Ultra-processed food industry (UPFI) actors have consistently opposed statutory regulation in health policy debates, including at the WHO. They do so most commonly with claims that regulatory policies do not work, will have negative consequences or that alternatives such as self-regulation work well or better. Underlying this are often assertions that industry is aligned with principles of evidence-based policymaking. In this study, we interrogate if this holds true by exploring the extent and quality of the evidence UPFI respondents employed to support claims around regulatory policy, and how they did this.First, we identified all submissions from organisations who overtly represent UPFI companies to consultations held by the WHO on non-communicable disease policy between 2016 and 2018. Second, we extracted all relevant factual claims made in these submissions and noted if any evidence was referenced in support. Third, we assessed the quality of evidence using independence from UPFI, nature, and publication route as indicators. Lastly, where peer-reviewed research was cited, we examined if the claims made could be justified by the source cited.and independent research, none appropriately represented the source.Across 26 included consultation responses, factual claims around regulation were made in 18, although only 10 referenced any evidence at all. Of all 114 claims made, 39 pieces of identifiable evidence were cited in support of 56 claims. Of the 39 distinct pieces of evidence, two-thirds were industry-funded or industry-linked, with only 16 externally peer-reviewed. Over half of industry-funded or industry-linked academic articles failed to declare a conflict of interest (COI). Overall, of only six claims which drew on peer-reviewed UPFI respondents made far-reaching claims which were rarely supported by high-quality, independent evidence. This indicates that there may be few, if any, benefits from consulting actors with such a clear COI. There is growing evidence that regulatory policies are more effective than voluntary industry measures in addressing obesity and dietary non-communicable diseases which pose a growing threat to public health.Ultra-processed food industry (UPFI) actors claim to be aligned with evidence-based policymaking, but nonetheless consistently oppose evidence-based regulation in favour of voluntary approaches.Our research shows that, in response to WHO consultations, UPFI groups made many factual claims to oppose regulatory policies and promote alternative measures, but only cited evidence in support of just over half of all instances.Most respondents did not make extensive use of evidence.The majority of the evidence cited to support factual claims lacked key indicators of quality such as independence or external peer review.Where industry respondents cited peer-reviewed research evidence, they often failed to represent the source accurately.In line with previous research on commercial use of evidence, our study suggests that UPFI actors not only tend to provide evidence which lacks key quality indicators but also employ evidential practices which serve to create doubt about the public health evidence.As such, industry actors might not only feed lower-quality evidence into policy processes; mirroring discourse around science and evidence could also potentially bolster their credibility and that of their arguments.Thus, those developing public health policies or policy recommendations should reconsider if and how they engage with commercial actors.informed policymaking has emerged more recently, explicitly acknowledging that while the best available evidence should be used, decisions are not purely based on technical considerations.Approximately 10 million global deaths per year are attributable to unhealthy diets,The fundamentally political nature of policymaking is particularly noticeable where powerful commercial entities find themselves facing a threat of regulation: multinational companies involved in the sale of ultra-processed food and beverage products as well as their representative groups  have consistently engaged in ‘corporate political activity’ to prevent, delay, or weaken regulatory policies,50Even preceding an acute policy debate, corporate influence on science can shape the body of evidence on a topic, thereby influencing what is perceived as a problem and which solutions are considered. Research funded by UPFI entities or conducted by academics with a conflict of interest (COI) appears more likely to reach conclusions that are favourable or simply non-threathening to the donor industry.53nature of evidence used by commercial actors and of how this evidence or, more broadly, the concept of evidence are used. The available research suggests that unhealthy commodity industry actors predominantly use evidence that is not independent and externally peer-reviewed—thus lower in quality—and where they do use scientific evidence, tend to misrepresent the source.28 56–58 60 62On the other hand, a less studied facet of the interface between public health policy, evidence, and corporations is the strategic use of evidence within the policymaking process. Existing public health research on this topic has focused predominantly on tobacco controlThis paper aims to combine both of these analytical strands to explore how UPFI actors promote their NCD policy preferences at the WHO. It builds on a previous study where we document how UPFI associations opposed regulatory approaches such as marketing restrictions, mandatory front-of-pack labelling, and particularly SSB taxation in consultations held to inform WHO recommendations.To what extent did UPFI actors refer to evidence when making factual claims about policies?What types of evidence did UPFI actors refer to when making factual claims about policies? Was it independent and peer-reviewed?Where peer-reviewed research was cited to support factual claims, does the claim accurately reflect the source content?To address the last research question, we draw on concepts from agnotology, a term coined by ProctorTo explore how evidence has been used by commercial actors in global-level policy spaces, we analysed UPFI responses to WHO consultations on NCD policy. Specifically, we focused on arguments against the statutory regulation of unhealthy foods and non-alcoholic beverages, assessing the evidence cited in this context for markers of quality. To establish whether peer-reviewed evidence was represented accurately, we also conducted a verification-oriented cross-documentary analysis which compares claims made with the cited source documents.58 62web-based consultation of the WHO Independent High-level Commission on NCDs,consultation on the Member State-led draft outcome document for the WHO Global Conference on NCDs (‘Montevideo roadmap’),updating Appendix 3 of the WHO Global NCD Action Plan 2013–2020zero draft Shanghai Declaration on Health Promotion.We systematically searched the WHO Headquarters website for consultations held between 2016 and 2018 which covered dietary NCD policy, were global in scope, and for which all responses were published in full. Four consultations met our criteria: the Our analysis concentrated on statements which opposed regulatory approaches to dietary NCDs, as we could not identify any which supported the introduction of new statutory regulation. Using Atlas.tiNext, we coded whether any evidence was referenced in support of the claim, and extracted it into a spreadsheet. We adopted a broad definition of evidence as formal and informal written sources, such as reports, journal articles, press coverage, blogs, and opinion pieces. We included all instances where evidence was formally cited (at the end of a page or submission), or referred to in the text, provided enough information was available to identify it through a web search. Links to general websites were not included as they do not clearly refer to a distinct piece of evidence. Where coding decisions were challenging or uncertain, this was resolved in discussion between the first and second author.We conducted two separate analyses. First, we assessed the quality of all evidence referenced in support of factual claims on policy effects. Second, we assessed how peer-reviewed research was used in this context.et alet alIndependence was assessed by first searching if the consultation submission itself stated a link between an UPFI entity and the evidence cited. If this was not the case, we went on to screen the cited piece of evidence for a funding or conflict or interest statement. If none was declared, we conducted web searches for the authors in combination with the name of the organisation which cited the evidence in its consultation response, and the four largest packaged food and soft drink companies .nature of evidence was categorised as research, opinion, strategy documents or raw data. To assess the origins of evidence, we categorised the publication route as either peer-reviewed journals and other academic outlets, intergovernmental organisations and governments, or publications by private companies and organisations. We separately assessed if academic sources had been externally peer-reviewed. For industry-funded and industry-linked academic publications, we also noted whether the source declared a COI. We ran descriptive analyses in IBM SPSS version 26.81We adopted the criteria from Evans-Reeves et alTo examine how scientific evidence was used by industry actors, a verification-oriented cross-documentary analysis was conducted for all instances where peer-reviewed research articles were cited to support relevant factual claims. In each case, we compared the statement made with the cited source to assess whether the claim reflected the latter. During this process, we noted where agnogenic practices occurred, drawing on the typology developed by Ulucanlar UPFI actors made 114 separate factual claims in 18 of the 26 included submissions . Of thesOnly 71 of the 114 claims in 10 of the 26 responses actually referred to any evidence. Yet, only in 56 of the 71 factual claims citing evidence was this evidence identifiable: the remaining lacked key information such as title, year published, or author, and one item was paywalled market research data.The remaining 43 factual claims were made without any reference to a specific piece of evidence, despite some making strong statements with casual references to ‘the evidence’, as exemplified by the Italian business association Federalimentare[a]vailable scientific evidence on sugar does not support a causal link between sugars consumption and obesity and associated chronic disease. As an example, while sugar consumption decreased in UK, Australia and Canada, the obesity rate grew in their respective populations.Similarly, the International Food and Beverage Alliance (IFBA)[…] the UK salt reduction initiative, a public-private partnership led by the UK government which has resulted in the reduction of average daily salt intakes by 15% since 2001. Similar salt reduction initiatives and trans fat and calorie reduction strategies around the world have also proven effective.The 56 claims with identifiable references were made by only five business associations: the International Council of Beverages Associations (ICBA), IFBA, the Grocery Manufacturers Association (now Consumer Brands Association),The 56 claims citing identifiable evidence referred to 39 separate pieces of evidence. Of these 39 pieces of evidence, just nine (23.1%) were clearly independent, while 13 (33.3%) were industry-funded; 13 (33.3%) were industry-linked, four (10.3%) appeared independent but did not provide sufficient information to conclusively rule out industry links. In most pieces of evidence classified as industry-linked, one or more of the authors had received funding from UPFI entities  were research, eight (20.5%) were strategy documents, four (10.3%) were opinion pieces, and one (2.6%) was raw data. Notably, 19 of the 26 research-based sources were industry-funded or -linked.Of the 39 pieces of evidence, 18 (46.2%) were published by academic outlets, closely followed by private companies and organisations which had published 17 (43.6%). This included reports published by the submitting business associations