Contextual fit is an important variable in the implementation of evidence-based programs (EBPs). The objectives of the current study were to examine the psychometric properties of the adapted Self-Assessment of Contextual Fit (SACF) measure for HIV clinical care settings  and explore how perceptions of contextual fit varied across two different interventions  and 12 clinical sites.We collected SACF-HIV data as part of a larger cross-project implementation science study (ATN 153). The study sample includes 128 clinicians, community health workers, interventionists, adherence counselors, and other members of the prevention and care team who engage in the implementation of EBPs at 12 HIV prevention and clinical care sites in the USA. We assessed the internal consistency of the SACF-HIV using Cronbach’s alpha and examined the sub-dimensionality of the scale with an exploratory factor analysis. To explore concurrent validity, we examined Pearson’s correlation coefficients between the adapted scale and fit-related sub-scale scores from the Evidence-Based Practice Attitudes Scale-50 (EBPAS-50). Variation in perceptions of fit by intervention was examined using descriptive statistics.α=0.895). Factor analyses revealed two sub-scales—one capturing general insights regarding contextual fit, such as perceptions of skill, experience, and alignment with client needs (loadings ranging from .5 to .84), and a second centering perceptions regarding implementation support, such as resources and administrative support (loadings ranging from .89 to .97). Concurrent validity was supported by statistically significant correlations in the expected direction with EBPAS-50 fit-related sub-scales . SACF-HIV mean fit scores varied by intervention and the difference was statistically significant .Internal consistency of the adapted scale was strong (There are relatively few tools assessing perceptions of contextual fit in HIV clinical settings. These results suggest the 12-item adapted SACF is a reliable, valid global assessment of perceptions of contextual fit and implementation support. The SACF-HIV can be used by practitioners and researchers interested in understanding an implementation context when planning to prepare and support EBP implementation.TMI ClinicalTrials.gov NCT03681912; YMPH ClinicalTrials.gov NCT03488914 Contributions to the literatureAdds to the growing body of implementation science research in HIV clinical settingsAdapts a brief measure of perceived contextual fit specifically for HIV clinical settings preparing for the implementation of evidence-based practicesProvides reliability and validity data supporting a brief assessment tool for examining HIV clinical implementation contextsEnding the HIV Epidemic [Despite a fast-growing implementation science literature, the implementation of EBPs into HIV settings, particularly youth-serving HIV clinics, has received less attention . SuccessEpidemic , underscContextual fit, or the match between intervention elements, strategies, and procedures with the values, needs, skills, and resources of those using the intervention , is conscomplexity and compatibility for use in promoting colorectal cancer screening in federally qualified health centers (FQHCs). Few studies have examined fit measures in HIV prevention and care settings or assessed perceptions of fit in this context.A number of measures of contextual fit have been developed and tested in school, mental health, and health care settings. For example, Horner, Salentine, and Albin  developeIn this article, we report the results of modifying the SACF for use in HIV clinical settings (hereafter referenced as SACF-HIV); we chose to adapt SACF because of its focus on a youth-centered evidence-based strategy as well as a call for testing it in other contexts . This reThe aims of this study were to (1) adapt the SACF for use in HIV clinical settings and examine its psychometric properties and (2) explore perceptions of conceptual fit for two evidence-based interventions. Specific study questions include the following: What is the internal consistency reliability and factor structure of the SACF-HIV? What is the concurrent validity of the SACF-HIV scale? How do perceptions of fit vary, if at all, across two evidenced-based HIV prevention interventions? We hypothesized the scale would be adaptable for HIV-related interventions and settings and that it would be positively related to the EBPAS Fit sub-scale given some conceptual overlap in items regarding dimensions such as fit with client needs and treatment philosophy and other sub-scales assessing fit-related constructs, such as the EBPAS Appeal and Organizational Support sub-scales. Finally, we anticipated a negative or no correlation with sub-scales that assessed constructs in opposite ways (Burden) or those that did not include conceptual overlap, such as EBPAS Balance and Limits sub-scales.Data for this study stem from ATN 153 , a largeThe study sample was drawn from 12 sites taking part in Scale it Up. All staff and medical providers at the site at the time of baseline were eligible and invited to take part. The sample included clinicians, community health workers, interventionists, adherence counselors, and other members of the prevention and care team who engage in the implementation of evidence-based programs; administrative and research staff with key decision-making roles  who provide input on prevention and care services and site operations; and a clinical leader (“Site PI”) to represent the organizational leadership perspective.We assessed perceived contextual fit by adapting a version of the SACF in school measure developed by Horner and colleagues . As partFit with the values and needs of the client and clinician (α = .88) [Balance of skills and the role of science in treatment (α = .79), time and administrative Burden associated with learning EBPs (α = .77), EBP Limitations and their inability to address client needs (α = .92), Appeal of EBP (α = .80), and Organizational Support for learning an EBP (α = .85). Scale ratings ranged from 0  to 4 (very great extent). Cronbach’s alphas reported above reflect those of the scale authors [Balance) to .91 (Fit) [We assessed attitudes toward evidence-based practices using the EBPAS-50 . For thiα = .88) , the Bal authors ; in the 91 (Fit) .At baseline, each participating ATN site provided a list of the identified key stakeholders, including names, contact information (phone number and email), and role(s) within the clinic. These potential participants then received an enrollment email that invited them to join the EPIS study and provided consent information and instructions on joining. Potential participants who did not respond to the initial invitation received reminder emails every 2 weeks throughout the baseline study period to encourage participation. EPIS study enrollment included both qualitative interviews (conducted first) and quantitative surveys (sent following the interviews). We used Qualtrics to collect all survey data, with each participant receiving a unique survey link that included a personal and site identifier. Those not completing the survey within 2 weeks received multiple reminders during the data collection period.For aim 1 (psychometric properties), we assessed the internal consistency of the SACF-HIV for the TMI intervention only using Cronbach’s alpha and examined the sub-dimensionality with an exploratory factor analysis using principal axis factoring with Promax oblique rotation. Criteria for item inclusion on a factor included primary loadings above 0.40 and cross-loadings below 0.30 .Fit, Balance, Burden, Limits, Appeal, Organizational Support) for TMI only using Pearson’s correlation coefficient. For aim 2 (variation in perceptions of fit by intervention), we generated descriptive statistics summarizing the SACF-HIV items and the overall scale scores by EBP (TMI and YMHP). All analyses used SPSS statistical software .To explore concurrent validity, we examined the correlations between the SACF-HIV  and six existing sub-scale scores from the EBPAS-50 (N=128) were, on average, 40.90 years old (SD = 11.17). Most  identified as female and Black/African American  or White . Most  had attained advanced educational degrees  and reported an average of 7.04 years (SD = 6.25) experience in their current role and 11.02 years (SD = 7.69) in the field of HIV. Most (81%) were clinical staff ; the remaining participants were administrative staff (18.8%). About a third  had a caseload exceeding 90 patients, regardless of position, while about 14% reported not having a caseload. The study sample represents 49.6% of those contacted to join the study at baseline (baseline data collection occurred from June 2017 through June 2018).Participants  and a second factor with three items related to Implementation Support (loadings ranged from .89 to .97 for resources and administrative support). The variance explained by the final 2-factor solution was 62.44%. Cronbach’s alphas for the sub-scales were 0.877 for General Fit and 0.910 for Implementation Support.Cronbach’s alpha for the overall SACF-HIV TMI scale was 0.895 Table . The ini Fit , Appeal , and Organizational Support  and negatively and significantly correlated with EBPAS-50 Burden . It was not significantly correlated with EBPAS-50 Balance (r = .17) or Limitations (r = −.11).Table eneral Fit sub-scale followed a similar pattern to the overall SACF-HIV TMI .Correlation analyses with the SACF-HIV TMI GMI Table . The SACp < 0.05).Table The primary objectives of the current study were to examine the psychometric properties of the SACF-HIV, an adapted Self-Assessment of Contextual Fit measure for HIV clinical care settings, and explore variations in perceptions of contextual fit across two different types of interventions . The internal consistency of the overall scale and the two sub-scales were well within the range considered acceptable . One subGeneral Fit sub-score of the SACF-HIV TMI correlated positively with the EBPAS-50 Fit score; both sub-scales share conceptual overlap regarding perceptions of fit with client needs, clinical approaches, and treatment philosophy. The EBPAS-50 Fit sub-scale assesses other dimensions of fit, such as autonomy in using an EBP and perceptions of clients’ acceptance, which is reflected in the moderate correlation between the two measures. We found a small negative association with the EBPAS-50 Burden sub-scale; both scales assess burden, but in slightly different ways and using opposite valances . Similarly, we hypothesized that there would be little relationship between SACF-HIV and the EBPAS-50 Limits and Balance sub-scales for TMI given the lack of conceptual overlap between the measures, and this was confirmed. One may have anticipated a stronger positive correlation between the SACF-HIV Implementation Support sub-scale and the Organizational Support sub-scale on the EBPAS; however, the focus of the items varied somewhat—the SACF-HIV Implementation Support items centered on perceived