All inpatients admitted to the hospital were screened utilizing the GeneXpert RT-PCR platform  via nasopharyngeal swabs. This screening program revealed that 1 in ~28 asymptomatic patients were positive for SARS-CoV-2.10 Within the hospital itself, there were, on average, 3.8 new COVID-19 admissions per day in the 2 weeks preceding and the 2 weeks during the employee testing window.At the time of HCW testing, the community burden of SARS-CoV-2 remained high, with 515 active cases per 100,000 county residents.A voluntary SARS-CoV-2 testing program was offered to HCWs over a 2-week testing window. HCWs were excluded if they had symptoms of COVID-19 or previously tested positive for SARS-CoV-2. The program was made available to personnel who cared for COVID-19–positive patients in the ED or on the COVID-19 care unit. Screening was performed using nasopharyngeal swabs and the Cepheid GeneXpert RT-PCR assay. Staff were permitted to return to work while awaiting test results.In total, 499 staff members were eligible for screening. Among them, 121 personnel volunteered to undergo testing (24.2% of those eligible). The results of all 121 tests were negative for SARS-CoV-2. Breaking down the uptake in testing by role: 6 of 53 of eligible respiratory therapists (11.3%) were tested, 33 of 92 eligible providers (35.9%) were tested, 71 of 262 eligible registered nurses (27.1%) were tested, and 11 of 82 of the eligible patient care assistants (13.4%) were tested.The voluntary hospital staff testing program described here was implemented as a method of ensuring the safety of our personnel and patients from the established threat of asymptomatic transmission. Had any staff members received a positive test result, appropriate isolation measures would have been implemented to prevent viral spread, including a 10-day minimum administrative leave. The negative results of all tested individuals allowed these personnel to return to work in confidence and also informed the hospital’s decision to not continue routine testing of employees.Universal SARS-CoV-2 testing of all patients admitted to the hospital, regardless of symptomatology or reason for stayTesting of all patients undergoing surgical procedures 24–48 hours prior to operationIsolation of all positive patients into designated COVID-19 care unitsNegative pressure ventilation systems for all COVID-19 care floorsPersonal protective equipment requirements including surgical masks and universal precautions on all floors, with the addition of gowns and eye protection on COVID-19 unitsMandatory N95 mask or PAPR/CAPR use for any aerosol-generating procedures in COVID-19 units“No visitors” policy throughout the hospital, absent exigent circumstances (in accordance with Michigan’s March 14 executive order)Universal symptom screening of all staff arriving to work, excluding workers if they presented with any of the following symptoms: fever, cough, shortness of breath, chills, body aches, loss of taste, or loss of smell.The 0% positive test rate among asymptomatic staff, despite the local community and hospital system experiencing a large burden of COVID-19 cases, is a testament to the ongoing work underway to ensure safety throughout the hospital. The following precautionary measures were implemented at our hospital:This testing was arranged through a COVID-specific Colleague Health Hotline designated to have a very low threshold for testing.Adherence to this protocol has been of utmost priority throughout the hospital, in part because the ramifications of nosocomial transmission became evident early in the pandemic. The hospital had numerous instances of SARS-CoV-2–positive patients admitted to non–COVID-19 units, with a significant delay in diagnosis due to atypical clinical presentations. This repeated exposure to SARS-CoV-2–positive patients on non–COVID-19 units informed the decision to proceed with this protocol.The uptake in testing among potentially exposed healthcare workers was also measured. Moreover, the 24.2% of eligible healthcare workers pursuing testing was lower than we expected. It is unclear whether this represents reluctance to undergo a diagnostic nasopharyngeal swab or confidence in the organizational approach. Regardless, this relatively low uptake does not support routine testing as an effective method to improve workforce confidence or safety.In the months since implementation, adherence to the listed protective measures has been central to the safety of the hospital community and has contributed to the lack of positive testing among asymptomatic HCWs. As statewide regulations and social distancing restrictions begin to relax, it is essential to adequately protect our healthcare workforce. The infection control methods described have demonstrated how this organization was able to effectively protect this vital resource. Extensive testing of employees does not seem to be cost-effective or necessary when strong symptom screening and infection control policies are in place. As hospitals and communities prepare for the next phase of the pandemic, we recommend close monitoring of employee symptoms, rapid access to testing when symptoms develop, strong infection control practices, and broad testing of patients to effectively cohort patients as an alternative to testing asymptomatic employees.


