity at these sites allowed in total. Within civet there is also a maximum ambiguity allowed for a query sequence to be processed and a catchment found  as a prAt this point in the pipeline, there is no limit to the size of catchments and as the pandemic has been sampled so intensively in some areas, even relatively low SNP distances can lead to a large catchment. The user has the option to downsample the catchments prior to tree building and configure the maximum number of the background sequences to include in a given catchment tree . DownsamCivet generates a fully customisable report, summarising information about the queries of interest and the surrounding diversity. The report generated is a HTML file that can be viewed in a web browser, thus allowing the interactivity of web-pages. The components of the report include an interactive table summarising metadata of the query sequences, including any user supplied metadata; which catchment a query falls in; and the mutations of interest if specified. This table can be sorted, filtered and its columns can be dynamically configured, all within the distributable report. For each catchment, the civet report contains a table summarising the catchment content (prior to downsampling) and describes which lineages and countries are present in this local diversity neighbourhood (example shown in https://github.com/rambaut/figtree.js). The trees can be expanded out along the vertical axis and tip nodes can be coloured by any field specified with annotations—tree-annotations. Clades can be collapsed down by clicking on the parent branch and uncollapsed by clicking again. Each taxa in the tree is associated with additional metadata that can be displayed by selecting a tip (demonstrated in https://github.com/aineniamh/snipit). The report also contains a query timeline based on supplied temporal metadata, and interactive maps both for plotting the query sequence locations and for summarising the background diversity in the location of interest up to administrative level 2 for the UK and administrative level 1 for the rest of the world.The civet report displays the catchment trees using the interactive tree visualisation library FigTree.js  practices e.g. , 2, 29. The case study presented in https://cov-lineages.org/resources/civet/civet_case_study_1.html. Genome sequencing of SARS-CoV-2 samples from staff and patients revealed the outbreak consisted of two distinct clusters, or catchments, corresponding to PANGO lineages B.1.1 and B.1.418. https://jbloomlab.github.io/SARS-CoV-2-RBD_DMS/ last accessed 2021-08-10) [https://cov-lineages.org/resources/civet/civet_case_study_2.html). The search defined queries as sequences from the UK with the spike N501Y mutation from the beginning of September 2020 to the latest data in the background set (2020-10-21). Civet can also be used as part of routine local surveillance to summarise the diversity of viruses circulating in a local area or to flag and monitor clusters of interest. The N501Y mutation in the SARS-CoV-2 spike protein has been predicted to increase SARS-CoV-2 receptor binding domain ACE2 affinity (1-08-10) . As suchhttps://cov-lineages.org/resources/civet/civet_case_study_3.html. Civet also has the flexibility to inform surveillance efforts at the national level. In Virus genome sequencing can help reveal transmission chains and clusters of interest to aid outbreak investigations and surveillance efforts, as exemplified by the case studies above. With civet, academic researchers and public health scientists can easily run complex and robust phylogenetic analyses with a single command, contextualising sequences of interest in the large background dataset and visualising them alongside temporal, spatial and other epidemiological metadata in an interactive, distributable report. This frees users to place emphasis on interpreting the data and allows them to deliver information on a time-frame that is useful for public health responses.https://github.com/COG-UK/datapipe). This makes the latest SARS-CoV-2 genome data available to civet users on a daily basis. COG-UK data protection stipulates that data cannot be removed from CLIMB-COVID and often outbreak investigations involve sensitive, protected metadata. With civet, researchers can run analysis on CLIMB-COVID, distribute the report and keep their metadata protected. Civet has been popular and widely used within the framework of COG-UK, by academic researchers and scientists in public health agencies, for investigating SARS-CoV-2 clinical outbreaks and running local surveillance. A similar centralised server infrastructure could be set up for a national surveillance response or more local “locked down” compute environments [Throughout the SARS-CoV-2 pandemic, civet has been primed for use investigating SARS-CoV-2 clinical outbreaks and running local surveillance on CLIMB-COVID  as part ronments  and civeCivet can easily perform phylogenetic analysis on large datasets and provide reports for any countries with sequences to analyse. Default settings are configured for SARS-CoV-2, but civet is virus-agnostic and can be set up to run on other viruses of interest with an appropriate background dataset and reference sequence. Although civet is currently a command-line based tool, a clear extension to the software is to develop and provide a graphical user interface. This will enable users unfamiliar with the command line to run civet. We also plan to continue developing civet and adding extra features, including a country specific summary comparing counts of genomes sequenced over time with additional epidemiological data such as cases per country over time, which is already available on the Johns Hopkins University COVID-19 DataAPI . This paAs the ability to rapidly sequence pathogens at scale has become less technically challenging, in part due to the availability of robust protocols such as those by the ARTIC Network , the amoS1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 TextThe COVID-19 Genomics UK (COG-UK) Consortium. COVID-19 impact project (Trinidad and Tobago Group).(DOCX)Click here for additional data file.