clinic support whereas the EBPAS-50 Organizational Support items focused on individuals’ desire to learn the evidence-based intervention given organizational supports.Concurrent validity was supported by significant correlations in the expected direction with well-established fit or fit-related sub-scales as measured by the EBPAS-50 and no relationships with others. As an example, the overall score and the One of the challenges of assessing contextual fit is the lack of accord and research on core elements of contextual fit. Horner, a lead developer of the SACF, and colleagues  identifySome of these elements, such as precision, were provided as part of data collection (in the description of TMI and YMHP), but we did not assess perceptions of the extent to which implementers felt the core features of the interventions were well defined and this would vary based on the degree of familiarity individuals had with the interventions. Assessing perceptions regarding clarity of core components could provide guidance on further intervention refinement, training, and support. Furthermore, the assessment of cultural relevance is relatively limited and focuses on values alignment around how youth should be treated and what might be in their best interest; measurement of this element could delve into assessing other aspects of cultural relevance, such as social and emotional relevance, the extent to which the interventions are affirming of all young people, and the extent to which the interventions accommodate differing cultures and learning differences. Data on these aspects of cultural fit could be beneficial for intervention selection or refinement  and might also guide training and support during a preparation phase to ensure implementers are able to describe and promote critical intervention features to recruit and engage young people. The SACF-HIV we examined also does not assess perceptions of the evidence supporting the intervention—rather, this information was provided as part of the description of the intervention strategy. Measuring perceptions of an intervention’s underlying evidence could inform users at the exploration and adoption phases of implementation as well as the implementation phase .We found that the SACF-HIV captures variations in contextual fit by EBP strategy, which may be an important consideration for shaping training and support plans to scaffold EBP uptake and implementation. As an example, participants rated perceived comfort with delivering key components of TMI more highly than YHMP, and perceived comfort ratings aligned with perceived ratings of skills to implement the two interventions. Motivational interviewing (MI) may be more familiar to stakeholders given it is a client-centered approach that can be incorporated into clinical settings in a variety of ways . It alsoAssessing contextual fit has relevance across phases of intervention development and implementation. During intervention development, data on fit  could lead to refinements that boost proximal implementation outcomes such as acceptability , 16. At Other tools support the notion of examining fit early, such as the Hexagon Discussion and Analysis tool . Once a The study sample was relatively small, from an intervention study and restricted to key stakeholders from 12 clinics across the USA that generally represent academic partnerships who are likely more open to innovation due to their university affiliation. Insights regarding variation in perceptions of fit by intervention type are limited by the small sub-sample of clinics (three) that were selected to implement YMHP. Because of the small sample, we were unable to explore how, if at all, the factor structure of the instrument differed for that intervention compared to TMI. This study was restricted to baseline assessments only and we did not examine the association of fit with implementation or programmatic outcomes, an important focus for future studies. Nevertheless, the study contributes important data and insights regarding an adapted tool for assessing perceptions of contextual fit for use in HIV prevention and clinical care settings that serve adolescent populations.This study examined baseline perceptions of contextual fit in selected HIV prevention and treatment settings. Additional research could explore how, if at all, perceptions of fit change over time and what factors contribute to such changes. Further research is also needed on how changes in conceptual fit perceptions impact key EBP implementation or effectiveness outcomes, and what strategies might be effective in boosting fit . Finally, future research could also examine the generalizability of these findings in other HIV prevention and clinical care settings, such as those who serve adult populations.There is also a continuing need for research that examines and clarifies the multiple domains of contextual fit to guide implementation supports and policy in HIV clinical settings. This research would support further refinement and/or development of contextual fit measures.Contextual fit is an important variable in the implementation of evidence-based programs (EBPs), yet there are relatively few tools assessing perceptions of fit in HIV clinical settings. This study contributes to the literature by assessing an adapted measure of contextual fit originally developed by Horner and colleagues  for scho


Still a male-dominated field, science has often undervalued the work of women. We are not equally represented at higher career levels and face more professional obstacles than our male counterparts. One among many examples, Rosalind Franklin's name is the first that come to mind. In the discovery of the DNA double helix, despite her crucial X-ray diffraction work, she never got the same recognition as her male counterparts Watson and Crick .Only 12 women have won the Nobel Prize in Physiology or Medicine since 1901. The first one was Gerty Cori, in 1947, for elucidating pathways of glucose metabolism. She shared the award with her husband, Carl Cori. Albeit working together, her salary was much lower than his One of the most renowned scientists in history, neurobiologist Rita Levi-Montalcini had to deal with discrimination against women in science, widespread antisemitism and a father who was not supportive of women's education. Despite all these challenges, she won a Nobel Prize in 1986, with Stanley Cohen, for their discoveries of growth factors Women in psychiatry” of the Frontiers in Psychiatry aims to promote valuable works of scientist women in the field. This edition features two studies on pharmacotherapy of psychosis spectrum disorders (PSD), including schizophrenia—one research report that discussed the long-term pharmacotherapy patterns of patients with PSD and one original research which evaluated epigenetic age and DNA methylome in patients treated with the atypical antipsychotic clozapine; one systematic review on the antidepressant escitalopram and one clinical original research article and another pre-clinical original research article, both on molecular targets of drugs of abuse.Although the opportunities for women scientists are better now, there is still a long way to go before gender equality in science is fully achieved. This second edition of “Maric et al., “Maintenance therapy of psychosis spectrum disorders in a real-world setting: Antipsychotics prescription patterns and long-term benzodiazepine use,” the authors set out to shed light on the real-world prescribing pattern in PSDs in the Western Balkans, where many psychotropic medications are fully reimbursed. Guidelines to treat PSDs recommend monotherapy in detriment to polypharmacy. The authors found a high rate of antipsychotic polypharmacy (42.7%), especially in males. Benzodiazepines were the most common add-on prescribed drugs and clozapine the most prescribed antipsychotic. The study alerts about the health risks of long-term AP polypharmacy associated with BDZ.In the study of Pérez-Aldana et al., “Clozapine long-term treatment might reduce epigenetic age through hypomethylation of longevity regulatory pathways genes,” the authors investigated epigenetic age—a biomarker of aging associated with age-related conditions and diseases—and DNA methylome in patients from Mexico treated with clozapine compared with drug-naïve patients. Clozapine induced a higher proportion of hypomethylated CpG sites in the blood compared to hypermethylated ones. Pathways enriched at hypomethylated sites included the longevity regulatory pathway, which interacts with AMPK and insulin signaling pathways. In addition, clozapine reduced the epigenetic age. Altogether, they suggested that long-term clozapine treatment might increase the life expectancy in schizophrenic patients treated with this drug.In fact, monotherapy is usually preferred to treat PSD, with clozapine being the first-line agent in drug-resistant schizophrenia . In the Eichentopf et al. in “Systematic review and meta-analysis on the therapeutic reference range for escitalopram: Blood concentrations, clinical effects and serotonin transporter occupancy” performed a systematic review and meta-analysis on the therapeutic reference range for escitalopram (ESC), the active enantiomer of citalopram. ESC binds with high affinity to the serotonin transporter and belongs to the class of selective serotonin reuptake inhibitors (SSRI), used to treat depression, anxiety, obsessive compulsive disorder and panic attack (c attack . The autSubstance use disorders (SUD) result in repeated relapses due to structural and functional brain alterations that persist even after periods of abstinence . ApproveVamvakopoulou et al. in “Selective D3 receptor antagonism modulates neural response during negative emotional processing in substance dependence” investigated the effects of a dopamine D3 receptor (D3R) antagonist—GSK598809—on the neural response to negative emotional processing in individuals with AUD and SUD . Blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) was used to assess brain function during an evocative image task, following administration of GSK598809 or placebo. The study showed that the D3R antagonist modulated relevant brain circuitry and may have restored the hypodopaminergic function observed in drug addiction  subregion of the prefrontal cortex (PFC) that is partially similar with that reported for longer cocaine IV-SA. Elevated Homer2a/b and Akt1 activation in the PL-PFC seems to be common biochemical correlates of incubated cocaine-craving across IV-SA models, highlighting a role for Akt1 signaling in the PL-PFC in the incubation of cue-induced cocaine craving. The study also demonstrated that incubated cocaine craving is associated with activated CaMKII within the PL-PFC.Cue-induced drug craving is a well-known characteristic of addiction that intensifies (incubates) during protracted withdrawal, leading to relapse . AlthougRC wrote the editorial. RJ and AS contributed to the review of the editorial. All editors edited the Research Topic.