In these humanized mice, the human thymic graft first underwent acute recoverable involution caused presumably by transplantation stress, followed by an age-related chronic form of involution. Although both the early recoverable and later age-related thymic involution were associated with a decrease in thymic epithelial cells and recent thymic emigrants, only the latter was associated with an increase in adipose tissue mass in the thymus. Furthermore, human thymic grafts showed a dramatic reduction in FOXN1 and AIRE expression by 10 weeks post-transplantation. This study indicates that human thymus retains its intrinsic mechanisms of aging and susceptibility to stress-induced involution when transplanted into immunodeficient mice, offering a potentially useful in vivo model to study human thymic involution and to test therapeutic interventions.Thymic involution is an important factor leading to the aging of the immune system. Most of what we know regarding thymic aging comes from mouse models, and the nature of the thymic aging process in humans remains largely unexplored due to the lack of a model system that permits longitudinal studies of human thymic involution. In this study, we sought to explore the potential to examine human thymic involution in humanized mice, constructed by transplantation of fetal human thymus and CD34 The + cells (i.v.) achieves efficient human thymopoiesis and T cell development in immunodeficient mice  mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd., and were housed in a specific pathogen-free micro-isolator environment and used in experiments at ~5 weeks of age. Discarded human fetal tissues with gestational age of 17 to 20 weeks and human blood samples were obtained with informed consent at the First Hospital of Jilin University. Protocols involved in the use of human tissues and animals were reviewed and approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University, and all of the experiments were performed in accordance with the protocols.NOD.CB17-3 in size, under renal capsule) and fetal liver-derived CD34+ cells  from the same fetal donor into 2 Gy-irradiated NOD/SCID mice as previously described  using various combinations of the following monoclonal antibodies: anti-human CD45, CD3, CD4, CD8, CD45RA, CD45RO, CD69, CCR7, CD31 ; and anti-mouse CD45 (BD Pharmingen) and Ter119 . Peripheral blood was collected from tail vein into heparinized tubes, and mononuclear cells were purified by density gradient centrifugation with Histopaque-1077 . FCM analysis was performed on a FACS Fortessa (BD Biosciences). Dead cells were excluded from the analysis by gating out lower forward scatter and high propidium iodide–retaining cells. Data analysis was performed using FlowJo 10.3 software.Human thymic grafts were embedded in tissue freezing medium  and immediately frozen in liquid nitrogen and then stored at −80°C. Cryosections (3.5 μm) were prepared and fixed in cold acetone for 20 min. The sections were stained with Alexa Fluor 647 anti-human CD326 (EpCAM) antibody , followed by DAPI staining. The slides were imaged and processed using a fluorescence microscope. To evaluate fat deposition, thymic sections were stained with Oil Red O  and hematoxylin. Images of the sections were collected using a light microscope (Olympus Corporation) from four different fields at ×100 magnification. Image Pro Plus software was used to analyze the integrated optical density (IOD) of the Oil Red O-stained areas.Total RNA was extracted with Trizol , and cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix . Quantitative real-time PCR was performed using a SYBR Green Kit (TransGen Biotech) with a StepOnePlus Real-Time PCR System . Quantitative real-time PCR primer sequences used in this study are shown in DNA was purified from human PBMCs and humanized mouse PBMCs and spleen cells using the QIAamp DNA Blood Mini Kit according the manufacturer's instructions . Quantitative real-time PCR was performed using AceQ Universal U+ Probe Master Mix V2 (Vazyme Biotech) with a StepOnePlus Real-Time PCR System . Quantitative real-time PCR primer and probe sequences used in this study are shown in t-test. Differences with a P-values of <0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 5 software and Microsoft Office Excel 2013.All data are presented as mean ± S.D and statistical significances between two groups were calculated using unpaired, non-parametric, two-tailed Student's + naïve T cells and recent thymic emigrants (RTEs) by flow cytometry (FCM), in which CD4+ naïve T cells and RTEs were identified as CD4+CD45RA+CD45RO− and CD4+CD45RA+CD45RO−CD31+, respectively . These data are in agreement with previous reports  were analyzed for the ratios of CD4ectively . We foun T cells , while s T cells . Consist T cells , the slo T cells . Similardecrease , with a decrease . For the reports , 17 confotential .