The fast pace of urbanisation may benefit or be detrimental to the socio-economic status of urban areas. Understanding how the configuration of urban areas influences the socio-economic status of their inhabitants is of crucial importance for urban planning. In theory, urban scaling laws and polycentric development are two well-known concepts developed to increase our understanding of urbanisation and its socio-economic effects. In practice, however, they fall short to explain the socio-economic status of urban regions. The urban scaling concept is constructed from a theoretical perspective, but functional relationships between urban centres are not taken into account in scaling models. In contrast, the concept of polycentricity is developed from a practical perspective and incorporates the socio-economic effect of relationships between urban centres in the process of urban development. However, polycentricity lacks a theoretical foundation, which would explain the socio-economic status of urban regions. In this study, we assess whether combining both concepts improves the ability to explain personal incomes in metropolitan areas in Switzerland. We first delineated metropolitan areas by implementing a modularity maximisation algorithm on the settlement network. Nodes in this network are Swiss municipalities and links are inter-municipal commuter flows. We found a strong relationship between the hierarchical organisation of functional connections within metropolitan areas and the socio-economic status of these areas. Both concepts were complementary and combining them proved to enhance the ability to explain socio-economic status. The combined model is a theoretical progress, which complements the traditional approaches and increases our understanding of cities and urbanisation processes. The human population living in cities is predicted to increase from 50 per cent in 2008 to 75 per cent by 2030 . This faIn general, it is believed that urban development and configuration are driven by two processes , 17. On The theory of urban scaling poses that urban productivity increases with size  –24, whicIn contrast to urban scaling laws, functional relations between urban centres within a metropolitan area are explicitly considered in spatial planning . ParticuIn order to capture both size and connections between functional centres of an urban system, a settlement network paradigm has been suggested to study the socio-economic status of such systems . FunctioIn summary, both urban scaling law as well as the level of polycentricity have been linked to the socio-economic status of urban systems. This raises the question whether these two concepts are complementary to one another or whether they are actually alternative descriptions of a single underlying process. To answer this question, we assessed whether the deviations from urban scaling law (SAMIs) are correlated to our indicator of polycentricity (i.e. GRC) and whether socio-economic variables can be better explained by a combination of both concepts. The combined model aims to explain two important variables associated with the socio-economic status of urban areas, the mean and the median income. The mean income provides an overview of the overall economic status of a metropolitan area. However, the median income is considered a more robust indicator for well-being and other development processes and is favoured over mean income in social welfare studies . A compaIn this study, we focus on metropolitan areas in Switzerland. Our settlement network consists of 2944 Swiss municipalities as nodes, which are linked by commuter flows derived from the Swiss national passenger transport model (NPVM) . For eacTo analyse the effect of both size and functional connections on the socio-economic outcomes of metropolitan areas, we delineated metropolitan areas as the relevant socio-economic units of Switzerland. There are multiple methods to delineate urban or metropolitan areas –52 and tThe metropolitan areas spatially coincided with the official Swiss labour market regions , 60. TheNot only the community structure but also the topology of each settlement network within a community is influenced by the topography and the socio-economics of the communities. For instance, the settlement network surrounding Sion in the canton of Valais  is diffe0 is the normalisation constant, N(σ) is a region’s population size, and β is the scaling coefficient. Applying a logarithmic transform, According to the conventional urban scaling law, the socio-economic status of metropolitan areas is related to their population size with the equation:In order to relate population size in the metropolitan areas to mean per capita income, we performed a regression analysis between log(Income) and log(Population) revealing a significant correlation  of metropolitan region σ, and Y0, N(σ) and β are the same as in A second product of the scaling analysis is the construction of scale-adjusted indicators of metropolitan performance. According to recent studies , 26, botCR) of the municipalities within a metropolitan area. CR(i) is equal to 1 if the ith municipality can be reached directly from all other municipalities within a metropolitan area . If a municipality in the settlement network can be reached only via other municipalities, then it would be less central and CR(i) < 1  is used to measure the heterogeneity of the local reaching centralities  explain mean income in Swiss metropolitan areas. The multivariate analysis consists of three steps: First, we assessed the relationship between GRC and personal mean income . Second,2 = 0.28; 2 = -0.06). Given that both metropolitan size as well as metropolitan hierarchy were significantly correlated to mean income  and both explanatory variables were significant  for different modularity scores . Our analysis shows that there is a positive and highly significant correlation between adjusted R-squared of the combined model and the modularity score of the delineated metropolitan areas . This suIn this study, we found strong effects of settlement patterns on the socio-economic status of metropolitan areas. These effects have important consequences for the planning and monitoring of settlement developments. Two major theories for studying settlement patterns are the urban scaling law , 25 and We found that both the population size as well as the hierarchical connection pattern of centres in metropolitan areas well explained the variation in mean income in metropolitan areas in Switzerland Figs  and 4a. Hierarchy is a fundamental characteristic of complex systems  –71 and iIn our study, the hierarchy was correlated to SAMIs , which hThe network of urban flows, such as commuter flows, proved to be useful in revealing the dynamic spatial structures of metropolitan areas, which was also found by Zhong et al. . Shen anAlthough a settlement network of commuter flows was used in our study, different types of metropolitan flows  could also be considered to measure the hierarchical organisation of metropolitan areas. A drawback to using flows is that there can be strong boundary effects caused by flows outside the study area. For instance, we chose to perform our study in Switzerland, but there are many trans-border commutes. Geneva, for example, is highly connected to France. Although disregarding these trans-border commutes potentially biased our results, we did not consider international commutes, mainly due to a lack of comparable data. Although other studies found that the detectability of scaling relationships was sensitive to the definition of boundaries , we haveThe two input variables  in our extended model can be compared to economic resources for productivity of a region . From thFor future generations, the socio-economic consequences of the ongoing global urbanisation largely depend on current urban planning decisions. Choosing between centralisation and decentralisation is a big dilemma in planning. The bottom-up process of urban development may result in a more monocentric and more hierarchical urban structure, because people tend to be drawn to larger cities for higher individual socio-economic gains . That isOur case study area is Switzerland, which consists of three major regions characterised by different types of landscapes. The densely populated Swiss Plateau is fringed in the north by the Jura Mountains and in the south by the Alps  for details ). This tThe Swiss landscape is not only differentiated by landscape typology but also by language regions with their specific cultural and ecological heritage. Four different languages are spoken in Switzerland. In the eastern part of Switzerland mainly German is spoken, while in the western part the main language is French. Italian and Romansh are spoken in the Canton Ticino, south of the Alps, and in the canton of Grisons in the south-east, respectively. The socio-economic status of these regions may be affected by regional specific factors, which can also affect the functional relationships within and between these areas.In our study, we constructed a network for the case-study area. The nodes in this network were the zones in the Swiss National Model for Passenger Transport (NPVM), corresponding to the municipalities (except in the large cities which are divided in multiple zones ). The weWe used income data for municipalities published by the Swiss Federal Tax Administration. In the few cases where municipalities were divided into several NPVM zones (i.e. in the larger cities), we assigned the same income data from the municipality to each of the NPVM sub-zones.Aij is the weight of the link between nodes i and j (number of commuters between nodes i and j), ki is the sum of all the links to node i, σi is the community in which node i is located, the function δ is equal to 1 if both nodes i and j are located in the same community and otherwise it is equal to 0, and finally We used the modularity maximisation method   to detecThe algorithm uses a greedy optimisation procedure to increase the modularity value by changing the partition structure in an iterative process. Each detected community was considered a metropolitan area. The income data for the detected metropolitan areas were calculated by aggregating the income data of individual zones.To quantify the hierarchy within a metropolitan area, we used the Global Reaching Centrality (GRC), developed by E. Mones . This meCR(i), is a normalised centrality measure of node i within a metropolitan area . CR(i) is calculated as follows:d is the number of steps through the network to reach node j from node i. If all the nodes in a network are reachable from node i, then CR(i) is 1, which is the maximum possible value for local reaching centrality and if the node i is not directly reachable from other nodes, CR(i) decreases.In order to calculate GRC, we first calculated the local reaching centralities of municipalities within a metropolitan area. Local reaching centrality, Vσ is the set of all nodes within σ [The heterogeneity of local reaching centralities in a network is measured by GRC, which is calculated as follows:within σ .Making use of simple hypothetical networks, we showed that a settlement network with one central node (i.e. monocentric) had a high hierarchy level, while a network with multiple central nodes (i.e. polycentric) had a lower hierarchy measure .In a monocentric network , where aMean incomei is the mean income in municipality i, Populationi is the population in municipality i, and Populationσ is the total population in metropolitan area σ.We used the following equation to calculate the mean income of the metropolitan areas:The median income in each metropolitan area could not simply be derived by aggregating the mean or median incomes from municipalities. Therefore, had to first reconstruct the personal incomes in each municipality. We assumed that the income distribution follows a log-normal distribution function as this is the case in many regions , 95. We μi and σi) for each municipality as follows:Median incomei is the median income of municipality i.Since we have the median and mean of the income data for each municipality we were able to calculate the pdf parameters  is the total personal income of metropolitan area σ and σ (N(σ)), which results in the following equation:Y(σ) is the per-capita mean income, Y0 is the new normalisation constant and N(σ) is the population size of urban area σ. The new scaling coefficient for the per-capita characteristic is β − 1, where β is the same coefficient as in In this study, we compared how well the mean income per metropolitan area could be explained by the area’s population size and the GRC. The relationship between population size and income is given with the urban scaling law:Using a logarithmic transformation, the equation takes a linear form as:μσ is the SAMI for metropolitan area σ. SAMIs can be calculated by re-writing Deviations of real per-capita values of a certain characteristic and those predicted by the scaling law  are refeWe calculated the SAMIs for all the metropolitan regions and related them to the mean personal income with linear regression.Y(σ)) was a function of two variables: the logarithm of the population and the hierarchical organisation of the network. This combined model can be formulated as:α is the coefficient of GRC in the model.In our final combined model, log. BIC is suggested as a model comparison criterion in studying scaling relationships [Bayesian information criterion (BIC) is an indicator to compare the quality of different models with one another –98. BIC ionships .P(data|m) is the probability of observing the data given the model. B12 can be estimated using the following equation:In order to compare two models, we calculated the Bayes factor as suggested in  and as uBIC = BIC1 − BIC2 = 6 implies that the model m = 2 is almost 20 times more likely to be the right model than m = 1.Based on the Kass and Raftery  suggest S1 Fig(DOCX)Click here for additional data file.S1 File(PY)Click here for additional data file.