themselves and evidence from think tanks and research firms such as Oxford Economics89 92how scientific evidence was used to support the factual claims around policy effects. We discuss examples under each core category of claims: questioning regulatory policies  and promoting alternatives to regulation (claim categories 3 and 3.1).In this section, we address Factual claims that regulatory policies do not work or will have negative consequences, although made in 12 responses from seven organisations, were only backed by peer-reviewed research evidence in three responses, all made by one organisation, ICBA. They cited three independent research articles to support five claims that SSB taxation does not work or will have negative consequences,105et al,Appendix 3 to WHO Global NCD Action Plan 2013–2020,To question the effectiveness of the policy, they cited a review by independent scientists, Bes-Rastrollo et al]. In this summary, it was found that of the six published studies to date where data had been measured (as opposed to modeled), five found no effect of taxation whatsoever, while the sixth found less than a 1.8 kg difference in body weight after 20 years.recent review summarizing all data related to taxation of sugars found that taxation did not affect obesity rates . In other words, consumers switched from soft drinks to milkshakes, smoothies and other similarly calorie-dense products – resulting in more calories consumed.et al concluded that one year after the introduction of the Berkeley SSB tax, ‘prices of SSBs increased in many, but not all, settings, SSB sales declined, and sales of untaxed beverages  and overall study beverages rose in Berkeley’.104In fact, Silver et alAppendix 3 of the Global NCD Action Plan,Discussing an independent observational study by Colchero et al], it is important to note that the calorie consumption from beverages has declined only slightly – roughly between two and six fewer calories per day in a diet of more than 3000 calories per day in Mexico , which is a daily caloric decrease of less than one half of one percent.Although one widely-publicized study indicates that purchases of taxed beverages decreased by an average of six percent in 2014  were likely underrepresented (as were the working poor or very poor) since the study was based on Nielsen panel data covering 53 cities each with 50,000 or more residents; (3) the data was based on purchases and not consumption; and (4) the study was not controlled for other environmental factors .It is noteworthy that ICBA directed no such critical assessment towards the favourable, typically lower-quality evidence it cited throughout this submission.ICBA invoked six articles to question the well-established58IFBA and the Grocery Manufacturers Association were the only organisations to use peer-reviewed research articles in support of self-regulation or co-regulation. They did so in eight instances, and all articles were either industry-funded or industrylinked.93–97et alet al article, aimed to ‘provide a detailed description of EPODE methodology, including its broad and overarching approach to strengthening and enriching CBIs [community-based interventions] aimed at preventing childhood obesity’.95IFBA,By exploring UPFI use of evidence in global health governance for the first time, we add to an emerging body of literature investigating how unhealthy commodity industries use evidence to oppose public health regulation.and independent research, all of which misrepresented the original source to some degree. These six claims were all made by the same organisation, ICBA, to oppose SSB taxation. The group, which represents soft drinks producers, submitted some of the longest consultation responses with the most references to evidence, which goes a long way towards explaining the skew of our sample towards SSB taxation.It is noteworthy that, despite claims to support EBPM and language which mimics scientific reasoning,Overall, the arguments made by UPFI respondents to oppose statutory regulation do not align with the public health evidence. Claims that regulation to address dietary NCDs does not have the desired effect, predominantly levelled at SSB taxation, contradict independent evidence which supports the potential of taxes to favourably influence dietary behaviours.124Independent evidence also indicates that self-regulation is not sufficient to address the issues of obesity and dietary NCDs.When assessing the independence of cited evidence, we found it remarkable that the majority of industry-funded or industry-linked academic articles did not declare a COI. While some simply did not contain a COI section, others explicitly declared that they had no COI. Of those that did declare an interest, this was at times combined with the acknowledgements. This exemplifies why current reporting practices are inadequate and highlights the urgent need for enforced and structured COI reporting processes within and beyond public health.130There are indications that commercial actors draw on a shared set of preferred evidence and consultancies across levels of governance and policy settings. For instance, reports by the research firm Oxford Economics64 132In addition to the agnogenic practices described above, casual mentions of ‘the evidence’ or ‘science’ without reference to concrete evidence, as well as vague expressions of alignment with EBPM, appear to form part of attempts to position industry as a legitimate actor in public health policy. Sitting beyond the instrumental role of evidence, this rhetorical facet may play a role in lending discursive power and credibility to policy actors.Overall, our findings confirm existing research on the use of evidence by unhealthy commodity industries in public health policy,Our results on evidence use were heavily driven by a small set of actors who referenced large amounts of evidence, whereas the majority referenced little or no evidence. This may limit their generalisability of our findings. While we went beyond declarations in the cited sources to identify industry links to the evidence, our web-based investigation is unlikely to have identified all extant connections. Our research focused on UPFI actors and does not compare how non-industry actors such as Member States or civil society used evidence in their submissions. We concentrated on this subset of respondents due to the inherent conflict between the interests of the UPFI and public health, which has manifested in UPFI opposition to policies needed to address the considerable burden of obesity and NCDs.34 63 139Our findings suggest that UPFI actors’ rhetorical alignment with EBPM


Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes.ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions , excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only.ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored.The online version contains supplementary material available at 10.1186/s12862-021-01858-x. Eukaryotic phosphatases play essential roles in regulating many cellular processes and can be classified in several ways, based on catalytic mechanism, substrate specificity, ion requirements and structure. One four-group classification distinguishes phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases , protein tyrosine phosphatases and aspartic acid-based phosphatases . The eukS. cerevisiae ALPH protein (YNL217W), a Zn2+ dependent endopolyphosphatase of the vacuolar lumen [2+ and possibly Mg2+ [Trypanosoma brucei: we recently found that ALPH1 is the only or major trypanosome mRNA decapping enzyme [7 methylguanosine (m7G) cap present at the 5´end of most eukaryotic mRNAs. This finding was surprising, as all other known eukaryotic mRNA decapping enzymes belong to a different enzyme family, the nudix hydrolases (the prototype is Dcp2). Trypanosomes lack orthologues to Dcp2 and all decapping enhancers associated with Dcp2, but use the ApaH like phosphatase ALPH1 instead. T. brucei ALPH1 consists of the catalytic domain with N- and C- terminal extensions of about equal sizes, that appear unique to Kinetoplastida and bear no similarities to any known domains.ApaH like phosphatases (ALPHs) evolved from the bacterial ApaH protein and were present in the last common ancestor of eukaryotes , 5. Theyein YNL21W, a Zn2+bly Mg2+ . The enzbly Mg2+ . The secg enzyme , the enzInterestingly, recent data indicate, that the bacterial precursor protein of ALPH, ApaH, may have an analogous function to Trypanosome ALPH1 in decapping of bacterial mRNAs. In vitro, ApaH cleaves diadenosine tetraphosphate (Ap4A) into two molecules of ADP , 12, butPuzzled by these novel functions of bacterial ApaH and trypanosome ALPH1 in mRNA decapping, we here set out to investigate whether mRNA decapping is a major function of eukaryotic ALPHs, or whether this function is restricted to trypanosomes. We developed a Python algorithm for the identification of ALPHs in all available eukaryotic reference proteomes and identified 441 ALPHs in 332/827 proteomes. We show that most ALPH proteins consist exclusively of the catalytic domain and many have predicted transmembrane regions or signal peptides and predicted non-cytoplasmic localisation, indicating functions distinct from mRNA decapping. We show in vitro mRNA decapping activity for three of these non-cytoplasmic ALPH proteins, indicating that, similar to bacterial ApaH, eukaryotic ALPHs accept non-physiological substrates and thus are likely to have a rather wide substrate range. The data indicate that there may be selection against the presence of cytoplasmic ALPH proteins in eukaryotes, possibly to protect mRNAs from unregulated degradation. Apart from the kinetoplastids, only 71 organisms possess ALPH proteins with cytoplasmic localisation predictions. In contrast to the Kinetoplastida, these ALPH proteins consisted (with 2 exceptions) of the catalytic domain only and all organisms have an orthologue to Dcp2 (with 3 exceptions likely caused by genome incompleteness).ALPHs belong to the PPP family of phosphatases and possess the four conserved signature motifs motif 1–4) of this family, GDxHG, GDxxDRG, GNHE, and HGG, sometimes with conservative substitutions . One dis of this Branchiostoma floridae has ALPH) and from Ecdysozoa  and fully absent from the few available proteomes of Amoebozoa (9 proteomes tested) and Metamonada (3 proteomes tested). A phylogenetic tree built from the catalytic domains of ALPHs mostly reflects the eukaryotic tree  of ribed in   domain, a domain of bacterial origin with unknown function in eukaryotes. The ALPH of Rhizopogon vesiculosus has a second ALPH domain.The majority of available ALPH sequences (292) are from Fungi, because the number of available proteomes is high (298) and 80% of these proteomes contain at least one ALPH. We investigated these ALPH sequences further by looking for predicted domains (Interpro ), signalS. cerevisiae : two high-throughput studies indicate vacuolar localisation [The most interesting finding was the presence of predicted trans-membrane regions and/or signal peptides within the C-terminal region in 78.1% of all fungal ALPH proteins: 184 ALPH proteins have predicted membrane helices (mostly one), a further 41 ALPH proteins have predicted signal peptides, 3 ALPH proteins have both predicted and only 64 ALPH proteins have neither predicted  contain ALPH. ALPHs are present in subgroups of Cnidarians (2/3), Echinodermatans (1/2), Lophotrochozoens (8/11), Placozoans (1/2) and Ichthyosporeans (1/2). 