The level of copy number alteration (CNA), termed CNA burden, in the tumor genome is associated with recurrence of primary prostate cancer. Whether CNA burden is associated with prostate cancer survival or outcomes in other cancers is unknown. We analyzed the CNA landscape of conservatively treated prostate cancer in a biopsy and transurethral resection cohort, reflecting an increasingly common treatment approach. We find that CNA burden is prognostic for cancer-specific death, independent of standard clinical prognosticators. More broadly, we find CNA burden is significantly associated with disease-free and overall survival in primary breast, endometrial, renal clear cell, thyroid, and colorectal cancer in TCGA cohorts. To assess clinical applicability, we validated these findings in an independent pan-cancer cohort of patients whose tumors were sequenced using a clinically-certified next generation sequencing assay (MSK-IMPACT), where prognostic value varied based on cancer type. This prognostic association was affected by incorporating tumor purity in some cohorts. Overall, CNA burden of primary and metastatic tumors is a prognostic factor, potentially modulated by sample purity and measurable by current clinical sequencing. Cancer cells carry different types of mutations that are associated with the cell starting to multiply uncontrollably. Certain changes only affect one or a few letters of the genetic code. Others, known as copy number alterations, or CNA, involve larger portions of the genome that can either be lost (deletions) or duplicated (amplifications). Tumors in different patients carry variable amounts of these deletions or amplifications, which together are known as the CNA burden.New technologies allow scientists to scan the genomes of tumors and examine the type of mutations present in each patient. The results can help to decide on the best course of action. For example, in prostate cancer, patients whose tumors have a high CNA burden are at greater risk of relapse after treatment. However, it has been unclear whether these people also have lower survival rates, and if CNA burden can predict outcome of other types of cancers.Hieronymus et al. conducted genetic analyses on over a hundred samples from prostate cancer patients who were not treated with surgery or radiation. The results showed that a higher CNA burden in the tumors is correlated with more deaths due to the disease. The findings in prostate cancer were also true across different types of cancers. These conclusions also emerged when Hieronymus et al. then looked at genomic data obtained from patients with various cancers using a different DNA sequencing test, which is certified for clinical use. This demonstrates that CNA burden could be a useful marker in clinical settings to help assess risk in cancer patients. Somatic copy number alterations (CNAs) are nearly ubiquitous in cancer  and alteThe CNA burden of a tumor is the degree to which a tumor's genome is altered as a percentage of genome length and represents a fundamental measure of genome copy number alteration level. As such, tumor CNA burden, rather than individual CNAs, may be associated with cancer outcomes. While tumor mutational burden (TMB) predicts response to immunotherapy across multiple cancer types , tumor CIn addition to questions about the prognostic potential and overall landscape of CNA in conservatively treated prostate cancer, it is unknown whether CNA burden is prognostic for prostate cancer survival, rather than only recurrence and metastasis. Nor is it known whether the prognostic significance of tumor CNA burden extends to other cancer types. Here we set out to address these questions, as well as whether tumor CNA burden can be prognostic in a clinical practice setting, including (i) in cancers treated conservatively rather than through immediate surgery or radiation, (ii) in biopsy or resection samples, and (iii) using a clinical targeted sequencing that allows rapid and cost-effective measurement of tumor CNA burden.To address these questions, we first examine the genomic CNA landscape of conservatively treated prostate cancer in more than a hundred diagnostic biopsy and resection specimens from a conservatively treated cohort; this cohort consisted of patients with localized prostate who were not treated with surgery or radiation within six months of diagnosis. We demonstrate that tumor CNA burden is associated with cancer-specific death, independent of standard clinical predictors. To explore the prognostic significance of tumor CNA burden more broadly in other cancer types, we find that tumor CNA burden is also associated with disease-free and overall survival in TCGA cohorts of primary breast, endometrial, renal clear cell, thyroid, and colorectal cancer in addition to prostate cancer, with the degree of association varying in some cancer types. We then establish the clinical feasibility of measuring tumor CNA burden using the FDA-cleared MSK-IMPACT clinical next generation sequencing (NGS) assay and confirm that tumor CNA burden is associated with overall and disease-specific survival in both primary and metastatic tumors across cancer types. In all, we demonstrate that tumor CNA burden is a prognostic factor associated with cancer recurrence and death in multiple cancer types, including in conservatively treated prostate cancer which would benefit from increased risk stratification.To explore the genomic copy number landscape of conservatively treated prostate cancer, we set out to analyze copy number alteration (CNA) in cancer obtained non-surgically through biopsy and transurethral prostate resection (TURP) using a widely studied, conservatively treated primary prostate cancer cohort . This reThe copy number alteration landscape of the conservative treatment cohort revealed canonical copy number alterations of prostate cancer, including gain of chromosome 8q and losses on chromosomes 6 p, 8 p, 13q and 16 p, though with lower frequency than seen in prostate cancer cohorts analyzed by our group (MSKCC cohort)  and TCGASince tumor CNA burden is associated with prostate cancer recurrence and metastasis in prostatectomy cohorts , we sougWe next asked whether tumor CNA burden was associated with outcome after adjusting for established prognostic variables, including Gleason sum score and the UCSF Cancer of the Prostate Risk Assessment (CAPRA) score  which coLarge, clinically annotated cancer genomic efforts such as TCGA now provide an opportunity to examine whether CNA burden is prognostic for primary cancer outcomes across many cancer types. In the TCGA primary prostate cancer cohort , tumor CThe prognostic significance of tumor CNA burden in prostate cancer led us to ask whether tumor CNA burden is prognostic in other cancer types. Towards this end, we examined published TCGA cohorts for multiple cancer types with available disease-free survival and overall survival data, including breast , endometWe next wanted to determine whether CNA burden’s prognostic associations could be observed using panel-based targeted sequencing assays that are increasingly entering clinical use, in contrast to CGH array-based determination of tumor CNA burden. The Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay is a clinical laboratory improvement amendments (CLIA)-certified sequencing-based assay  of severWe find that tumor CNA burden assayed by targeted clinical sequencing is significantly associated with overall survival in primary prostate tumors  , it willAnother interesting feature of the association of tumor CNA burden with outcome demonstrated here is that it has prognostic significance independent of tumor mutation burden (TMB). This is consistent with recent work in glioblastoma, breast, lung, and ovarian cancer showing that CNA-derived signatures have more prognostic power than somatic point mutation-based signatures, as measured by concordance index . Thus, tThe prognostic significance of tumor CNA burden raises intriguing questions regarding the underlying biology. Tumor CNA burden may be a simple measure that correlates with the extent of oncogenic driver alterations. Yet, we show that tumor CNA burden retains its prognostic significance after adjustment for a number of known oncogenic alterations in primary prostate cancer, including PTEN loss associated with increased tumor CNA burden . In metaSCR_007303).Of the TAPG1 cohort , FFPE prSCR_012763, StataCorp, College Station, TX).Tumor CNA burden (tumor CNA burden) was analyzed as percent CNA burden, defined as the length of the genome altered by copy number alteration multiplied by 100. For regression analyses, tumor CNA burden was scaled as per five percent so that the estimates of our hazard ratios were more interpretable. All statistical analyses were performed using Stata 13 . First, we assessed whether there was an association between tumor CNA burden and CSS by utilizing a univariate Cox model, censoring patients who did not die at the date of their last follow-up and patients who died of other causes at their death date. Secondly, in order to assess whether there is information from tumor CNA burden over and above biopsy Gleason score, we utilized a multivariable Cox model, adjusting for biopsy Gleason sum categorized as ≤6, 7, and ≥8. Finally, to assess whether there is an association between tumor CNA burden and CSS after accounting for the preoperative predictors of CSS, we utilized a multivariable Cox model, adjusting for the UCSF-CAPRA score, a preoperative risk score calculated by incorporating the patient’s age at diagnosis, PSA at diagnosis, primary and secondary Gleason score at biopsy and clinical tumor stage. As percent of positive biopsy cores was not available for the cohort, a modified CAPRA score was utilized not incorporating this information. Among our cohort of 107 patients, 47 patients were missing clinical tumor stage; multiple imputation was used to impute the missing values. Statistical analyses were performed utilizing the measured and imputed values combined across 10 imputations using Rubin’s method. Furthermore, to evaluate the discriminative accuracy of the model including tumor CNA burden, we calculated bootstrap optimism-corrected Harrell’s C-index. It should be noted that the discrimination of the CAPRA score is lower in the TAPG1 conservative treatment CNA cohort than seen in some other prostate cancer cohorts, and this may impact the degree to which tumor CNA burden increases the concordance index. All data used for these analyses are available in stcompet command in Stata.For illustrative purposes, we utilized competing risk methods to estimate the probability of death from prostate cancer in the setting of death from other causes. Cumulative incidence was shown for patients who died from prostate cancer, or died from other causes, stratified on tumor CNA burden in relation to the median tumor CNA burden among the cohort, using the BRCA1, BRCA2, ATM, TP53, RB1, and PTEN), using separate models for each alteration. As the overall point mutation burden was not available for all patients, 34 patients with primary prostate cancer and 11 patients with metastatic prostate cancer were excluded from this portion of the analysis in their respective cohorts.For analysis of the prostate cancer MSK-IMPACT cohort , the pubSCR_002877, http://www.cbioportal.org/study?id=msk_impact_2017, samples and annotation used at time of analysis available as metan command in Stata. Both fixed effects and random effects estimates were calculated. Fixed effects weights were calculated using inverse-variance weighting, metan weights were calculated using the DerSimonian and Laird method.For analysis of our pan-cancer IMPACT cohort  , breast https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103665).Data access. The conservative treatment TAPG copy number cohort array data was deposited in NCBI GEO ; Mark A Rubin (Reviewer #3).Thank you for submitting your article "Tumor copy number alteration burden is a pan-cancer prognostic factor associated with recurrence and death" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The manuscript analyzed the CNA landscape of conservatively treated prostate cancer in a historic biopsy and transurethral resection cohort and found that increased abundance of CNA in tumors is correlated with recurrence and mortality. This finding was further extended across multiple cancer types by analyzing data from TCGA and other cohorts. Given the prevalence of CNA throughout cancer genomes, this study highlights the potential for incorporating overall CNA burden assessment as a molecular prognostic factor in treatment decision making.eLife.The reviewers agreed that the approach is rigorous and addresses important aspects