NORAD is a conserved long noncoding RNA (lncRNA) that is required for genome stability in mammals. NORAD acts as a negative regulator of PUMILIO (PUM) proteins in the cytoplasm, and we previously showed that loss of NORAD or PUM hyperactivity results in genome instability and premature aging in mice . Recently, however, it was reported that NORAD regulates genome stability through an interaction with the RNA binding protein RBMX in the nucleus. Here, we addressed the contributions of NORAD:PUM and NORAD:RBMX interactions to genome maintenance by this lncRNA in human cells. Extensive RNA FISH and fractionation experiments established that NORAD localizes predominantly to the cytoplasm with or without DNA damage. Moreover, genetic rescue experiments demonstrated that PUM binding is required for maintenance of genomic stability by NORAD whereas binding of RBMX is dispensable for this function. These data provide an important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance. Noncoding RNA activated by DNA damage (NORAD) is particularly noteworthy, due to its unusually abundant expression in mammalian cells and tissues and strong evolutionary conservation across mammalian species. Initial studies of NORAD revealed that this lncRNA is required to maintain genomic stability in mammalian cells (NORAD to bind to and negatively regulate PUMILIO RNA binding proteins (PUM1 and PUM2) in the cytoplasm  have emerged as regulators of diverse biological processes. Among these transcripts, an cells , and proytoplasm . PUM proytoplasm . Notably tissues . Accordiand mice , while Put cells .NORAD was proposed  complex in the nucleus that includes Topoisomerase I (TOP1) and other proteins that are critical for genome maintenance. Importantly, PUM and RBMX interact with different sites on NORAD and function in distinct subcellular compartments. Thus, while it remains to be determined whether the NORAD:RBMX interaction is necessary for regulation of genomic stability, both PUM and RBMX may play important, non-mutually exclusive roles in the genome maintenance functions of NORAD.Recently, an alternative mechanism for the regulation of genomic stability by proposed . Proteomresponse . SubsequNORAD functions to maintain genome stability in human cells and directly tested the requirement for PUM and RBMX interactions in this activity. RNA fluorescent in situ hybridization (FISH) using a panel of probes spanning the entire length of NORAD, as well as cellular fractionation studies, definitively demonstrated that this lncRNA localizes predominantly to the cytoplasm and does not detectably redistribute to the nucleus upon induction of DNA damage. Genetic rescue experiments in NORAD knockdown cells established that PUM binding is essential for maintenance of genomic stability whereas interaction with RBMX is completely dispensable for this function. Further experiments demonstrated that RBMX is not required for induction of NORAD following DNA damage nor its cytoplasmic localization. Together, these studies establish the importance of the NORAD:PUM axis in regulating genomic stability in mammalian cells and provide a foundation for further dissection of the mechanism and physiologic role of this pathway.Here, we further examined the mechanism by which NORAD reported a predominantly cytoplasmic localization of this lncRNA in human cell lines, based on RNA FISH using pools of fluorescently-labeled oligonucleotide probes and subcellular fractionation experiments  was used in both the previous , gave rise to a nonspecific nuclear signal that was present in both NORAD+/+ and –/–NORAD HCT116 cells. The remaining 10 probes produced a highly consistent, predominantly cytoplasmic, punctate localization pattern in wild-type cells that was absent in –/–NORAD cells  experiments performed in HCT116 cells that identified the NORAD:RBMX interaction . Isoforms of PUM1 and PUM2, along with RBMX, were indeed identified as significantly-enriched interacting partners of NORAD compared to RMRP (NORAD-interacting partners.Previous crosslinking-immunoprecipitation coupled with high throughput sequencing (CLIP-seq) studies demonstrated that an cells  and mousan cells . In ligheraction . Since t to RMRP . Notably to RMRP . These rNORAD, with PUM2 overexpression phenocopying, and PUM1/2 knockdown suppressing, the effects of NORAD deletion , which represents the most conserved of five repeated segments within this lncRNA termed NORAD domains (NORAD (nt 33–898), which also harbors one PRE, was also generated (5′ fragment).Genetic epistasis experiments have strongly implicated a role for PUM1/2 in the regulation of genomic stability by deletion . Neverthng sites . For eac binding , was mut deleted   with a single-guide RNA (sgRNA) targeting the endogenous NORAD promoter. As expected, in cells infected with a non-targeting control sgRNA (sgControl), increased total levels of NORAD were observed upon expression of NORAD rescue constructs in trans   . Upon siNORAD transcripts to endogenous PUM1, PUM2, and RBMX. Pull-downs of each of these proteins resulted in the expected enrichment of wild-type NORAD, but not GAPDH, relative to immunoprecipitation with control IgG  to assess binding of wild-type and mutant trol IgG . The PREonstruct .NORAD constructs by quantifying the number of aneuploid cells in each population using DNA FISH for marker chromosomes 7 and 20, as described previously (NORAD rescue populations after infection with non-target sgRNA (sgControl), indicating that overexpression of the NORAD constructs in trans did not trigger genomic instability. Furthermore, as expected, knockdown of endogenous NORAD in GFP-control cells resulted in a significant accumulation of aneuploid cells. The frequency of aneuploidy observed under these conditions was very similar to that observed previously in –/–NORAD HCT116 cells  abolished NORAD function in this assay. Finally, we found that the ND4 segment containing several highly conserved PUM binding sites, but not the 5′ fragment encompassing the RBMX binding site, exhibited significant rescue activity in this assay. Overall, these data provide compelling evidence that PUM, but not RBMX, binding to NORAD is necessary for the maintenance of genomic stability by this lncRNA.To further assess the role of RBMX and PUM binding in genome maintenance by c errors  and the wn cells . Examinanockdown . ConsistNORAD, we were able to confirm binding of this protein to the 5′ end of NORAD, as reported  were cultured in McCoy’s 5a media (Thermo Fisher Scientific) supplemented with 10% FBS  and 1X AA (Gibco). The cell line was authenticated by ATCC using short tandem repeat (STR) analysis in November 2017. All cell lines were confirmed to be free of mycoplasma contamination. HCT116 eviously .5 HEK293T cells per well in a six-well plate. The following day, cells were transfected with 1.4 μg of pHR-SFFV-dCas9-BFP-KRAB (Addgene plasmid #46911), 0.84 μg of psPAX2 (Addgene plasmid #12260), 0.56 μg of pMD2.G (Addgene plasmid #12259), 8.4 μl of FuGENE HD (Promega), and 165 μl Opti-MEM (Thermo Fisher) according to the manufacturer’s instructions. Medium was changed the next day. Two days after transfection, medium was collected and passed through a 0.45 μm SFCA sterile filter. Recipient HCT116 cells were transduced overnight using medium supplemented with 8 μg/ml polybrene (EMD Millipore). Cells expressing BFP were enriched by FACS and single-cell clonal lines were derived.To generate the HCT116 CRISPRi cell line, lentivirus expressing dCas9/BFP/KRAB was produced by first seeding 6 × 10NORAD were synthesized by in vitro transcription using a DIG-labeling mix (Roche). Primers used for amplification of the DNA template for each probe are provided in 5 cells were grown on poly-L-lysine coated coverslips for 24 to 36 hr. For RNA FISH experiments with DNA damage treatment, cells were grown for 24 hr and treated with either 1 μM doxorubicin or 200 nM camptothecin for an additional 12 hr. Samples were rinsed twice in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min, washed again in PBS, and permeabilized in 0.5% Triton X-100 for 10 min. Samples were then washed twice with PBS and rinsed with DEPC-treated water prior to incubation in prehybridization buffer  for 1 hr. 10 ng/μl DIG-labeled RNA probe was diluted in hybridization buffer (prehybridization buffer with 5% dextran sulfate) and used for hybridization at 55°C for 16 to 20 hr. Following hybridization, samples were washed, treated with RNase A, and blocked using Blocking Reagent (Roche). DIG-labeled probes were detected using mouse monoclonal anti-DIG primary antibody  and a Cy3-labeled goat anti-mouse IgG secondary antibody . Immunofluorescence and western blot analysis of the DNA damage marker γ-H2AX was performed using anti-γ-H2AX (Ser139) antibody . Samples were mounted using SlowFade Diamond Antifade with DAPI mounting media (Invitrogen) and imaging was performed using a Zeiss LSM700 confocal microscope. ImageJ was used for further image analysis.RNA FISH was performed as described previously  with the6 cells were collected for subcellular fractionation, which was performed as previously described  on ice for 5 min and centrifuged at 500g × 2 min. The supernatant containing the cytoplasmic fraction was separated from the pelleted nuclear fraction. RNA was then isolated from both fractions using the Qiagen RNeasy kit and equal cell equivalents of nuclear and cytoplasmic RNA were used in subsequent qRT-PCR reactions. All samples were tested for NORAD as well as NEAT1 (nuclear control) and GAPDH (cytoplasmic control). The sum of the nuclear and cytoplasmic