Nature Genetics,1 Han and colleagues employed a dual genetic lineage tracing approach in combination with a model of heart failure (HF) to identify a key sub-population of endocardium-derived fibroblasts, which gives rise to excessive myofibroblast formation in a Wnt pathway-mediated manner and contributes to cardiac fibrosis.1 The identified fibroblast sub-population and Wnt signaling pathway could be novel targets for the therapy of HF.In a recent study published in Improvements in risk management and cardiovascular intervention have significantly reduced age-specific cardiovascular disease-related mortality. In contrast, the rate of HF-related hospitalisations has increased over the past decades and HF is a disease with highest social and economic cost in industrialized countries. HF is defined as impairment of the heart’s blood pumping function and its severity is assessed based on ejection fraction. HF often occurs after myocardial infarction (MI), with infarct size and quality of ventricular remodeling after MI influencing ventricular dysfunctions. Causes of HF also include hypertension, atrial fibrillation, and cardiomyopathies Fig. . EspeciaPost-infarct healing and ventricular remodeling are crucial to preserve cardiac function and determine the development of HF. However, the underlying factors are largely unknown and there exists a need to improve our mechanistic understanding to ultimately improve the mid-to-long-term outcomes of HF patients. It is important: i) to identify specific phenotypes of vulnerable patients, who benefit from specific interventions; ii) to identify strategies to limit aggravation of cardiac damage by targeting specific cell populations; iii) and for MI-related HF, to further optimize infarct healing to prevent adverse remodeling.1 Han and colleagues used genetic lineage tracing to label and track endocardium-derived fibroblasts (EDFs) in the heart. They generated mice with a dual tamoxifen-inducible genetic tracing-system  to target endocardium-derived cells and fibroblasts  to discern endocardium- (GFP + ) from epicardium (tdTomato + )-derived fibroblasts  to mimic pressure overload in the heart. To determine the role of EDFs, they generated a sophisticated dual color-labeled model , verifying DTR-mediated ablation of endocardium-derived fibroblasts by genetic tdT color-labeling. Importantly, Sirius red staining revealed genetic deletion of these cells reduced the extent of cardiac fibrosis after TAC and improved heart function.The authors further investigated the role of EDFs in cardiac fibrosis by generating fibroblast-diphtheria-toxin receptor (DTR) mice (Ctnnb1, the gene encoding β-catenin and the key component of the Wnt pathway, was deleted in EDFs. This led to lower β-catenin protein levels in GFP + fibroblasts. Following TAC, Sirius red staining indicated less myocardial fibrosis in Ctnnb1 mutant compared to control mice. The Wnt pathway is best known for its role in morphogenesis and embryogenesis and has been found to become activated in organ injury and regeneration. However, its specific role in cardiac fibrosis is incompletely understood. The study by Han and colleagues now provides compelling evidence that Wnt signaling plays an important role in the expansion of EDFs during cardiac fibrosis and directly links it to HF.To explore the mechanisms involved in EDF expansion during cardiac fibrosis, the authors prepared GFP + and GFP- fibroblasts and compared their gene expression signature. They found positive regulation of the canonical Wnt signaling pathway and proliferation in GFP + fibroblasts, suggesting the Wnt pathway as a potential driver. To study its causal role in fibroblast expansion, 3 In addition to the TGF-β and Wnt/β-catenin pathways, the MAPK pathway, mitochondrial and metabolic signals, or the complement system have been implicated in cardiac fibroblast activation, differentiation, and dysregulation.4 Hence, a network of pathways likely controls cardiac fibrosis4 and understanding their precise interplay may facilitate the development of diagnostic and therapeutic strategies for the treatment of cardiac fibrosis.2The TGF-β cascade is another primary pathway involved in cardiac fibroblast activation and differentiation. Transient activation of TGF-β favorably affects the repair of the infarcted heart through its anti-inflammatory and pro-fibrogenic properties. In contrast, prolonged activation exacerbates adverse remodeling and cardiac dysfunction Fig. . In respAlthough the study by Han et al. is limited to the pathogenesis of cardiac fibrosis following pressure overload, as mimicked in the TAC model, it provides compelling insights into the role of EDF in cardiac fibrosis. However, the restricted regional distribution of these cells also implies that non-endocardium-derived fibroblasts may contribute to cardiac fibrosis in other regions of the adult heart. Still, the data by Han et al. overall suggest that targeting endocardium-derived fibroblasts and/or Wnt signaling in this fibroblast-subtype may have diagnostic and therapeutic implications in HF. It is likely that the conclusions are translatable to other models of HF such as MI-triggered adverse ventricular remodeling. Indeed, reactivation of Wnt signaling upon ischemic injury is well described. The work by Han and colleagues also highlights the importance of complex genetic tracing strategies in understanding cellular fate and function in complex biological systems.4 and a variety of markers/modalities are employed for diagnostics.4 To improve diagnostic precision in HF patients, ‘the concept of treatable trait, a certain physiologic derangement characterized by biomarkers or biological changes that portend a predictable response to a therapy’ has been proposed.5 Although biomarkers often refer to defined laboratory tests, the term is used more broadly. It is important that the trait is measurable and corresponds with the modifiable insult or physiological process, which causes the adverse effect and is linked to a treatment response.5 As such, and despite the limitations of disease-mimicking mouse models, the cell type of endocardium-derived myofibroblasts identified by Han and colleagues in conjunction with the Wnt pathway driving the expansion of these pro-fibrotic cells could be promising diagnostic and therapeutic targets and may in the future qualify as treatable traits for adverse cardiac remodeling and HF.Heart failure is a syndrome related to an impairment of the heart’s ventricular ejection fraction. The underlying cause is often multifactorial