Decades of effort have yet to deliver an efficacious vaccine for pathogens such as HIV, despite major revelations about HIV antigens and development of new vaccine platforms. Innovative outside-the-box approaches are needed to overcome immunological roadblocks to the success of past and current vaccine regimens. We discovered that natural killer (NK) cells are a potent obstacle to vaccine success through perforin-dependent suppression of activated CD4 T cell responses early after vaccination. This killing reduces the quantity and quality of antibody responses. Yet, the features of NK cells that enable this activity or determine susceptibility of specific subsets of T cells remain ill-defined.We used transgenic virus-specific mouse CD4 T cells to detect very early changes in responding T cells immediately proximal to immunoregulatory killing by NK cells. Transgenic T cells were seeded in recipient mice, depleted or not of NK cells, and then infected with lymphocytic choriomeningitis virus. At early time points post-infection, these T cells were isolated for single-cell RNA-sequencing or high-dimensional flow cytometry analyses. These data revealed transcriptomic and proteomic features of subsets of activated CD4 T cells whose presence was enabled or enhanced by the absence of NK cells.Perforin-dependent killing of CD4 T cells by NK cells is measurable on days 2 and 3 post-infection. Analysis of the pool of responding T cells on day 3 revealed phenotypic skewing away from follicular helper T cell-type responses to Th1-type responses in the presence of NK cells. Single cell RNA sequencing and high-dimensional spectral flow cytometry reveal clusters of T cells that are markedly enriched in the absence of NK cells.We created a model system permitting characterization of the population of T cells targeted by NK cells after infection or immunization. Elucidation of features of the targeted T cells will provide mechanistic insights in the receptor-ligand system enabling this biology, improve our understanding of how NK cells influence the pool of responding T cells, and facilitate discovery of the corresponding subset of NK cells responsible for this activity. These discoveries are likely to facilitate development of methods to enhance vaccine responses.All Authors: No reported disclosures.


Phylodynamic methods reveal the spatial and temporal dynamics of viral geographic spread, and have featured prominently in studies of the COVID-19 pandemic. Virtually all such studies are based on phylodynamic models that assume—despite direct and compelling evidence to the contrary—that rates of viral geographic dispersal are constant through time. Here, we: (1) extend phylodynamic models to allow both the average and relative rates of viral dispersal to vary independently between pre-specified time intervals; (2) implement methods to infer the number and timing of viral dispersal events between areas; and (3) develop statistics to assess the absolute fit of discrete-geographic phylodynamic models to empirical datasets. We first validate our new methods using simulations, and then apply them to a SARS-CoV-2 dataset from the early phase of the COVID-19 pandemic. We show that: (1) under simulation, failure to accommodate interval-specific variation in the study data will severely bias parameter estimates; (2) in practice, our interval-specific discrete-geographic phylodynamic models can significantly improve the relative and absolute fit to empirical data; and (3) the increased realism of our interval-specific models provides qualitatively different inferences regarding key aspects of the COVID-19 pandemic—revealing significant temporal variation in global viral dispersal rates, viral dispersal routes, and the number of viral dispersal events between areas—and alters interpretations regarding the efficacy of intervention measures to mitigate the pandemic. Phylodynamic methods encompass a suite of models for inferring various aspects of pathogen biology, including: (1) patterns of variation in demography through time , 2016;  or amongThese phylodynamic methods adopt an explicitly probabilistic approach that model the process of viral dispersal among a set of discrete-geographic areas . The obsThe vast majority  extend discrete-geographic phylodynamic models to allow both the average and relative dispersal rates to vary independently across pre-specified time intervals; (2) enable stochastic mapping under these models to estimate the number and timing of viral dispersal events between areas, and; (3) develop statistics to assess the absolute fit of discrete-geographic phylodynamic models to empirical datasets. We first validate the theory and implementation of our new phylodynamic methods using analyses of simulated data, and then provide an empirical demonstration of these methods with analyses of a SARS-CoV-2 dataset from the early phase of the COVID-19 pandemic.phylogenetic model that allows us to estimate a dated phylogeny for the sampled viruses, biogeographic model that describes the history of viral dispersal over the tree as a continuous-time Markov chain. For a geographic history with Phylodynamic models of dispersal include two main components : a phyloll areas .both the relative and average dispersal rates to vary independently across two or more pre-defined intervals. Here, we describe how to compute transition probabilities, perform inference, simulate histories, and assess the absolute fit of the interval-specific models.We could specify alternative biogeographic models based on the assumed constancy of the dispersal process. For example, the simplest possible model assumes that the average dispersal rate, ic model  that assic model  might alThe transition-probability matrix, Under a constant-rate model, the transition-probability matrix for a branch is al rates —which alUnder a phylodynamic model with interval-specific relative dispersal rates —which alWe combine the two approaches described above to compute transition-probability matrices under an interval-specific model that allows both the average dispersal rate and the relative dispersal rates to vary independently among intervals. Let a given branch span puted as:vl=∑p=1nμequation , and appequation  as normaBEAST source code to implement the above equation for computing We modified We estimate parameters of the interval-specific models within a Bayesian statistical framework. Specifically, we use numerical algorithms—Markov chain Monte Carlo (MCMC) simulation—to approximate the joint posterior probability distribution of the phylodynamic model parameters—the dated phylogeny, We have also implemented numerical algorithms—stochastic mapping—to simulate histories of viral dispersal under the interval-specific models; these methods allow us to estimate the number of dispersal events between a specific pair of areas, the number of dispersal events from one area to a set of two or more areas, and the total number dispersal events among all areas. Stochastic mapping—initially proposed by Nielsen —is commoLet a given branch start at time To simulate the states at each time point, we first compute a transition-probability matrix for each interval:Second, we simulate histories within each interval. For a given time interval, we simulate histories conditional on the start and end states generated in the first step using the uniformization algorithm described by relative fit of two or more candidate phylodynamic models to a given dataset using Bayes factors; this requires that we first estimate the marginal likelihood for each model (which represents the average fit of a model to a dataset), and then compute the Bayes factor as twice the difference in the log marginal likelihoods of the competing models . Comparing the fit of these competing phylodynamic models to the data offers two benefits: (1) confirming that our inference model adequately describes the process that gave rise to data will improve the accuracy of the corresponding inferences , and; (2) comparing alternative models provides a means to objectively test hypotheses regarding the impact of events on the history of viral dispersal . We can assess the g models .absolute fit of a candidate model using posterior-predictive assessment  the tipwise-multinomial statistic. The parsimony statistic is calculated as the difference in the parsimony score for the observed areas and the simulated areas across the tips of the tree . We compute parsimony scores using the parsimony function in the R package, phangorn  We calculate these summary statistics for phangorn ; (2) we We performed a simulation study to explore the statistical behavior of the interval-specific phylodynamic models. Specifically, the goals of this simulation study are to assess: (1) our ability to perform reliable inference under interval-specific models; (2) the impact of model misspecification, and; (3) our ability to identify the correct model. To this end, we simulated 200 geographic datasets under each of two models: the first assumes a constant Interval-specific phylodynamic models are inherently more complex than their constant-rate counterparts, and therefore contain many more parameters that must be inferred from geographic datasets that contain minimal information; these datasets only include a single observation . These considerations raise concerns about our ability to reliably estimate parameters of interval-specific phylodynamic models. Encouragingly, when the inference model is correctly specified  and established a global distribution—with reported cases in his date —despite his date . This cry period , orange.c 71μ4Q; , dashed c 71μ4Q;  to the eIn addition to revealing differences in the global viral dispersal rate, our interval-specific phylodynamic models allow us to explore how relative dispersal rates vary through time. Specifically, our analyses allow us to identify the dispersal routes by which the SARS-CoV-2 virus achieved a global distribution during the early phase of the COVID-19 pandemic. We focus on dispersal routes involving China both because it was the point of origin, and because it was the area against which travel bans were imposed. Inferences under the constant-rate , thereby enhancing the accuracy of our epidemiological inferences based on these models.Phylodynamic methods increasingly inform our understanding of the spatial and temporal dynamics of viral spread. The vast majority of discrete-geographic phylodynamic studies assume—despite direct (and compelling) evidence to the contrary—that disease outbreaks are intrinsically constant:  average  or relatrelative  dispersaOur simulation study demonstrates that (in principle): (1) we are able to accurately identify when phylodynamic models are correctly specified, overspecified, or underspecified ; (2) wheboth the global rate of viral dispersal and the relative rates of viral dispersal vary over four distinct intervals) significantly improves the relative and absolute fit to our study dataset compared to constant-rate phylodynamic models : (1) our interval-specific phylodynamic model  intervals.Our interval-specific models promise to enhance the accuracy of phylodynamic inferences not only by virtue of their increased realism, but also by allowing us to incorporate additional information (related to events in the history of disease outbreaks) in our phylodynamic inferences. The ability to incorporate independent/external information is particularly valuable for phylodynamic inference—where many parameters must be estimated from datasets with limited information—which has also motivated the development of other innovative phylodynamic approaches for incorporating external information . The potImportantly, comparison of alternative phylodynamic models provides a powerful framework for testing hypotheses about the impact of various events  on viral dispersal dynamics. Our empirical study allows us, for example, to assess the impact of domestic mitigation measures imposed in the Hubei province of China. This simply involves comparing the relative fit of our data to two candidate phylodynamic models: independently across intervals. For example, under an alternative scenario for either the relative and/or average rates of viral dispersal.We have focused on interval-specific models where each interval involves a change in both the average and relative dispersal rates. For example, the scenario depicted in Nevertheless, this flexibility comes at a cost: interval-specific models are inherently more complex than their constant-rate counterparts, with many parameters that must be estimated from minimal data . Accordingly, careful model selection and validation is necessary to avoid specification of an over-parameterized model  model realism, incorporating additional information, and providing a powerful and flexible means to test alternative models/hypotheses—our phylodynamic methods will greatly enhance our ability to understand the dynamics of viral spread, and thereby inform policies to mitigate the impact of disease outbreaks.msac159_Supplementary_DataClick here for additional data file.