7 of these 18 Holozoan ALPH proteins have a predicted signal peptide or transmembrane helix at their C-termini and with the exception of ALPH from Lottia gigantea, all proteins have non-cytoplasmic localisation predictions, mostly to the mitochondria (13/18) -binding superfamily N- terminal of the catalytic domain and ALPH of Leptotrombidium deliense may be interacting with actin, as it has a Kaptin domain C-terminal of its ALPH domain.ALPHs are underrepresented in Holozoans Fig. . In partarians 2/, Echinodarians 2/, EchinodAll the 42 organisms containing an ALPH protein with a cytoplasmic localisation prediction also have a Dcp2 homologue  Oomycetes, including for example Phytophthora parasitica: all the strains of this plant pathogen have three ALPH isoforms. Predicted signal peptides or transmembrane helices are present at the C-termini of many Chloroplastida ALPHs (11/19), as well as in ALPH proteins of the Alveolata Vitrella brassicaformis, the diatom Phaeodactylum tricornutum and the Haptista Emiliania huxleyi; in all cases the presence of transmembrane helices or signal peptides correlates with a predicted non-cytoplasmic localisation , Apicomplexa (20 proteomes), Ciliata (3 proteomes) or Dinoflagellata (1 proteome). ALPH is present in 3 of 4 species of red algae, 11 of 18 species of green algae (Chlorophyta), in the filamentous green algae Chlamydomonas reinhardtii has a predicted Transposase IS605 domain , the ALPH of Helicosporidium has a coatomer delta subunit domain and the three large and almost identical ALPH proteins of Chlorella sorokiniana have a PsbQ-like domain (IPR023222), an Arm-like repeat and a second ALPH domain. A PsbQ-like domain is typical for proteins of the photosystem II, consistent with localisation predictions of the three Chlorella sorokiniana ALPH proteins to the chloroplast.The majority of ALPH proteins from Diaphoretickes is very short and, in particular ALPHs of Oomycetes, consist almost exclusively of the catalytic domain with very short N- and C-terminal extensions. Of all 63 Diaphoretickes ALPH proteins, only five have additional domains: the ALPH of Arabidopsis thaliana (in some cases only as a fragment as proteomes were not complete) , they all have N-terminal extensions of a similar size. The in vitro mRNA decapping activity of T. brucei ALPH1 does not require the N-terminus [ALPHs of Kinetoplastida fall into two groups Fig. A: each og enzyme . These Aterminus , and theLeishmania, Leptomonas, Crithidia and Endotrypanum have extensions within the catalytic domain that are mainly caused by enlarged distances between motif 1 and 2  and between motif 4 and 5 (up to 156 nucleotides). The only Kinetoplastida that has no ALPH apart from ALPH1 is Leishmania tarentolae; blast searches identified a protein at the expected position in the genome which lacked both motif 5 and motif 6, indicating a recent loss of the protein.In addition to the ALPH1 homologue, all but one Kinetoplastid have between one and three additional ALPH proteins that consist exclusively of the catalytic domain Fig. A. These VHG/GDIHG for ALPH1/non-ALPH1) and in motif 6 (IDTG/LDSG for ALPH1/non-ALPH1)  Fig. B. When aH1) Fig. A.T. brucei ALPH1, fused to eYFP either C- or N- terminally, localises to the cytoplasm, P-bodies and to the posterior pole of the cell and this cytoplasmic localisation is consistent with the essential function of ALPH1 in mRNA decapping AT. brucen system . ALPH2 sIHG, GDLVGKG, GNHD, HAG, VFFGH, LDTG) are more similar to the non-ALPH1 group and this is also suggested by the absence of N- and C-terminal extensions  . PerkinsThe phylogenetic data from us and others  show thaSphaeroforma arctica, ALPH of the green algae Auxenochlorella protothecoides (A0A087SQ73) and ALPH2 from Trypanosoma brucei . As a control, ALPH of Sphaeroforma arctica was also produced as an inactive mutant by mutating a conserved amino acid in the metal ion binding motif  , further strong evidence for the specificity of the decapping activity.We produced recombinant ALPH proteins from randomly selected organisms of the three eukaryotic kingdoms that have ALPHs: we used ALPH of the Ichthyosporea IG:GNVIG ). All fonic acid , 43. The2+) Fig. B. All thThe data show that all ALPH enzymes tested accept capped mRNA as a substrate in vitro. Importantly, the tested ALPH proteins have non-cytoplasmic localisation predictions, indicating that mRNA cannot be their physiological substrate. Instead, the findings suggest that ALPH proteins may have a similar broad substrate specificity as previously shown for bacterial ApaH. The data support the hypothesis that the preference for none or non-cytoplasmic ALPHs in many eukaryotes could serve to prevent uncontrolled mRNA degradation.7 methylguanosine (m7G) cap, linked via an inverted 5´-5´bridge [mRNAs are essential molecules in both eukaryotes and prokaryotes and need to be protected from unregulated exonucleolytic degradation. 5´-3´exoribonucleases require monophosphorylated RNA 5´ends and the major protection strategy is therefore to avoid such monophosphates. Most eukaryotic mRNAs are modified at their 5´end by an m5´bridge . A small5´bridge , 46. Bac5´bridge , and als5´bridge , the 3-d5´bridge  or dinuc5´bridge –26. Arch5´bridge .7G cap and non-canonical metabolite caps [Trypanosoma brucei was shown to be the only or major mRNA decapping enzyme in these parasites [In common to the increasing number of different mRNA caps and mRNA 5´ end modifications discovered throughout all kingdoms of life is the pyrophosphate bond, and therefore the requirement of a pyrophosphatase to remove the cap or the polyphosphate. Enzymes of four different families can cleave pyrophosphate bonds at the 5´end of mRNAs . DXO andite caps –55 as weite caps  and NudCite caps  that actite caps . Enzymesarasites . Bacteriarasites –26 and lThe major aim of this work was to find out how widespread mRNA decapping by ApaH like phosphatases is. To identify ALPH proteins in all available eukaryotic proteomes, we have developed a Python based algorithm based on recognising the six conserved motifs of ALPH. We detected 441 ALPH proteins in 332 of 827 organisms. The full details of these 441 ALPH proteins are summarized in Additional file T. brucei ALPH2, non-cytoplasmic predictions were confirmed experimentally [T. brucei mRNA decapping enzyme ALPH1 has unique N- and C-terminal extensions and the C-terminal extensions are conserved across all Kinetoplastida ALPH1 homologues and it is therefore tempting to speculate that they contribute to enzyme regulation. 87 of the 120 ALPH proteins with cytoplasmic predictions consist of the catalytic domain only  and the ALPH protein of Ectocarpus siliculosus (disordered regions)  enzymes of the nudix domain family [+ caps [All three ALPH proteins that we tested had mRNA decapping activity in vitro, even though none of these enzymes is likely to use mRNA as their physiological substrate as all three have (predicted or proven) mitochondrial localisation. This broad substrate range of ALPHs is not unexpected: the related bacterial ApaH protein cleaves NpnN nucleotides –15 as wen family : the majn family  and can  [+ caps . Recent  [+ caps , 62, 63;The broad substrate range of ALPHs that includes mRNA caps provides an explanation for its loss or non-cytoplasmic localisation in most eukaryotes. The last common ancestor of all eukaryotes had ALPH. This assumption is supported by the presence of ALPH proteins in all eukaryotic kingdoms found in this and earlier  studies.Trichomonas this can be identified by Blast and in Giardia by more sophisticated in silico methods [Naegleria, but not in Euglena. The question whether DCP2 was present in the last common ancestor and selectively lost in Kinetoplastida, or whether it evolved subsequently to the separation of the Discoba cannot be answered without experimental data.One open question is whether all organisms outside the Kinetoplastida use DCP2 orthologues as their major mRNA decapping enzyme. While DCP2 has been functionally characterised in yeast , human  and plan methods , 67. WheIn the last few years, more and more mRNA cap variants have been discovered in both prokaryotes and eukaryotes, and more and more enzymes that act in mRNA decapping, including ApaH like phosphatases and bacterial ApaH. Our analysis of eukaryotic ApaH like phosphatases is an important contribution towards a more comprehensive picture of mRNA decapping.Despite being present in all eukaryotic kingdoms, ApaH like phosphatases are poorly understood with only a few studies available. Here, we present a new algorithm for the identification of ALPH proteins and we provide a comprehensive dataset of ApaH like phosphatases of all available reference proteomes. Trypanosomes and possibly a few other organisms use ALPH proteins for mRNA decapping; however, the physiological functions of the vast majority of eukaryotic ALPH proteins remain to be discovered. In vivo experiments are essential, as the wide substrate range of ALPH will impede the identification of physiological substrates in vitro.A novel algorithm was developed using Python 3 to identify ALPHs in a set of proteome fasta files. The algorithm initially used narrow matrices to identify the 6 conserved motifs  based on the data of [ALPHs with missing motifs due to wrongly annotated start codons or sequencing errors will not be detected, with the exception of an unidentified amino acid included in the extended matrix . For the Kinetoplastida, such ALPHs were manually included based on the available genome information from TriTrypDB , 29; forFinally, all proteins were manually inspected: even though the extended sequence matrix would allow the presence of motifs with little homology to the consensus motif, we found that the extended matrix was used rather rarely  and if so, the extended motifs had still most amino acids from the narrow matrix. The Python programme is available in Additional file T. brucei ALPH2 = Tb927.4.4330) or with minor N-terminal truncations to exclude the transmembrane regions  in Arctic Express DE competent cells (Agilent). At the N-terminus, the proteins were fused to a tag consisting of a 6xHis-Tag, a Thrombin cleavage site, a SUMO tag and a TEV cleavage site, resulting in the following additional protein sequence:All ALPH proteins were expressed either full length  gave slightly different trees, but, importantly, with all methods, the ALPH1 isoforms group together in one clade that never contains non-ALPH1 isoforms.Proteomes were downloaded from Uniprot, using all available reference proteomes of UniProt_release 2019_02 (For Archaea: 2019_11). . AdditioTrypanosoma brucei Lister 427 procyclic cells expressing a TET repressor (pSPR2.1) were used for all experiments [2. Transgenic trypanosomes were generated using standard procedures [eriments . Cells weriments  at 27 ℃ ocedures . All expZ-stack images (100 stacks at 100 nm distance) were taken with a custom build TILL Photonics iMic microscope equipped with a sensicam camera (PCO), deconvolved using Huygens Essential software . All images are presented as Z-projections (method sum sliced) produced by ImageJ software.7G-AACUAACGCUAUUAUUAGAACAGUUUCUGUACUAUAUUG . The sample was incubated for 1 h at 37 °C. Control reactions were done without enzymes. The reaction was stopped by ethanol precipitation: 1 µl 3 M RNAse-free sodium acetate (pH 5.5) (Ambion), 1 µl RNAse-free glycogen (ThermoFisher Scientific) and 30 µl cold 100% ethanol was added, the sample incubated at −20 °C for 30 min, followed by centrifugation . The pellet (containing the RNA oligo) was washed once in 80% ethanol, air-dried, resolved in 5 µl RNAse-free water (ThermoFisher Scientific) and either stored at −80 °C or directly prepared for loading to the gel.Each decapping reaction was done in 10 µl volume (with RNAse-free water) containing 50 mM Tris–HCl (pH 7.9), 100 mM NaCl, 1 mM metal ions (variable), 40 units Ribolock RNAse inhibitor (ThermoFisher Scientific), 0.25 µM recombinantly-produced ALPH enzyme and 0.39 µM capped RNA oligo m7G cap structure, this way increasing the difference in gel mobility between capped and uncapped oligos [RNA Samples were separated on urea acrylamide gels that contained acryloylaminophenyl boronic acid , 43: bord oligos , 43. ForAdditional file 1:Figure S1. Phylogenetic tree of ALPH in eukaryotes.Additional file 2: Python Software.Additional file 3: Table S1. List of all organisms that were screened for the presence of ALPH  and detailed information on all ALPH proteins that were identified in this study.Additional file 4: Table S2. a) Documentation of the last step of software improvement: the ‘missing motif screen’ on the complete set of proteomes. b) extended control matrix. c) list of ALPH proteins with truncated N- or C-termini.Additional file 5: Table S3. The 285 available reference proteomes for Archaea were downloaded from UniProt on 7th of January 2020. They were screened with the Python programme for the presence of ALPH. Only one proteome contained one ALPH.