An important cervical cancer prevention strategy in low- and middle-income countries (LMICs) has been single-visit screen-and-treat (SV-SAT) approach, using visual inspection with acetic acid (VIA) and ablative treatment with cryotherapy to manage precancerous lesions. While SV-SAT with VIA and cryotherapy have established efficacy, its population level coverage and impact on reducing cervical cancer burden remains low. In Kenya, the estimated cervical cancer screening uptake among women aged 30–49 is 16% and up to 70% of screen-positive women do not receive treatment. Thermal ablation for treatment of precancerous lesions of the cervix is recommended by the World Health Organization and has the potential to overcome logistical challenges associated with cryotherapy and facilitate implementation of SV-SAT approach and increase treatment rates of screen-positive women. In this 5-year prospective, stepped-wedge randomized trial, we plan to implement and evaluate the SV-SAT approach using VIA and thermal ablation in ten reproductive health clinics in central Kenya.The study aims to develop and evaluate implementation strategies to inform the national scale-up of SV-SAT approach with VIA and thermal ablation through three aims: (1) develop locally tailored implementation strategies using multi-level participatory method with key stakeholders , (2) implement SV-SAT approach with VIA and thermal ablation and evaluate clinical and implementation outcomes, and (3) assess the budget impact of SV-SAT approach with VIA and thermal ablation compared to single-visit, screen-and-treat method using cryotherapy.Our findings will inform national scale-up of the SV-SAT approach with VIA and thermal ablation. We anticipate that this intervention, along with tailored implementation strategies will enhance the adoption and sustainability of cervical cancer screening and treatment compared to the standard of care using cryotherapy.NCT05472311. Contributions to the literatureApplication of an evidence-based, contextually appropriate, and replicable implementation strategy to improve screening and treatment of precancerous lesions of the cervix in low- and middle-income country settings.Rigorous evidence of impact on screening and treatment uptake using the single-visit and screen-and-treat approach with VIA and thermal ablation.Generate data on budget impact of the strategy to support scale up and sustainability planning.Cervical cancer is almost entirely preventable with current technologies, yet it remains the fourth most common cause of cancer incidence and mortality among women globally . Ninety The current WHO guideline recommends women over 30 years old be screened for cervical cancer every 3 years via visual inspection with acetic acid (VIA), Lugol’s iodine, or cytology when human papillomavirus (HPV) testing is not available as a primary screening modality . The sinIn the SV-SAT approach, women who screen positive for precancerous lesions are immediately treated with ablative therapy such as cryotherapy or thermal ablation, which minimizes loss to follow-up or attrition of screen-positive women , 18. AccImplementation strategies of evidence-based interventions (EBIs) such as SV-SAT with VIA and thermal ablation can facilitate identification of multi-level determinants of cervical cancer screening and treatment uptake and tailor EBIs to the local contexts to enhance their adoption and sustainment. We hypothesize that this intervention, along with tailored implementation strategies, will enhance the adoption and sustainability of cervical cancer screening and treatment compared to the standard of care using cryotherapy. Our specific aims are to:Aim 1) Develop locally tailored implementation strategies using multi-level participatory method with key stakeholders Aim 2) Implement the intervention and evaluate clinical and implementation outcomes using the RE-AIM framework.Aim 3) Assess the budget impact of the intervention compared to single-visit, screen-and-treat method using cryotherapy.We anticipate that the findings from this project will inform wider implementation and scale-up SV-SAT approach with VIA and thermal ablation and provide critical evidence to guide national policy and serve as a model for cervical cancer prevention efforts in similar LMIC settings. (NCT05472311).cure/treatment in Kiswahili, the Kenyan national language) aims to develop and evaluate implementation strategies to inform the national scale-up of SV-SAT approach with VIA and thermal ablation. We will initially use qualitative research with a participatory approach to develop an implementation strategy to introduce the intervention  in ten reproductive health clinics in central Kenya. Subsequently, we will evaluate the implementation of the intervention following the stepped-wedge cluster randomized trial design. The conceptual framework for this trial was informed by the implementation research logic model  Hospital levels 3–5 according to the Kenyan level system, public or private medical facilities; (2) current use of VIA as the primary screening method; and (3) cervical cancer screening volume over a 4-month period (greater than 1000) . AdditioThree to four clinics will be randomly assigned to one of three 6-month implementation waves at the stakeholder meeting at the conclusion of year 1. The clinics will be stratified by size.The intervention will include cervical cancer screening and treatment using SV-SAT with VIA and thermal ablation. To build on existing data from the region and the investigative team, we will use participatory methods to conduct qualitative interviews with clients, front-line health providers, facility managers, and policy makers in the Ministry of Health to assess and address barriers and facilitators to uptake and provide information on the introduction of the intervention through stakeholder engagement. Based on informal engagement with frontline health care workers and managers in the study area, we anticipate openness to the intervention. Existing barriers will be identified early in project implementation to address any unforeseen challenges to implementation. At the end of year 1, we will hold a stakeholder workshop to map out context-specific strategies that address potential barriers to intervention uptake and account for heterogeneity in provider and system inputs across facilities. Information gathered during these research activities will help investigators refine the essential components of the