expression level of each transcript was set to 100%, and the percentage of each transcript localized to each compartment was determined. NEAT1 and GAPDH, respectively, showed the expected nuclear and cytoplasmic localization in each experiment, confirming successful fractionation.Cells were seeded in triplicate and 1 × 10escribed . BrieflyNORAD and RMRP RAP-MS experiments (https://massive.ucsd.edu) using the identifier: MSV000082561. Peptide identification and quantification was performed using Proteome Discoverer (Thermo Fisher) with three search engines combined . MS/MS spectra were searched against the human Uniprot database. Search parameters included: trypsin enzyme specificity with a maximum of 2 missed cleavages tolerated, False Discovery Rate (FDR) set to 0.01 at both peptide and protein level, ±10 ppm for precursor mass tolerance with a shorter window for fragment mass tolerance for the first search, and carbamidomethylation of cysteine modification and iTRAQ labels on N-termini and lysine residues as fixed modifications and oxidation of methionine and N-termini acetylation as variable modifications. All peptide and protein identifications had scores surpassing the combined search engine significance threshold for identification. Protein abundance was calculated as the intensity given from precursor quantification and was then normalized to the total peptide amount. To correct for total abundance differences between samples, protein and peptide abundance values in each sample were corrected by a constant factor such that the end total abundance was equivalent across all samples. Fold change was calculated as the log2 difference of average scaled protein abundance in NORAD samples and RMRP sample. For statistical analysis, we used the limma package (https://www.r-project.org/) to calculate the adjusted p-value using a moderated t-test and Benjamini Hochberg method to control the FDR.The raw mass spectra files from iTRAQ-labeled eriments  were dow package  in R (htRNA was isolated from cells using the RNeasy Mini Kit (Qiagen), or, for RIP experiments, Trizol (Invitrogen), and treated with RNase-free DNase (Qiagen). RNA was reverse transcribed with PrimeScript RT-PCR mix (Clonetech), and Power SYBR Green PCR Master Mix (Applied Biosystems) was used for qPCR. Biological replicates represent independently grown and processed cells. Technical replicates represent multiple measurements of the same biological sample. Primer sequences are provided in NORAD was amplified from a modified pcDNA3.1 vector containing the NORAD cDNA (NORAD polyadenylation site. The PRE-mutant (PREmut) construct containing 18 PRE mutations (TGT to ACA) was synthesized by GENEWIZ. The 5′ deletion construct (∆1–898), 5′ fragment (nt 33–898), and ND4 were amplified from the full-length NORAD construct using primers provided in AAVS1/PPP1R12C targeting vector  modified by replacing the puromycin resistance gene with a hygromycin resistance gene and digested with KpnI and MfeI to remove the GFP cassette. These vectors, as well as a control GFP vector, were then inserted into the AAVS1 locus of HCT116 CRISPRi cells using a previously described TALEN pair targeting the AAVS1/PPP1R12C locus . sgRNA sequences are provided in AAVS1/NORAD construct insertion and sgRNA expression, cells were grown in 1 μg/ml puromycin and 500 μg/ml hygromycin.Single guide RNAs (sgRNAs) targeting a sequence upstream of the endogenous AAVS1/NORAD constructs and depleted of endogenous NORAD with CRISPRi as described above. 20 × 106 cells were washed in cold PBS and UV crosslinked on ice in a Spectrolinker XL-1500 (Spectronics) at 254 nm (400 mJ/cm2). Cells were then scraped, centrifuged, snap-frozen in liquid nitrogen, and stored at −80°C. RIP was performed following a modified eCLIP protocol . Beads were then resuspended in 100 μL Wash Buffer #2, and 70 μL was used for RNA extraction and the remainder for western blotting. Proteins were extracted by incubation in Laemmli buffer for 10 min at 70°C. Antibodies used for western blotting were anti-PUM1 , anti-PUM2 , and anti-RBMX .PUM1, PUM2, and RBMX RIP experiments were performed in HCT116 CRISPRi cells stably expressing protocol  as folloNORAD construct rescue experiments was assessed 18 to 21 days after knockdown of endogenous NORAD. DNA FISH was performed as described previously . Slides were analyzed using an AxioObserver Z1 microscope. For each sample, 100 anaphase nuclei were imaged and assessed for the presence of chromosome segregation defects. Samples were counted in an experimenter-blinded manner. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.NORAD" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Igor Ulitsky (Reviewer #1); Philip Grote (Reviewer #2).Thank you for submitting your article "PUMILIO, but not RBMX, binding is required for regulation of genomic stability by noncoding RNA The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.NORAD, a mouse loss-of-function model of which was previously published in eLife. In the new manuscript, the authors focus on human cells, where they can reliably test the effects of loss-of-function of NORAD an genome stability by measuring fraction of HCT116 cells that become aneuploid, following loss of NORAD combined with expression of different rescue constructs. In this setting, the authors test different sequence variants and measure the fraction or aneuploid cells. This system allows the authors to compare the relative contribution of two groups of regions within NORAD – the ~20 PUMILIO binding sites, and the region that binds RBMX. RBMX was recently shown to bind NORAD by Munschauer et al., which showed that this binding is important for proper replication fork progression, and it was suggested in that paper that this binding is important overall for the roles of NORAD in genome stability. The new results convincingly show that PUMILIO binding is important for the aneuploid cell% phenotype . The authors also convincingly show that NORAD is mostly cytoplasmic in unstressed and in DNA-damaged cells, and that in contrast to the previous analysis by Muschauer et al., and consistently with all previous RIP and CLIP data, NORAD binds PUM1 and PUM2 PUMILIO proteins.In the manuscript, Mendell and colleagues describe an advance in their study of NORAD function and modes of action is of high interest, and the relative importance of the binding to PUMILIO in the cytoplasm and to RBMX in the nucleus for genome stability, as well as localization of NORAD, are a matter of controversy following the conflicting papers. The experiments are generally performed to a high standard and analyzed appropriately. The comments below mainly refer to the way the results are presented in context of prior work from the authors and from Munschauer et al., and are intended to provide a better distinction within the manuscript between the new results and discussion about the results of others.The topic of Essential revisions:NORAD has a dual function: as a modulator of PUMILIO factors and as an "assembler" of the NARC1 complex. For a more definitive result, the same phenotypic assays as described in the Munschauer paper would be required. Ideally this should be done, but if the same assay and same readout are not possible then statements (in the Title and Abstract and elsewhere) regarding RBMX binding not being important for genome stability would need to be toned down.The readout of genome integrity described by Munschauer et al. (% chromosome segregation defects) is not identical to the one described in this submission  and so formally cannot invalidate Munschauer et al.'s conclusions. Please comment on the possibility that, even with all the new results, Additional comments:eLife was in mouse, but should also clarify that the new results are mostly in human cells.• The Abstract mentions that much of the prior work published in • Abstract. What is meant by the data providing "a foundation for further investigation of this pathway"? If not specific then this statement should be removed.• Introduction section: The first sentence on lncRNAs is too generic and is not true for the majority of lncRNAs – remove/edit.NORAD … capacity to bind a large fraction of PUM1/2 within the cell". Is there evidence that more than a few percent of PUM1/2 molecules per cell are bound at any time? Justify. What value is this large fraction?• "• "definitively demonstrated that this lncRNA localizes predominantly to the cytoplasm and does not traffic to the nucleus upon induction of DNA damage". The authors do not exclude that some nuclear relocalization does happen. So this sentence  should be toned down.• Results section paragraph three: The authors should show that these conditions indeed lead to DNA damage.NORAD RNPs in the nucleus and in the cytoplasm).• Results section: "We conclude that the disparate localization pattern observed using a commercially available RNA FISH probe set  most likely represented a non-specific signal." Speculation, such as this, has a better fit to the Discussion section and here other possibilities should be discussed as well  before CRISPRi? Are NORAD levels roughly double? This should be mentioned, as these transiently increased levels may affect the phenotype in cells after CRISPRi.• In the same section: What happens to NORAD expression or localization”: Speculation regarding how RBMX affects NORAD levels also should be placed in the Discussion.• Subsection “RBMX is not required for NORAD:RBMX study finds PUM only ranking 185th in the RAP-IP experiment and below the significant threshold? Why does applying the strategy with three search engines make this difference?