of biomarker evaluation including taking into account clinical and pathology parameters. The reviewers also felt that the manuscript is clear and well-written. However, the reviewers also expressed a concern about the novelty of the results in the light of previously published work, as elaborated below. The reviewers would like to see your response to these concerns in a point-by-point response letter and in a revised manuscript before committing to a decision regarding appropriateness for publication in Essential revisions:1) The authors state that they are surprised by the findings that CNVs have such a strong association with clinical progression for prostate and other cancers. Yet, this is known in the prostate cancer research community and has been previously reported on in different eras based on available technology. Therefore, it would be reasonable for the authors to consider their finding in the context of the prior knowledge. For example, comparative genomic hybridization (CGH) technology led to some of the first genome-wide observations regarding copy number alterations and disease state . CGH identified somatic copy number alterations in a high percentage (~75%) of localized PCA. Losses were found to be 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). These early genome-wide studies also suggested that the pattern of copy number alterations changes with disease progression. For example, gains of 7, 8q, and X were more often observed in the CRPC state . SNP arrays also very nicely accessed CNV . These platforms were used by the TCGA studies to perform a comprehensive copy number analysis for localized PCA . As well as a prior meta-analysis that showed recurrent CNVs were associated with disease progression . (Note: This paper is referenced in the text but for a more focused point). These are just a selection. There are others.2) Related to the above comment, if the major point is that these new datasets add substantially more information, it would be nice to show a down-sizing experiment in silico wherein decreasing the coverage means the signal that supports these associations is lost. What is the minimal coverage needed to make these associations? This is important due to prior work that used older technologies to make similar claims.3) There is recent published work that appears to be very similar. Pearlman et al., (2018) report, "pan-cancer metastasis potential score (panMPS) based on observed CNAs". panMPS predicts metastasis and metastasis-free survival in cohorts of patients with prostate cancer, triple negative breast cancer and lung adenocarcinoma, and overall survival in the Metabric breast cancer cohort and three cohorts from The Cancer Genome Atlas (TCGA), including prostate, breast and lung adenocarcinoma. And Ross-Adams et al., (2015) explored risk in association with CNV and integrating transcriptomic data, albeit on smaller prostate cancer data sets. These studies and work reported in bioRχiv from the ICGC groups on structural variation might also be valuable to include in a discussion of recent relevant prior work in this field.4) Is the association between CNA burden and survival in prostate cancer explainable by differences in purity? Impure samples will have low apparent CNA burden.5) The manuscript points out that "aneuploidy" but not "CNA burden" has previously been associated with outcome. In many cases, "aneuploidy" referred not to overall changes in tumor ploidy, e.g. due to genome doubling, but indeed to something similar to CNA burden. "Chromosomal instability" has also been used to describe the same phenomenon and has been associated with prognosis in multiple cancers. For example: Murayama-Hosokawa et al., 2010 ; Walther et al., 2008; Mouradov et al., 2013; and Berg et al., 2015 ; Karlsson et al., 2007 (breast cancer); and Carter et al., 2006 (multiple cancer types).6) The overall association between CNA burden and survival may be obscuring negative associations between specific CNAs and survival. Have the authors evaluated the association between specific CNAs and survival, after controlling for overall CNA burden? Essential revisions:1) The authors state that they are surprised by the findings that CNVs have such a strong association with clinical progression for prostate and other cancers. Yet, this is known in the prostate cancer research community and has been previously reported on in different eras based on available technology. Therefore, it would be reasonable for the authors to consider their finding in the context of the prior knowledge. For example, comparative genomic hybridization (CGH) technology led to some of the first genome-wide observations regarding copy number alterations and disease state . CGH identified somatic copy number alterations in a high percentage (~75%) of localized PCA. Losses were found to be 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). These early genome-wide studies also suggested that the pattern of copy number alterations changes with disease progression. For example, gains of 7, 8q, and X were more often observed in the CRPC state . SNP arrays also very nicely accessed CNV . These platforms were used by the TCGA studies to perform a comprehensive copy number analysis for localized PCA . As well as a prior meta-analysis that showed recurrent CNVs were associated with disease progression . (Note: This paper is referenced in the text but for a more focused point). These are just a selection. There are others.We thank the reviewers for highlighting this and appreciate the well demonstrated connection of specific CNVs and CNV signatures  to disease state in prostate cancer. We have expanded our consideration of this in the Introduction and Discussion section. As the reviewers note, CNV patterns or clusters have been associated with Gleason 8+ disease  and recurrent disease . Indeed, our group initially found CNA patterns associated with shorter time to biochemical recurrence in prostate cancer . Nonetheless, previous studies have primarily looked at the association of individual or sets of CNVs with pathology and disease state, rather than outcomes themselves and time to outcome.The novelty of the work presented in our study lies in the association of (1) high quality clinical endpoints across cancer types and in a pan-cancer cohort with (2) the overall level or burden of genomic CNA of a tumor, agnostic to the identity of the component CNAs. Moreover, we adjust for prognostic pathological features which have previously been linked to CNVs, showing that association of CNA burden with outcome is independentof the previously published association with pathology features or disease state. We have expanded on this in the discussion to address the reviewers’ comments.The key novel insight is the following: specific prognostic CNVs are likely to be cancer specific, whereas here we show the prognostic potential of a generalized measure of overall copy number dysregulation, as well as the feasibility of measuring this dysregulation using a current clinical NGS test. We acknowledge that the concept of CNA burden as a prognostic factor could seem rather obvious, at first glance, if viewed as a summation that includes all specific prognostic CNVs. But CNA burden could include both positive and negative prognostic CNVs that counterbalance this. Our work demonstrates that the prognostic utility of overall CNA burden is not lost in the noise of CNAs that are unassociated or negatively associated with poor outcome.2) Related to the above comment, if the major point is that these new datasets add substantially more information, it would be nice to show a down-sizing experiment in silico wherein decreasing the coverage means the signal that supports these associations is lost. What is the minimal coverage needed to make these associations? This is important due to prior work that used older technologies to make similar claims.This is an interesting question that raises a number of issues about coverage, resolution, and depth of genome sampling in assaying tumor copy number, both in newer sequencing approaches and older approaches such as array-based methods. The effective coverage of the genome is similar between targeted sequencing by IMPACT  versus older aCGH approaches (used for our conservative treatment cohort) as well as whole exome sequencing. Specifically, the method of calculating burden from the IMPACT targeted sequencing data incorporates SNP probes throughout the genome. We have updated the description of how CNA burden is calculated from IMPACT to clarify this. To illustrate the similarity in breadth of coverage between these technologies, we have added the prostate cohort analyzed by targeted IMPACT assay to Figure 1A. As shown with this addition, the coverage is similar with the newer clinically approved technology.Rather than running a downsizing experiment, we took advantage of a pan-cancer IMPACT cohort for which whole exome sequencing was also performed (n=1005) and ran an upsizing experiment. The CNA burden determined by targeted IMPACT assay and by whole exome sequencing is highly correlated . We have added this analysis to the Results section and Discussion section. While the suggestion for a further downsizing experiment is interesting, we feel the IMPACT versus WES comparison addresses the main point raised by the referee. Efforts to further downsize from IMPACT to determine the minimal coverage required for prognosis determination is beyond the scope of this initial report and would best benefit from incorporation of other prognostic clinical markers.3) There is recent published work that appears to be very similar. Pearlman et al., (2018) report, "pan-cancer metastasis potential score (panMPS) based on observed CNAs". panMPS predicts metastasis and metastasis-free survival in cohorts of patients with prostate cancer, triple negative breast cancer and lung adenocarcinoma, and overall survival in the Metabric breast cancer cohort and three cohorts from The Cancer Genome Atlas (TCGA), including prostate, breast and lung adenocarcinoma. And Ross-Adams et al., (2015) explored risk in association with CNV and integrating transcriptomic data, albeit on smaller prostate cancer data sets. These studies and work reported in bioRχiv from the ICGC groups on structural variation might also be valuable to include in a discussion of recent relevant prior work in this field.We thank the reviewers for bringing our attention to the recent work by Pearlman et al., (2018). We have added a discussion of this study and how it derives a 295-gene frequency-weighted CNA signature derived by supervised analysis of prostate metastases that is prognostic for metastasis and overall survival in prostate and breast cancer. It is interesting to note that CNA burden outperforms their signature in one prostate cohort, while their signature outperforms CNA burden in the other prostate cohort they analyze (one in which PSA and stage are not prognostic). As a set of CNAs selected from prostate cancer, it may have less generalizable prognostic significance beyond the two cancers examined. We expand on this with some of the recent studies showing that CNA frequency, independent of identity, is associated with survival in pediatric AML  and NSC lung cancer . We have also added some discussion of prepublication results from ICGC and others (available on BioRχiv) on somatic CNA in cancer with regards to CNA, prognosis, and mechanism.4) Is the association between CNA burden and survival in prostate cancer explainable by differences in purity? Impure samples will have low apparent CNA burden.This is an important question which we have addressed with several additional analyses. First, we looked at the association between CNA burden and outcome in the TCGA prostate cohort after adjusting for purity using the ABSOLUTE purity metric, which was reported with the first publication of this cohort and represents a standard computationally-determined measure of purity. We find that CNA burden is still prognostic for recurrence after adjusting for sample tumor purity in the TCGA cohort (Table 2 and Results section). It is also still prognostic after adjusting for tumor purity along with Gleason and/or mutation burden, but these additional multivariate models are overfitted and we therefore elected not to include them in the revised manuscript. Next, we analyzed the IMPACT cohorts after adjusting for purity. Importantly, purity in these cohorts is more variable since these samples are not subject to the quality control requirements mandated by TCGA . We found that CNA burden in prostate tumors falls below the significance of association with overall survival when adjusting for purity determined by FACETS. However, purity-adjusted