+ cell transplantation, and analyzed for human CD4+ naïve T cells and CD4+ RTEs  thymocytes was detected in the human thymic grafts, a significant decline in the ratio of DP thymocytes, which was associated with an increase in CD4 single positive (SP) and CD8 SP thymocytes, was clearly detected between 16 and 22 weeks  in the human thymic grafts. Immunofluorescence staining revealed that EpCAM-positive TECs increased by ~3-fold between 10 and 16 weeks, and then declined progressively  and autoimmune regulator (Aire) in the human thymic grafts. We found that 16 weeks , likely 22 weeks .TRECs are stable episomal, non-replicative DNA circles generated during T-cell receptor gene rearrangement in developing T-lymphocytes in the thymus. Therefore, TRECs are a marker for recently formed T-lymphocytes . We measin vivo model for longitudinal studies. The lack of a suitable in vivo model is also a bottleneck in developing interventions to treat thymic involution. In this study, we explored the potential to study human thymic involution in hu-mice constructed by transplantation of human thymic tissue and hematopoietic stem/progenitor cells into immunodeficient mice.The thymus is a primary lymphoid organ where T cells are generated. Thymic involution, or the shrinking of the thymus with age, is an important factor that inhibits thymic output, leading to immune defects in vertebrates. Although thymic atrophy or involution has been extensively investigated in animal models, in humans the process and its underlying mechanisms remain relatively unknown, largely due to the lack of an Thymic involution with age is a chronic process that results in progressive reduction in thymic output, leading to degeneration of adaptive immunity in aged individuals. In addition to age-related involution, the thymus may undergo acute (recoverable) atrophy under certain stress conditions, such as infection, pregnancy, and chemotherapy. In hu-mice, we found that human thymus first undergoes acute recoverable involution, followed by an age-related chronic form of involution. The first thymic involution was likely caused by transplantation stress, and recovered by week 14. Although the early recoverable atrophy (likely induced by transplantation stress) and the later age-related thymic involution were both associated with a decrease in TECs and RTEs, only the latter was associated with an increase in adipose tissue mass in the thymus. This is consistent with previous reports in mice that age-related thymic involution is closely associated with adipocyte expansion in the thymus .FOXN1 transcription correlates with age, with a sharp decline after adolescence (+ TECs declines rapidly within a few weeks after birth and remains constant up to 2 years of age, while FOXN1 downregulation continues on a per-cell basis through the end of life (FOXN1 and AIRE decreased substantially by 10 weeks after transplantation, when compared to the original fetal thymic tissue. Since FOXN1+ and AIRE+ TECs are highly sensitive to toxic or damaging insults (FOXN1 appears in the sixth week of gestation in humans, serving as a key regulator of TECs development and differentiation in the fetal and adult thymus . Snapsholescence . Mouse s of life . AIRE is of life , 26. We  insults , 27, botWe noted that thymic output, as determined by measuring RTEs, is considerably lower in hu-mice compared to humans. It has been reported that human T cells can divide in the periphery without losing their naïve phenotype . Thus, ain vivo system for understanding human thymic involution and testing therapeutic interventions.In conclusion, this study suggests that human thymus in hu-mice undergoes both stress-induced acute thymic involution and age-related chronic thymic involution. We acknowledge that the hu-mouse host environment is not identical to that of a human. However, previous studies from our group and others have confirmed that human thymic grafts remain functional in this hu-mouse model , 9, 28. All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethics Committee of the First Hospital of Jilin University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the First Hospital of Jilin University.Q-YT, J-CZ, J-LG, and YL performed experiments and analyzed data. L-YY, XW, and Y-GY discussed the data and critically reviewed the manuscript. Q-YT and L-GS conceptualized the project and wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is thought to be transmitted mainly by person-to-person contact  were calculated to examine differences between the survey cohorts and to examine potential associations between reported characteristics . Jupyter Notebook  was used to conduct statistical analyses.Among respondents in the U.S. cohort , 16.8% knew someone who had positive test results for COVID-19, compared with 42.0% of respondents in NYC and 10.8% in Los Angeles ; 5.9% ofBroad support for recommended COVID-19 mitigation strategies was found nationwide . OverallWidespread adherence to recommended COVID-19 mitigation strategies was reported in all three cohorts. Overall, 77.3% of adults nationwide reported self-isolating,Overall, 84.3% of adults in the U.S. survey cohort believed their state’s COVID-19 community mitigation strategies were the right balance or not restrictive enough, compared with 89.7% in NYC and 79.7% in Los Angeles. As well, 74.3% of respondents in the United States reported they would not feel safe if these restrictions were lifted nationwide at the time the survey was conducted, compared with 81.5% in NYC and 73.4% in Los Angeles. In