Transcranial alternating current stimulation (tACS) is a non-invasive brain stimulation technique allowing to induce changes in oscillatory activity. Theta activity has been reported to play a major role in maintenance of information in working memory (WM).The current study had the initial goal to check the effect of theta tACS on accuracy and resting state EEG in a set of match-to-sample WM tasks.In the first experiment, we tested 31 participants in the WM task after 20-min tACS applied at Fpz and CPz at 6 Hz, 1 mA.). In the second experiment, we compared the after-effects and online effects of the stimulation in a sample of 25 individuals. Five similar 25-min blocks filled with the same working memory task were distributed over 3 days. We assessed the same group of participants in all three sessions. On the Training day, the participants performed one block without stimulation. On the Sham-Verum day (SV), the first block with Sham stimulation followed by the second block with Verum stimulation. On the Verum-Sham day (VS), the blocks order reversed.After-effects of the stimulation did not produce any significant changes either in behavior (accuracy in the task) or resting-state EEG (theta frequency band spectral power in the first experiment. In the second experiment, 6 Hz tACS delivered before the WM task was not able to produce any observable changes in working memory performance. The same hold true for online stimulation.Theta frequency tACS applied to Fpz-CPz electrodes is not an efficient method to improve WM.


This paper explores the extent of community-level stock-out of essential medicines among community health workers (CHWs) in low- and middle-income countries (LMICs) and identifies the reasons for and consequences of essential medicine stock-outs.A systematic review was conducted and reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Five electronic databases were searched with a prespecified strategy and the grey literature examined, January 2006–March 2021. Papers containing information on (1) the percentage of CHWs stocked out or (2) reasons for stock-outs along the supply chain and consequences of stock-out were included and appraised for risk of bias. Outcomes were quantitative data on the extent of stock-out, summarized using descriptive statistics, and qualitative data regarding reasons for and consequences of stock-outs, analyzed using thematic content analysis and narrative synthesis.p < 0.01) higher rate than the health centers to which they are affiliated . A comparison of the period 2006–2015 and 2016–2021 showed a significant (p < 0.01) increase in CHW stock-out level from 26.36% [CI 95%: 26.22–26.50] to 48.65% [CI 95%: 48.02–49.28] while that of health centers increased from 7.79% [95% CI 7.16–8.42] to 14.28% [95% CI 11.22–17.34]. Distribution barriers were the most cited reasons for stock-outs. Ultimately, patients were the most affected: stock-outs resulted in out-of-pocket expenses to buy unavailable medicines, poor adherence to medicine regimes, dissatisfaction, and low service utilization.Two reviewers screened 1083 records; 78 evaluations were included. Over the last 15 years, CHWs experienced stock-outs of essential medicines nearly one third of the time and at a significantly (Community-level stock-out of essential medicines constitutes a serious threat to achieving universal health coverage and equitable improvement of health outcomes. This paper suggests stock-outs are getting worse, and that there are particular barriers at the last mile. There is an urgent need to address the health and non-health system constraints that prevent the essential medicines procured for LMICs by international and national stakeholders from reaching the people who need them the most.The online version contains supplementary material available at 10.1186/s12960-022-00755-8. Community health workers (CHWs) are paraprofessionals or lay individuals based in the community who provide health services to urban and rural communities . The rolUnderstanding the extent of, reasons for and consequences of stock-outs of essential medicines at the community level is critical for optimizing CHW programs . Some prWe conducted a systematic review of published and grey literature on the extent of, causes of, and consequences of stock-outs at the CHW level and report it here in line with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . Where lThe initial search was conducted between August and September 2018 and updated in March 2021. We reviewed peer-reviewed articles and grey literature published in four electronic databases: [A team of supply chain (SC) experts was engaged in soliciting evidence-based, unpublished literature. Grey literature was accessed by requesting documents and reports from supply chain organizations . Different platforms, such as the Interagency Supply Chain Group (ISG), Child Health Task Force (CHTF), International Association of Public Health Logisticians (IAPHL), the Community Health Community of Practice, etc., were also approached.“the complete absence of a required medicine at a storage point or delivery point for at least 1 day.” A study was included if it contained (a) information on the number or percentage of CHWs stocked out or (b) reasons for CHW stock-outs or (c) consequences of CHW stock-out.In this study, we defined CHWs as paraprofessionals or lay individuals based in the community who provide health services to urban and rural communities . SeveralRelevant data were systematically extracted from selected studies and tabulated in a Microsoft Excel spreadsheet. The following information was extracted from each study: (1) Study characteristics  Quantification of stock-out (including number and percentage of CHWs or HFs stocked out), (3) Reasons for stock-out and (4) consequences of stock-out.The Risk of Bias in Non-Randomized Studies of Interventions (ROBINS-I) assessment tool , 20 was This tool comprises seven bias domains: (1) confounding; (2) selection of participants; (3) classification of intervention; (4) deviation from interventions; (5) missing outcome data; (6) measurement of outcomes; and (7) selection of reported result overall. Risk of bias was rated as: ?—no information; 1—low risk; 2—moderate risk; 3—serious risk; 4—critical risk; and not applicable (N/A)—if the study did not consider the criterion. A study was rated low risk of bias if it was ranked low for all domains; at moderate risk if ranked moderate in at least one domain; at serious risk of bias if it was ranked serious in at least one domain; and at critical risk of bias if it was ranked critical in at least one domain. Overall assessment for any single study was not less severe than the most severe assessment allocated for a single domain for that study.Qualitative studies relevant to the two themes (reason for and consequence of stock-out) were rated using the GRADE Confidence in the Evidence from Reviews of Qualitative research  approach to ascertain the confidence level that can be placed in the findings . Rating Two review authors independently rated all the included studies and resolved any disagreement by reciprocal consulting .We analyzed only articles published since 2006 to ensure that findings are contemporarily relevant. This cutoff date provides 15 years of data to analyze and corresponds to the period in which CHWs gained renewed attention following the WHO declaration of health workforce shortage .We compared the stock-out rate in the period 2006–2015) before the declaration of sustainable development goals (SDG) and after (2016–2021) to assess quantitative changes in the extent of stock-out. Descriptive statistics of stock-out rates among CHWs and health centers were computed based on the numbers reported by various studies. A two-sample test of means was applied to determine whether a statistical link existed between the CHW and health center stock-out rates over the entire period and for each of the two entities  over the pre and post SDG era  in stock-out levels between CHWs and health centers for the entire review period and each of CHWs and health centers between the two comparison periods  of included papers with qualitative findings described procurement challenges as reasons for stock out  of included papers with qualitative findings described distribution challenges as reasons for stock-out  of included papers with qualitative findings describing storage challenges as reasons for stock-out at CHW and health center levels  of included papers with qualitative findings described challenges relating to community-level stock management as reasons for stock-out  from case referrals that CHWs couldn’t treat due to stock-out , 30, 51.Fifteen articles considered the consequences of stock-out on CHWs to include demotivation from community members’ complaints about lack of medicines , 80, 81 Stock-out at national levels led to CHWs experiencing a long wait for medicine supply after training , therebyTwenty-six papers considered the consequences of stock-out on end-users. Community-level stock-outs meant end-users were not offered services and were referred to the health center by CHWs , 73, 85.Some end-users resorted to alternative vendors   includinUsing ROBINS-I, 12 of the 28 studies with quantitative findings were evaluated to have a serious overall risk of bias, nine moderate, six low and one had no information for assessing risk of bias.As shown in  higher stock-out rate among CHWs (28.93%) in comparison to the health centers to which they are affiliated (9.17%). This may be explained by the fact that CHWs are often not included in health worker registries used for supply forecasting [This review explores the extent, reasons, and consequences of community-level stock-out to provide evidence to guide last-mile supply chain strengthening. It shows that most studies focused primarily on sub-Saharan African countries, perhaps reflecting the popularity of community case management approach or uneven burden of stock-out in sub-Saharan Africa. This review indicates that CHWs were out of stock nearly one third of the time and shows a significantly (ecasting  or are lecasting .Of note, the health center stock-out rate from our review is lower than rates of between 45.60% and 70.60% documented by WHO in their study of public health sector health facilities across 36 LMICs in 2008 . The lowWorrisome, however, is the increase in stock-out rate during the period 2016–2021 at both CHW and health center levels, despite growing political commitment to CHWs. Increased commitment must translate to tangible interventions and policy reviews to address all the health and non-health system challenges contributing to stock-outs. If left unattended, it could cause severe setbacks to the universal health coverage and equitable achievement of post-2015 agenda.This review notes problems relating to distribution as a significant cause of stock-out in LMICs and acknowledges that the private sector could play key roles in strengthening this portion of the supply chain . NonetheWe contacted several professionals to access unpublished literature to enrich our findings. That said, the included studies have used various reporting metrics to present stock-outs at the CHW and health center levels which limited possibilities for analysis. Standard criteria for reporting stock-out are recommended for future studies.While we included only articles published from 2006 to 2021, we compensated for this by comparing our findings with the extent of stock-out and common themes in the reasons for and consequences of stock-out in the period pre-dating 2006 and found no remarkable variance (Additional file Community-level stock-out of essential medicines constitute a serious threat to universal health coverage and equitable improvement of health outcomes. This evidence suggests they are getting worse, rather than better and that CHWs and end-users are disproportionately affected. To ensure equitable and sustainable access to essential medicines, there is a need for intensive remediation of the barriers outlined.Additional file 1. Appendices 1–7.