implementation strategy to be evaluated in Aim 2.n = 20). These health providers will be purposively sampled by profession to ensure representation of nurses, clinical officers, and doctors who provide reproductive health care, as well as a subset with management roles (n = 10), and two female patients recently screened for cervical cancer in each potential study facility (n = 20). Sample size determination in qualitative research rests on maximizing potential for saturation . Based on experience, we fully envision reaching saturation with (n = 5) policymakers stationed at the National Cancer Institute and the Division of Reproductive Health at the Ministry of Health of Kenya. Our investigative team has extensive experience conducting in-depth interviews with health providers.In-depth interviews will use semi-structured topic guides, to explore barriers and facilitators to potential adoption, delivery, and sustainment of the intervention. We will also interview frontline health providers carrying out cervical cancer screening in reproductive health clinics  and patient flow will be obtained. Infrastructure supplies and staffing data collection will be collected at three time points in the ten RH study facilities; before and after each implementation wave and at the end of the maintenance phase. Routine data on the proportion of women screened, screened positive, and treated for abnormal lesions will be abstracted monthly, by research staff, covering a minimum period of 6 months prior to introduction of the intervention at any facilities and continuing through the end of the maintenance phase. During pre-implementation periods, clinics will contribute data to the “control” condition in the stepped wedged trial.At 10 reproductive health clinics in central Kenya, we will introduce the intervention and site-specific implementation strategy developed in Aim 1 in a stepped-wedge fashion and rigorously evaluate how effectively this strategy is disseminated and implemented. In three successive 6-month waves, we will roll out the intervention at three to four clinics. A maintenance phase of varying lengths will be conducted across all waves.The stepped-wedge study design is powered based on the primary effectiveness outcome, which is the proportion of screen-positive women who receive same-day treatment. According to data on cervical cancer screening and preventive treatment for Kiambu county from 2017 to 2019, 20% of cervical cancer screening clients who screened positive received cryotherapy or LEEP. Routine data from our proposed study facilities show an average of 30 clients per month screened at each facility in the first 3 months of 2020. Assuming 6-month waves, 30 clients screened per month and a 5% screen positive rate, we expect an average of 9 screen-positive women per facility per wave. Assuming an intracluster correlation coefficient of 0.2, alpha = 0.05, and 20% treatment coverage pre-intervention, we will have 80% power to detect a three-fold increase to 60% treatment coverage with the intervention (R [v 3.6.3] package swCRTDesign [v 3.1]). We estimate that 90% of screen-positive women will be eligible for ablative treatment.Before the implementation of the intervention, we will train health providers in participating reproductive health clinics, with a refresher course on VIA and thermal ablation treatment techniques as part of single visit as per IARC guidelines. The training curriculum will be adapted to address gaps identified in year 1. On-site hands-on training will be done at each facility with support of research training team for a duration of 4 days. Outreach community activities will be done prior to scheduled on-site training to provide an adequate number of clients for screening and demonstration of thermal ablation. Each trained provider will be required to complete a set number of procedures before they receive a proficiency certificate. The selected reproductive health clinics have some of their providers trained on VIA and cryotherapy. Our team of investigators has three reproductive health experts with thermal ablation training skills who will develop and train a team of trainer of trainers at each reproductive health clinic. Regular training at each clinic will be provided with engagement of a Kenyan consultant gynecologist to provide consultant training services for the trainer of trainers for the initial 2 clinics. Thereafter, the research team and the county trainer of trainers will provide on-site hands-on training. Training will be led by specialists skilled in colposcopy, loop electrosurgical excision procedure (LEEP), cryotherapy, and thermal ablation. Quarterly CME and refresher training during implementation will be done. In addition to thermal ablation training, CME-type training on cervical cancer prevention interventions and refresher didactic training on VIA will be performed.We will provide bi-monthly technical support from the study team with assessment of fidelity and tailoring of the implementation strategy for 6 months at all sites. Depending on their wave assignment, sites will move to a maintenance phase of 18 (wave 1), 12 (wave 2), or 6 (wave 3) months of length as described in Fig. We will convene a committee that will serve an advisory team at the facility level. This committee will work closely with the research team to provide input on on-project implementation processes and receive updates provided by the research team. The research team will liaise with the committees to schedule regularly scheduled meetings. We will evaluate the impact of the intervention on the clinical and implementation outcomes using the RE-AIM framework Table .Table 1SFor reach of the intervention, we will assess the number of providers trained within each clinic. We hypothesize that the intervention will increase the number of women receiving cervical cancer screening and treatment services in all facilities. We will abstract program data and estimate the number of women aged 25–49 years  seeking services in reproductive health clinics who are screened per month .We will assess effectiveness by determining whether the intervention is associated with an increase in the number of screen-positive women identified per month and proportion of screen-positive women who complete treatment (target = 60%) comparing pre-implementation period to intervention periods. This will include women treated with thermal ablation, cryotherapy, LEEP, or any other procedure.The primary analysis will be an individual level analysis comparing the