CNA burden is still significant in the pan-cancer primary tumor cohort and approaches significance in the metastatic tumor cohort (Supplementary file 3 and Results section).This variability may in part be influenced by the smaller hazard ratio of CNA burden determined by IMPACT (sequencing panel) assay versus arrays (TCGA and TAPG/conservative treatment cohort) in these respective cohorts. It is also worth noting that a subset of samples in the IMPACT prostate cohort could not be included in the purity-adjusted analysis due to computational limitations in the purity calculation. Due to this limitation, we saw a decrease in the significance of association between mortality and CNA burden even before adjusting for purity, given with this smaller number of cases .In summary, tumor purity is a potentially significant variable that modulates CNA prognostic significance, and it will be important to continue to explore its effect especially in the context of additional molecular prognostic factors. It is interesting that within the IMPACT cohort, purity itself is associated with outcome and may warrant further investigation in such work. We have added a discussion of these considerations to the Discussion section.5) The manuscript points out that "aneuploidy" but not "CNA burden" has previously been associated with outcome. In many cases, "aneuploidy" referred not to overall changes in tumor ploidy, e.g. due to genome doubling, but indeed to something similar to CNA burden. "Chromosomal instability" has also been used to describe the same phenomenon and has been associated with prognosis in multiple cancers. For example: Murayama-Hosokawa et al., 2010 ; Walther et al., 2008; Mouradov et al., 2013; and Berg et al., 2015 ; Karlsson et al., 2007 (breast cancer); and Carter et al., 2006 (multiple cancer types).We thank the referees for calling attention to thee related concepts of CNA burden, aneuploidy, and chromosomal instability. Aneuploidy has predominantly been defined as having a non-diploid DNA index, generally assayed by cytometry . The ploidy metric (determined by CLONET) used in our manuscript to adjust for aneuploidy in our multivariable regression model reflects the same information as DNA index conventionally used to measure aneuploidy. Indeed, ploidy estimates from SNP and sequencing data, such as the one we use, have been successfully benchmarked against FACS and SKY-determined DNA index in human tumors and cell lines . Therefore, we believe our conclusion that the prognostic significance of CNA burden is independent of aneuploidy reflects the traditional measure of aneuploidy in prognostic significance.However, we understand that aneuploidy has various, often more mechanistic, definitions , some of which are not captured by ploidy as numerical DNA index. To address the reviewers’ points, we have clarified how the ploidy used in our analysis was determined and its relationship to ploidy and DNA index used in prognostic studies of aneuploidy or chromosomal instability (Results). We have also qualified our conclusion from this finding in the discussion to take into consideration the way aneuploidy was measured. We have also expanded the discussion of how future work will be needed to determine if CNA burden is prognostic independent of cancer type-specific prognostic classifications, including MSI-positive status in colorectal cancer which, as the reviewers note, is largely exclusive with CIN-positive status and may be involved in the prognostic interplay between CNA burden and CIN status .6) The overall association between CNA burden and survival may be obscuring negative associations between specific CNAs and survival. Have the authors evaluated the association between specific CNAs and survival, after controlling for overall CNA burden?We looked for the association between survival and selected specific CNAs in multivariable regression models with CNA burden in prostate cancer . This analysis was focused on the question of whether the specific CNAs we examined accounted for the prognostic significance of CNA burden, and we found that they did not. These specific CNAs did have negative associations with survival  for the most part (or no significant association) and their negative associations with survival were not strengthened by the adjustment for CNA burden through its inclusion in the model.The question about whether there are specific CNAs that have a positive association with survival that might be obscured by CNA burden is interesting and worthy of future exploration. We believe that many individual prognostic CNAs are likely to be cancer type-specific, and current cohorts unfortunately do not have sufficient size to run individual regressions for all CNAs to uncover these in a rigorous fashion.


Systematic review (SR) guidelines recommend extending literature search to gray literature in order to identify all available data related to the review topic. We aim to conduct an overview of SRs on population health in the Middle East and North Africa (MENA), to assess the methodology of these SRs, to produce an evidence map highlighting methodological gaps in SRs regarding gray literature searching, and to aid in developing future SRs by listing gray literature sources related to population health in MENA.We will conduct an overview of SRs based on the Cochrane Handbook for Systematic Reviews of Interventions. This overview will be reported following PRISMA 2009 guidelines. Using comprehensive search criteria, we will search the PubMed database to identify relevant SRs published since 2008. Our primary outcomes are gray literature sources and study-level quality in the gray literature. We will include MENA countries with Arabic, English, French, and/or Urdu as primary official languages and/or media of instruction in universities. Two reviewers will independently conduct a multilevel screening on Rayyan software. Extraction of relevant data will be done on Statistical Package for the Social Sciences (SPSS) software. The methodological quality of included SRs will be assessed using the Assessment of Multiple Systematic Reviews (AMSTAR) tool. Any disagreements will be resolved by discussion and consensus.t tests will be used to determine whether the differences between subgroups are statistically significant. Additionally, an evidence gap map will be constructed to highlight characteristics and quality of the gray literature used in SRs on population health in MENA and emphasize existing gaps in gray literature searching. We will also list gray literature sources identified in the included SRs stratified by country and research topic.We will estimate the overall proportion of SRs that used gray literature as one of their data sources. Subgroup analyses will be conducted to identify characteristics of these gray literature sources. Chi-squared and This overview will comprehensively assess the overall quality of the SRs on population health issues in MENA. Our findings will contribute to the improvement of population health research practices in MENA.The systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) on 26 October 2018 .The online version of this article (10.1186/s13643-018-0751-4) contains supplementary material, which is available to authorized users. In healthcare, systematic reviews (SRs) and meta-analyses have become increasingly essential. Researchers, clinicians, and policymakers read them to keep up to date with their field and to make decisions , 2. ThusNon-English gray literature has been demonstrated to not affect results of reviews of intervention studies included in Cochrane Review Groups . We willOur protocol has been registered on PROSPERO (PROSPERO registration number CRD42017076736 ) and is The research questions we want to address are (i) what are the sources of gray literature used in the SRs on population health in MENA, (ii) what proportion of these SRs use gray literature, (iii) what are the characteristics of gray literature sources included in these SRs, (iv) are there differences between the SRs that use gray literature and those that do not, (v) are there differences between SRs using gray literature according to the type of gray literature, and (vi) what is the proportion of good quality studies from gray literature included in SRs on population health in MENA?Our study is an overview of published systematic reviews. As such, we will include only SRs . NarratiThe following eligibility criteria are designed in compliance with the population-intervention-comparator-outcome (PICO) model . PopulatThe 27 identified MENA countries –18, theiWe will include SRs on population health in MENA published since 2008—the publication year of the first version of the Cochrane Handbook for Systematic Reviews of Interventions . We willIn order to provide an exhaustive list of gray literature sources, we will check the reference list of any review on available population health data sources in MENA identified during the multilevel screening process. If we identify relevant citations of gray literature sources related to population health in MENA, we will add them to our provided list of gray literature sources.We will conduct a literature search (by AA) on PubMed  Additio. Key seaAll reviews, SRs, and meta-analyses identified on PubMed will be imported into Endnote  and duplUsing the Assessment of Multiple Systematic Reviews (AMSTAR) tool, we will assess the methodological quality of included SRs , 57.We wWe will summarize SRs’ methodology with a descriptive approach using tables presenting key characteristics of gray literature sources and quality of studies included from gray literature. From the SR-level quality assessment, we will estimate the overall proportion of SRs that used gray literature as one of their data sources. We will also provide a list of gray literature sources identified in the included SRs stratified by country and by research topic. This list format will be similar to the Canadian Agency for Drugs and Technologies in Health checklist “Grey Matters” in the field of drugs and technologies in health . This Cat test and chi-squared test [p value < 0.05). Bonferroni correction will be used to address the multiple testing issue regarding false positive.In the subgroup analysis, we will assess the differences between SRs that use gray literature and those that do not. We will estimate the proportion of SRs that used gray literature according to whether these SRs were produced by institutions located in MENA or outside MENA. We will compare language and format of gray literature sources included in SRs produced by institutions in MENA and among those produced by institutions outside MENA. Student’s red test  will be Additionally, for each MENA country, we will review the five latest SRs containing gray literature searching. From the SRs, we will report on primary study-level quality assessment. We consider that SRs’ authors are the experts in their research topics; as such, we will rely on their study-quality assessment. We are not aiming to compare the study-level quality between SRs but to estimate the overall proportion of good quality studies from the gray literature related to population health using the primary-study quality assessment. This proportion of good-quality primary studies included in our overview will be estimated after excluding duplicate studies using Endnote software . When anWe will construct an evidence gap map defined as a visual depiction of the characteristics of evidence in a particular field  highlighOur overview will be reported following the preferred reporting items for overviews of systematic reviews  As overvOur overview of SRs will enable us to identify, map, and compare the use of gray literature sources in published SRs on population health in MENA. The findings of this overview of SRs will have implications for research and evidence-based clinical and policy decisions. Our results will provide insight on available gray literature sources related to population health in MENA. The overview will describe to what extent researchers are missing reliable information when conducting SRs without including gray literature or when including a specific type of gray literature source. Our project will inform researchers, clinicians, and policymakers about the importance of including an extensive gray literature search in a region where official languages and/or media of instruction in universities include languages other than English.Ethical approval will not be needed, as this is a systematic review. Data used will not be individual patient data; therefore, there will be no concerns about privacy.Additional file 1:PRISMA-P* 2015 checklist: recommended items to address in a systematic review protocol .