• Can the authors please elaborate why the NORAD in Figure 2C at 12h is significant.• There are no biological replicates in Figure 2D; why has Figure 2C biological replicates and not Figure 2D? Also, please provide statistics if the burst of • Please provide for the 2 independent clones in Figure 4C the analysis as in Figure 3B; does the degree of overexpression differ between the clones?• Results paragraph one; perhaps "Importantly, a single cell line (human colon cancer cell line HCT116) was used in both of these studies"?NORAD is a predominantly cytoplasmic RNA in HCT116 cells"• Results paragraph four – insert "predominantly": "NORAD interactome”: Please confirm whether the RAP-MS experiments used HCT116 cells also.• Subsection “PUM1, PUM2, and RBMX are components of the NORAD is necessary for genome stability” paragraph three: NORAD "interaction with RBMX was abolished". Is this correct?• Subsection “Binding of PUMILIO, but not RBMX, to • Figure 4C Explain horizontal dotted line and add significance estimates for the different comparisons.• Figure 4—figure supplement 1. Provide full legend that explains why there are 2 bands in the anti-PUM1 WB and try to improve on the anti-PUM2 WB.NORAD constructs”: typo "and ND4 and were amplified"• Subsection “Generation and AAVS1 knock-in of  Essential revisions:The readout of genome integrity described by Munschauer et al. (% chromosome segregation defects) is not identical to the one described in this submission  and so formally cannot invalidate Munschauer et al.'s conclusions. Please comment on the possibility that, even with all the new results, NORAD has a dual function: as a modulator of PUMILIO factors and as an "assembler" of the NARC1 complex. For a more definitive result, the same phenotypic assays as described in the Munschauer paper would be required. Ideally this should be done, but if the same assay and same readout are not possible then statements (in the Title and Abstract and elsewhere) regarding RBMX binding not being important for genome stability would need to be toned down.NORAD. These new data have been added to Figure 5 of the revised manuscript. Similar to what we reported previously , and later confirmed by Munschauer et al., NORAD knockdown resulted in an increase in the percentage of anaphase cells that display defective chromosome segregation. Expression of full-length NORAD, or NORAD lacking the RBMX binding site, rescued this phenotype whereas mutation of the PUMILIO binding sites fully abrogated NORAD function in this assay.We have now performed the identical assay to that performed by Munschauer et al. (quantification of the frequency of chromosome segregation defects), which further demonstrates that RBMX binding is not essential for maintenance of genomic stability by NORAD.It is worth noting that we initially chose the DNA FISH assay for the present study because it is a more sensitive readout of genome instability. DNA FISH reports the cumulative effect of chromosome segregation defects over many cell divisions whereas the analysis of mitoses provides a more limited snapshot of the defects present at a single time-point. Nevertheless, we appreciate the reviewers’ suggestion to perform both assays, which have yielded essentially identical results, thereby providing an even more definitive demonstration that PUMILIO binding is essential, whereas RBMX binding is dispensable, for genome maintenance by NORAD has a dual function: as a modulator of PUMILIO factors and as an "assembler" of the NARC1 complex”. First, our data establish that assembly of the NARC1 complex is neither necessary nor sufficient for the genome maintenance function of NORAD. Since deletion of the RBMX binding site does not impair NORAD function, assembly of NARC1 is not necessary for genome stability. Moreover, the PUMILIO binding site mutant, which retains the ability to interact with RBMX, behaves as a null in genome stability assays, thus demonstrating that assembly of NARC1 is not sufficient for genome maintenance. In light of these observations, the proposed ability of NORAD to assemble the NARC1 complex is not relevant to NORAD’s genome maintenance function and therefore we chose not to investigate it deeply in this study. Nevertheless, we have performed a limited number of experiments to examine whether NORAD is indeed necessary for the assembly of NARC1, as determined by assessing the interaction of RBMX with TOP1 . As shown below, in co-immunoprecipitation assays in NORAD wild-type and knockout cells performed using epitope-tagged RBMX , we detect no difference in RBMX:TOP1 interaction in the absence of NORAD. We would prefer not to add these data to our manuscript, however, because this experiment is tangential to our main conclusion, which is that NARC1, whether or not it forms, has no role in the genome maintenance function of NORAD. Clearly, substantial additional work would be necessary to establish the conditions, if any, under which NORAD stimulates assembly of this complex and its functional relevance.The reviewers have also requested that we comment on the possibility that “Additional comments:• The Abstract mentions that much of the prior work published in eLife was in mouse, but should also clarify that the new results are mostly in human cells.We have edited the Abstract to indicate that the present study utilized human cells.• Abstract. What is meant by the data providing "a foundation for further investigation of this pathway"? If not specific then this statement should be removed.NORAD, the data reported in this study provide an “important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance”, as it is now phrased in the Abstract.By clarifying the mechanism of action of • Introduction section: The first sentence on lncRNAs is too generic and is not true for the majority of lncRNAs – remove/edit.bona fide regulators.The first sentence of the Introduction has been edited to: “Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes.” This no longer infers that this is true for the majority of lncRNAs, simply that some act as • "NORAD … capacity to bind a large fraction of PUM1/2 within the cell". Is there evidence that more than a few percent of PUM1/2 molecules per cell are bound at any time? Justify. What value is this large fraction?NORAD copy number per cell, the number of PUM binding sites per NORAD molecule, and the number of PUM1/PUM2 proteins per cell , it is accurate to state that NORAD “has the capacity to bind a large fraction of PUM1/2 within the cell.” As described in that previous publication, there are sufficient PUM binding sites provided by NORAD to bind up to 100% of the cellular PUM1/PUM2 pool. However, we agree that it is not clear what fraction of PUM1/2 is actually bound to NORAD at any given time, so we have rephrased the sentence to acknowledge that “…it is not yet known whether NORAD limits PUM activity through a simple sequestration model or whether additional mechanisms contribute to PUM inhibition.”Given our previous measurements of • "definitively demonstrated that this lncRNA localizes predominantly to the cytoplasm and does not traffic to the nucleus upon induction of DNA damage". The authors do not exclude that some nuclear relocalization does happen. So this sentence  should be toned down.NORAD is predominantly cytoplasmic with or without DNA damage, without an increase in nuclear signal under any tested conditions, we believe that this is an accurate description of the results.We have rephrased the sentence in the Introduction to: “…this lncRNA localizes predominantly to the cytoplasm and does not detectably redistribute to the nucleus upon induction of DNA damage.” Given that the localization pattern of NORAD is a predominantly cytoplasmic RNA in HCT116 cells and does not detectably redistribute to the nucleus upon DNA damage.Similarly, we revised the relevant sentence in the Results and Discussion section to read: “These comprehensive RNA FISH and subcellular fractionation experiments provide definitive evidence that • Results section paragraph three: The authors should show that these conditions indeed lead to DNA damage.We have added γ-H2AX immunofluorescence and western blot data to Figure 2—figure supplement 1 of the revised manuscript, which confirms that doxorubicin and camptothecin treatment lead to DNA damage.