primary efficacy outcome (probability of screen-positive women being treated the same day) pre- vs post-intervention. We will estimate differences using weighted least square methods appropriate for this facility-randomized three-wave stepped-wedge design, where all clinics begin in the control condition. We will consider the intervention effect to be fixed throughout the post-intervention period, but random intercepts per facility and wave. Within the post-intervention period, we will examine whether the effect waned in the maintenance phase using an analysis comparing the intensive vs maintenance phases. To analyze secondary outcomes of probability of providers being trained and number of women screened (REACH outcomes) and number screen-positive (secondary efficacy outcome), we will use the same analysis approach. Exploratory analysis will examine potential effect modification of facility-level factors such as patient volume, patient to provider ratio, and women screened. Potential multi-level adjustment variables include individual-level factors such as age.To assess adoption, we will assess (1) facility readiness to implement the intervention, (2) the proportion of clinics incorporating thermal ablation into routine care through updated policies, and (3) proportion of trained providers performing the single-visit screen-and-treat approach to the screen-eligible women and thermal ablation for screen positive women.n = 30) and 6 frontline health workers per intervention facility (n = 60). Analysis will test whether sufficient inter-rater reliability and inter-rater agreement exist to aggregate individual responses to the facility level. If tests do not justify aggregation, we will use a measure of intra-facility variability in readiness rather than a facility-level mean in our analysis. The resulting analysis will provide readiness profiles for each facility as they initiate implementation, which will complement adoption, implementation, and effectiveness data in understanding the broader impact of the intervention.To assess readiness for intervention adoption, we will apply the validated ORIC assessment scale translated into Kiswahili and adapted to the implementation context after each wave begins. ORIC is a 12-item Likert-type scale, broken into domains of change commitment (4 items) and change efficacy (8 items), and has demonstrated reliability, content validity, structural validity, structural invariance, and known-groups validity in field application. We will apply the ORIC to 3 management team members of each intervention facility  at each clinic and in-depth interviews with health managers (n = 10) and screen-positive women  and spread out over the intensive implementation wave. Interviewees will be purposively selected to capture diversity in health facilities . Interview guides will include questions adapted from the CFIR question bank (cfirguide.org) to gather data about selected constructs from 4 of the established CFIR domains , the largest confidence interval for HPV clearance will be ± 8.1%.We will assess fidelity to thermal ablation training by assessing for HPV clearance among HPV-positive women. We will collect HPV cervical swab prior to and 6 months post-treatment to assess for clearance of type-specific HPV infection (target 60% HPV clearance) post-treatment on all screen positive women treated with thermal ablation. HPV samples will be shipped to the UW/UON HIV/STD Research laboratory in Mombasa. Assuming at least 200 women consent to the HPV sub-study and 70% of them test positive for HPV prior to thermal ablation  in a decision analysis model to estimate and compare the costs incurred and averted. Cost and impact data will be used to estimate the budget impact of SV-SAT with each treatment strategy. Our primary analyses will take the programmatic perspective, and secondary analyses will include costs incurred by patients to access services and duration of time used by providers to provide services. Our approach, analyses, and results reporting will align with published economic evaluation guidelines, which will maximize the transparency and generalizability of our findings to other settings , 32. We To capture heterogeneity in costs and efficiency across settings, we will collect cost data in a representative sample of six reproductive health clinics. We will use an activity-based micro-costing approach to measure start-up, implementation, personnel, supply, capital, overhead, and patient costs in each clinic with standard practice (SV-SAT with cryotherapy) and with implementation of the intervention Table . Measure34We will summarize data on costs to estimate the total cost of the intervention per woman. Our micro-costing approach will allow us to identify and exclude costs specific to research activities, focusing only on programmatic costs. To reflect variable costs and activities in different phases of implementation of the intervention, we will stratify costs by initial start-up, implementation and maintenance phases. For each strategy, we will break costs down into the categories in Table To account for differences in reach and effectiveness by treatment option, we will develop a decision tree model to estimate the expected number of CIN2 + and cervical cancer cases that would arise with SV-SAT with thermal ablation relative to SV-SAT with cryotherapy given the size of the eligible patient population in Kenya. To estimate these health outcomes, we will incorporate study data on reach and effectiveness with each strategy (Aim 2) and published data on HPV natural history and progression to cancer. Costs will be estimated using study data on the cost per woman screened and treated with the intervention and published data of the costs of cervical cancer screening and treatment. To account for variability and uncertainty in key parameters, we will conduct sensitivity analyses by jointly varying these parameters within observed or probable ranges. The model will be programmed using TreeAge Software  . Using dThe intervention of SV-SAT with VIA and thermal ablation can be a crucial component in reaching cervical cancer elimination in LMICs. Our study proposes to develop and evaluate implementation strategies to inform the national scale-up of the intervention. Findings from our study will inform scale-up of SV-SAT with VIA and thermal ablation to both national and county governing bodies and support decision-making regarding allocation of resources towards training of health care providers and providing equipment.