addition, among those who reported that they would not feel safe, some indicated that they would nonetheless want community mitigation strategies lifted and would accept associated risks .Reported prevalence of self-isolation and feeling safe if community mitigation strategies were lifted differed significantly by age, employment status, and essential worker status among adults in the U.S. survey cohort . The perReported prevalences of always or often wearing a cloth face covering in public and maintaining ≥6 feet of physical distance also varied significantly across respondent demographics and characteristics. Respondents who were male, employed, or essential workers were significantly more likely to report having been in public areas in the past week. Among respondents who had been in public areas during the preceding week, significantly higher percentages of women, adults aged ≥65 years, retired persons, and those living in urban areas reported wearing cloth face coverings. A significantly higher percentage of adults aged ≥65 years and nonessential workers reported maintaining 6 feet of physical distance between themselves and others and abiding by the recommendation to avoid gatherings of 10 or more persons than did others. Adherence to recommendations to maintain 6 feet of physical distance and limit gatherings to fewer than 10 persons also differed significantly by employment status and race, respectively, with employed persons less likely than were retired persons to have maintained 6 feet of distance and black persons less likely than were white or Asian persons to have limited gatherings to fewer than 10 persons.There was broad support for stay-at-home orders, nonessential business closures, and adherence to public health recommendations to mitigate the spread of COVID-19 in early- to mid-May 2020. Most adults reported they would not feel safe if government-ordered community mitigation strategies such as stay-at-home orders and nonessential business closures were lifted nationwide at the time the survey was conducted, although a minority of these adults who did not feel safe wanted these restrictions lifted despite the risks.There was a significant association between age and feeling safe without community mitigation strategies, with younger adults feeling safer than those aged ≥65 years, which might relate to perceived risk for infection and severe disease. As of May 16, adults aged ≥65 years accounted for approximately 80% of reported COVID-19–associated deaths, compared with those aged 15–24 years, who accounted for 0.1% of such deaths (Other variations in attitudes, behaviors, and beliefs by respondent characteristics have implications for implementation of COVID-19 mitigation strategies and related prevention messaging. For example, a lower percentage of respondents in the U.S. survey cohort reported wearing cloth face coverings and self-isolating than did those in NYC and Los Angeles. However, although use of cloth face coverings in NYC and Los Angeles were similar, NYC experienced substantially higher COVID-19-related mortality during the initial months of the pandemic than did Los Angeles (The findings in this report are subject to at least four limitations. First, behaviors and adherence to recommendations were self-reported; therefore, responses might be subject to recall, response, and social desirability biases. Second, responses were cross-sectional, precluding inferences about causality. Third, respondents were not necessarily representative among all groups; notably a lower percentage of African Americans responded than is representative of the U.S. population. In addition, participation might have been higher among persons who knew someone who had tested positive or had died from COVID-19, which could have affected support for and adherence to mitigation efforts. Finally, given that the web-based survey does not recruit participants using population-based probability sampling and respondents might not be fully representative of the U.S. population, findings might have limited generalizability. However, this survey did apply screening procedures to address issues related to web-based panel quality.Widespread support for community mitigation strategies and commitment to COVID-19 public health recommendations indicate that protecting health and controlling disease are public priorities amid this pandemic, despite daily-life disruption and adverse economic impacts (Stay-at-home orders and recommended personal protective practices were disseminated to mitigate the spread of COVID-19 in the United States.During May 5–12, 2020, a survey among adults in New York City and Los Angeles and broadly across the United States found widespread support of stay-at-home orders and nonessential business closures and high degree of adherence to COVID-19 mitigation guidelines. Most respondents reported that they would feel unsafe if restrictions were lifted at the time of the survey.Routine assessment of public priorities can guide public health decisions requiring collective action. Current levels of public support for restrictions and adherence to mitigation strategies can inform decisions about reopening and balancing duration and intensity of restrictions.