Stroke incidence and prevalence are increasing, causing substantial socioeconomic burdens and loss of healthy life-years worldwide . Timely Establishing novel therapeutics to promote the recovery and management to avoid complications along with adequate rehabilitation in acute and subacute phases is important because the time window for recovery after stroke is limited . After aThe Research Topic “Transitional and Long-term Continuous Care & Rehabilitation After Stroke” included 14 manuscripts, ranging from a study protocol, case report and observational study to randomized controlled trials, a mini-review, systematic review, and meta-analysis. These timely contributions offer an interesting insight from acute rehabilitation to transitional and long-term care in patients with stroke.Wang et al. conducted a randomized controlled trial investigating the effect of early out-of-bed mobilization within 48 h of stroke symptom onset compared to the conventional rehabilitation group in the unique patient group who underwent mechanical thrombectomy. In this study, early mobilization reduced non-fatal complications 3 months post-stroke and improved the activities of daily living at 3 months and 1-year post-stroke, although the effects on mortality and mRS were comparable with that of conventional rehabilitation.Effective rehabilitation and medical management to promote functional recovery and reduce complications are important, and three related papers in this area were presented. A large randomized controlled trial (AVERT) showed that early mobilization within 24 h of stroke onset was feasible but not effective in terms of favorable outcomes defined as a modified Rankin Scale (mRS) score of 0–2 at 3 months post-stroke . HoweverChang M. et al.  in their systematic review and meta-analysis of five studies.Dysphagia is a common impairment after stroke that is considered important due to its association with the incidence of pneumonia as confirmed by Mele et al. retrospectively analyzed the data of 413 patients with stroke from a single neurorehabilitation center and reported that statin therapy was not associated with recovery and functional outcomes but associated with a lower incidence of bone fractures.Statins have a potential to promote recovery after stroke by their possible pleiotropic effects; however, their results in human studies are controversial –13. MeleGeed et al. reported screening and enrollment data from the Critical Periods After Stroke Study (CPASS), which recruited patients within 30 days of stroke ” aiming to expedite home discharge after stroke by providing adequate management and rehabilitation services after discharge in the patient's home. ESD has been known to reduce long-term dependency and length of the hospital stay; however, most studies on ESD have been conducted in western countries . MoreoveKim et al. conducted a face-to-face cross-sectional survey to investigate the unmet needs for rehabilitative management in common health-related problems after stroke. Approximately half of the respondents reported at least one unmet need, and the total number of unmet needs was significantly associated with a poor health-related quality of life after adjusting for age, sex, and mRS.Stroke survivors can have long-term stroke-related impairments or complications associated with poor quality of life. Zhang et al. conducted a multicenter randomized controlled trial to investigate the effect of dry needling at the myofascial trigger point  on hand spasticity in 210 patients with chronic stroke. The dry needling demonstrated significantly better immediate spasticity relief than sham needling and control groups, and the spasticity relief persisted at 4 weeks after baseline. Fan et al. presented a randomized, double-blind, sham-controlled trial protocol to investigate the short-term effects of radial extracorporeal shock wave therapy on flexor spasticity of the upper limb in 100 patients with >1 month first-ever stroke and elbow joint spasticity grade >1, determined using the modified Ashworth scale.Post-stroke spasticity is associated with complications such as contractures pressure ulcers or pain and poor quality of life , 19. AdePark et al. prospectively enrolled patients with <3 months after stroke, who ingested dietary supplement power containing 6 g of branched-chain amino acids twice a day for 1 month. They selected the control group by a retrospective medical chart review to balance the age and stroke lesions with the intervention group. The branched-chain amino acid supplementation group demonstrated better improvement in skeletal muscle index measured by dual-energy X-ray absorptiometry and in functional scales such as the modified Barthel index.The incidence of sarcopenia in patients with stroke ranges from 14 to 54% and is associated with poor outcomes . Park etSingh et al. reported a case of a 52-year-old woman with a 9-year chronic stroke, who received 20 therapy sessions (45 min/session) with robotic exoskeleton assistance to extend the wrist and flex the fingers in the affected upper limb triggered by electromyography activity of the extensor digitorum communis. She demonstrated improvements in the Fugl–Meyer Assessment score, Barthel index, and modified Ashworth scale with relevant changes in neurophysiologic parameters. Finally, Angerhöfer et al. reviewed the need for brain–computer interface (BCI)-enabled neurorehabilitation in severe upper limb paresis. Specifically, the authors discussed how BCI-based long-term treatment strategies could be established in Germany and outlined the challenges in this process. This review will be an excellent reference for researchers who are interested in effective BCI applications and their clinical implementation.Novel therapeutics for stroke motor recovery or compensating the impaired function are needed especially for patients with more severe impairments considering their poor recovery potentials . Singh eAll 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. The handling editor TP declared a past collaboration with the author(s) W-SK and CP.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intracardiac echocardiography is a recent tool that allows visualization of the displaced tricuspid valve, the true AV groove, and the atrialized right ventricle. We present a 3-dimensional electroanatomic map built using intracardiac echocardiography and the CARTOSOUND Accessory pathways (APs) insert in the true AV groove, but difficult electrogram (EGM) analysis with low-amplitude fractioned signals recorded in the atrialized right ventricle (RV) and a prominent ridge in the groove2 can create many challenges in mapping and ablation. The AV groove has been defined in the past with a microelectrode catheter in the right coronary artery and right atrial (RA) angiography.3 We used intracardiac echocardiography (ICE) and a dedicated module  to delineate the right AV anatomy. In Figure 1, we present a 3-dimensional electroanatomic map of a 50-year-old patient with EA who underwent a successful right-sided mid-septal AP ablation. The locations of the true annulus where AV signals were recorded (white dots) and a mid-septal AP that was ablated  are shown; the yellow dots represent the His-bundle potential. The green lines display the contour of the RA marked on an ICE image , and the purple dots indicate the hinge points of the tricuspid valve leaflets. The RA profile is consistent with the RA opacification on the angiogram image. A significantly enlarged RA is seen. Figure 1D includes the baseline 12-lead electrocardiogram with ventricular pre-excitation, particularly evident in leads I and III, which disappears after ablation; intracardiac signals at the successful ablation site are also displayed. Use of ICE should become a routine practice in patients with abnormal hearts like those with EA, in whom EGMs do not match the anatomy, to help the electrophysiologist in the challenging task of navigating catheters.Ebstein’s anomaly (EA) is a genetic condition characterized by apical displacement of the septal and posterior leaflets of the tricuspid valve.