• Results section: "We conclude that the disparate localization pattern observed using a commercially available RNA FISH probe set  most likely represented a non-specific signal." Speculation, such as this, has a better fit to the Discussion section and here other possibilities should be discussed as well .eLife allows a versatile format for Research Advance articles and we have chosen to write our manuscript with a combined Results and Discussion section. We believe that this is the most effective means to communicate the results and conclusions of this study. In light of our use of this format, reasonable speculation to provide plausible explanations for disparate results is appropriately located immediately following the presentation of each main experiment. We have edited the text as follows to make it clear that the specific sentence to which the reviewer refers is speculation:“We speculate that the disparate localization pattern observed using a commercially-available RNA FISH probe set  most likely represented a non-specific signal.”NORAD with or without DNA damage .We do not believe that differences in HCT116 clones provides a likely explanation for the disparate localization patterns observed. The HCT116 cells used in our current and previous localization studies were purchased from ATCC and do not represent a laboratory-specific clone. We and others have reported an identical, cytoplasmic localization pattern in U2OS cells and MEFs . Moreover, fractionation studies in a large panel of cell lines, including the experiments reported here, have similarly demonstrated cytoplasmic localization of NORAD RNPs in the nucleus or cytoplasm provides a plausible explanation for the disparate localization patterns observed. We and others have previously reported predominantly cytoplasmic localization of NORAD using RNA FISH with tiled oligonucleotide probe sets , which is the same type of probe used by Munschauer et al. Our new data generated using in vitro transcribed probes spanning the entire length of NORAD further support cytoplasmic localization. Equally importantly, the extensive subcellular fractionation studies reported here and elsewhere  further demonstrate cytoplasmic localization of NORAD with or without DNA damage, providing a completely independent experimental approach to support this conclusion.Similarly, we do not believe that differential accessibility of different probe sets to • Results section: "Since these RAP-MS experiments utilized pulse labeling with 4-thiouridine to crosslink NORAD to protein interactors, a bias towards detection of proteins that bind to newly synthesized NORAD would be expected, likely explaining the enrichment of nuclear interactors observed." This is also speculation and should be moved to the Discussion.As described above, this manuscript is formatted with a combined Results and Discussion section. As such, reasonable speculation and data interpretation follows the presentation of each main experiment. Regarding the specific sentence to which the reviewer refers, we have further edited the text to make it clear that we are speculating here:NORAD to protein interactors, a bias towards detection of proteins that bind to newly synthesized NORAD would be expected, potentially explaining the enrichment of nuclear interactors observed.”“Since these RAP-MS experiments utilized pulse labeling with 4-thiouridine to crosslink • Subsection “Binding of PUMILIO, but not RBMX, to NORAD is necessary for genome stability”: It should be mentioned whether the 5' region the authors use contains any PUMILIO binding sitesThis fragment does contain one PRE and we have modified the text accordingly:NORAD (nt 33-898), which also harbors one PRE, was also generated (5' fragment).”“An RBMX binding site fragment, representing the 5' end of NORAD is also apparent in Figure 3A.A PRE in this segment of • In the same section: What happens to NORAD levels in the edited cells (with the extra NORAD variants in the AAVS locus) before CRISPRi? Are NORAD levels roughly double? This should be mentioned, as these transiently increased levels may affect the phenotype in cells after CRISPRi.These data are provided in Figure 3C and we now explicitly describe these results in the text as follows:NORAD were observed upon expression of NORAD rescue constructs in trans  . Upon silencing of the endogenous NORAD locus, however, near physiologic expression levels for all constructs were achieved, with the exception of the 5' fragment which exhibited ~3-fold overexpression, perhaps indicating increased stability of this transcript segment when expressed in isolation.”“As expected, in cells infected with a non-targeting control sgRNA (sgControl), increased total levels of NORAD expression in control cells does not result in genomic instability. These data were included in our original manuscript and are now shown in Figure 5B of the current version. In the text, we describe these results as follows:Moreover, we showed that increased NORAD rescue populations after infection with non-target sgRNA (sgControl), indicating that overexpression of the NORAD constructs in trans did not trigger genomic instability.”“Importantly, no significant increase in aneuploidy was observed in any of the • Subsection “RBMX is not required for NORAD expression or localization”: Speculation regarding how RBMX affects NORAD levels also should be placed in the Discussion.As described above, this manuscript is formatted with a combined Results and Discussion section. As such, reasonable speculation and interpretation follows the presentation of each main experiment.• Can the authors please elaborate why the NORAD:RBMX study finds PUM only ranking 185th in the RAP-IP experiment and below the significant threshold? Why does applying the strategy with three search engines make this difference?NORAD signal compared to signal observed in RMRP pull-down. We do not think RMRP pull-down is the ideal control since different proteins will exhibit different levels of binding to RMRP which may or may not represent background. Instead, we believe that NORAD knockout would have been a better control. Regardless, they chose to set a log2 fold-increase of 1.6 (NORAD/RMRP pull-down), with p < 0.05, as their arbitrary significance threshold. Notably, we observe PUM1 immediately below the enrichment cut-off of log2 1.6, with a more significant p-value than RBMX . Nevertheless, we would prefer not to comment further on their analysis since the data are clear that i) PUM1 and PUM2 are detectable interactors by RAP-MS and ii) these interactions have been confirmed in multiple independent CLIP datasets from human and mouse cells.Because the Munschauer et al. study analyzed the RAP-MS experiment using proprietary software that we do not have access to (Spectrum Mill v6.01 pre-release from Agilent Technologies), we are unable to reproduce their analysis and therefore we cannot state definitively why PUM proteins were not more highly ranked in their reported results. However, several aspects of their analysis are notable and may have contributed to their findings. First, we detect two isoforms for both PUM1 and PUM2. We do not know which isoform they refer to as PUM2  and they do not comment on the PUM1 ranking . Second, their ranking is based on fold increase in • There are no biological replicates in Figure 2D; why has Figure 2C biological replicates and not Figure 2D? Also, please provide statistics if the burst of NORAD in Figure 2C at 12h is significant.NORAD expression after doxorubicin/camptothecin treatment is indeed statistically significant.We now provide biological triplicates for the experiments in Figure 2D and Figure 2—figure supplement 1D (subcellular fractionation after treatment with doxorubicin/camptothecin plus actinomycin D). In addition, we provide statistics in Figure 2C and Figure 2—figure supplement 1C, showing that the burst of • Please provide for the 2 independent clones in Figure 4C the analysis as in Figure 3B; does the degree of overexpression differ between the clones?NORAD constructs in both replicates is now provided in Figure 3C of the revised manuscript. Both clones exhibit very similar levels of expression of NORAD rescue constructs.The expression levels of all • Results paragraph one; perhaps "Importantly, a single cell line (human colon cancer cell line HCT116) was used in both of these studies"?We have edited this sentence as follows:NORAD trafficking as the cause of these discordant results.”“Importantly, a single cell line (human colon cancer cell line HCT116) was used in both the previous  and more recent studies , arguing against a cell-type specific difference in • Results paragraph four – insert "predominantly": "NORAD is a predominantly cytoplasmic RNA in HCT116 cells"Edited as suggested.• Subsection “PUM1, PUM2, and RBMX are components of the NORAD interactome”: Please confirm whether the RAP-MS experiments used HCT116 cells also.HCT116 cells were indeed used in the RAP-MS experiments and this is now indicated in the text.• Subsection “Binding of PUMILIO, but not RBMX, to NORAD is necessary for genome stability” paragraph three: NORAD "interaction with RBMX was abolished". Is this correct?NORAD exhibit minimal enrichment after RBMX pull-down relative to IgG (~1.0-1.3 fold enrichment). This magnitude of enrichment is indistinguishable from experimental noise. To more accurately describe these results, we revised the text to state that “interaction with RBMX was not detectable above background.”As shown in Figure 4A of the revised manuscript, the 5' deletion and ND4 fragments of • Figure 4C Explain horizontal dotted line and add significance estimates for the different comparisons.The horizontal line indicates the highest level of aneuploidy observed in the sgControl samples. Because this is a complex figure with many data points, we feel that inclusion of this line aids the reader in distinguishing samples with elevated aneuploidy from those without. We have added a similar line to the new Figure 5D (% chromosome segregation defects). Both lines are defined in the figure legend. In addition, significance estimates are provided for all genome stability assays .• Figure 4—figure supplement 1. Provide full legend that explains why there are 2 bands in the anti-PUM1 WB and try to improve on the anti-PUM2 WB.We have added a legend to Figure 4—figure supplement 1 that explains that PUM1 migrates as a doublet in HCT116 cells, possibly indicating post-translational modification or alternative splicing. We have confirmed that both bands represent PUM1 protein using siRNA knockdown experiments (not shown). We have replaced the PUM2 and RBMX blots in this figure with higher quality western blots.• Subsection “Generation and AAVS1 knock-in of NORAD constructs”: typo "and ND4 and were amplified"Thank you for highlighting this typo, which we have now corrected.