All of Us and the UK Biobank have introduced a new research paradigm where data are stored centrally in cloud-based Trusted Research Environments (TREs). To characterize the advantages and drawbacks of different TRE attributes in facilitating cross-cohort analysis, we conduct a Genome-Wide Association Study of standard lipid measures using two approaches: meta-analysis and pooled analysis. Comparison of full summary data from both approaches with an external study shows strong correlation of known loci with lipid levels (R2 ~ 83–97%). Importantly, 90 variants meet the significance threshold only in the meta-analysis and 64 variants are significant only in pooled analysis, with approximately 20% of variants in each of those groups being most prevalent in non-European, non-Asian ancestry individuals. These findings have important implications, as technical and policy choices lead to cross-cohort analyses generating similar, but not identical results, particularly for non-European ancestral populations.Recently, large scale genomic projects such as  The emergence of large-scale genomics projects has led to genetic studies across cohorts. Here, the authors conduct genome-wide association studies meta-analyzing in trusted research environments or pooling together and find similar, but not identical results. All of Us Researcher Workbench (AoU RW) and the UK Biobank Research Analysis Platform (UKB RAP), for large-scale clinical and genomic data-sharing and analysis4. In general, a TRE is a secure computing environment which provides approved researchers with tools to access and analyze sensitive health data. TREs offer many benefits, including (1) increased protection of study participant data, (2) decreased barriers to access and analyze data, (3) lower cost of shared data storage, and (4) increased collaboration across the scientific community7. The positive impact of TREs is clear, as is their potential to facilitate population- and global-scale health research9.Traditional data sharing processes require researchers to download copies of data to their own systems. More recently, health research is shifting to use Trusted Research Environments (TREs), such as the 11. Researchers wishing to safely and appropriately analyze data across different TREs face technological hurdles and policy requirements to do so12. Several approaches to data analysis across enclaves have been proposed. These include a meta-analysis whereby researchers perform analysis in separate TREs and then meta-analyze de-identified results outside of an enclave, and pooled analysis whereby researchers create and analyze merged data within a single enclave  was used to explore cross-analysis of UK Biobank and All of Us whole genome sequence data and UK Biobank whole exome sequence data twice - (1) by meta-analyzing GWAS results from separate TREs and (2) by analyzing pooled data in a single TRE. The goals, recruitment methods, scientific rationale and genomic data for All of Us and UK Biobank have been described previously2. In All of Us, we leveraged 98,622 whole genome sequenced samples alongside 200,643 whole exome sequenced samples from the UK Biobank. Although whole genome sequence data are available for UK Biobank, pooled analysis would require the data to be moved to a common enclave, which is not permitted by its access policy. The 200k exome release from UK Biobank was therefore explicitly chosen for use in this project because it was the last release of individual-level UK Biobank sequence data permitted to be analyzed outside of the UKB RAP, and therefore available for use in both pooled and meta-analyses performed on the AoU RW. Since our project was focused on comparing the computational approaches rather than on discovering new associations, maximal sample sizes were not needed.We performed a genome-wide association study on circulating lipid levels involving All of Us and UK Biobank TREs (see supplement for further details). Phenotypes were prepared separately. We curated lipid phenotypes  using the cohort builder tool within the AoU RW. We obtained phenotype information on one or more lipid measurements from electronic health records for 37,754 All of Us participants with available whole genome sequence data. In the UK Biobank, one or more lipid measurements from systematic central laboratory assay were available for 190,982 participants with exome sequence data17. Covariate information  and data on lipid-lowering medication for these corresponding samples were extracted from All of Us survey and electronic health record data and UK Biobank self-reported data. The lipid phenotypes were adjusted for statin medication19 and normalized (see supplement).For the meta-analysis, GWAS of lipid levels were performed separately in the 20 on the subset of variants within the UK Biobank exonic capture regions  were significantly associated (p < 5E-08) with LDL-C  was included as an additional covariate to mitigate potential batch effects from the different sequencing approaches and informatics pipelines used in All of Us and UK Biobank (see supplement). 464 variants were significantly associated (p < 5E-08) with the LDL-C phenotype from 284 loci (r2:0.5)  as well as ~170,000 other individuals, most of whom were of European ancestry. Effect sizes from both of our analyses are highly correlated with the two previously published standards  of associations in common with the Selvaraj study. When compared with the Hindy study, an average of ~3 more genome-wide significant SNPs were retained with the pooled analysis  was slightly lower for all lipid phenotypes using the pooled approach  and African ancestral samples, we were able to identify multiple variants unique to these ancestral groups using the pooled approach. Among the ancestry-specific variants from the pooled analysis we identified 5 rare variants specific to African ancestry and 1 from American ancestry. We also observed that the 64 variants uniquely significant in pooled analysis had more significant CADD scores (Phred-scores >= 20) when compared to those uniquely significant in meta-analysis , with much of the signal observed in the American ancestral group . The variants identified from pooled analysis (Phred-scores >= 20) were rare and present in non-European ancestry and these variants harbored