Evidence-based practice in psychology consists of two quality parameters. One of these quality parameters is efficacy and the other is efficiency. In this article, it is argued that the only relevant parameter for determining quality in clinical care is efficiency. Moreover, emphasising efficacy in psychotherapy practice is symptomatic of a scientocentric ideal in evidence-based practice in psychology. The proper understanding and use of scientific findings entail leaving this scientocentric ideal. In addition, it is crucial that efficiency is related to the ethical aims that constitutes psychotherapy practice. Effectiveness and efficiency” set new standards for medical practice. He argued that without the guidance of randomised controlled trials, medical doctors delivered suboptimal, and sometimes even harmful, services. Because randomised controlled trials were believed to be bias-free, and consequently did not share the biases that led medical doctors to provide suboptimal treatment, Cochrane used the term “effectiveness” to denote the scientific merits of a given treatment. Cochrane recognises that he uses the term effectiveness to denote what is commonly coined “efficacy” (which he avoids simply because he dislikes the word). More specifically, effectiveness refers to the causal effects of a treatment. A treatment is deemed “effective” if scientific findings indicates that it causes a given change in the health status of its recipients. According to Cochrane, randomised controlled trials are superior in bringing about information of causal factors. Due to their lack of bias, randomised controlled trials let researchers draw unequivocal inferences (of whether a treatment is effective or not). Thus, there is an implicit methodological hierarchy in Cochrane’s ideal for clinical science, but Cochrane’s ideal for science and practice are not identical.There is an often-overlooked political ambition of Cochrane’s thinking, which was to provide effective health-care treatment available for everyone. This political aim led him to coin a second term. Efficiency includes factors such as the economic cost of various treatment options and the optimal use of staff and equipment. Efficiency is the translation of scientific research in clinical setting, taking the most important external  factors into consideration. By distinguishing effectiveness and efficiency, Cochrane underscored that clinical science differs from clinical practice. Whereas the models of science constructively reduce complexity, the reduction of complexity in clinical practice is associated with the risk of suboptimal clinical care.Another important distinction in Cochrane’s thinking is that between cure and care. Cochrane used the term cure to denote the combination of effectiveness and efficiency. One reason for combining effectiveness and efficiency in a concept is to highlight that effective treatments have variable efficiency in different contexts. Thus, one could either identify more effective cures or change the clinical setting to increase efficiency. He also introduced the term care, which are the affectionate aspects of patient treatment  . While rThe legacy of Cochrane prevails in evidence-based medicine and other evidence-based health-care practices. The effectiveness-efficiency distinction exists  in most evidence-based medicine models . HoweverIn contrast to Cochrane’s model, the more recent evidence-based medicine models are tripartite and consists of best external evidence, clinical expertise and patient values . This isEvidence-based medicine was the template for the American Psychological Association’s policy statement for evidence-based practice in psychology . The autHowever, there are also problems with regards to the definition of best evidence, in evidence-based practice in psychology. At first sight, the notion of evidence in the policy statement is quite inclusive. It contains a section with the heading “multiple types of research evidence.” Under this heading it is argued that “[m]ultiple research designs contribute to evidence-based practice, and different research designs are better suited to address different types of questions” , p. 274.treatment efficacy and clinical utility:However, another section of the policy-statement describes “specific interventions” . In thisTreatment efficacy is defined as the systematic and scientific evaluation of whether a treatment works. Clinical utility, on the other hand, is defined as the applicability, feasibility, and usefulness of the intervention in the local or specific setting where it is to be offered. Clinical utility also includes determination of the generalisability of an intervention whose efficacy has been established  . If a psCochrane’s distinction between care and cure has been confused by the authors of the policy-statement for evidence-based practice in psychology. The very distinction is obsolete in psychotherapy, because care  is the better part of cure  . A grim clinical utility. In the policy-statement it is argued that:The other quality parameter is At a minimum this includes attention to generality of effects across varying and diverse patients, therapists, settings, and the interaction of these factors; the robustness of treatments across various modes of delivery; the feasibility with which treatments can be delivered to patients in real-world settings; and the costs associated with treatments , p. 275.practice, the only relevant parameter is clinical utility.Clinical utility denotes the ability to combine different sources of knowledge to find the optimal treatment in real-life settings. In addition, clinical utility includes economic factors. Leaving economic factors aside, it should be clear that that the definition of an optimal real-world treatment is by no means straightforward. There is a large number of fact and value entanglements in psychotherapy practice making the notion of utility equivocal. Clinical practice concerns itself with the unique individual patient. The most relevant question when facing an individual is whether one has the knowledge, skill and resources to help that very individual. If the clinician has knowledge about effects that do not pertain to that very individual, lack the skill to convert knowledge into practice and/or do not have the resources to provide the treatment, knowledge about a given form of treatment is irrelevant. In that sense, knowledge about treatment efficacy qua treatment efficacy is useless for clinical practice. It is useful if and only if it leads to clinical utility. Thus, insofar that evidence-based practice in psychology aims at regulating psychotherapy per se serves as quality indicator of clinical practice. However, treatment efficacy is only be a quality indicator for research and not for practice.The move from empirically supported treatments  to evideThe proper understanding of efficiency comes with a major caveat. The sound use of scientific research in psychotherapy practice depends on the conceptualisation of psychotherapy. The propositions of psychotherapy research are value-laden and the various psychotherapy schools and interventions merges with ethics . PsychotWhen defining “real-world effects” one has to take into consideration that psychotherapy aims at realising a vision of a better life . This enis. Ultimately, the distance from science (as it is defined in evidence-based practice in psychology) to clinical practice is considerable, because the former deal with empirical regularities at a group level and the latter deal with the realisation of a better life at the level of the individual. Without sorting out these misconstructions, there is little hope that evidence-based practice in psychology could serve as a satisfactory regulatory principle.Because the policy-statement for evidence-based practice in psychology aims at regulating practice through scientific and extra-scientific parameters, it should encompass the difference between science and practice. In the current version of the policy-statement there is a failed attempt to achieve this. This does not only lead to a series of misunderstandings as to what makes psychotherapy “work,” but also with misunderstandings of what psychotherapy The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accurate classification of variants in cancer susceptibility genes (CSGs) is key for correct estimation of cancer risk and management of patients. Consistency in the weighting assigned to individual elements of evidence has been much improved by the American College of Medical Genetics (ACMG) 2015 framework for variant classification, UK Association for Clinical Genomic Science (UK-ACGS) Best Practice Guidelines and subsequent Cancer Variant Interpretation Group UK (CanVIG-UK) consensus specification for CSGs. However, considerable inconsistency persists regarding practice in the combination of evidence elements. CanVIG-UK is a national subspecialist multidisciplinary network for cancer susceptibility genomic variant interpretation, comprising clinical scientist and clinical geneticist representation from each of the 25 diagnostic laboratories/clinical genetic units across the UK and Republic of Ireland. Here, we summarise the aggregated evidence elements and combinations possible within different variant classification schemata currently employed for CSGs . We present consensus recommendations from CanVIG-UK regarding (1) consistent scoring for combinations of evidence elements using a validated numerical ‘exponent score’ (2) new combinations of evidence elements constituting likely pathogenic’ and ‘pathogenic’ classification categories, (3) which evidence elements can and cannot be used in combination for specific variant types and (4) classification of variants for which there are evidence elements for both pathogenicity and benignity. Accurate classification of variants in cancer susceptibility genes (CSGs) is key for the correct estimation of cancer risk and management of patients with potential cancer predisposition.A variety of evidence types can contribute to assertions of pathogenicity or benignity, for example, the number of independent cases with a characteristic phenotype, familial segregation data, frequency in population controls and functional analyses. Historically, appropriation of disparate evidence elements could differ widely between diagnostic laboratories and produce discrepant classifications. To advance consistency in diagnostic variant interpretation, in 2015, the ACMG published a framework for variant classification.CanVIG-UK was established in 2017 as part of the UK-ACGS activity supporting adoption and dissemination of the ACMG framework for variant interpretation.The ACMG framework has greatly improved the consistency with which variants in CSGs are classified within the UK molecular genetics laboratory community. Nevertheless, within the framework, several areas are ambiguous, undefined or make reference to the need for ‘expert judgment’.Frequently encountered within CanVIG-UK are variants for which there is uncertainty and inconsistency regarding the combination of multiple evidence elements, in particular those for which (1) the evidence elements available do not conform to any of the combinations specified in the original ACMG framework; (2) a proposed combination of evidence elements is of contentious legitimacy; and (3) there are conflicting