Health-related organisations disseminate an abundance of clinical and implementation evidence that has potential to improve health outcomes in low- and middle-income countries (LMICs), but little is known about what influences a user decision to select particular evidence for action. Knowledge brokers (KBs) play a part as intermediaries supporting evidence-informed health policy and practice by selecting and synthesising evidence for research users, and therefore understanding the basis for KB decisions, can help inform knowledge translation strategies. The Theoretical Domains Framework (TDF), a synthesis of psychological theories, was selected as a promising analysis approach because of its widespread use in identifying influences on decisions to act on evidence-based healthcare guidelines. This study explored its application in the context of KB decisions regarding evidence for use in LMICs.The study analysed data collected from participants of a 2015 global maternal and newborn health conference in Mexico. A total of 324 conference participants from 56 countries completed an online survey and 20 from 15 countries were interviewed about evidence use and sharing after the conference. TDF domains and constructs were retrospectively applied and adapted during coding of qualitative data to enhance understanding of the KB decision process in selecting evidence for action.Application of the TDF involved challenges related to overlapping constructs, retrospective use, and complexities of global health settings and relevant knowledge. Codes needed to be added or adapted to account for how KBs’ internal reflections on external factors influenced their actions in selecting evidence to share and use, and the decisions they made during the process. Four themes of the rationale for changing the TDF were identified during analysis, namely Influences from Beyond the Organisation, Knowledge Selection as a Process, Access and Packaging of Knowledge, and Fit for Use.Theories of individual behaviour, such as those in the TDF, can enhance understanding of the decisions made by actors such as KBs along dissemination and knowledge translation pathways. Understanding how KBs reflect on evidence and interact with their environment has the potential for improving global dissemination efforts and LMIC-to-LMIC exchange of implementation evidence.The online version of this article (10.1186/s12961-019-0463-9) contains supplementary material, which is available to authorized users. Determining the factors that influence successful dissemination and uptake of evidence-based, context-appropriate health practices has critical importance for reducing maternal and newborn mortality in low- and-middle income countries (LMICs), which carry the greatest burden of preventable deaths , 2. The Despite keen interest by the health research community and global commitment, no one standard for KT emerges as widely accepted among the many proposed theories and frameworks –8. AmongThe way in which literature addresses critical elements of KT suggests the importance of individual perspective and choices about evidence. While dissemination of knowledge is a crucial aspect of KT, publication alone without interpersonal communication has been shown to be less effective in promoting evidence-informed decision-making , 9. IndiIn the context of public health, individual or organisation knowledge brokers (KBs) serve as intermediaries between research producers and consumers to facilitate KT – production and context-appropriate use of evidence to inform decision-making in health policy and practice –14. KBs Use of psychological theory to understand individual decisions about evidence – such as those facing KBs – has origins in studies on internal and external factors influencing use of social science research in the 1970s  and applWhether used alone or with other frameworks, the TDF has been shown to be useful by offering a broad range of constructs that may influence individual decisions to make use of evidence , 18.While the complexity of healthcare settings and associated decision-making is widely accepted, such complexity is more significant in global health, where wide variations in provider roles, culture and socioeconomic factors  provide The context of the study was the Global Maternal Newborn Health 2015 Conference held in Mexico City, October 18–21, 2015. Multiple organisations and programmes working globally to improve maternal and newborn health (e.g. Saving Newborn Lives at Save the Children) collaborated in convening the conference. Conference organisers designed the event for sharing evidence and planning future action in health research, policy and practice to improve health outcomes, particularly in LMICs . To meetThe target population for the study was participants in the 2015 Global Maternal Newborn Health Conference held in Mexico City in 2015.Authors used a mixed methods explanatory sequential design . QuantitQuantitative data were collected using a self-administered online survey. Qualitative data were collected using three methods, namely (1) including open-ended questions in the survey ; (2) conducting semi-structured interviews with selected survey respondents; and (3) reviewing documents related to the conference (e.g. session descriptions in the programme) to understand the context of respondents’ comments about particular knowledge they shared and used. Both the survey and interviews were conducted in English. Verbal translation was provided for one interview respondent who communicated through an interpreter.The 22-question survey was intended to determine if respondents shared knowledge from the conference and with whom, and if they used it and how they used it, among other subjects. The 15 interview questions were intended to obtain additional details about respondent experiences sharing and using knowledge from the conference and influences on their decisions regarding sharing and use . The distribution list was used as a convenient way of reaching the target population since conference organisers did not maintain a database of contact information for actual conference attendees. The team sent the initial e-mail message 9 months after the conference with reminders sent 2 and 3 weeks later. The survey closed after 1 month.Respondents were recruited through an e-mail invitation to complete a survey that was sent to a communications distribution list used by conference organisers to reach potential attendees. A subset of the list had attended the conference (www.skype.com) or phone and audio recorded with permission, then transcribed. The lead author and trained interns conducted the interviews in English. One interview respondent used an interpreter. Interviews were completed between 10 and 14 months after the conference.The interview candidates were purposively selected from the pool of survey respondents. Among survey respondents, 124 (38.3%) answered ‘Yes’ to a question asking if they would be willing to participate in a 30-min interview about their knowledge sharing and use experience following the conference. From the pool, TN purposively selected a sample of 20 survey respondents to contact for semi-structured interviews based on maximum diversity of country, type of work, type of organisation, mode of participation (online versus in-person), abstract acceptance (Y/N), and use or sharing of knowledge from the conference (Y/N). The study budget and timeline determined the target number of interviews. The variables for participant diversity were selected with the rationale – based on the researchers’ 2012 and 2013 similar studies  – that tInclusion/exclusion criteria for survey respondents was based on their confirmation of attending the 2015 Global Maternal and Newborn Health Conference either in person or online. Inclusion/exclusion criteria for participation in the semi-structured interviews was entering a valid e-mail address in response to the survey question, “May we contact you for a 30-minute interview to hear more about your experience using and sharing information and knowledge from the conference?” Potential participants that supplied an e-mail address that bounced back as undeliverable or failure to respond to follow-up e-mails from the researchers also resulted in exclusion.The study protocol was reviewed by the lead author’s institutional review board and was determined to be exempt from further review. Nevertheless, interviewers obtained and recorded verbal consent by signing and dating consent forms, which was done during study as an ethical practice. Consent was not obtained from respondents who answered the survey as responses were not stored with personally identifiable information. The e-mail invitation to complete the survey said that respondents to the survey could be entered into a prize drawing for a Kindle Fire. There was no incentive to participate in the interviews.The online survey received responses to some or all questions from 324 of 1000 in-person conference participants (a 32.4% response rate) and additional online participants. Respondents represented 57 countries. A total of 64 candidates were contacted to be interviewed, of which 20 respondents from 15 countries were successfully interviewed. Participant countries consisted of both LMICs and higher-income countries as organisers invited participants based on the relevance of their evidence or work to global issues of maternal and newborn health, not on the country in which they were based. The study participants, as with the conference invitees, included a variety of health system roles relevant to facilitating understanding, sharing and use of the evidence presented at the conference. These roles included researchers, policy-makers, funders, programme implementers and healthcare professionals.Table The team analysed survey responses of conference participants using Microsoft Excel. Analysts imported interview transcripts and survey data into MAXQDA qualitative data analysis software (version 18) to facilitate coding open-ended questions and transcripts. Descriptive statistics (frequencies) were generated for demographic variables  and variables of knowledge use and sharing  to explore trends in characteristics among the study participants.-defined domains from the validated TDF  to establish the midwifery-led care, so midwife can link the community and the health facilities, and if the babies and mothers need the high quality or entrance care they can divert the mothers and the newborns in the tertiary health facilities level.” “Other modifications were based on researcher reflection that several of the definitions seemed better suited to exploring behaviour related to one practice in a specific setting, rather than a broad array of knowledge applied in diverse settings that was characteristic of the current study. The rationale for the TDF changes aligns with published guidance to focus application of a given framework to the scope of a study  in our…country at a central level there is no proper coordination between two divisions, maternal health and child health.” , Asia)“As the conference knowledge was intended for use in resource-limited environments, the study team also found it useful to be able to determine the extent to which the availability of resources influenced evidence use.So you pass the information to a lower cadre that should do the work, but very often they don’t have the stethoscope and…to check the blood pressure, or they don’t have the urine dipstick to check for the proteinuria.” “The significance of international influences became evident during the coding process and led to the addition of several non-TDF codes, namely Multi-Country Importance, Comparable Setting and Success, and International Relationships. These codes helped fill a gap in capturing the value that KBs placed on evidence generated or applied in other LMICs that had similar characteristics ....whenever we propose some kind of suggestion…public health officials…ask us…have you any good success stories, where from you collected this kind of idea. So we suggest to them that… we come to know about such kind of practices being used in other countries, and their economic situation and their electricity situation is the same as ours. So we can do such kind of [idea] easily and these are easily applicable...” “The abundance and breadth of data applicable to the TDF domain Environmental Context and Resources led the team to subdivide the domain into three areas of influence, namely (1) aspects of the organisation or setting, (2) characteristics of a country or health system, and (3) availability of financial, human or other resources. Each distinction aided understanding of the data and has implications for possible interventions to aid evidence use and sharing. For example, the ability to note country or health system contextual factors surfaced issues with the governing environment that impeded evidence use.It was my first time to attend the…international conference. To me it was like I didn’t know that maybe what I was looking at... I didn’t know that it was very, very important, and it’s not only in [my country]” “For this reason, the team expanded the domain and definition to include the process of acquiring knowledge (Knowledge and Learning).In other studies using the TDF, the knowledge linked to behaviour has already been selected and a specific action identified . Individuals apart from those targeted in the studies have