Recently, distinct mutational footprints observed in metastatic tumors, secondary malignancies and normal human tissues have been demonstrated to be caused by the exposure to several chemotherapeutic drugs. These characteristic mutations originate from specific lesions caused by these chemicals to the DNA of exposed cells. However, it is unknown whether the exposure to these chemotherapies leads to a specific footprint of larger chromosomal aberrations. Here, we address this question exploiting whole genome sequencing data of metastatic tumors obtained from patients exposed to different chemotherapeutic drugs. As a result, we discovered a specific copy number footprint across tumors from patients previously exposed to platinum-based therapies. This footprint is characterized by a significant increase in the number of chromosomal fragments of copy number 1–4 and size smaller than 10 Mb in exposed tumors with respect to their unexposed counterparts (median 14–387% greater across tumor types). The number of chromosomal fragments characteristic of the platinum-associated CN footprint increases significantly with the activity of the well known platinum-related footprint of single nucleotide variants across exposed tumors. Chemotherapies, in conjunction with radiotherapy and surgery still constitute the workhorse of the first line of treatment for many tumor types. Some chemotherapies exert their cytotoxic effect through DNA damage, which results, in many instances, in cell death. Particular types of DNA damage associated with certain chemotherapies lead to specific footprints of single nucleotide variants, which have been identified in tumor and healthy cells of exposed people. Here, we demonstrate that platinum-based chemotherapies also leave a type of larger mutational footprints in the genomes of exposed cells. These consist of an increase of chromosomal fragments of copy number between 1 and 4 and length below 10 Mb, possibly resulting from an increased frequency of double strand breaks in the chromosomes of the cells of exposed patients. This increase in structural variants induced by the exposure to platinum-based chemotherapies may have implications for the evolution of tumors in treated patients, as well as in the long-term side-effects of chemotherapies they may experience, due to damage accumulated in healthy cells. Chemotherapies and radiotherapy (in conjunction with surgery) constitute the first line of treatment of many tumor types. In the past half century they have contributed to an important reduction of the mortality related to these malignancies . These tThe type of DNA damage introduced by some of these anti-cancer therapies may also lead to structural variants in the tumor genome. For example, the exposure to radiation therapy is known to cause the introduction of indels and certain types of translocations ,13. HoweThroughout the evolution of tumors, the genome of their cells gain and lose chromosomal fragments of different length. As a consequence, the tumor genome appears like a collage of fragments of the chromosomes of its ancestral normal cell with different copy number and zygosity. One of the most dramatic events affecting the structure of a cell’s genetic material is the duplication of its entire genome. This event of whole-genome doubling (WGD) constitutes a hallmark of cancer genome evolution –16. By dTherefore, we reasoned that metastatic WGD tumors could be exploited to overcome the difficulties outlined above to study the influence of anticancer therapies on the landscape of SVs of tumor cells. We carried out a systematic analysis of 2709 WGD metastatic tumors from patients who, as part of the treatment of their primary malignancies, had been exposed to 85 anticancer therapies, sequenced by the Hartwig Medical Foundation (HMF cohort) . We idenOur starting hypothesis is that WGD tumors exposed to certain chemotherapies accumulate large SVs with specific features. Therefore, we first set out to identify tumors with WGD across two cohorts of cancer samples, one comprising 2778 primary tumors   and the The tumors in these cohorts clearly separate into two clusters when represented in a plane defined by their ploidy and their level of homozygosity Figs  and S1A.Moreover, while the interquartile range  of the ploidy of tumors with WGD corresponds to a range between 2.9 and 3.6 , there a-16, Figs However, as mentioned in the introduction, by relaxing the selective constraints on large SVs, the occurrence of WGD may provide the tumor with an indirect advantage: a more plastic genome. Large chromosomal segments are lost by tumors that experience a WGD, resulting in an average ploidy of 3.2 for both primary and metastatic malignancies . HoweverTo test this hypothesis, we leveraged whole-genome sequencing data obtained by the Hartwig Medical Foundation  on 1093 We classified the chromosomal segments in each cancer genome according to their copy number, zygosity and length into 48 groups  that havWe identified 10 CN categories that change significantly  between WGD tumors exposed or unexposed to four types of anticancer therapies . These a-4 in the comparison for the most significant CN category). Across lung tumors, all 22 WGD samples exposed to pyrimidine analogs are also exposed to platinum-based therapies (N = 120). Overlaps between all pairs of treatments across tumor types are presented in -5 to 0.034). We thus discarded the associations with anti-EGFR drugs or pyrimidine analogs. In the case of taxanes, CN categories that appear significantly different across exposed or unexposed tumors, were not replicated across tumor types, i.e., different CN categories appear across breast tumors and prostate tumors  were also exposed to platinum-based therapies. When the comparison is restricted to those exposed only to pyrimidine analogs (N = 26) and those unexposed to pyrimidine analogs or platinum (N = 120), the significance is lost  across colorectal and lung tumors . These CTo better capture this CN footprint of platinum-based therapies, we summed the number of fragments in each CN category in platinum-exposed and unexposed WGD tumors across cancer types, and computed the CN category-wise difference. The CN footprint of platinum-based therapies thus appears as an increase in the number of heterozygous and LoH chromosomal fragments of copy number 1–4 and size smaller than 10 Mb across platinum-exposed WGD tumors . This foPlatinum-based drugs  are bifunctional agents that create adducts in the DNA by establishing covalent links with one or two closeby bases. Adducts connecting two bases may be intra or interstrand . AlthougSince intrastrand links generated by platinum-based drugs are expected to distribute randomly along the genome of WGD tumors during exposure, we expected that chromosomal fragments resulting from their repair would also distribute evenly across the genome. To test this hypothesis, we computed the difference in the number of chromosomal fragments of copy number 1–4 and size smaller than 10Mb between WGD tumors exposed and unexposed to platinum-based drugs along each chromosome. As anticipated, we observed that chromosomal fragments smaller than 10 Mb (representative of the platinum CN signature) are evenly distributed along the genomes of WGD colorectal and lung tumors Figs . Had thiNext, we asked if an imbalance between insertions and deletions occurred during the error-prone repair of these interstrand links. To answer this question, we looked at the overall ploidy of platinum exposed and unexposed WGD tumors, and observed no significant differences between these two groups of cases, with the notable exception of lung tumors . This suWe then hypothesized that the same CN footprint will be apparent across WGD tumors from other primary sites, even if they don’t reach significance due to fewer numbers of exposed samples. Indeed, the same trends of increase of LoH and heterozygous chromosomal fragments smaller than 10 Mbase observed across WGD tumors of ovarian , esophagWhile the occurrence of the WGD results in a decrease of the selective pressure on chromosome fragmentation induced by platinum-based drugs–via repair of interstrand bridges–, we expect that such fragmentation also occurs across tumors without WGD. We thus focused on non-WGD tumors exposed to platinum-based therapies. As with WGD tumors, a trend towards the increase of chromosomal fragments below 10 Mb is observed across non-WGD metastatic lung tumors . InteresTo validate the platinum CN footprint we tapped onto an independent smaller cohort of advanced tumors (POG570) . The POGWe also reasoned that the more intense the exposure to platinum-based therapies, the greater the observed CN footprint will be. We previously demonstrated that higher activity of the platinum-related mutational (SBS and DBS) footprint results from longer times of exposure to these drugs . We thusIn summary, we postulate that a platinum CN footprint characterized by increased number of short (below tens of megabases) LoH and heterozygous chromosomal fragments may result from the increase in the number of double strand breaks stemming from platinum-induced interstrand bridges. While it is possible that the presence of this CN footprint increases the aggressiveness of platinum-exposed tumors, the mortality curves are not significantly distinct, Identifying and measuring the genetic footprints of chemotherapies in exposed cells makes an important contribution to the study of their effects on both tumor and normal cells. We recently tapped on our ability to detect the mutational footprint of several chemotherapies to estimate their mutational toxicity on the basis of regularly used treatment schemes ,11. We aHere, we identify the CN footprint of platinum-based drugs in metastatic tumors, as a result of the exposure to these drugs. The CN profile of platinum-based drugs appears as an increase in the fragmentation of the tumor genome, likely due to the gain of double strand breaks resulting from interstrand links. A clear connection between the ability to repair double strand breaks via homologous recombination repair and the effectiveness of platinum-based therapies has been shown by recent studies that segment patients on the basis of the observed activity of inferred chromosomal instability signatures ,29. FurtThough these footprints are apparent across tumors originating in different organs, and across both WGD and non-WGD tumors, their signal is still weaker than that of SBS mutational footprints ,7,30,31.Since chemotherapies are systemic treatments, their effects–not only the immediate ones, but also those occurring at longer terms–may also affect normal cells of the exposed person. We recently showed that the platinum-related mutational signature is detectable in hematopoietic cells that were normal at the time of exposure to the treatment, and which subsequently suffered a full clonal expansion to produce a full blown acute myeloid leukemia . It seemhttps://docs.icgc.org/pcawg/data/. A cohort of metastatic tumors was obtained upon request from the Harwtig Medical Foundation  across HMF tumors.Somatic copy number (CN) per chromosomal fragment (CN fragments) calculated from whole genome sequencing  were obtained from 2 different studies. We obtained primary tumors from the Pancancer Analysis of Whole-Genomes (PCAWG ) upon reion (HMF ); https:For each of the samples, genome ploidy represents the mean of the total ploidy of each of the called CN fragments weighted by their genomic length. The fraction of the genome under a loss of heterozygosity (LoH) in PCAWG is computed as the sum of the length of all CN fragments