functional severe consequences extending to missense, frameshift and stop-gain mutations.Next, we tested how the analytical approach impacted the ancestry frequency distributions of significant variants. We obtained ancestry data from gnomAD and referenced the popmax ancestry informationups Fig.  and 9. Wx faster rate with the number of siloed TREs in the meta-analysis versus the pooled analysis (see supplement).Cost and complexity are critical considerations impacting the use and usability of large-scale biomedical research data. We evaluated analysis complexity by examining the number of discrete computational steps required to complete a lipid GWAS Fig. . The numAll of Us data from a Hail matrix table to create a BGEN file for use with REGENIE ($220). The primary cost driver for the pooled analysis was the Hail processing needed to merge the UK Biobank and All of Us variant data ($360).Additionally, we report the cost comparison of the meta- versus pooled analyses. There are two aspects to the overall cost: (1) Cloud resource utilization (including the cost of data storage and cloud compute), and (2) the person-time needed to perform and review the results of each step. For cloud data storage costs, the respective TREs assume the considerable cost of hosting the primary formats of the genomic data, freeing researchers of this cost burden. Cloud compute costs are tool dependent. For analysis steps involving R, PLINK, or REGENIE the cloud compute resource costs are quite low - on the order of cents to a few dollars. Analysis steps involving Hail, by comparison, incur increased cloud compute cost. Hail processes data in a parallel fashion, leading to reduced wall-clock time to complete large-scale analyses. Hail is particularly useful whenever there does not already exist an optimized, purpose-built tool to perform the exact genomic data transformation needed. The primary cost driver for the meta-analysis was the Hail processing needed to extract relevant Person-time is highly dependent on the researcher’s familiarity with the datasets, methods, tools, and TRE capabilities. We found the amount of person-time for the meta-analyses was roughly twice that required for the pooled analyses. The person-time savings gained during pooled data harmonization, manipulation, and visualization within a single analysis environment, outweighed the cost of the additional steps required to merge the phenotype and genomic data.All of Us data using lipid GWAS as a case-study in computational approaches to analysis across TREs. Specifically, we looked at scientific and technical differences between meta-analysis of data in separate TRE silos, and pooled analysis of data in a single TRE. In each analysis we controlled for potential batch effects by including the source cohort as a covariate and limiting both pooled and meta-analyses to the subset of variants common in both the All of Us and UK Biobank cohorts. Each approach successfully replicated known genetic associations with plasma lipids. For both approaches, effect sizes found for each lipid trait are highly correlated with previously published studies. However, we did note several important scientific differences. First, pooled analysis enabled 1,496,404 additional variants to be included in the GWAS, compared with meta-analysis. Most of these variants were of lower minor allele frequencies, and thus this difference may be attributed to the fact that merging the two cohorts prior to applying the AC > 6 filter “rescued” rarer variants. We expect that the smaller overall number of variants retained for meta-analysis, because variants with an exceptionally low allele count are not considered by the analysis method, may negatively impact analysis of rare disease or rare variants. In these cases, a pooled approach may be preferred.We present two potential methods for the cross-analysis of UK Biobank and All of Us and UK Biobank TREs. In comparison, we found 464 variants significantly associated with LDL-C from pooled analysis of All of Us genome and UK Biobank exome sequencing data. We noted approximately 20% of variants significant in only the pooled analysis or significant in only the meta-analysis were most prevalent in non-European, non-Asian ancestry individuals. Prior foundational work has demonstrated that given otherwise equivalent datasets pooled and meta-analysis will generate theoretically and empirically equivalent results26. However real-world experience as illustrated above and by others29 has identified numerous differences between cohorts including phenotype ascertainment, genetic ancestry and population structure. Therefore, it is not surprising that these two analytical approaches yielded scientifically similar, but not identical, results. This has important implications for studying genetic variants in diverse individuals.Second, the analytical approach impacted the number and ancestry frequency distributions of variants significantly associated with our phenotype of interest. We report 490 variants significantly associated with LDL-C from meta-analysis of GWAS performed separately in x faster rate than for pooled, and therefore there is a significant increase in meta-analysis cost associated with the person-time required to develop and debug an analysis. That increased cost is high for two TREs, and even more significant as the number of TREs increases, which is expected as the amount of valuable global data increases.In addition to the scientific differences considered above, researchers seeking to analyze data across TREs face significant technical hurdles. Both complexity and cost scale with the number of data enclaves cross-analyzed. The pooled GWAS approach described was the least complex of the two investigated, requiring almost half as many discrete computational steps as meta-analysis. While analysis steps are displayed in a logical order in Fig. This study found several capabilities provided by existing TREs that facilitated cross-cohort analysis, and that if adopted by future TREs would facilitate incorporation of more data into future analyses. These include: (1) maintaining a single centrally funded copy of data that can be accessed in-place by researchers, 2) providing robust, integrated research support, (3) providing access to flexible, scalable infrastructure