The ferret transmission model is extensively used to assess the pandemic potential of emerging influenza viruses, yet experimental conditions and reported results vary among laboratories. Such variation can be a critical consideration when contextualizing results from independent risk-assessment studies of novel and emerging influenza viruses. To streamline interpretation of data generated in different laboratories, we provide a consensus on experimental parameters that define risk-assessment experiments of influenza virus transmissibility, including disclosure of variables known or suspected to contribute to experimental variability in this model, and advocate adoption of more standardized practices. We also discuss current limitations of the ferret transmission model and highlight continued refinements and advances to this model ongoing in laboratories. Understanding, disclosing, and standardizing the critical parameters of ferret transmission studies will improve the comparability and reproducibility of pandemic influenza risk assessment and increase the statistical power and, perhaps, accuracy of this model. The susceptibility of ferrets to influenza virus infection has been known for nearly a century. Ferrets and humans share similarities in lung physiology, cellular receptor distribution, and clinical signs of infection, making the ferret an attractive small mammalian model for laboratory study of influenza viruses . BeyAlthough some factors are considered to be controllable, much of the variability between groups cannot be easily overcome. For example, the absence of commercially available uniform caging for ferret transmission experiments (a reflection of country- and institution-specific size regulations and facilities constraints) represents a parameter that would be difficult to standardize. However, a greater understanding of the relative role and contribution of different variables can improve our ability to better contextualize and interpret results among laboratories. For example, does the virus dose affect transmissibility or otherwise influence detection of transmissible quasispecies in contact ferrets? Gustin et al. previously demonstrated the potential effects of the dose and the route of inoculation  on transmission potential  would beAs discussed at the workshop, the participating laboratories analyzed the protocols used for evaluation of influenza virus transmissibility via airborne particles in the ferret model, which highlighted the breadth of experimental designs. It was also clear that all variables that are probable contributors to differential results are not routinely disclosed in peer-reviewed publications or other platforms where results are discussed. Lack of disclosure of all variables can complicate the ease of comparing findings between laboratories, warranting a closer look at the feasibility of using a more comparable study design between multiple laboratory groups for risk-assessment studies. As such, a push toward comprehensive description of specific experimental conditions would aid this effort and would probably be valuable when risk assessments performed in different laboratories are compared.Beyond the experiment- and facility-based variability we describe, the lack of a standard protocol for the number of experimentally infected animals and the number of recipient ferrets (donor:recipient ratios) included in ferret transmission studies can affect the ability to interpret results among groups and represents a substantial controllable parameter. The ideal standard for risk assessment activities seems to be a 1:1 donor:recipient experimental setup to assess virus transmissibility via the airborne route, where each virus-infected ferret is exposed to only 1 recipient ferret. This design facilitates ease of interpretation of results and provides added rigor from a statistical perspective. This design also restricts exposure to virus-laden particles expelled from each donor ferret to its respective recipient, ensuring that any detected transmission event would have originated from exposure to separate donors. However, because of space limitations, these experiments are often conducted in replicates inside a single physical containment area with a shared ventilation system  while still maintaining a 1:1 donor:recipient ratio. If transmission is mediated by virus-laden particles expelled by donors, increasing the number of donors within a single containment area is likely to increase the concentration of virus-laden particles in the air, thereby increasing the observed transmissibility. Specifically, it is not known whether transmission kinetics would be comparable if 3 independent experiments were performed with 1 donor to 1 recipient (each ferret housed singly) versus 1 experiment with 3 donors and 3 recipients per containment area. Air sampling devices that allow monitoring of the quantities of virus-laden particles in the air throughout the experiment would help refine the experiment outcomes and are likely to become part of these experimental designs in the future.Further impeding efforts to compare results among laboratories, many experiments include an additional contact ferret co-housed with an experimentally infected ferret to evaluate virus transmissibility in a direct contact setting while still assessing the respiratory droplet transmission to a recipient ferret housed in an adjacent cage. In this design, several ferrets may serve as donors . Moreover, the donor and direct contact–infected ferrets are likely to shed virus-laden particles at different times, further complicating the results of the transmission experiments. Ideally, the effects of different experimental designs should be investigated in systematic experiments, and researchers should strive to disclose this information as comprehensively as possible.During workshop discussions, most researchers agreed that it would be helpful for the field to coalesce around a fixed 1:1 donor:recipient ratio  for risk-assessment transmission experiments. Introduction of direct contact ferrets into the experimental setup would probably extend the amount of time that virus-laden particles can be released in the air. Virus amplification by direct contact ferrets may also lead to virus adaptation and emergence of variants with increased respiratory droplet transmission potential. Applying a 1:1 donor:recipient ratio would increase the consistency of the experimental design under which risk-assessment experiments are conducted across multiple laboratories, differentiating them from broader, more heterogeneous research-based assessments that would include more experimental designs and variables. However, individual laboratories have built up datasets and experience over the years while performing risk-assessment studies with different strains of influenza viruses; thus, adopting a common protocol may be difficult to achieve within a short time.Although viruses that are readily transmitted by the airborne route will exhibit robust transmission in a direct contact setting, some influenza viruses that are not transmitted efficiently via respiratory droplets are nonetheless transmitted between ferrets placed in direct contact, which facilitates transmission via multiple modes. Studies evaluating virus transmissibility between ferrets placed in direct contact may be influenced by many of the experimental drivers discussed here; when using this model, further contributions to variability are introduced by animal behavior and housing practices. Although scoring for the Centers for Disease Control and Prevention Influenza Risk Assessment Tool includes data derived from the direct contact transmission model in risk assessment, it is not fully clear how to interpret the relative pandemic risk resulting from viruses that transmit in a direct contact setting but not via respiratory droplets. As discussed above, a greater understanding of what confers virus transmissibility in both models will improve our ability to interpret results from more permissive direct contact models with the more stringent respiratory droplet transmissibility. This knowledge will improve our ability to appropriately include and aggregate results from both types of transmission studies in influenza virus risk assessments.Although the ferret transmission model has greatly improved our understanding of influenza virus transmissibility, there are limits to what this model can contribute to risk assessment and how results are interpreted. In particular, inefficient virus transmission  remains a difficult outcome to understand. It is often unclear whether this event results from genetic changes in the virus during the transmission event, reflects the transmitted infectious dose, or results from other contributing factors; concurrent contextualization of these results with other laboratory paramaters  can often provide additional insight. Similarly, interpretation of seroconversion in the absence of detectable virus in respiratory secretions or detectable virus in the absence of seroconversion can be difficult. Moving toward a consensus on the implications of inefficient transmission events would be helpful because, currently, efficient and inefficient transmission are not well defined.Another major limitation of current ferret transmission studies is the small group size, which is driven by cost, size of the animals and their associated housing requirements, and ethical and practical constraints. For this reason, statistical analyses of data from transmission experiments are infrequently performed  or deep-sequencing approaches have provided, and probably will continue to provide, additional information to aid in the interpretation of inefficient virus transmission events, elucidate transmission bottlenecks, and differentiate between within-group variability and larger differences in experimental setup and design (An additional avenue for improved understanding of virus transmissibility via respiratory droplets are aerobiology-based approaches. These approaches include analysis of the exhaled breath of infected ferrets and the amount and size distribution of virus-containing aerosols released by infected ferrets (In vivo ferret transmission studies are not performed in isolation. The incorporation of these data into larger research efforts has greatly expanded our understanding of the complex determinants of influenza virus transmission in mammals. For example, hemagglutinin acid stability and the hemagglutinin–neuraminidase balance have been linked with virus transmissibility via the airborne route in ferrets, as have receptor binding preference, gene constellation, neuraminidase stalk length, and other parameters (The plasticity of the ferret model permits a wide range of experimental approaches to assess influenza virus transmissibility. This plasticity represents a great advantage when designing research experiments to evaluate viral, host, environmental, and other factors that contribute to transmission between mammals. However, it might be beneficial for studies conducted primarily for risk-assessment purposes to be performed under conditions as uniform as possible. For example, moving toward a standardized 