with a minor copy number allele equal to zero divided by the fraction of the genome with discernible CN . In the case of HMF, the LoH estimation is equivalent, but since the CN is not provided as an absolute number, but rather as a continuous value, LoH segments are those with a minor CN smaller than 0.5.The clinical data obtained for tumors in the HMF cohort included the primary cancer site and the information regarding the pre-treatment (treatment in this manuscript) received by each patient as part of the management of their primary malignancy. This treatment data see  was usedThe whole-genome doubling status (WGD) of each sample is determined based on the average ploidy of the genome and its fraction of LoH . In bothAs a representation of a variety of tumors and treatments, we selected all the combinations of tumor types and treatment comprising at least 50 samples  in orderThis study uses a total of 48 CN categories that classify each chromosomal CN segment according to its absolute copy number , zygosity , and length  following the classification implemented by Christopher D. Steele and colleagues  Fig 2B)Fig 2B). We filtered out samples with no treatment record and those tumor type-treatment combinations with less than 20 WGD cases. As a result, we obtained a total of 15 treatments and 10 tumor types based on their primary location. The analysis results in 39 combinations of tumor type-treatment to interrogate the impact of anticancer therapies on the selected CN category. For each CN category across each tumor type-treatment group, an independent Wilcoxon’s signed-rank test was used to estimate a p-value of the difference of the prevalence of the CN category between exposed and unexposed WGD tumors .In order to avoid the overlap present across many tumor-treatment pairs, only one treatment is selected for the multi-test correction from those with an overlap of more than 90% of the samples in either treated or untreated groups. All obtained values were corrected by the effect of the multiple tests using the Benjamini & Hochberg false discovery rate.We performed a manual curation of significant results to determine the appropriate treatment for those considered as overlapping (see previous paragraph). For colorectal cancer, pyrimidine analogs and platinum cohorts present an overlap of 88 and 97% of cases respectively, and show significant associations between some CN categories and the treatments . We seleWith the number of the 48 CN categories in each tumor , we first generated a matrix of counts using the SigProfilerMatrixGenerator version 1.1. Then, CN signatures were extracted using the sigProfilerExtractor version 1.1  and the default set of parameters. The solution recommended by the tool based on signature stability and the average error of reconstruction of the CN profiles comprised 9 CN signatures. To determine if any of these CN signatures was associated with the exposure to any treatment, we carried out a Wilcoxon signed-rank test of the activity of each signature  across exposed and unexposed WGD tumors, for each tumor type separately .Then, we analyzed (part of the sigProfiler package) the equivalence between these 9 CN signatures and the CN signatures extracted across primary tumors in a recent study . BrieflyTo integrate all CN categories with different prevalence between platinum exposed and unexposed tumors into a platinum CN footprint, we first combined the prevalence (count) of chromosomal fragments in each CN category across all platinum exposed and unexposed WGD tumors, irrespective of their tumor type. Then, we summed over each CN feature the prevalence observed for exposed tumors, and separately that observed across unexposed tumors. Finally, we subtracted the vector representing the cumulative platinum CN profile of unexposed tumors from the vector representing the cumulative platinum CN profile of exposed tumors. Thus, in the resulting vector, positive components correspond to CN categories with higher prevalence across platinum exposed tumors. We selected these to form the platinum CN footprint . For praTo determine the intensity of the platinum CN footprint along the genome, we divided the genome into regions of 1Mb and calculated the intensity of the CN footprint per WGD tumor in each region Then, we subtracted intensity in each region for platinum non-exposed tumors from that of platinum exposed tumors. The resulting exposed-to-unexposed differences in intensity per genomic region are presented in We extracted a new set of SBS signatures on its 3 base nucleotide context (SBS96) across HMF and PCAWG tumors. In order to increase the number of samples from the same tumor type and to perform a more accurate attribution of signatures, we aggregated metastatic and primary samples from the same cancer types. The matrix of counts of SBS in their trinucleotide contexts (SBS96 context) was constructed using SigprofilerMatrixGenerator version 1.1. , and theThe final signatures’ extraction result corresponds to the one recommended by the SigProfilerExtractor. Across the suggested sets of signatures, the platinum signature was selected as the one with a cosine similarity higher than 0.9 with any of the previously described platinum signatures (SBS31 and SBS35) in COSMIC version 3.2.The presence of the platinum SBS signature on exposed samples is limited to those tumors that underwent a clonal expansion fixing the mutations of the treatment ,8,11. WeFinally, we compared the number of mutations represented by that activity with the intensity of the platinum CN footprint (see above). The samples were divided into tertiles according to their amount of detected platinum SBS  and the significant differences in the intensity of the platinum CN footprint across groups was estimated with a two-tailed Wilcoxon-Mann-Whitney test.For each of the patients, the time to death represents the time between the date of the annotated biopsy and the date of death. Those patients without a date of death are considered alive and the last follow-up is fixed at the date when the current version of the data was published . The survival analysis follows a Cox proportional hazards regression model for each tumor type of 4 different groups of samples: (I) Platinum-exposed with WGD, (II) Platinum-exposed without WGD, (III) Platinum-unexposed with WGD, and (IV) Platinum-unexposed without WGD.https://www.bcgsc.ca/downloads/POG570/). Patients who received platinum-based drugs  as part of the treatment of the primary tumor were identified. Only the colon, breast or lung cohorts satisfied the requirement of at least 10 platinum exposed and unexposed tumors, and were thus selected for analysis. The length of chromosomal fragments identified in each tumor were computed as the difference between their start and end genomic coordinates. Then, the chromosomal fragments of CN 1–4 and length below 10 Mb in each tumor (platinum CN footprint) were counted and the counts of exposed and unexposed tumors compared. To compute the number of days elapsed between the end of the platinum treatment and the biopsy across exposed tumors, the relative date of treatment end was subtracted from the relative biopsy date.We obtained files containing the clinical and treatment data and CN calls of 570 metastatic tumors in the POG570 cohort  from a rS1 FigPrimary and metastatic WGD tumors a) Separation between WGD and non WGD tumors across the primary (PCAWG) and metastatic (HMF) cohorts on the basis of their ploidy and level of homozygosity. Similar to (PDF)Click here for additional data file.S2 FigSignificant comparisons  are highlighted with asterisks below each bar.(PDF)Click here for additional data file.S3 FigComparison with CN signatures a) Comparison of the activity of CN signatures extracted de novo from HMF WGD tumors between samples exposed and unexposed to different anticancer treatments. No signature appears with significantly different activity between exposed and unexposed WGD tumors. b) Equivalence (linear combination reconstruction) between CN signatures extracted de novo from the HMF cohort and CN signatures previously extracted from primary tumors (ref. 21). c) Comparison of the activity of CN signatures extracted from primary tumors (ref. 21) between samples exposed and unexposed to different anticancer treatments. No signature appears with significantly different activity between exposed and unexposed WGD tumors in the HMF cohort.(PDF)Click here for additional data file.S4 FigNumber of chromosomal fragments with copy number 1–4 and length smaller than 10 Mb across WGD lung (a) and colorectal (b) tumors exposed or unexposed to platinum-based drugs. Individual points represent the number of chromosomal fragments in a chromosome in an exposed or unexposed tumor. P-values represent the significance of a two-tailed Wilcoxon-Mann-Whitney test.(PDF)Click here for additional data file.S5 FigThe distributions of both groups of tumors from each organ are compared using a two-tailed Wilcoxon-Mann-Whitney test.(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Figa) Days elapsed between the end of platinum treatment and the biopsy of metastatic or recurrent tumors across the POG570 and HMF cohorts. Significantly longer time lapses for breast and lung tumors  are apparent across the HMF cohort. As a result, there is a higher likelihood of clonal expansion between treatment and biopsy (thus increasing the probability to detect platinum-related SBS and CN) in HMF metastatic tumors. This, together with the larger sample size probably explains why the platinum CN footprint is not as clear across POG570 colon tumors. One platinum exposed colorectal patient in the POG570 cohort did not have available CN data. b) Number of chromosomal fragments of size below 10 Mb identified across platinum-exposed and unexposed colon, breast and lung tumors. These numbers are significantly greater across exposed breast and lung tumors (two-tailed Wilcoxon-Mann-Whitney) and slightly greater  across colon tumors. The platinum CN footprint is thus replicated in the POG570 cohort, except for colon tumors.(PDF)Click here for additional data file.S8 FigEach curve represents a group of patients bearing either platinum-exposed or unexposed and WGD or non WGD tumors. The p-values were obtained using a Kaplan-Meier test.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file. This paper was transferred from another journal. As a result, its full editorial history  may not be present.13 Jan 2023Attachment127768_1_rebuttal_2523273_rlqw2r.pdfSubmitted filename: Click here for additional data file. 24 Jan 2023Dear Dr Gonzalez-Perez,We are pleased to inform you that your manuscript entitled "Copy number footprints of platinum-based anticancer therapies" has been editorially accepted for publication in PLOS Genetics. 