decided that the knowledge is relevant to address a timely problem relevant to a local context. For this study, labels and definitions needed to be adapted to reflect the process KBs undergo in identifying and deciding what to do (if anything) about knowledge to which they had been exposed. For example, the domain Knowledge relates to whether someone has awareness of information to the extent that they could take a proscribed action. With KBs, however, perceiving that they had learned something new appeared to be a facilitating precursor to focusing attention on and acting upon the knowledge.Okay, one of the major reasons [I shared the knowledge about newborn resuscitation technique] is that… if you go on a deep analysis of some of the causes [of high] neonatal mortality rates, most of these, they are somehow preventable deaths if we apply correct skills.” “Expanding the definition of Skills enabled the study team to capture both narrowly defined clinical skills (e.g. resuscitation technique) and broader reasoning skills  involved in the KB process.Similarly, the domain Skills was clearly relevant to KB descriptions of learning a specific clinical skill such as newborn resuscitation; however, it omitted a crucial aspect of the knowledge selection process, namely the ability to critically appraise knowledge as scientifically or otherwise valid and contextually relevant.Working with the nurses, we tried to share with them the importance of making sure that they provide quality services to the antenatal clinic. This is one of the lessons I learned at the conference. I try to make sure that at any time I speak to nurses and midwives, I emphasize the issue of the content of care they give to their client.” “Another need for extending domain definitions related to the later stages of the knowledge selection process, during which KBs considered the possibilities of future action based on knowledge. Given that the KBs in this study also mentioned seeing themselves as responsible for applying knowledge, the team wanted to capture KB traits of initiative and action-orientation as facilitators in the process. To address this need, the team expanded the definition of the domain Intentions from planning on adhering to specific guidelines to a broader willingness to take the initiative on knowledge action.I often have information for me because most of the time I live in the state capital. I don’t go to villages or rural area, where people that need the information live. So when you come back [from a conference] you have to identify people to actually go to those villages or rural areas who will be able to pass this information down the line…to people that need the information to change their life.” “KBs also mentioned the need for them to filter information from the conference that had been packaged for an audience working globally rather than locally.With many of the studies using the TDF or relating to KBs taking place in higher-income countries, access to knowledge in a needed format and language is seldom depicted as a significant issue. With the conference knowledge being intended for use in resource-limited settings, the study team identified the need to capture external influences related to knowledge access in LMICs. These influences included access to knowledge synthesis products and electronic resources (in lieu of print resources). To capture these concepts, the team added the codes Accessibility and Knowledge Presentation, which appeared relevant as both a facilitator and barrier. In one example, the respondent mentioned the challenge in arranging for a flow of information from urban to rural settings for local use.“Because it is a cultural practice [in our country] that people apply something on the umbilical cord of a newborn child. And if you provide them with the something which is safe [chlorhexidine] and which will prevent sepsis… so that’s why it was a decision that instead of one day it should be a seven day application. So we adapted it to our cultural practices.” In many studies using the TDF, the evidence in question has been previously determined to be a good fit for the context. With the global nature of the conference, the evidence presented could potentially be shared and used in settings worldwide. KBs described their thought processes in identifying potentially actionable evidence and assessing whether and how it could be adapted for local use. In order to capture this reasoning, the study team added codes for Adaptability of Evidence and Local Applicability.Overall, codes categorised as internal facilitators appeared most influential on respondents, followed by external facilitators, external barriers and, finally, internal barriers. Figure TDF-based codes and inductively derived codes were equally useful in identifying relevant internal facilitators and barriers. Inductively and deductively derived non-TDF codes were most helpful in identifying relevant external facilitators and barriers, although Environmental Context and Resources is a concept that appears both in the TDF and other literature.The top relevant domains and relative importance of internal versus external influences appeared to be mostly consistent across respondent characteristics such as type of work, type of organisation and region. Slight differences in internal versus external influences appeared when comparing respondents by type of work. For example, for respondents working in Health/Service Delivery, external barriers were more influential than for respondents working in other types of work. One nuanced difference among the rich descriptions, though not affecting estimations of relevance, concerned the Emotion domain. Expressions of passion for improving health outcomes in an LMIC appeared almost exclusively with respondents who both worked and lived in an LMIC country. Respondents based in one country, but working on activities in other countries, did not express passion to the same extent.Using the TDF provided a starting point in this study to identify the internal barriers and facilitators to evidence sharing and use described by KBs; however, its use was somewhat challenging and required adaptation to address broad external factors and adequately explain knowledge brokering behaviour.Analysis of qualitative data using TDF-derived codes helped identify factors that relate to effective knowledge brokering, which may be because of the TDF focus on human behaviour and its underpinnings in psychological theory. Interpersonal communication has been identified in the literature as an essential element in knowledge brokering and in advocating use of evidence in public health decision-making , 31–34. The TDF was useful in exploring KB characteristics as well. Studies have examined individual aspects of KBs, and there have been calls for additional research on desirable KB personal attributes . PositivMost findings of influences on evidence use surfaced by the TDF agree with ones mentioned in the literature, whilst a few disagreed. The high relevance of Belief About Consequences coincides with findings showing that healthcare professionals tend to act on knowledge if they believe it will have positive consequences for the care of patients (or not act upon it in the case of anticipated negative outcomes) , 38–40. The authors experienced several difficulties with applying the TDF, some of which the literature also reports. The time-consuming aspect of utilising the TDF posed a problem that has been mentioned in other research , 29, 41,Other challenges concerned how the study design incorporates use of the TDF. The current study used the TDF retrospectively on existing data, instead of prospectively for instrument design and analysis. While the TDF has been successfully used retrospectively (e.g. systematic review of implementation interventions ), such aFinally, the authors experienced challenges in distinguishing between internal and external factors and, similarly, between barriers and facilitators. For example, the way that respondents reflect on external factors could be said to be determined by their internal attributes. In one case, a public health decision-maker displayed an internal skill in crafting messaging about a health practice in response to an external factor, that is, resistance of local healthcare providers. Similarly, a respondent’s view of whether an external factor was a barrier or facilitator may have been determined by internal factors. For example, in a case of a healthcare service provider describing cultural practices for newborn cord stump care, the provider described the practices as an opportunity to substitute an evidence-based approach in a culturally acceptable way rather than a barrier.Use of the TDF offered a starting point for exploring KB decision processes, but might not be as useful if study aims focus on implementation of a particular intervention, for which there are other frameworks. Notable among these implementation frameworks are the Tailored Implementation for Chronic Diseases – Determinants of Practice Checklist , PromotiThe study had limitations related to sampling, respondent bias, study timeline and TDF use. First, self-selection of study participants may have led to over-reporting of facilitators and under-reporting of barriers due to social desirability bias – volunteers for the study may have wanted to show appreciation for being included in the conference, particularly those who received sponsorship to attend. In turn, over-reporting and under-reporting may have influenced estimations of which TDF domains were most relevant to KBs. Insufficient samples of respondents for key demographic characteristics such as region and type of work limited the ability of the authors to identify how domain relevance may have varied by strata. Second, the length of time between the conference and interviews (10–14 months) may have introduced a recall bias. The extended timeline also made it infeasible to obtain comments from study participants about the findings. Finally, the authors’ adaptation of the TDF meant that they were no longer working with a validated version of the TDF, and the changes were not validated.Additional research should be conducted to build on the theoretical contributions of the TDF to explore internal and external factors influencing evidence sharing and use in LMICs. Additionally, integration of TDF with commonly used implementation frameworks should be explored for interventions in LMICs that have knowledge brokering or dissemination as a critical component. Use of the TDF in building KB capacity in key influential areas, such as the interpersonal skills involved in knowledge brokering, should also be explored.Theories of individual behaviour such as those in the TDF can help understand when, where and for whom knowledge brokering is effective in increasing evidence-informed health policy and practice in LMICs. Understanding how KBs in LMICs reflect on evidence and interact with their environment has potential for improving global dissemination efforts and LMIC-to-LMIC exchange of evidence and implementation approaches.Box 1 TDF domains 1. Knowledge2. Skills3. Social/professional role and identity4. Beliefs about capabilities5. Optimism6. Beliefs about consequences7. Reinforcement8. Intentions9. Goals10. Memory, attention and decision processes11. Environmental context and resources12. Social influences13. Emotion14. Behavioural regulationAdditional file 1:Survey questions. (PDF 90 kb)Additional file 2:Interview script. (PDF 69 kb)