1:1 donor:recipient ratio in risk-assessment studies would probably enhance the comparability of results found by different research groups and would enable inclusion in meta-analyses .Current knowledge regarding the viral, host, and environmental parameters that drive transmission outcomes is limited. Understanding these parameters would be beneficial for infection control, and future studies should aim to validate these factors empirically. As data regarding the exact role of each of the potential parameters discussed in this article are developed, improved documentation of variables  associatMany unanswered questions are relevant for understanding the pandemic risk posed by novel and emerging influenza viruses that lie outside a standardized risk-assessment experimental setup. Some studies that would provide additional valuable information include modulation of distance between cages when assessing virus transmissibility via the airborne route, shortening the duration of exposure between inoculated donors and contact ferrets (A useful exercise might be for several research groups to test and compare results of a transmission experiment by using 1 selected virus strain prepared by 1 laboratory. Many practical considerations would need to be discussed, including the particular strain to test and the anticipated transmission phenotype of this virus. Complete standardization of ferret transmission studies conducted worldwide is not possible; however, exercises that seek to examine the relative contributions of laboratory-specific drivers of experimental variability may identify critical parameters or conditions. Similarly, meta-analyses of published data, with included disclosure of the parameters listed in Influenza viruses will continue to jump the species barrier and cause human infection and disease. Virus-transmissibility assessments in the ferret model represent one of numerous activities conducted to aid pandemic preparedness efforts in the event that a pandemic virus does emerge in the future. Continued refinement of the ferret model, concurrent with advances in identification of viral, host, and environmental factors that influence transmission risk (


Originally designed for measuring isotope abundances and elemental masses, mass spectrometry is becoming a mainstay across life sciences. As electrospray ionization of biomolecules turns 30 and the Orbitrap mass analyzer 20, we take this opportunity to highlight the role of both inventions in stirring mass spectrometry from physics into biology and discuss the advances and challenges that may impact the future applications of biomolecular mass spectrometry. The rate of scientific discovery is often dramatically accelerated by new methodological approaches or instruments. A few examples immediately come to mind: X-ray crystallography enabled the visualization of proteins in three-dimensional space; massively parallel sequencing transformed the fields of genomics, transcriptomics and epigenetics by increasing throughput and decreasing sequencing costs by orders of magnitude; and CRISPR-Cas now allows genetic manipulations to be carried out with a level of speed and accuracy that would have qualified as science fiction a mere 10 years ago. 2019 marks the decennial anniversaries of two such innovations in the application of mass spectrometry that have transformed several fields of biological and clinical research: electrospray ionization of large biomolecules, and their high-resolution detection with the Orbitrap mass analyzer.1; who showed that inserting an aqueous sample solution in a conductive capillary and applying an electrical current allows generating gaseous ions of the sample compounds that can be measured by mass spectrometry. Following the demonstration that ESI enables the mass spectrometric analysis of labile biomolecules, Fenn and his coworkers brought ESI-mass spectrometry into the biology limelight with a review article published in 19892. Ten years later, Alexander Makarov introduced the Orbitrap mass analyzer, presenting a new approach enabling robust mass measurements at high resolution3. Although it was not until 2005 that a commercial Orbitrap mass spectrometer became available, Makarov’s presentation in 1999 provided the first glimpse into the underlying principles of what is today one of the most widely used mass analyzers for proteomics and analytical chemistry.Electrospray ionization (ESI) was originally described in 1984 by the 2002 Nobel laureate John B. Fenn and his colleague Masamichi Yamashitacollection of articles published in Nature Communications that showcases applications of ESI- and Orbitrap-mass spectrometry in the life and clinical sciences. The collection features recent research, review and perspective articles, as well as papers that have attracted our readers’ attention over several years. It covers proteomics, approaches to localize and quantify post-translational modifications, mass spectrometry-based structural biology, and mass spectrometric analyses of other biomolecules such as carbohydrates, nucleic acids and metabolites. In two historical perspectives, the Orbitrap inventor Alexander Makarov recounts how this mass analyzer came into being and Matthias Mann, John Fenn’s graduate student and one of the first researchers to analyze proteins by mass spectrometry2, revisits the invention of ESI and the subsequent emergence of proteomics5.To highlight the anniversaries of these two key enabling advances in the life sciences, we recently unveiled a www.nature.com/milestones/mass-spec). As both Mann5 and Makarov4 point out, key elements of state-of-the art mass spectrometers were invented long before the application of mass spectrometry to biomolecules. Following the works of the physicists Joseph John (J.J.) Thomson and Wilhelm Wien on positive rays and cathode rays7, mass spectrometry arose as an analytical technique that was first applied to measuring stable elemental isotopes in the gas phase8. Owing to the development of additional methods to ionize and fragment analytes, new mass analyzers to measure their mass-to-charge ratios, and increasingly comprehensive compound libraries, mass spectrometry soon became an established tool in chemical research. However, mass spectrometric analysis of proteins and nucleic acids remained out of reach because their more labile peptide or sugar-phosphate backbones were lost during the transfer into the gas phase. This changed— allowing mass spectrometry to enter the realm of biology— with the invention of ESI2, where charged droplets of sample solution gradually desolvate until only ‘naked’ molecular ions remain; and matrix-assisted laser desorption/ionization (MALDI)10, which relies on embedding biomolecules in an energy-absorbing crystalline matrix ionized with a laser pulse of appropriate energy and wavelength.Notwithstanding the importance of ESI and the Orbitrap analyzer, there are many other influential developments that paved the way towards biological mass spectrometry . On its way to becoming a FAIR research field, biomolecular mass spectrometry must overcome the obstacles outlined above, which will require concerted efforts from all parties involved in the scientific process. This presents a formidable challenge, but it is well worth the effort. As 2019 also marks the fifth anniversaries of the human proteome drafts—two landmark projects that critically relied on open data17—the jubilees of 2019 remind us that bringing mass spectrometry forward requires both ingenious concepts and public sharing of the amazing science they spawn.For both new applications and methodological advances in mass spectrometry, it is increasingly clear that their full potential will be realized only if methods, data, code, and software are reported in accordance with the community guidelines and made findable, accessible, interoperable, and reusable (FAIR


Atrial fibrillation (AF) is commonly associated with heart failure. A bidirectional relationship exists between the two—AF exacerbates heart failure causing a significant increase in heart failure symptoms, admissions to hospital and cardiovascular death, while pathological remodeling of the atria as a result of heart failure increases the risk of AF. A comprehensive understanding of the pathophysiology of AF is essential if we are to break this vicious circle. In this review, the latest evidence will be presented showing a fundamental role for calcium in both the induction and maintenance of AF. After outlining atrial electrophysiology and calcium handling, the role of calcium-dependent afterdepolarizations and atrial repolarization alternans in triggering AF will be considered. The atrial response to rapid stimulation will be discussed, including the short-term protection from calcium overload in the form of calcium signaling silencing and the eventual progression to diastolic calcium leak causing afterdepolarizations and the development of an electrical substrate that perpetuates AF. The role of calcium in the bidirectional relationship between heart failure and AF will then be covered. The effects of heart failure on atrial calcium handling that promote AF will be reviewed, including effects on both atrial myocytes and the pulmonary veins, before the aspects of AF which exacerbate heart failure are discussed. Finally, the limitations of human and animal studies will be explored allowing contextualization of what are sometimes discordant results. Two highly prevalent forms of cardiovascular disease are atrial fibrillation (AF) and heart failure, and in spite of recent advances in treatment these conditions remain important causes of morbidity and mortality. AF is an abnormal heart rhythm affecting more than 30 million patients worldwide  resulting in membrane depolarization. The shape of the atrial action potential differs from that of the ventricle, at least in larger species, where generally the atrial action potential is shorter and more triangulated )  Bers, .ICa(L) triggers the release of a larger quantity of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm via ryanodine receptors (RyRs) in a process known as calcium-induced calcium release -tubules allow dyads to occur not only at the cell periphery but throughout the cell interior, such that calcium rises in a more synchronous manner in ventricular myocytes Figure . CalciumFollowing the release of calcium from the SR, free intracellular calcium is then available to bind to troponin C, causing myocyte contraction. Relaxation is brought about by the removal of calcium from the cytosol which occurs via two main mechanisms: reuptake into the SR by the sarcoplasmic reticulum calcium ATPase pump (SERCA) regulated by the inhibitory proteins phospholamban and sarcolipin, and removal from the cell via the sodium-calcium exchanger (NCX) on the plasma membrane Figure . Minor cIn the steady state, calcium influx and efflux are maintained in balance. Any increase in calcium influx e.g., during beta adrenergic stimulation, is met by a corresponding increase in calcium efflux  or spontaneous calcium release . If the depolarizations induced by EADs or DADs are large enough they may lead to one or more additional premature action