evidence elements, that is, towards both pathogenicity and benignity.We thus sought within CanVIG-UK to improve consistency in combination of evidence elements within the ACMG framework, addressing the following five objectives:To establish an objective numerical system for combining evidence elements.To evaluate the maximum number of evidence items attainable via new ACMG-based specifications.To define specifically permitted and non-permitted combinations of evidence elements.To define all new combinations of evidence elements attainable using new ACMG-based specifications.To establish consistent practice in classification of variants with discordant (conflicting) evidence elements.In the original 2015 ACMG framework, four evidence strength levels were defined: supporting (P), moderate (M), strong (S) and very strong (VS).et al to conform well to a Bayesian structure, namely, that prior probability × likelihood ratio = posterior probability, where the evidence strength levels were related in an exponential series to a base of 2.08.Although developed empirically through clinical consensus, the ACMG framework was subsequently demonstrated by Tavtigian et al in a recent adaptation of their original metastructure.Derived from the Tavtigian Bayesian metastructure and consistent with the UK-ACGS Best Practice Guidelines for Variant Classification in Rare Disease 2020, we defined an ‘exponent scoring system’, similar to the approach taken by Tavtigian Conversion of the tally-based 2015 ACMG framework into a numerical exponent scoring system (derived from the Tavtigian Bayesian metastructure) is a useful evolution of the ACMG framework allowing rapid calculation of variant classification category. It is designed to augment, not replace the five-level classification system. In addition, the exponent scoring system:Enables delineation of previously undescribed legitimate combinations of evidence elements of equivalent numerical posterior probability.et al.Highlights the incongruity in the numerical posterior probability for some combinations of evidence elements in the original 2015 ACMG framework, as previously described by Tavtigian Allows objective stratification of the evidence strength for likely pathogenic variants (exponent score sum range 6–9). The exponent system provides a clearer and more consistent numerical language with which to continue dialogue regarding management quandaries about likely pathogenic variants being of ‘lower confidence’ and ‘higher confidence’. This typically comprises contexts in which a higher bar of evidence is argued to be desirable .Likewise allows objective stratification of evidence strength for ‘uncertain’ variants (exponent score sum range 0–5) .Defining the swathe of VUSs for which ‘active’ variant monitoring is indicated .Differential evidence requirements in oncology between the therapeutic context and the context of familial risk prediction. It has been argued in oncology that evidence of therapeutic actionability for some somatic biomarkers in use may be significantly weaker than for others, for example, atypical BRAF variants as indication for vemurafenib administration or an Allred score of 4 as indication for tamoxifen administration.Key to implementation of the Bayesian metastructure (and indeed the 2015 ACMG framework) is recognition that the overall (posterior) probability of pathogenicity is dependent not just on the aggregate likelihood ratio of the contributory evidence elements but also on the prior probability of pathogenicity. The exponent sums presented correspond to the respective posterior probability thresholds only in the context of a specified prior probability of 0.1 (10%). This approximates to clinical analysis of a limited gene set in a proband with a suspected Mendelian disorder.The exponent scoring system (derived from the Tavtigian Bayesian metastructure) is a legitimate means of summing evidence elements from the 2015 ACMG framework.Where there is a prior probability of ~0.1 (10%), an exponent sum of ≥6 equates to a classification class of likely pathogenic  posterior probability of pathogenicity), ≥10 of pathogenic (>0.99 (99%) posterior probability of pathogenicity), ≤−1 of likely benign (<0.1 (10%) posterior probability of pathogenicity) and ≤−6 of benign (<0.001 (0.01%) posterior probability of pathogenicity).A higher exponent sum is required to attain the equivalent posterior probabilities of pathogenicity where the prior probability of an underlying Mendelian mechanism is significantly less than 0.1 (10%) eg, variants identified as ‘additional findings’ on sequencing for an alternative indication.Five-point (hot) VUSs in well-studied CSGs (BRCA1/BRCA2 and mismatch repair (MMR) genes) should be discussed by a multidisciplinary team for potential inclusion on clinical reports. There are rapid-paced international research endeavours relating to these genes through which upclassification is quite possible.In the 2015 ACMG framework, 30 different evidence elements were specified (including three different evidence strength levels for PP1).10.1136/jmedgenet-2020-107248.supp1Supplementary dataFor each of the ACMG-based specifications used for CSG variant interpretation, the number of potential evidence elements at each strength level was increased compared with the original 2015 ACMG framework. Accordingly, a greater number of possible combinations of evidence elements will be attainable than was possible using the original 2015 ACMG framework.et alWithin the 2015 ACMG framework and subsequent ACMG-based specifications, several of the evidence elements are (1) non-independent of each other and/or (2) incompatible with regard to the specific variant types to which they can be applied. Accordingly, the combinations of evidence elements actually attainable in practice are restricted, and there is inconsistency regarding which evidence elements are used together. We identified pairs of evidence elements for which the legitimacy of combination is debatable and established, within CanVIG-UK, consensus on the legitimacy of combination . We thenGene-specific ACMG-based specifications vary in the combinations of evidence items they permit. It is unclear to what extent this variation reflects between-gene clinical–biological heterogeneity versus differences in approaches between expert groups.Pairs of evidence elements should be combined as per In the 2015 ACMG framework, eight evidence combinations were provided for pathogenic, six for likely pathogenic, two for likely benign and two for benign. Using our updated counts of total possible numbers of evidence elements , using tet al and the UK-ACGS Best Practice Guidelines for Variant Classification in Rare Disease 2020, we confirmed that two combinations in the 2015 ACMG framework were incongruous with the exponent scoring system, (1) one ascribed as pathogenic for which the exponent score is only eight and (2) one ascribed as likely pathogenic for which the exponent score is 10.As previously described by Tavtigian It was the overall consensus of the CanVIG-UK group to retain the requirement for at least two items of evidence to provide buffer against false-positive classification of a variant as likely benign/likely pathogenic via a spurious evidence item. Thus, although a sufficient exponent sum is attained with a single evidence item, classification out of VUS is not permitted in these instances: (1) likely pathogenic with a single very strong evidence element, (2) likely benign with a single supporting/strong evidence element.The exponent sum threshold for a classification category can be attained via any combination of two or more concordant evidence elements.Variants should not be classified as pathogenic, likely pathogenic, benign or likely benign on the basis of a single evidence item, except BA1 .et al paper, regardless of the extent of conflicting evidence; (3) we could use the net exponent score but with rules-based restriction regarding the maximum number of evidence elements ‘discordant’ with the final classification.In the original 2015 ACMG framework, the recommendation was to classify as uncertain any variant for which there was discordancy in the evidence elements.These three approaches were reviewed within CanVIG-UK through application to a number of exemplar variants. Consensus opinion was for option 3 as laid out in the following consensus recommendations and in In variant classification, we seek to balance the clinical benefit of classification of a variant out of the ‘VUS’ category against the harms of erroneous misclassification. Variants classified as likely pathogenic have a 90%–99% likelihood of pathogenicity: this group should thus contain a ~5% (1%–10%) frequency of variants that are truly benign and have been misclassified as likely pathogenic. Variants classified as likely benign have a 0.1%–10% likelihood of truly being pathogenic. Thus, while downclassification of variants in CSGs will and should occur periodically in practice, it is important to recognise the consequent clinical disruption, particularly if multiple unaffected family members have undertaken risk-reducing surgery.There would be a clear rationale for agnostic use of the net exponent score if all evidence was of unquestionable provenance and guaranteed to be wholly correct. In practice, in clinical observations, laboratory assays and/or published reports, there is always potential for error in evidence generation or communication. The presence of discordant evidence elements will occur by chance through statistical distribution of true results; it may also be an indicator towards error within one of the evidence items. Discordant results between clinical findings and laboratory results may also be an indicator of intermediate penetrance. Overall in CanVIG-UK, we adopted a strategy whereby classification from VUS to another class was permitted, providing the conflicting evidence did not exceed a single supporting evidence element.net exponent sum for benignity. Pathogenic variants will necessarily be rare ; the frequency of benign variants will vary from very common to very rare.PM2 (absence in controls) provides evidence of rarity, rather than evidence against benignity and can thus be ignored when calculating the Where there is discordant evidence, regardless of the net exponent score, the classification class cannot exceed likely pathogenic or likely benign. Variants with discordant evidence items should not be classified as pathogenic or benign.Conflicting evidence items should be combined to calculate a net exponent sum using the ‘exponent score system’ (derived from the Tavtigian Bayesian metastructure). In the presence of discordant evidence, providing there is no more than one discordant evidence element at no more than supporting level, if the net exponent sum is >6, the variant can be assigned the variant class of ‘likely pathogenic’; if the net exponent sum is <−1, the variant can be assigned the variant class of ‘likely benign’.net exponent sum for benignity.PM2 can be ignored when calculating the Clinical variant interpretation is a rapidly evolving field. The 2015 ACMG framework has provided an invaluable common framework for which there has been wide international adoption and improved congruity of approach. ClinGen gene-specific ACMG-based specifications in cancer susceptibility are currently finalised for PTEN, TP53 and CDH1.