The European Society of Radiology (ESR) considers the use of evidence-based referral guidelines in clinical practice essential to ensure the appropriate utilisation of medical imaging for patients. Since 2014, the ESR has been working with the American College of Radiology (ACR) to develop referral guidelines for Europe, based on the ACR Appropriateness Criteria (AC) and appropriate use criteria developed by the ACR Rapid Response Committee.Any changes to existing recommendations, and any additional guidelines, should be based on evidence as far as possibleExpert opinion, judgement, European practice standards, should only function as a supplement when necessaryAppropriateness recommendations should give no consideration to national or institutional circumstances, costs, or availability of equipment.This paper sets out the methodology used by the ESR’s Referral Guidelines Subcommittee to adapt the ACR criteria for use in the European clinical decision support (CDS) platform ESR iGuide. The ESR adheres to the ACR’s original methodology as far as applicable, and has established additional methodological guidance for its experts, establishing several key principles:The cooperation between the ACR and ESR on appropriate use criteria and imaging referral guidelines provides added value to both societies as European studies and guidelines from European subspecialty societies of radiology are fed into the process and the exchange of views among the experts makes the guideline development and review process more robust.The ESR’s aim is to ensure referrers and patients benefit from the best possible guidance for appropriate imaging. ESR develops guidelines designed for use in a decision support softwareThe ESR bases their recommendations on a review and adaptation of the ACR Appropriateness CriteriaRecommendations are based on the best-available evidence supplemented where necessary with expert opinion and practice standardsGuidelines are periodically released in coordination between ACR and ESRThe European Society of Radiology (ESR) considers referral guidelines for medical imaging essential for improving the appropriateness and justification of radiological procedures. To address gaps in the availability of imaging referral guidelines and the lack of guideline use in Europe, the ESR decided to develop a clinical decision support system for European imaging referral guidelines. The most efficient way to introduce guidelines for use in a clinical decision support system at the European level is to leverage existing efforts. Having assessed several options, the ESR entered into a cooperation with the American College of Radiology (ACR) in 2014. Their Appropriateness Criteria (AC) were already in use in the decision support system ACR Select, and following an open call for proposals the ESR selected the National Decision Support Company (NDSC) platform, also used for ACR Select, to deliver the ESR’s guidelines.The ESR initially appointed a project group for a rapid review process of the ACR Select content. The content was divided into nine topic areas, and nine radiologists selected by the ESR reviewed and adapted the CDS guidelines for use in ESR iGuide within one year.This group was transformed into the permanent ESR Imaging Referral Guidelines Working Group (IRG WG) in 2016, subsequently turned into the ESR Referral Guidelines Subcommittee (RGSC) in 2018, as part of the ESR’s Quality, Safety and Standards Committee (QSSC). The purpose of this RGSC is to maintain the content of ESR iGuide and review and adapt future versions of the ACR Select content for use in Europe.This document sets out the process and methodology according to which the ESR RGSC reviews and modifies the ACR Select content.The ACR Select content is composed of the ACR AC for diagnostic imaging, which are evidence-based and developed according to a widely accepted, transparent methodology –4. The AThe ACR Select content includes additional guidelines developed by the ACR’s Rapid Response Committee (RRC), created using the evidence base of the ACR AC supplemented by expert consensus and opinion, and the experience of using CDS in clinical practice. The purpose of this additional content is to widen the coverage of the guidelines to areas where the strength of evidence may be insufficient to develop an ACR AC topic.The methodology in use for the ACR AC has evolved from the work of the Rand Corporation in the late 1980s. Numerous specialty societies have adopted and used the general approach, including the methodology of the ACR.The work of the ESR’s RGSC of reviewing and adapting the ACR’s clinical imaging guidelines (CIG) builds upon the methodology used for the development of the ACR Appropriateness Criteria, which is in line with the RAND method. The ESR’s methodology’s aim is to ensure that the review and adaptation process is evidence-based to the largest extent possible. Appropriateness ratings reflect medical necessity and the diagnostic value of diagnostic imaging studies.Inherent in guidelines is the focus on balancing the possible benefit against the possible risk. It is clear that there are at the very least potential negative consequences of radiation; the concerns are greater in younger patients, due to the latency of these potential adverse effects. The cost of the imaging exam is another important variable. Depending on the nature of the healthcare system, this may not be a concern to the individual undergoing the imaging exam, but it is always a concern to the system as a whole. If the likelihood is very low or negligible that a specific imaging exam in a specific clinical setting is going to provide useful information - for example a routine chest radiograph in an otherwise healthy young adult non-smoker - then even a modest cost is hard to justify. For most guidelines, both cost and radiation, then, are always part of the risk-benefit equation. The aim of CIG is to provide the best possible advice in specific clinical settings, realising that with the clear limitations in knowledge, the many specific clinical variables , and available expertise and equipment, a definitive recommendation may not always be possible.In the European setting, the ESR’s guidelines should be equally valid across a variety of different health systems. Therefore, while radiation risk is one of the variables to consider in the justification of an imaging study in a given clinical situation, the consideration of the cost of these studies must necessarily be disregarded in the ESR content version. The resulting guidelines should be generic to be widely applicable, and can be localised to account for specific variables such as cost and availability when guidelines are implemented in a particular setting.if any, imaging exam is most likely to be helpful in the diagnosis and care of this patient in this clinical setting. Therefore, the diagnostic value, in relation to the potential radiation risk, should be the deciding factor for each appropriateness rating.There are also reasons other than medical necessity to consider imaging. These include patient preference; a patient may want a CT scan for back pain, simply for personal reassurance. Also, many healthcare providers feel that they can both reassure themselves and their patients and perhaps lessen the risk of a malpractice accusation if an imaging exam is requested even if there is no real concern for negligence. There may also be financial incentives to getting an imaging study, if the referring provider has a fiduciary interest in the imaging equipment. It may simply be expeditious: it can be faster to get an imaging study than to carefully evaluate a patient in a busy emergency room, or explain at length to an anxious patient that an imaging study is unlikely to be clinically useful. The use of CIG is an effective means to deal with many of these non-medical reasons for obtaining imaging. Again, the entry point for CIG is the question: which, The membership of the ESR RGSC is determined in accordance with the ESR’s statutes. The chair of the RGSC proposes members in consultation with the chair of the QSSC. All nominees must be approved by the ESR Executive Council.As of 2019, the membership of the RGSC consists of experts for each of the 10 topic areas in ESR iGuide, and members’ expertise  must correspond to these topic areas. For each topic area, the expert(s) are representatives from the appropriate European subspecialty society of radiology and/or chosen from the general ESR membership.Breast ImagingCardiac ImagingGastrointestinal ImagingMusculoskeletal ImagingNeurologic ImagingPaediatric ImagingThoracic ImagingUrologic ImagingVascular ImagingWomen’s ImagingTopic areas:Each of the appointed experts reviews and modifies the guidelines in his assigned topic area, conducts literature and evidence reviews and consults colleagues and other specialists at his discretion.The methodology for the ACR Appropriateness Criteria is the basis for the work of the ESR RGSC and is adhered to as far as applicable. The basic principles are that any changes ESR experts make to the guidelines should be based on scientific evidence to the largest extent possible. Expert opinion, judgement and experience of European practice standards can function as a supplement where necessary. No consideration should be given to national, local, or institutional circumstances such as national regulations, cost or reimbursement rates, or the availability or quality of imaging equipment, as the resulting guidelines should be generic and equally applicable anywhere in Europe. To account for local circumstances, the ESR iGuide can be modified for that particular situation during implementation.The guidelines contain over 1,600 indications (March 2018) for which different radiological examinations have been evaluated in terms of their diagnostic value and appropriateness. The ESR also applies the most widely used rating scale from 1 to 9, with 1–3 defined as “not usually appropriate”, 4–6 defined as “may or may not be appropriate” and 7–9 defined as “usually appropriate” according to the ACR methodology. Each indication is applicable for defined patient groups . Like the ACR, the ESR follows the RAND/UCLA Appropriateness Method User’s Manual [The content is divided into 10 radiological subspecialty or topic areas,As part of the review process, ESR experts are expected to survey the ACR evidence. Based on the strength of evidence and assessment by the ACR for each AC topic, provided in evidence tables with information on the study type, number of patients, study objective, study results, and an assessment of the studies quality, reviewing experts consider additional literature not yet covered in the ACR AC development process when determining whether to change an appropriateness rating. This literature should be high quality, peer-reviewed, and publicly available, and can include guidelines issued by European subspecialty societies of radiology. Appropriateness ratings for the European edition of the guidelines are changed if the evidence is considered sufficient, and/or if expert consensus and/or opinion are sufficiently strong to make a guideline compatible with European standards of practice.For all changes, an explanatory comment is added to provide a reason for changing the rating. The ESR has kept the RGSC deliberately small to ensure an efficient process, but ESR experts are encouraged to work with or consult colleagues during the review at their discretion to solicit different opinions and discuss changes.The experts review each indication falling into their area of expertise. A number of scored (i.e. rated based on appropriateness) imaging exams are associated with each examination.Review of indication nameReview exam names associated with the indicationsReview of ACR documentation: narrative, rating table, evidence table, referenced literatureif not: proposal for changing indication nameIndication names are based on ACR Commons terminology or ICD, so changes to indication names should only be made if an indication is unclear or confusingDetermination if indication name is adequate for EuropeDetermination whether the associated exams are relevant for the clinical indicationif not: changing, removing, or adding examsExam names and modifiers should follow the ACR Commons terminology6.Review of the appropriateness rating of exam for every indication based on ACR evidenceAgreement with appropriateness rating: no further actionEvidence needs to be strongest for changes to ACR AC topics and for changes across appropriateness category (as opposed to rating changes within a category)Literature search: assessment of available additional studies, guidelines, other relevant publicationsRevision of appropriateness rating to reflect the additional evidence foundCitations are edited to add the literature considered by the ESRIn addition: expert comment to explain the changeIn the absence of sufficient literature evidence, all changes to appropriateness ratings are marked as “Changed based on ESR expert opinion”; preferably, this should only be done to non-ACR AC topics and should only be rating changes within the same categoryChange in appropriateness rating: review of additional literature; citation of literature to justify appropriateness change; references to be added to review sheet7.age range: 0–150 yearssex: male, female, both/unknownReview of the applicable patient characteristicsThe review and adaptation process is as follows:Changes to appropriateness ratings: referenced literature evidence and explanatory comments; at expert discretion only in exceptional casesChanges to indication names: based on evidence or at expert discretionChanges to exam names: based on evidence or at expert discretionChanges to age range for which a guideline applies for: based on evidence or at expert discretionChanges to sex for which a guideline applies for: based on evidence or at expert discretionNew rules (indications plus rated exams): based on referenced literature evidence and explanatory commentsTo summarise, the content can be adapted by ESR experts in the following ways:relevance of all indications/exams (according to ACR methodology for topic selection)avoidance of unnecessary overlap/duplication between indications In addition, the entirety of the content within a topic area should be reviewed to ensure:All ESR content changes are communicated to the ACR RRC for consideration and discussed in periodic meetings between the RGSC and RRC. Furthermore, ESR changes are communicated to the ACR panels for consideration in future revisions of the ACR Appropriateness Criteria.The ESR releases new editions of the ESR iGuide content approximately once a year. The first two content versions included changes to the appropriateness category of the reviewed imaging studies in less than 10% of cases, indicating a very high degree of consensus between the ACR and ESR expert groups.The ESR reviews new versions of the ACR Select content after they are released in the United States. All modifications, additions and deletions are documented so that ESR experts understand exactly what has changed since the previous edition.In the future, the ESR and ACR plan to have the ACR RRC and the ESR RGSC working simultaneously on new CDS content versions, and releasing two editions with different ACR and ESR appropriateness ratings at the same time.