potentials.Triggered activity refers to additional impulses triggered by an initial action potential, known as afterdepolarizations. Although several mechanisms can lead to afterdepolarizations, many of these relate to abnormalities in calcium handling. There are two types of afterdepolarization termed “early” or “delayed” afterdepolarizations. Early afterdepolarizations  but increased delayed rectifier currents compared to atrial myocardium , decreased forward mode INCX or an increase in potassium currents  and enhance calcium efflux through NCX, simultaneously shortening the plateau of the action potential and extending terminal repolarisation, while small calcium transients produce a longer plateau but shorter terminal repolarization. Atrial alternans can be measured in patients using standard electrophysiological catheters and correlates with vulnerability to AF   decreases per beat  likely contributes to action potential shortening   which is decreased in the early stages of rapid atrial pacing both in terms of current density and LTCC expression (Table ICa(L)  offset any increase in INCX. Secondly, the rate of arrhythmogenic calcium release in the form of sparks is unaltered since increased INCX is balanced by protective changes such as decreased RyR expression and reduced phosphorylation by calcium calmodulin dependent kinase two (CaMKII), decreased intracellular sodium, and increasing intracellular calcium buffering  in mice leads to hyperphosphorylation of RyRs at the CaMKII site and an increase in leak  and the expression of LTCCs are reduced in paroxysmal AF  density is generally decreased, consistent with protection against calcium overload, this is offset by rapid atrial rates during paroxysms of AF which would be expected to promote calcium loading. Additionally decreased ICa(L) could have further pro-arrhythmic effects due to a shortening of the action potential duration, decreasing the refractory period and therefore the wavelength of potential re-entrant circuits.Similar to the acute response to rapid atrial pacing, both the current density of ICa(L) and t-tubule loss. These changes appear to initiate a stepwise progression of remodeling toward persistent AF in which an atrial substrate develops which can support increasing durations of AF. This remodeling includes a positive feedback mechanism in which activation of CaMKII phosphorylates RyRs increasing diastolic leak and encouraging further rises in intracellular calcium  who had an increased incidence of post-operative AF  and protecting the atria from excessive calcium loading could potentiate the development of paroxysms of AF via calcium dependant arrhythmias.Overall, paroxysmal AF is associated with increased RyR diastolic leak, increased SERCA function despite unchanged SERCA expression, decreased The duration of the paroxysms of AF tends to prolong until persistent AF develops, defined clinically as episodes of arrhythmia that last more than seven days before returning to sinus rhythm spontaneously, if at all  seen early in response to rapid atrial stimulation continues in response to repeated episodes of AF  might decrease over the time course of the disease  between paroxysmal and persistent AF  maintaining current amplitude. These differences may also explain why some studies report the nadir of ICa(L) is reached quickly before remaining stable, while others reported a slow downward trend inversely proportional to increasing amounts of time in AF  in AF in some species  loss. While t-tubule loss could increase as AF develops and contribute to the progressive loss of ICa(L) the available data does not support this concept. Following 7 days of rapid atrial pacing in the dog t-tubules were reduced by 60%  contributes to the progressive shortening of action potential duration seen in persistent AF , if calcium silencing fails then cytosolic calcium concentrations inevitably rise. This rise activates intracellular signaling pathways such as the pro-fibrotic Wnt pathway via calcium sensitive enzymes such as protein kinase C, CaMKII and calcineurin, and leads to myocyte hypertrophy, fibrosis and atrial dilatation  a failure in the ability of the calcium cycling mechanism to adapt to increased rate, (ii) the presence of pre-existing structural heart disease or, (iii) early loss of atrial t-tubules promoting arrhythmogenic calcium release. Other, extra cardiac factors, may also also determine the vulnerability to AF and include roles for obesity  contributed to by loss of atrial t-tubules, increased SR calcium content despite unchanged or lower calcium reuptake by SERCA, and increased diastolic calcium leak. These effects may be mediated by atrial stretch caused by elevated intra-atrial pressure  has generally been found to decrease in heart failure  has also been found in those with dilated atria and other forms of structural heart disease associated with an increased susceptibility to AF  appears to be due to a reduction in the expression of LTCCs  on the t-tubules that remain in heart failure is also reduced . The decrease in atrial ICa(L) is contributed to by a reduced baseline response to beta stimulation  in heart failure  where inhibiting ICa(L) in control cells reproduces the increase in SR calcium content. Here, decreased ICa(L) can paradoxically increase SR calcium content  and increased SR calcium content interact to give rise to the systolic calcium transient is unclear, with both increased and decreased calcium transient amplitude reported (Table ICa(L) may decrease calcium transient amplitude  or reverse mode INCX which together with increased SR calcium content could increase calcium transient amplitude  and INCX but decreased late INa  and increased late INa  , although pathological remodeling of contractile proteins contributes to this process  in heart failure mirrors that seen in both paroxysmal and persistent AF. SR calcium content increases in heart failure as some studies report in early or paroxysmal AF although this is not found in persistent AF. There is limited data on the effects of heart failure on atrial RyRs but, unlike in AF, heart failure does do not appear to lead to RyR phosphorylation. However, the net effect of these modifications leads to a similar endpoint of increased RyR leak and afterdepolarizations in heart failure and AF. Although the pulmonary veins are the source of triggers for AF in the majority of patients with lone AF (Haissaguerre et al., The abnormalities in atrial calcium handling that occur in response to heart failure have some similarities with those that occur in AF Table . The decA major difference between the AF that occurs alongside heart failure and the early stages of AF that occurs without evidence of overt heart disease is the atrial structure. As we have seen, heart failure leads to atrial dilatation and fibrosis, promoting heterogeneous conduction, wavebreak, and a substrate that supports multiple simultaneous re-entrant circuits. The atrial structural remodeling found in heart failure occurs more rapidly and to a greater extent than in AF without heart failure (Li et al., Overall, the pathophysiology of AF in those with heart failure shares many similarities to the advanced AF seen in those without heart failure. Heart failure patients often present with AF some distance along their journey of atrial remodeling.Although there is strong evidence to support the role of calcium remodeling as a whole in AF and heart failure, some aspects of this process show wide variation in the experimental data which often produces conflicting results. There are a number of potential reasons for the differences in findings, and herein we provide a critical review of these.Obtaining human atrial myocytes to study calcium handling can be performed by using samples of atrial appendage that are routinely discarded following cardiac surgery (Voigt et al., To avoid the limitations of using human tissue, animal models are frequently used which include rodents and large mammals such as dogs, sheep, or goats. However, spontaneously occurring AF in these animal models is rare (Heijman et al., Animal models of AF benefit from the fact that the frequency and duration of arrhythmia can be quantified allowing a time course of remodeling to be determined. However, it is uncertain whether the atrial changes induced artificially by rapid atrial pacing which produce persistent AF within days to weeks are truly comparable to the human condition in which persistent AF typically develops over months to years. Furthermore, the atrial arrhythmias seen in rodents may be a poor comparator for human AF due to the differences in ion channel expression between rodents and man (Gussak et al., The results from animal models of atrial electrophysiology in heart failure may also be confounded by the technique used to induce ventricular dysfunction. Many large animal models use ventricular tachypacing to produce a dilated cardiomyopathy. While this technique is likely to be representative of end stage heart failure, it must be borne in mind that tachycardiompathy accounts for only a minority of the causes of heart failure seen in humans (Mosterd and Hoes, Overall, interpreting the results of studies of calcium handling in AF requires careful attention to the methods used including species, duration of AF, and confounding comorbidities and medications.Calcium plays a fundamental role in the pathophysiology of AF, as well as in the bidirectional relationship between AF and heart failure. Remodeling of calcium cycling in response to rapid atrial stimulation can be seen within days in the form of calcium signaling silencing. However, these initially protective changes eventually become pathological as paroxysmal AF develops and can facilitate the progression to persistent AF. A recurring theme throughout this progression is increased diastolic leak of calcium from RyRs which promotes afterdepolarizations. The rise in intracellular calcium concentrations also activates pro-fibrotic pathways leading to structural remodeling in the atria that produces a substrate that can support the complex re-entry seen in AF. This review has provided a comprehensive stepwise description of the fundamental aspects of calcium cycling remodeling.Heart failure leads to remodeling of calcium cycling and structural changes within the atria which support persistent AF. In return, AF exacerbates heart failure by reducing cardiac output, due in part to a loss of atrial contraction but also by affecting ventricular calcium cycling. A vicious circle ensues whereby one condition begets the other. Questions remain surrounding the complex interaction between AF and heart failure, but the undeniable importance of calcium cycling remodeling in both conditions should prompt the exploration of these mechanisms as potential novel therapeutic targets.ND prepared the first draft of the manuscript, which was critically revised by CP, JC, GM, DE, AT, and KD. JC produced the figures. All authors agree with its final content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The electrocardiogram recorded at presentation  node, but some have also been reported in other structures such as the His-Purkinje system, Kent bundle and the atria. Alternating Wenckebach periods are usually attributed to block at different levels due to transverse dissociation [