The prevalence of nonalcoholic fatty liver disease (NAFLD) has been significantly increased due to the global epidemic of obesity. The disease progression from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH) is closely linked to inflammation, insulin resistance, and dysbiosis. Although extensive efforts have been aimed at elucidating the pathological mechanisms of NAFLD disease progression, current understanding remains incomplete, and no effective therapy is available. Bile acids (BAs) are not only important physiological detergents for the absorption of lipid-soluble nutrients in the intestine but also metabolic regulators. During the last two decades, BAs have been identified as important signaling molecules involved in lipid, glucose, and energy metabolism. Dysregulation of BA homeostasis has been associated with NAFLD disease severity. Identification of nuclear receptors and G-protein-coupled receptors activated by different BAs not only significantly expanded the current understanding of NAFLD/NASH disease progression but also provided the opportunity to develop potential therapeutics for NAFLD/NASH. In this review, we will summarize the recent studies with a focus on BA-mediated signaling pathways in NAFLD/NASH. Furthermore, the therapeutic implications of targeting BA-mediated signaling pathways for NAFLD will also be discussed. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and is rapidly emerging as the leading cause of end-stage liver disease due to the global epidemic of obesity, type 2 diabetes, and metabolic syndrome . It is wBAs are exclusively synthesized from cholesterol in the hepatocytes. BAs are the key organic components of bile stimulating bile flow . IntraheThe gut microbiome is composed of a diverse range of microbes. These microbes play critical roles in maintaining intestinal barrier function, modulating metabolic processes and immune responses . One majde novo synthesis from cholesterol in the liver. The human BA pool size is about 4–6 g. The daily amount of BAs secreted into the intestine is approximately 12–30 g in adults. The average number of daily BA recycling is between 3 and 5 times [Enterohepatic circulation is an essential physiological process that circulates BAs from the liver to the bile, followed by entry into the intestinal lumen, absorption by the enterocytes in the ileum, and transport back to the liver via the portal vein . The eff 5 times .BAs are exclusively synthesized from cholesterol in the hepatocytes as the dominant catabolic pathway of cholesterol. The two major biosynthetic pathways of BAs have been well characterized, known as the classical pathway and alternative pathway . The claBA synthesis is tightly regulated. The major feedback suppression mechanism controlling BA homeostasis has been well characterized ,38. BA-iClostridium [Once the BAs are secreted into the gastrointestinal tract, a small portion of BAs escape the reabsorption and will be deconjugated and modified by gut bacteria via various biotransformations, such as oxidation, esterification, epimerization of hydroxyl groups, and desulfation to generate a variety of secondary BAs. In humans, more than 50 secondary BAs have been identified in fecal samples . The inistridium . In micestridium  . BSEP is located at the canalicular membrane of hepatocytes and is responsible for the secretion of BAs into the canalicular lumen. The genetic mutations of ABCB11 are linked to progressive type II familial intrahepatic cholestasis [+-taurocholate cotransporting polypeptide  and Na+-independent organic anion-transporting polypeptide . Compared to NTCP, the driving force responsible for OATP-mediated uptake is not well understood. In the basolateral membrane, the multidrug resistance-associated protein 2 , MRP4 (gene symbol ABCC3), and organic solute transporter α/β  provide alternative excretion routes for BAs into the systemic circulation [BAs are amphipathic molecules containing both hydrophilic (α-hydroxyl group) and hydrophobic (β configuration of H on C5) faces  Figure . The diflestasis . The majculation . In the + with BA at the apical membrane. Once the BAs are uptaken by intestinal epithelial cells, their efflux is carried out by OSTα/β. In addition, it has been shown that MRP3 may also be involved in BA efflux, at least in rodents [Most BAs in the intestine are reabsorbed at the terminal ileum, where the bile acid transporters are highly expressed. The ileal active transport system is the primary route for conjugated BA uptake, especially for the more hydrophilic and taurine-conjugated BAs. ASBT, also known as the ileal BA transport (IBAT), is able to cotransport Na rodents .BAs have long been known as detergents to facilitate lipid and nutrient absorption in the intestine. Since the identification in 1999 of the nuclear hormone receptor (NHR), FXR, as a BA-activated receptor, BAs have been extensively studied as critical signaling molecules regulating lipid, glucose, and energy metabolism ,49,50,51FXR (gene symbol NR1H4) is expressed in the liver, intestine, colon, kidney, adrenal gland, and ovary. The highest expression levels of FXR are detected in the liver and gastrointestinal tract. FXR is optimally activated by unconjugated BAs and regulates BA synthesis in a tissue-specific manner . In hepaTGR5 is initially identified in macrophages as the first GPCR activated by BAs . TGR5 isS1PR2 is one of the five S1PRs, which was originally discovered as endothelial differentiation G-protein-coupled receptor 5 (EDG5) . During It has been reported that TLCA activates muscarinic receptor 3 (M3), but not M1 and M2 in gastric chief cells. The sulfated TLCA binds to M1, but not M2 and M3. TLCA-mediated activation of M3 inhibits acetylcholine-induced increases in inositol phosphate formation and activation of mitogen-activated protein kinase (MAPK). However, the concentration needed for TLCA to inhibit acetylcholine-mediated effects is at a mM range .The liver is the largest secretory gland in the human body and plays a crucial role in lipid, glucose, and energy metabolism. Due to the complexity and limited understanding of NAFLD disease pathogenesis, no regulatory-approved therapy is available, and liver transplantation remains the only therapeutic option for end-stage liver disease patients . There iThe maintenance of metabolic homeostasis both in the liver and gut is critical for many normal physiological processes. The gut and liver are constantly communicating with each other via numerous metabolites. BAs, as the crucial messengers, not only regulate hepatic metabolic functions but also impact the gut microbiome. Vice versa, the gut microbiome plays a critical role in maintaining intestinal homeostasis and exerts its influence on the liver by modifying BA composition, changing the BA pool, and regulating immune response ,76,77. DRecent studies also showed that the circulating BA profile is significantly changed and is correlated to NAFLD disease severity . The ratThe gender-specific differences in gut microbiota composition have been well established . ChangesGPCRs and NRs are major pharmacological targets . The bio−/− and SphK2−/− mice, HFD-feeding rapidly induced hepatic lipid accumulation and dysregulation of BA metabolism [Activation of S1PR2 by conjugated primary BAs plays an essential role in the maintenance of hepatic lipid metabolism. TCA/S1PR2-mediated activation of ERK and AKT signaling pathways and nuclear SphK2 is critical to maintaining hepatic lipid homeostasis. Activation of nuclear SphK2 has a significant impact on gene transcription by modulating histone acetylation. In S1PR2tabolism ,68. In ctabolism . The exptabolism . Activattabolism ,107. ConThe pathogenesis of NAFLD is multifactorial. Recently, it has been proposed to rename NAFLD as metabolic-associated fatty liver disease (MAFLD) based on clinical evidence indicating the close association of NAFLD and metabolic syndrome . The gut


Capacity strengthening initiatives aimed at increasing research knowledge and skills of investigators in low- and middle-income countries (LMICs) have been implemented over the last several decades. With increased capacity, local investigators will have greater leadership in defining research priorities and impact policy change to help improve health outcomes. Evaluations of models of capacity strengthening programs are often limited to short-term impact. Noting the limitations of traditional output-based evaluations, we utilized a broader framework to evaluate the long-term impact of the Vanderbilt Institute in Research Development and Ethics (VIRDE), a decade-old intensive grant development practicum specifically tailored for investigators from LMICs.To assess the impact of VIRDE on the research careers of alumni over the past 10 years, we surveyed alumni on research engagement, grant productivity, career trajectory, and knowledge gained in grant writing. Descriptive statistics, including means and total counts, and paired sample t-tests were used to analyze the data.Forty-six of 58 alumni completed the survey. All respondents returned to their home countries and are currently engaged in research. Post-VIRDE grant writing knowledge ratings were significantly greater than pre-VIRDE. The number of respondents submitting grants post-VIRDE was 2.6 times higher than before the program. Eighty-three percent of respondents submitted a total of 147 grants post-VIRDE, of which 45.6% were awarded. Respondents acknowledged VIRDE’s positive impact on career growth and leadership, with 88% advancing in career stage.Gains in grant writing knowledge and grant productivity suggest that VIRDE scholars built skills and confidence in grant writing during the program. A substantial proportion of respondents have advanced in their careers and continue to work in academia in their country of origin. Results show a sustained impact on the research careers of VIRDE alumni. The broader framework for research capacity strengthening resulted in an expansive assessment of the VIRDE program and alumni, illuminating successful program elements and implications that can inform similar capacity strengthening programs. Despite commitment by national governments and international funding, health outcomes in many low- and middle-income countries (LMICs) lag behind other nations . ImproviMany models of research capacity strengthening have been implemented over the years . ProgramReviews of capacity strengthening programs have noted difficulties in equipping scholars to work within structural challenges at institutional and country levels. Such challenges include a lack of funding, salary support, protected time to conduct research, research personnel and infrastructure, and mentorship –43. WithNoting the challenges that LMIC researchers encounter in sustaining a research career and inadequacies of traditional productivity metrics, Cooke created a framework to guide and evaluate research capacity strengthening programs . The fraIn 2011, the Vanderbilt Institute in Research Development and Ethics (VIRDE) program was launched to support junior investigators in LMICs. VIRDE utilized Cooke’s six principles at the individual level to build a unique program that trains investigators in grant writing and research ethics, with an aim for program scholars to attain funding support, sustain their research career, and become research leaders who positively impact health outcomes through policy change. The four-week, intensive, mentored practicum takes place in-person at Vanderbilt University in Nashville, Tennessee, USA. VIRDE includes state-of-the-art didactic sessions promoting competencies in grant writing and the responsible conduct of research. These sessions are coupled with dedicated time for hands-on, mentored practical skills building in grant writing, culminating in a grant application that is ready for submission for external funding. Scholars also engage in laboratory rotations, graduate-level courses, and regular meetings with a personalized mentoring committee.Evidence-based quality improvement is an essential element of the VIRDE program. Annual program evaluations inform program modifications and enhancements for the next iteration. Additional bi-annual check-ins with program alumni have contributed to quality improvement efforts and served to maintain networks and support alumni. To assess long-term impact, a program evaluation questionnaire utilizing Cooke’s