and tools suited to large-scale data analysis  is a population-based cohort of approximately 500,000 participants recruited from 2006 to 2010, that has existing genomic and longitudinal phenotypic data. Baseline assessments were conducted at 22 assessment centers across the United Kingdom, with sample collections including blood-derived DNA. Secondary use of this data was approved by the Massachusetts General Hospital Institutional Review Board (protocol 2021P002228) and was facilitated through UK Biobank application 7089. The All of Us Researcher Workbench (AoU RW) and the UK Biobank Research Analysis Platform (RAP), the genotypes were filtered to include only variants within the exome capture region with an alternative allele frequency of 6 or more. Whole genome sequenced (WGS) data from All of Us alpha 3 release was available as a Hail matrix table on the AoU RW. The alpha3 genotypes were filtered to include only variants within the same exome capture region with an alternative allele frequency of 6 or more. As initial quality control, variants with Hardy-Weinberg equilibrium exact test p-value below 1e-15 or missing call rates exceeding 10% were removed. QC also checked for samples with missing call rates exceeding 10%, but none were found. To mitigate batch effects, in the pooled analysis the prepared genotypes were filtered to include only those variants found in both cohorts and in the meta-analysis the results were filtered to include only those indicated found to be in both cohorts.Whole exome sequencing (WES) from the 200 K exome release is the most recent release of genomic data permitted by UK Biobank policy to be analyzed outside of the UK Biobank Research Analysis Platform (RAP). The 200 K exome release includes approximately 10 Million exonic variants with >95% of targeted bases covered at a depth of 20X or greater. On both the All of Us from the phenotype resources of these cohorts. LDL-C was either directly measured or calculated by the Friedewald equation when triglycerides were <400 mg/dL. Given the average effect of lipid lowering-medicines, when lipid-lowering medicines were present, we adjusted the total cholesterol by dividing by 0.8 and LDL-C by dividing by 0.7, triglycerides remained natural log transformed for analysis. The lipid phenotypes were then inverse rank normalized by the residuals, scaled by the standard deviation and adjusted for the covariates. We included PC1-10, age, age2 and sex at birth as covariates in our study. To mitigate batch effects, for the pooled analysis we also included a covariate of ‘cohort’.The primary outcomes in this study included LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), total cholesterol (TC) and triglycerides (TG) as phenotypes. We curated and harmonized the lipid measurements and statin drug exposures for both UK Biobank and p-value not exceeding 1 × 10−15 for both step 1 and for the genome wide associations. We carried out meta-analysis of the siloed GWAS results from each cohort using the METAL package with the Standard Error scheme, where the methods weights effect size estimates using the inverse of the corresponding standard errors. The UKB siloed analysis was carried out on the UKB RAP, and the All of Us siloed analysis and the pooled analysis were carried out on the AoU RW. All the steps were implemented in R or Python notebooks. Complete details on the various steps carried out in the project are provided in the supplementary information.Single variant genome wide association studies (GWAS) were carried out using REGENIE v2.2.4. We implemented REGENIE Step1 NULL model generation using quality-controlled variants with a minor allele count (MAC) of 100. We applied the leave one chromosome out (LOCO) method for GWAS while adjusting for the covariates stated above. We used variant and sample missingness at 10% followed by Hardy-Weinberg equilibrium Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplemental Data 1–3Reporting Summary


This Frontiers volume on Decision Neuroscience presents nine articles highlighting the importance of attention as a fundamental mechanism underpinning decision making. A crucial distinction is between goal-directed top-down (TD) and stimulus-driven bottom-up (BU) processes  and mean fixation duration (MFD). Their findings provide in review evidence for the independent contributions of preference for fairness (System 1) and self-interest maximizing (System 2) inclinations to UG decisions and shed light on the underlying processes.The study conducted by Yang et al. undertake an eye-tracking study to explore the impact of an incentive strategy on decision confidence. They find that incentives do not affect intertemporal choice on the gain domain. By contrast, in the loss domain, subjects in the incentivized group were more likely to choose the larger-later options. This points to the disproportionate influence of loss stimuli in intertemporal choice.Azulay et al. examine how empathy and stress measured by the Trier Social Stress Test may influence altruistic giving proxied by behavior in the Dictator Game. While acute stress by itself does not affect the level of altruistic giving, they find that individual differences in trait empathy moderate the effects of stress on giving. Elevations in stress-induced cortisol result in more generous giving, but only in individuals high in empathy. We note that attention is a prerequisite for the sentiment of empathy: when we are stressed or emotionally exhausted, our ability to pay attention may be impaired.Chew and Ebstein begin with a description of how the frog catches flies instantly upon sensing them. Relying on retinal neurons which are activated by small, dark, and moving objects exclusively, the frog's “eye speaking directly to brain” circuitry obviates the need for higher cognitive processes accompanied by a complete loss of flexibility. This hard-wired sensory-perceptual process motivates a survey of early papers on the brain's bottom-up (stimulus driven) and top-down  attention networks leading the authors to hone in on two pairs of neurotransmitters involved in valuation and attention processing—dopamine and serotonin (DA-5HT) and acetylcholine and norepinephrine (ACh-NE). Drawing on Zhong et al.'s  model ofAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.