Fgf10 and Gdnf leads to lung and kidney agenesis, respectively. However, FGF10 plays a significant role during kidney branching and both the FGF10 and GDNF pathway converge on the transcription factors ETV4/5. Although the involved signalling proteins have been defined, the underlying mechanism that controls lung and kidney branching morphogenesis is still elusive. A wide range of modelling approaches exists that differ not only in the mathematical framework  but also in the spatial scale . Due to advancing imaging techniques, image-based modelling approaches have proven to be a valuable method for investigating the control of branching events with respect to organ-specific properties. Here, we review several mathematical models on lung and kidney branching morphogenesis and suggest that a ligand-receptor-based Turing model represents a potential candidate for a general but also adaptive mechanism to control branching morphogenesis during development.Many organs require a high surface to volume ratio to properly function. Lungs and kidneys, for example, achieve this by creating highly branched tubular structures during a developmental process called branching morphogenesis. The genes that control lung and kidney branching share a similar network structure that is based on ligand-receptor reciprocal signalling interactions between the epithelium and the surrounding mesenchyme. Nevertheless, the temporal and spatial development of the branched epithelial trees differs, resulting in organs of distinct shape and size. In the embryonic lung, branching morphogenesis highly depends on FGF10 signalling, whereas GDNF is the driving morphogen in the kidney. Knockout of  Branching morphogenesis is a common developmental process by which arborized structures with a high surface-to-volume ratio are created. In vertebrate organ development, branching morphogenesis describes how an epithelial organ bud branches into its surrounding mesenchyme. Epithelial-mesenchymal interactions via ligand-receptor signalling are well-established regulators of developmental processes such as growth and patterning . Yet, itEven though the signalling networks relevant to organogenesis have been defined and gene functions and interactions have been studied intensively, it remains unclear how macroscopic features of branched organs, including size, network topology and spatial patterning are encoded. Mathematical modelling has proven to be a valuable method for examining the impact of signalling interactions in developmental processes, such as branching morphogenesis. Consequently, a wide range of modelling approaches have been established that can differ not only in the mathematical framework but also in the spatial scale .In the following, we will focus on the role of fibroblast growth factor (FGF) 10 during branching morphogenesis of the vertebrate kidney and the lung and discuss to which extent mathematical models can help our understanding of branching morphogenesis. Lung and kidney branching has been studied in model organisms other than the mouse and the rat and orthologs of the genes described here have similar roles in other species, but are not further discussed in the context of this mini-review .-/-Fgf10 mice display kidney dysgenesis with impaired ureteric bud (UB) development and medullary dysplasia , a morphogen related to the TGF-beta growth factor family, is critical for kidney organogenesis. The absence of GDNF as well as of its receptor tyrosine kinase RET and co-receptor GFRα1 lead to kidney agenesis or severe hypodysplasia . InteresF Figure    . Ret is  ampulla . Fgfr2 it the UB .Fgf10 does not abolish branching and in the -/-; Sprouty1-/-Gdnf mutants UB branching occurs albeit both Fgf10 and Fgfr2 being expressed uniformly in the MM and the UB, respectively  could also have an impact. Tissue recombination experiments have been used to study epithelial-mesenchymal interactions and how the mesenchyme influences branching morphogenesis and cell fate specification of a branching epithelium .Wnt11, Ret and Pax-2  as key signalling factors Figure  Cellier. In ordeSolving the model on static idealised 2D and 3D domains shows that the described interactions give rise to Turing patterns that correspond to FGF10 or GDNF signalling patterns, respectively, representing the branching modes of lateral branching, bifurcations and trifurcations . As repoTaking into account the dynamical processes during branching morphogenesis, 2D time-lapse movies of cultured lung and kidney explants and a 3D sequence of lung buds have been used to test alternative models and showed that only the ligand-receptor Turing model reproduces the areas of outgrowth for these physiological geometries Figures  Menshyk.The emerging Turing pattern is typically very dependent on the initial conditions. Combining tissue-specific expression of the considered signalling factors with the ligand-receptor based Turing model allowed for robust outgrowth behaviour despite noisy initial conditions due to the impact of the geometry (Therefore, the ligand-receptor based Turing mechanism potentially constitutes a common mechanism for regulating branching morphogenesis in both lungs and kidneys. However, Turing patterns only explain the branch point selection but not the regulation of branch lengths, widths or angles. In the lung, the length and width of branches seems to be controlled by a bias in cell division . MoreoveIn this review, we focused on the signalling interactions during lung and kidney morphogenesis and discussed several modelling approaches. Based on the robustness and flexible applicability to different branched organs, we conclude that the ligand-receptor based Turing mechanism represents a potential candidate for a general regulatory mechanism for branching morphogenesis. However, several studies exist confirming that mechanical stresses influence branching morphogenesis . Since tAlthough mathematical models have advanced our understanding of branching morphogenesis, none have yet proven to fulfil all criteria to explain the branching behaviour on a holistic level. This may be attributed to a lack of biological methods to obtain quantitative information on relevant modelling parameters and computational limitations regarding analysis of large imaging data. The latter is also relevant for solving complex mathematical models, which requires tremendous computational resources. However, combining the constant improvements in all domains will improve the conclusions that can be drawn from modelling approaches. We are hopeful that data based modelling will continue to improve our understanding of branching morphogenesis.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Striatal cholinergic interneurons (CINs) are the main source of acetylcholine in the striatum and are believed to play an important role in basal ganglia physiology and pathophysiology. The role of CINs in striatal function is known mostly from extracellular recordings of tonically active striatal neurons in monkeys, which are believed to correspond to CINs. Because these neurons transiently respond to motivationally cues with brief pauses, flanked by bursts of increased activity, they are classically viewed as key players in reward-related learning. However, CIN modulatory function within the striatal network has been mainly inferred from the action of acetylcholine agonists/antagonists or through CIN activation. These manipulations are far from recapitulating CIN activity in response to behaviorally-relevant stimuli. New technical tools such as optogenetics allow researchers to specifically manipulate this sparse neuronal population and to mimic their typical pause response. For example, it is now possible to investigate how short inhibition of CIN activity shapes striatal properties. Here, we review the most recent literature and show how these new techniques have brought considerable insights into the functional role of CINs in normal and pathological states, raising several interesting and novel questions. To continue moving forward, it is crucial to determine in detail CIN activity changes during behavior, particularly in rodents. We will also discuss how computational approaches combined with optogenetics will contribute to further our understanding of the CIN role in striatal circuits. Choline to CINs . Hence, The improvement of parkinsonian tremor by both dopaminergic agonists and anticholinergic drugs led to the dopamine (DA)-ACh balance hypothesis, where DA and ACh are believed to play opposite roles in the striatum . Even thvia muscarinic receptors (mAChRs): M1 receptors are present on D1 and D2 MSNs and M4 receptors are preferentially expressed by D1 MSNs. Activation of mAChRs modulates an array of voltage-gated channels and intracellular pathways in MSNs. Determining the combinatorial effect of these actions, potentially even opposing each other, is highly challenging and has recently been covered at length by excellent recent reviews  are expressed by interneurons and extrinsic afferent terminals, MSNs respond to ACh exclusively  reviews . A hallm reviews . We can  reviews . HoweverThe widespread excitatory input from the cortex targeting D1 and D2 MSNs sets the activity of the direct and indirect striatofugal pathways which play a fundamental role in movement planning and action selection. Understanding how CINs modulate the dynamics of corticostriatal processing and MSN activity is therefore essential to uncover basal ganglia function. Cholinergic modulation of long-term corticostriatal plasticity has been addressed in excellent reviews  and willvia the inhibition of mAChRs located on the glutamatergic terminals, suggesting the existence of tonic cholinergic presynaptic inhibition  is associated with a decrease in D1 and D2 MSN excitability that might involve a lowering of M1 mAChRs activation  the light parameters  used to manipulate CIN firing are not physiologically relevant, (ii) CINs do not impact basal ganglia-related behaviors in physiological conditions and/or (iii) the behavioral tasks used are not appropriate to reveal CIN functions in rodents. What we know about the function of CINs comes from studies carried out in primates, describing the correlative changes in electrical activity of presumed-CINs during behavior. Optogenetics, mainly applied in rodents for technical reasons, is perfectly suited to go beyond correlational analysis and to investigate the causal implication of CINs in behavior. However, we first need to accurately describe the firing properties of these cells in rodents to be able to manipulate their activity in an appropriate way.in vivo extracellular firing activity  and broad spike waveform (i.e. spike duration > 2 ms). These electrical properties are easily distinguishable from all the other striatal cell populations and represent a good signature of CINs as confirmed later by in vivo juxtacellular labelling , or from unknown origin might also synchronize CIN population . Among tApart from the classical conditioning experiments, the contribution of CIN activity has