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Gordenin, Ph.D.Academic EditorPLOS GeneticsDavid KwiatkowskiSection EditorPLOS Geneticswww.plosgenetics.orgTwitter: @PLOSGenetics----------------------------------------------------Comments from the reviewers (if applicable):Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: My previous comments have been adequately addressed with this manuscript revision.Reviewer #2: Authors have responded to my comments satisfactorily.Reviewer #3: This resubmission of the research by Gonzalez et al represents a thorough improvement on the previous work. Of particular note, the comparison to a new independent cohort, which although limited in size, reproduced the main finding. The comparison to Steele et al is also appreciated along with the addition of supplementary tables with sample information and the inclusion of sample numbers throughout the figures which improved the clarity of the paper.I recommend publication of this improved manuscript.Below are some minor typos which should be fixed prior to publication.Some minor commentsFig 1b cancer samples. Would be a nice comparison to know the differences in the cohorts here.Fig 1c legend typo LoOHFig3a in figure variatonFig 4 legendTwice says “with copy number 1-4 and length smaller than 10 Mbwith length between 10 Kb and 10 Mb”**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: None**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  3 Feb 2023PGENETICS-D-23-00053 Copy number footprints of platinum-based anticancer therapies Dear Dr Gonzalez-Perez, We are pleased to inform you that your manuscript entitled "Copy number footprints of platinum-based anticancer therapies" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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However, the experimental development of a photoelectric system capable of combining atomic-level visualization with simultaneous electrical current measurement with picoampere-precision still remains a great challenge due to light-induced drift while imaging and noise in the electrical components due to background current. Herein, we report a novel photoelectric TEM holder integrating an LED light source covering the whole visible range, a shielding system to avoid current noise, and a picoammeter, which enables stable TEM imaging at the atomic scale while measuring very small photocurrents (pico ampere range). Using this high-precision photoelectric holder, we measured photocurrents of the order of pico amperes for the first time from a prototype quantum dot solar cell assembled inside a TEM and obtained atomic-level imaging of the photo anode under light exposure. This study paves the way towards obtaining mechanistic insights into the operation of photovoltaic devices by providing direct information on the structure–activity relationships that can be used in device optimization.Herein, a novel  A photoelectric system is capable of simultaneous atomic-level visualization and pico-ampere-precision. In situ transmission electron microscopy (TEM) encompasses a broad range of techniques where high-resolution imaging and spectroscopy are carried out while applying various stimuli to the sample material. Recent studies mainly focus on the application of a single stimulus, which may be heat, stress, electrical biasing, or ultrashort photon pulses, on the material.1 By applying electric fields on a ferroelectric architecture in operation, Pan's group directly observed the kinetics and dynamics of electric domain switching and how the defects and interfaces impede full ferroelectric switching of a thin film.2 Using in situ TEM, Gao et al. reported the dynamic observation of the growth of a conducting filament in nanoscale resistive memories and oxygen vacancy migration resulting in electrically induced change in resistance in cerium oxides.3,4 A nanoscale electrochemical device was assembled inside a TEM to observe the lithiation behaviour of a nanowire in situ during electrochemical charging, which served to advance the fundamental understanding of the electrochemical reactions and the mechanism of degradation of the material during operation.5 Inspired by the success of in situ TEM technology in combination with electrical and electrochemical analysis, photocatalytic reactions were monitored inside a TEM to understand the structure–activity correlations in the photocatalyst during a catalytic reaction while introducing light as a stimulus.6 Light-illumination sample holders have also been designed for environmental TEM (ETEM) to investigate the photodecomposition of a photocatalyst such as the transformation of Cu2O to Cu under UV irradiation in a H2O environment.7 A fibre-based photoexcitation system for ETEM, which allows to control the temperature, gas environment, and light, has been developed by the Crozier's group, and this photoexcitation system has been used to investigate the changes taking place on the surface of anatase nanocrystals during photo-induced splitting of water molecules in the vapor state.8 These studies contribute towards an in-depth understanding of the structure–activity correlations at the atomic level. However, often, the information about the relationship between the structure and functionality is incomplete while using only a single-field as a stimulus. In the study on photoelectric devices, the major challenge is to directly correlate the observed light-induced current with local structural in-homogeneities and dynamics.9in situ tools to provide multi-field coupling conditions have, therefore, been recently developed to accurately characterize the properties of individual devices in real time.10 The electrical transport coupled with optical and piezoelectric properties of individual ZnO nanowires was studied in situ by introducing ultraviolet illumination during TEM imaging.11 The photoelectric performances of individual CdS nanowires and CdS/ZnO heterostructures were characterized using a novel laser-compatible high-resolution in situ TEM technique.12,13 These findings provide mechanistic insights into how strain affects the photoelectric properties of materials and offer guidelines for the design of future flexible electronic, optoelectronic, and photovoltaic devices. However, the power conversion efficiency and long-term performance of photovoltaic devices and photodetectors are still very low, and there are many fundamental problems that need to be solved to further improve their performance. It is now recognized that atomic level in situ observation of materials is critical to relate the structure of photoelectric devices to their performance. To date, there is no multimodal optical-electric system that can directly correlate the atomic structure with photoelectric performance because of two reasons: the introduction of light introduces considerable drift in TEM imaging, and the noise in the electrical circuit is detrimental to making precise photocurrent measurements.Novel in situ photoelectric holder enables to use light sources with different wavelengths and also ensures ultrasensitive pico ampere-precision measurement without compromising the microscope's performance, especially with respect to imaging at the atomic scale. Examples of these mutually compatible measurements include use of a custom-designed photodetector with a quantum dot sensitized solar cell while successfully imaging the changes in its structure under light exposure at the atomic level inside a TEM. Of particular technological importance is the capability to have a fundamental understanding of the structure and photoelectric performance at the atomic level and provide specific guidelines for the design of high-efficiency photoelectric devices.Herein, we report a multimodal photoelectric TEM sample holder capable of imaging materials at the atomic-scale while simultaneously measuring photocurrents with pico ampere precision. This et al.14 Mercaptopropionic acid (MAP)-capped CdSe QDs in an aqueous solution were prepared using previously reported procedures.15 The NWs were immersed in a solution of QDs for 2 h after which, they were rinsed sequentially with water and ethanol. The precipitate of ZnO NW/CdSe QDs was dried and stored for further in situ TEM experiments. CdS nanoparticles were prepared by a simple successive ionic layer adsorption and reaction technique,16 and the precipitated powder was obtained and used for in situ characterization.ZnO nanowires (NWs) were prepared using a simple hydrothermal method similar to that previously reported by Cheng Bin in situ experiment, the CdS and CdSe QD-coated-ZnO NWs were mounted on a copper electrode by immersing a copper grid in the corresponding solution in ethanol. The movable metallic electrode is a W and Pt tip, controlled by a piezo-tube to make contact with the CdS and CdSe QD-coated ZnO NW mounted on the copper grid. The photoelectric properties of the individual nanowire QDSSCs were measured inside the TEM  using a custom-made Nanofactory in situ-scanning tunneling microscope (STM)-TEM electrical probe, which was modified to incorporate a white LED to function as a light source. A pA Keithley 6430 source meter was used to measure the solar cell performance by tracing the photocurrent vs. time and current–voltage curves. Before each experiment, we used an irradiance meter  to calibrate the light source by slightly tuning the input voltage for obtaining the same light intensity.In our in situ photoelectric set up including a light-emitting diode (LED) as the light source and the electrode tip for sample loading replaces the electrical measuring system of the STM holder; the shielding system connected with a pico ampere meter is designed to measure ultra-small currents. The process used to deposit the LED on sapphire (Al2O3) is represented in Fig. S1.The custom-designed photoelectric TEM holder combined with an external electric measurement system designed based on the commercially available Nanofactory: TEM-STM holder is shown in in situ TEM. The actual image of the photoelectric holder with the blue-light-on is shown in Fig. S2.The circuit diagram for the photocurrent measurement in the shield is shown in In in situ inside the TEM. In in situ in the TEM are operational when irradiated by light . To confirm that the photocurrent originated only due to photo-stimulation, we also characterized the effect of electron beam of different intensities on current measurements under dark conditions  response that is the most important parameter. Under real conditions, the operation of a solar cell is under a certain forward bias. To confirm that our homemade holder is capable of biasing the sample under illumination, we obtained the TEM image of the assembled device and measured the I–V curves under dark and under exposure to light, as shown in in situ fabrication of a photodetector device based on nanoparticles under light irradiation of different wavelengths, and the typical low magnification TEM image of the CdS nanoparticle photoelectric device is represented in Fig. S5b.s Fig. S4. At a loin situ photoelectric characterization. The unique control of the power input for the light source prevented the light source from influencing the microscope performance while illuminating the sample with light of different wavelengths covering this entire visible spectrum. Using the movable probe, photoelectric nano-devices were successfully fabricated in situ during TEM imaging, and photocurrents on the pico-ammeter scale were detected while simultaneously imaging the crystal structure at the atomic level. This technique opens up the possibility to develop simultaneous in situ electrical, optical, and structural measurements and manipulation of the samples inside TEM without sacrificing the image quality. Our novel probe should be particularly useful to obtain a direct correlation between the structure at the atomic level and the performance of optically active materials and devices, namely, photovoltaic cells, quantum dots, photo resistors, and nanomaterials. Moreover, the new information thus obtained can trigger the development of new materials and devices.In summary, a special TEM specimen holder was designed, manufactured, and tested. Based on a commercially available STM-TEM holder, the custom-designed sample holder enabled There are no conflicts to declare.RA-008-C7RA10696C-s001RA-008-C7RA10696C-s002RA-008-C7RA10696C-s003RA-008-C7RA10696C-s004RA-008-C7RA10696C-s005RA-008-C7RA10696C-s006RA-008-C7RA10696C-s007