principles was sent to all VIRDE alumni during the tenth year of the program. This paper illustrates the results of the program evaluation, assesses the impact of the program over the last decade, and describes lessons learned.The VIRDE program was designed for biomedical and public health investigators in LMICs. Initially the program was developed to bolster research independence of junior faculty affiliated with Vanderbilt Institute for Global Health (VIGH) partner institutions in Africa, Asia, and Latin America, but has since expanded to include scholars affiliated with additional institutions. Partner institutions in LMICs manage the initial round of the selection process, identifying appropriate candidates. Selected applicants then complete an online application, including submission of their curriculum vitae or National Institutes of Health (NIH)-style biosketch, a draft proposal based on an NIH R21 grant format, and support letters from their in-country mentors. Prior to selection, applicants are required to give a proposal presentation online to the VIRDE Selection Committee. Priority is given to applicants who submit a compelling grant proposal idea and identify committed mentors from their home institution and Vanderbilt. Scholarships for VIRDE participants are supported by institutional funds and VIGH collaborative US government and foundation-sponsored training and research grants with LMIC partner institutions.Cooke’s six principles of research capacity strengthening were incorporated into the VIRDE curriculum through seminars, mentoring teams, and on-going post-program collaborations. The VIRDE curriculum includes over 40 contact hours of specially tailored seminars in grant writing, grant administration, research ethics, and career development taught in person or virtually by Vanderbilt faculty and senior staff, VIRDE alumni, and LMIC partner faculty. Examples of grant writing seminars include funding strategies, effective writing of standard grant sections, and administrative elements of grant management. Research ethics seminars cover topics related to the fundamentals of research ethics, ethical research design, international standards of clinical trials, and ethics of authorship and collaborative partnerships. Career development seminars focus on leadership and strategies for success as an academic researcher.During the program, scholars participate daily in several hours of didactic workshops led by VIRDE faculty. When not in workshops, scholars hone their grant proposal and engage in various capacity strengthening activities at Vanderbilt, including attending courses and seminars, visiting laboratories and research cores, and meeting with potential collaborators. Scholars are supported by dedicated mentor teams from their home country and Vanderbilt-based mentors via meetings held several times each week during the course. With guidance from their mentors, scholars develop a grant proposal in stages by completing weekly writing assignments. The course concludes with a mock NIH-style grant review of the proposals by a committee comprised of Vanderbilt faculty, VIRDE alumni, VIRDE scholars, and invited experts. Scholars leave the program with a strong draft of a grant proposal and a submission plan. Upon returning to their home country, scholars continue to receive support from their mentoring team and administrative technical assistance from Vanderbilt staff.n = 58). REDCap, a secure online platform for survey development and database management [To assess the impact that the VIRDE program has had on the research careers of program alumni over the past 10 years, the authors developed an online program evaluation that was sent to all VIRDE alumni .n = 48), 68.8% were male and 31.3% were female , Nigeria (20.8%), Mozambique (16.7%), or Ghana (12.5%). Other countries included Brazil, Bangladesh, China, Kenya, Pakistan, and Tanzania. All but one respondent has remained in their home country since completion of VIRDE.agreed” or “strongly agreed” with the statement “VIRDE contributed to my career advancement.”Most respondents (77.1%) reported being employed in academia. Smaller percentages reported working in government institutions (20.8%) or non-governmental organizations (14.6%). Almost all respondents (92.0%) reported currently working at the same type of organization as they did during VIRDE, with many (88.0%) having advanced in their career stage since their participation in VIRDE. All respondents except one “All respondents reported they currently work in some research capacity, although the hours devoted to research differed. The majority of respondents (62.5%) reported spending approximately 30 to 59% of their professional work time on research. Smaller proportions of respondents reported spending greater than 60% or less than 30% of their time on research (27.1 and 10.4% respectively).a personal desire to do research” (70.2%). Other factors selected by at least half of respondents included “a positive research culture at their institution” (64.6%) and “mentorship” (50%).Respondents were asked to select factors that enabled them to pursue research. The most common selected factor was “Mentoring during VIRDE” and “VIRDE lectures” were the most selected responses . “Personal motivation” (56.5%), “protected work time during VIRDE” (45.7%), “interactions with VIRDE peers” (39.1%), and “network connections developed during VIRDE” (32.6%) were also listed as enablers of their success.Respondents were requested to select factors that had the greatest impact on their ability to successfully develop research grants while participating in VIRDE. “p < 0.001). The average grant knowledge score pre-VIRDE was 3.0 and post-VIRDE was 4.4, an increase of 44%. The largest knowledge gains were in “reviewing a grant” (77.5% increase), “creating a biosketch or CV” (52.1%), and “writing the resources and environment section” (50.4%).Respondents reported on a scale of 1 (strongly disagree) to 5 (strongly agree) their grant writing knowledge across ten competencies pre- and post-VIRDE . Paired sample t-test for composite comparison of pre/post test scores: p < 0.001). The average research ethics score pre-VIRDE was 2.4 and post-VIRDE was 3.2, a 33% increase. In the pre-VIRDE section, there were 78 “uninformed” responses, compared to just 4 in the post-VIRDE section. The biggest changes were in “US policies and regulations on research ethics” (55.3% increase), “responsibilities of mentors and students in research” (44.8%), and “Good Clinical Practice (GCP) Standards” (41.8%). Additionally, 86.7% of participants indicated that VIRDE impacted their knowledge of these ethics topics “a lot.”Respondents also reported their pre- and post-VIRDE knowledge about research ethics across 18 competencies on a scale of 1 (uninformed) to 4 (expert) Fig. . The resp < 0.001.Respondents ranked their level of knowledge on 18 research ethics competencies using a four-point Likert-scale . Paired sample t-test for composite comparison of pre/post test scores: Respondents also reported increases in mentoring and teaching of others in their home institutions about grant writing. Only two scholars (4.3%) reported teaching courses about grant writing prior to VIRDE, and that number increased to 12 (25.5%) after VIRDE. Similarly, prior to VIRDE, eight respondents (17.0%) mentored others in grant writing, which increased to 31 (67.4%) post-VIRDE.Before VIRDE, 36 respondents (76.6%) reported collaborating on research at organizational, regional, and/or international levels. After VIRDE, 46 (97.9%) respondents reported collaborating on at least one of these levels. The rate of collaboration increased by 27.3% at the organizational level, 40.0% at the regional level, and 44.8% at the international level.Of the 47 respondents, only 15 (31.9%) had ever submitted a grant proposal for funding prior to their participation in VIRDE Table . Pre-VIRWe analyzed the types of funding agencies that supported the awarded grants. United States Government (USG) agencies supported around 40% of grants both before and after VIRDE. However, post-VIRDE there was increased diversity in other sources of funding, such as foundations and pharmaceutical companies, and about 18% came from national organizations, which was an increase over pre-VIRDE. Almost two-thirds (63.6%) of the respondents’ first awarded grant post-VIRDE was a one-to-two-year grant.We evaluated demographic differences between respondents who did submit 39, 83.0%) and those that had not yet submitted  grants after VIRDE  held leadership roles at their organizations. Among those working in academia, 13 (35.1%) respondents serve or served as a Department Chair, Dean, Deputy Dean of Research, or in another similar administrative leadership capacity. No respondents held solely a national or international leadership position, but nine (19%) held leadership roles at organizational, national, and international levels. More individuals reported that their research outputs impacted policy changes post-VIRDE  than pre-VIRDE .results from our multi-center stroke prevention trial will change the narrative on and practice of stroke prevention in children with sickle cell disease in resource-constrained regions.”Post-VIRDE, 20 respondents reported that their research contributed to policy change. In open-ended responses, they described changes made at three levels: clinic management and hospital; local and national; and regional and international. In clinic management and at the hospital, respondents helped develop new guidelines and protocols for the clinical management and treatment of diseases across a variety of medical specialties. At local and national levels, their research helped influence the adoption of new standards of care for sickle cell disease, change HIV treatment guidelines, advance a testing platform for syphilis during pregnancy, and provide free hydroxyurea for stroke prevention. Further, respondents helped improve tobacco control policies and establish a national non-communicable disease research database. Internationally, one respondent’s research helped change the World Health Organization (WHO) guidelines on the medical eligibility criteria for contraceptive use. Another respondent is helping to establish a digital surveillance platform for anti-microbial resistance in sub-Saharan Africa, while another wrote that “n = 43) were centralized around the skills and knowledge respondents gained that influenced their ability to write fundable grants. As one respondent explained, these improved research outputs “enhanced my career and promotion prospects” and other respondents were able to “advance from lecturer to senior lecturer.” These sentiments were referenced in similar ways by 13 respondents (30.2%). Seventeen respondents (39.5%) wrote about their success in receiving grants and 9 (20.9%) wrote about increased knowledge and skills to apply for grants and receive research funding. Through VIRDE, they “have learnt new approaches to searching different funding sources, (and the) technical issues of NIH grants.” Other respondents commented on awards they received to attend additional training programs and enrolment in doctoral programs because of their increased competencies from VIRDE.When asked about the impact that VIRDE had on their career advancement, open-ended responses  a grant mobilizer and community leader in research and health program delivery.”Some respondents wrote about increased motivation, perseverance, and focus, with descriptions like “Based on evaluation data, VIRDE scholars develop skills and knowledge in grant writing and research ethics during this month-long immersive training program. While at VIRDE, they grow their professional networks and return to their home institutions where many successfully write and submit grants. For some scholars, this grant-funded research has resulted in changes to health care practice and policies. Alumni mentor and teach grantsmanship to colleagues, sustaining and promoting a research culture in their institutions.Given the differences in research environments between LMICs and HICs, outcome measures commonly used in HICs, including number of publications in peer-reviewed journals, conference presentations, and funded grant applications, may not reflect accurately research capacity and productivity for scholars in LMICs. Cooke’s framework for research capacity strengthening