also been tested during operant tasks. In these experiments, the animal has to execute an action to obtain a reward. These studies have revealed the involvement of CINs in more complex behavioral aspects such as contextual , temporaIn addition, recent works have taken advantage of transgenic ChAT-Cre mice to genetically identify CINs and record their activity with two-photon calcium imaging and fiber photometry, during spontaneous locomotion in head-fixed animals . In doinCINs modulate striatal activity during behavior. A theoretical study of the putative function of these neurons in motor learning and their possible role in pathophysiology through modeling could drive experimentally testable predictions and thereby guide further experimental investigation. Previous modeling efforts involving CINs remain relatively sparse. They range from simulating intracellular and ion-channel dynamics linked to cholinergic signaling to the effect of CIN activity modulation on behavior.On the microscopic scale, two modeling studies have highlighted the tight coupling between DA neurons and CINs due to the dopaminergic modulation of both the intrinsic currents generating tonic firing  and the On the macroscopic scale, the influence of CINs on behavior can be either immediate, due to the modulation of striatal output by CINs, or delayed/persistent, due to ACh-dependent plasticity in the striatal network that leads to long-term changes in striatal response to its external inputs. A recent study by To go beyond this current state of theoretical investigations, one may ask the following questions. What are the respective/specific roles of DA and ACh during learning? How redundant are these signals? Are they separable in time or space? What is the specific motor impairment expected due to the abnormal CIN activity following DA depletion and could some PD symptoms be linked to CIN signaling dysfunction? These questions may be answered by integrating current experimental evidence and DA-ACh interactions and its effect on striatal dynamics revealed by previous theoretical work in a circuit model of the basal ganglia-thalamo-cortical loop. This model may display action selection properties and DA-driven reinforcement learning , as wellOur current understanding of the role of CINs in striatal function derives mostly from extracellular recordings of TANs in monkeys. Because these neurons transiently respond to motivationally relevant cues with brief pauses, flanked by bursts of increased activity, they are classically viewed as key players in reward related learning. However, how CINs, and particularly the pause in their tonic firing, modulate striatal output has yet to be demonstrated. It is also undisputable that CINs play a key role in relation to dysfunctional aspect of basal ganglia information processing such as in PD and it seems important that future works keep dissecting the causal role of CINs in striatal circuits.NMal, AL, NMau, and CB drafted, provided critical revision of the article, wrote, and approved the final version of the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The 20–70% participation of diabetes patients in lifestyle interventions (LSI) worldwide seems to be rather sub-optimal, in spite of all intents of such interventions to delay further progress of the disease. Positive effects through LSI are expected in particular for patients who suffer less from diabetes-related limitations or other chronic diseases. Seeing that diabetes prevalence and with it mortality are increasing, LSI have become an inherent part of diabetes treatment standards. Various qualitative studies have been carried out to identify participation barriers for LSI. However, these have not resulted in more detailed knowledge about the relative importance of factors with an inhibiting impact on participation. Since it cannot be assumed that all of the influencing factors have equivalent values, it is necessary to investigate their individual importance with regard to a positive or negative decision about participating. There are no systematic reviews on patient preferences for LSI programs in diabetes prevention. As a result, the main objectives of this systematic review are to (i) identify existing patient preference elicitation studies related to LSI for diabetic patients, (ii) summarize the methods applied and findings, and (iii) appraise the reporting and methodological quality of such studies.We will perform systematic literature searches to identify suitable studies from 14 electronic databases. Retrieved study records will be included based on predefined eligibility criteria as defined in this protocol. We will run abstract and full-text screenings and then extract data from all selected studies by filling in a predefined data extraction spreadsheet. We will undertake a descriptive, narrative synthesis of findings to address the study objectives, since no pooling for quantified preferences is for methodological reasons implementable. We will pay special attention to aspects of methodological quality of preference elicitation by applying established evaluation criteria of the ISPOR and some own developed criteria for different elicitation techniques. All critical stages within the screening, data extraction, and synthesis processes will be conducted by two pairs of authors. This protocol adheres to PRISMA and PRISMA-P standards.The proposed systematic review will provide an overview of the methods used and current practice in the elicitation and quantification of patients’ preferences for diabetes prevention lifestyle interventions. Furthermore, the methodological quality of the identified studies will be appraised as well.CRD42018086988PROSPERO The online version of this article (10.1186/s13643-018-0884-5) contains supplementary material, which is available to authorized users. Diabetes mellitus is a highly prevalent chronic disease affecting approximately 9 to 10% of the global adult population . DespiteLifestyle interventions (LSI) are a central part of diabetes care, in particular in type 2 diabetes mellitus –9. It haThe choice of particular clinical goals and selection of lifestyle and pharmacologic treatments to reach those goals is a multifaceted process that requires using valid and reliable methods that transparently report on data elicitation processes, potential strengths and shortcomings, and efficiently eliciting patient preferences and values as well by integrating these with evidence of treatment effectiveness as well as provider preferences . VariousPreferences are comparative judgements between entities . In thisHealth preference research is connected with the following assumptions : (i) it In a systematic review to identify the use of preference elicitation methods in healthcare decision-making , studiesThe International Society for Pharmacoeconomics and Outcomes Research (ISPOR) adopted a broad approach for MCDA, including in the consideration of MCDA methods that help deliberative discussions using explicitly defined criteria. A pragmatic definition is given by Mühlbacher and Kaczynski : “MCDA cA preceded feasibility search determined a plethora of studies and some scoping or systematic reviews concerning patient preferences in connection with various medication interventions for diabetes disease –67. It sWhile there are systematic reviews regarding treatment preferences in people with diabetes, a systematic review adressing LSI preferences is mising. The main objective of this systematic literature review is to identify existing studies eliciting patient preferences for LSI programs for diabetes prevention, summarize the methods applied and findings, and appraise their methodological quality. We will review identified studies with regard to their methodical quality to provide an overview of current practices in patient preference elicitation for LSI programs in diabetes prevention reported in the literature and to find out which program aspects are included and how important they are for surveyed patients and professionals. Since pooling preferences elicited with different methods in various samples is not applicable, we will summarize the findings of the identified studies by narrative comparisons.This protocol adheres to the Preferred Reporting Items in Systematic Reviews and Meta-analyses (PRISMA) statement  and PRISWe will include studies reporting quantified elicitation of preferences on lifestyle programs for diabetes prevention with characteristics given in Table The following literature databases will be searched: PubMed (via NLM), MEDLINE (via Ovid), Journals@Ovid (via Ovid), EMBASE (via Ovid), EconLit (via EBSCOhost), Web of Science (via Thomson Reuters), Science direct (via Elsevier), and PsycINFO (via Ovid).To identify potential existing systematic reviews with regard to our research topic, we will also search the Cochrane Library (via Wiley) including the NHS CRD databanks Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment (HTA), and NHS Economic Evaluation Database (NHS EED). We will also include grey literature databases in our search: CINAHL (via EBSCOhost), Catalogue ZB MED (via ZBMed), Current Contents (via ZBMed), DissOnline (via ZBMed), Econis (via ZBMed), and Econbiz (via ZBW) and finally for the German region the “Karlsruher virtueller Katalog” as well.We will not restrict the timeframe of the search nor will we apply any search filters  to guaraThe hits of the systematic literature search will be collected and stored in EndNote . The abstract and full-text screening will be carried out using the EndNote Surface. During the data extraction, we will fill in our own spreadsheet forms. All information will be shared and be made available within the working group consisting of two tandems.We will select studies in several steps. First, we will identify all relevant studies by conducting the search in the scientific publication databases listed in the “During the title/abstract screening, we will collect systematic literature reviews addressing LSI in diabetes prevention as a potential reference source of interest. After the full-text screening, we will export the reference list of all publications identified for the data extraction. Moreover, we will check forward citations of these publications by tracking them in different citation databases. We will apply PubMed’s “related articles” feature for the same papers to collect the first 10 hits. The restricted number of hits is voluntarily chosen to reduce the workload. All references gathered in this step will be subject to the same steps as described in the “Research question and samplePerspective of preference elicitationMethod of preference elicitationMethod of preference quantification and authors’ rationale for its choiceCriteria, attributes, and values of preferencesSubgroup analysis (as pre-specified in the studies)Results of preference quantificationTwo reviewers will independently extract data from all included studies after the full-text review by filling in a predefined data extraction spreadsheet. The data extraction spreadsheet was first piloted. The data extraction spreadsheet is provided in addition to the paper documents Additional file 2:PRISMA-P checklist. (DOCX 37 kb)