provided an objective, non-traditional tool to assess the VIRDE program. Success of alumni and the program were broadly measured by scholars’ knowledge and skill enhancement, increased research collaborations, sustained engagement in research, and impact of research grants on health care practice and policies.Gains in grant writing and research ethics knowledge as well as an increase in mentoring and teaching of these skills suggest that VIRDE scholars built skills and confidence in grant writing during the program. Respondents reported their largest gains in grant writing knowledge were related to reviewing a grant proposal and in the writing of specific sections of a grant proposal. Lectures and assignments throughout the one-month practicum focused on honing the scholars’ skills in developing each section of a grant application, culminating in a mock NIH-style grant review. Throughout the process of developing a grant proposal, scholars received frequent guidance from their mentoring committee. Reported gains in knowledge along with high ranking of VIRDE mentorship and lectures affirm the utility of hands-on, mentored practice combined with instruction.We found a correlation between participation in VIRDE and increased research collaborations with international partner institutions. Partnerships are the foundation of the VIRDE program. VIRDE scholars are selected from institutions with existing collaborations with Vanderbilt University, as such it is not surprising that respondents reported being engaged in international collaborations. However, we found that post-VIRDE, the number of scholars engaged in international as well as institutional and national collaborations increased. The greatest increase in collaborations was at the international level, which could be attributed to VIRDE’s multidisciplinary and multi-country cohorts of scholars who work closely together during the month-long training, as well as scholars’ access to new mentors and exposure to research facilities at Vanderbilt during VIRDE.The VIRDE program was founded upon a vision of training continuity and research sustainability. Survey results highlight the ripple effects of knowledge transfer as VIRDE alumni increased their engagement in teaching and mentoring in grant writing at their institutions. Sharing knowledge about grant writing has a two-fold impact. First, colleagues, mentees, and trainees learn about and improve their skills in grant writing, thus widening the impact of the program without additional program resources. Second, through teaching and mentoring, VIRDE scholars solidify their knowledge and establish themselves as leaders in grant writing. VIRDE alumni receive all training resources, including course slides. Providing course materials and continued technical assistance to alumni equips them to develop their own grantsmanship trainings adapted to their setting, which has been accomplished by some alumni.Through increased grant funding, opportunities are created for alumni to both focus on and sustain their research careers. The data revealed that nearly three times more grants were submitted post-VIRDE, including 24 scholars who submitted their first grant proposal. In addition, post-VIRDE the diversity of funding organizations from which scholars applied to for grant funding increased, demonstrating new paths to sustainability and research independence for many individuals. Almost two-thirds of scholars who submitted grant proposals were awarded at least one grant and overall, half of submitted grants were funded. Respondents highlighted renewed focus, motivation, and perseverance in their pursuit of a research career as a result of participating in VIRDE. Such character growth can have a lasting positive impact on a scholar’s career trajectory. Incorporating mentoring and training to enhance these traits may contribute to scholars’ successes in grant writing, leadership, and research dissemination.A substantial proportion of respondents have advanced in their career stage, serve in leadership roles, and continue to work in academia in their country of origin, contributing to a low rate of “brain drain.” Brain drain is common in LMICs as trainees often leave their country for career opportunities abroad. However, our data suggests that VIRDE equips scholars with skills, confidence, and networks needed to create the opportunities that lead to sustaining a research career while remaining in their home countries. Applicants are vetted first by an in-country team comprised of principal investigators who have existing partnerships with Vanderbilt before applying to VIRDE. This in-country process likely increases the probability of supported scholars who are connected to local networks to remain in academic research. Local collaboration for training is helpful to create sustainable, supportive research environments for scholars post-training.Applicants are vetted for their ability to generate innovative research that is relevant to their setting. While traditional research dissemination metrics focus on publications in academic journals, we focus on the ability to sustain a research career through grant funding and for research to improve practice and health outcomes. Through evidence generated in their grant funded research, scholars reported changing practice, implementing policies, and impacting patients’ quality of life. Notably, scholars described that their research contributed to improving medical diagnostic and treatment protocols and other health-related policies in their regions, demonstrating the relevance and importance of their research. Scholars also contributed to international policy decision making groups, broadening the impact of their research to improve practice in other regions. These output metrics could be attributed to scholars’ increased grant productivity, collaborations, and confidence post-VIRDE, revealing the ability of this program to serve as a catalyst for scholars to develop research that is close to practice and responds to community and patient needs.We have highlighted elements beyond traditional metrics that indicate success of alumni and this program. These metrics, which include enhancing grant writing skills; expanding collaborations and roles as mentors, teachers, and leaders in grant writing; remaining in research; submitting grants; and impacting local and international practice, build upon Cooke’s framework and could be used by other programs to measure success. Key program elements that contributed to the program’s success include 1) a rigorous application and selection process led by LMIC partners requiring applicants to submit a draft grant proposal as part of the application process; 2) a curriculum based on applicable grant-writing skills taught by senior research faculty and staff combined with proposal development milestones; 3) committed in-country and U.S. based mentorship teams during and after VIRDE; 4) protected writing time away from competing work duties; and 5) an immersive cohort experience that provided participants with expanded professional networks.a personal desire to do research,” “a positive research culture at their institution,” and “mentorship.” Success as an independent investigator also requires intrinsic motivation, determination, and perseverance, especially in a LMIC setting where respondents reported systemic challenges.VIRDE is a one-month program and grant development and submission can take months to years. Hence, based on the evaluation results, participating in VIRDE could be considered as a catalyst for success in grant writing. We acknowledge that VIRDE may not be the sole reason for their successes. Therefore, we queried scholars on their perception of VIRDE’s impact on their career and research productivity, as well as other factors that contributed to their success outside of VIRDE. Among the factors that enabled them to pursue research, at least half of respondents selected “As we plan for the next 10 years, we are using this data to develop programming that continues to be relevant and effective. To date, only 31% of VIRDE participants were women. We recognize that VIRDE’s current format, requiring participants to spend one-month away from home, poses a challenge for gender equity among participants. Additionally, a one-month program in the U.S. can be costly and a prohibiting factor for researchers in LMICs. At present, VIRDE requires a funding source for participant scholarships as well as institutional support for program instructors and mentors. VIRDE scholars have predominantly been supported by NIH training grants, therefore limiting the number and breadth of participants. To address gender equity and cost, we are exploring incorporating online modules to decrease expense and duration of time in the U. S for those who cannot attend the entire month in-person. Further, for partner institutions which have a nucleus of VIRDE alumni and a more mature research portfolio, we are exploring transitioning some, or all, of the program elements to be taught in-country. We found that post-VIRDE, 25% of alumni taught grant-writing courses and 67% provided grant writing mentoring, demonstrating feasibility of expanding VIRDE through alumni trainers. With these cadres of research scholars trained in grant writing, VIRDE will continue to build individual grant writing skills among future participants while supporting alumni to develop and lead courses in the next phase of VIRDE. With networks of individual research scholars trained in grant writing, strengthening institutional research systems is the next step. We are working with alumni to develop programs at their institutions that strengthen indigenous grants management support .We noted limitations in our data collection and analysis. Data related to knowledge and behavior were self-reported. Therefore, we relied on an accurate representation from respondents about their level of knowledge, participation in activities, and grant submission and awards. We also asked respondents to recall their knowledge and activities pre-VIRDE and differentiate that information from post-VIRDE knowledge and behavior. Participant recall on retrospective data may be biased. Additionally, respondents may have provided biased responses knowing that the survey came from the program director with whom many alumni are still in contact and that responses may be identifiable due to the relatively small sample size. Respondents may have wished to present their knowledge, skills, and outputs in a positive light. However, we believe that knowing the survey’s origins may have increased their willingness to participate in the survey, thus increasing the response rate. The survey included over 200 answer fields and survey fatigue may have affected responses towards the end of the survey. Since not all alumni responded, the actual program impact could be different. In reflecting on the survey design, respondents were not asked about challenges and areas for improvement associated with participating in the program, writing grants, or their research career. Such questions would have provided critical feedback and additional insight to VIRDE and grant writing for these researchers. Finally, these results are applicable to VIRDE scholars and findings may not be generalizable to the larger population.Our program evaluation data indicate that the VIRDE program contributed to increases in scholars’ knowledge in grant writing and research ethics. With increased knowledge, skills, and confidence in those areas, teaching and mentoring activities increased. After the program, scholars also increased their collaborations at local, national, and international levels and held more leadership roles. Fulfilling the aims of the program, scholars’ rates of grant submissions and awards increased, reflecting scholars’ ability to grow and sustain their research career. Scholars’ funded research also influenced community health outcomes and national and international health policies. Over the past decade, VIRDE has strengthened the research grant writing capacities of nearly six dozen biomedical investigators from LMICs, and has impacted positively on trainees’ health structures and research systems on many levels. Our experience can be used to inform the development, implementation, and long-term evaluation of similar research capacity training programs in under-resourced settings.