In addition, controversy exists about the degree to which suicidal and non-suicidal self-harm share a common genetic aetiology. In the present study, we conduct genome-wide association studies (GWAS) on lifetime self-harm ideation and self-harm behaviour  using data from the UK Biobank . We also perform genome wide gene-based tests and characterize the SNP heritability and genetic correlations between these traits. Finally, we test whether polygenic risk scores (PRS) for self-harm ideation and self-harm behaviour predict suicide attempt, suicide thoughts and non-suicidal self-harm (NSSH) in an independent target sample of 8,703 Australian adults. Our GWAS results identified one genome-wide significant locus associated with each of the two phenotypes. SNP heritability (hsnp2) estimates were ~10%, and both traits were highly genetically correlated (LDSC rg > 0.8). Gene-based tests identified seven genes associated with self-harm ideation and four with self-harm behaviour. Furthermore, in the target sample, PRS for self-harm ideation were significantly associated with suicide thoughts and NSSH, and PRS for self-harm behaviour predicted suicide thoughts and suicide attempt. Follow up regressions identified a shared genetic aetiology between NSSH and suicide thoughts, and between suicide thoughts and suicide attempt. Evidence for shared genetic aetiology between NSSH and suicide attempt was not statistically significant.Family studies have identified a heritable component to self-harm that is partially independent from comorbid psychiatric disorders. However, the genetic aetiology of  Moreover, self-harm behaviours are now recognized by the American Psychiatric Association as independent conditions for further study. Namely, non-suicidal self-injury and suicidal behaviour disorder were recently introduced in the section 3 of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V)2. The lifetime prevalence estimate for suicide thoughts is ~10%, while suicide attempt and non-suicidal self-harm (NSSH) affect ~2.5 and ~5% of the population, respectively5. Higher rates have been reported amongst children and adolescents6. The key difference between suicidal and non-suicidal self-harm is that the former implies an intent to die as a consequence of the act. Non-suicidal self-harm acts include equally dangerous behaviours such as cutting, burning or poisoning, but are underlined by a different motivation such as seeking attention or the desire to feel pain.Every year nearly one million people take their own lives8. Multiple studies have documented that the presence of a psychiatric disorder considerably increases the risk for both suicidal and non-suicidal self-harm11 but a sizeable genetic component (33-51% of variance) of suicide risk is not explained by underlying psychiatric conditions16. Importantly, controversy exists about whether NSSH and suicide attempt are part of the same liability spectrum20. Notably, twin studies have identified a genetic correlation between suicide thoughts and NSSH7, but the extent to which shared genetic factors underlie NSSH, suicide thoughts and suicide attempt (i.e. a self-harm liability continuum hypothesis) still remains elusive. Investigating broad sense self-harm could improve our understanding of the aetiology and underpinnings of the liability to both suicidal and non-suicidal self-harm.Twin and family studies indicate that NSSH, suicide thoughts and suicide attempt are moderately heritable31, robustly associated genetic variants are still elusive32. A number of studies assessing the genetic predictability of suicidality have suggested a shared aetiology between depression and suicidality, and suggestive evidence for suicidality-related associations35. Investigating the genetic architecture of self-harm regardless of suicidal intent, could be valuable to gain biological insights into the relationship between self-harm and suicide. In the present study, we explore the genetic aetiology of lifetime self-harm ideation and lifetime self-harm behaviour  using a GWAS and PRS approach. An overview of the phenotypes and terminology used throughout this manuscript is available in Table Although several genome-wide association studies (GWAS) on suicidality have been published to dateself-harm ideation and self-harm behaviour)36. Self-harm was assessed as described previously37. DNA extraction and genotyping are described in ref. 38. Notably, genotyping was performed using two highly related arrays: the UK BiLEVE Axiom array and the UK Biobank Axion Array.The discovery sample consisted of ~156,700 participants from the UK Biobank (as of February 2019) with self-harm data . The self-harm ideation item was: “Have you contemplated harming yourself ?” . Notably, both these phenotypes make no distinction between suicidal and non-suicidal self-harm. As such, they include both suicidal and non-suicidal self-harm. A liability threshold model  estimated the traits to be highly correlated ρ = 0.89 .self-harm ideation and lifetime self-harm behaviour. Association analyses were performed using BOLT_LMM39, based on a linear mixed model and allele dosages  accounting for the first 20 genetic ancestry principal components, standard covariates , and correcting for cryptic relatedness and population stratification using a genetic relatedness matrix as the random effects variance covariance structure. Approximately 48,000 individuals of non-European ancestry were excluded from the analyses. A stringent, but standard quality-control (QC) protocol40 was applied: variants with low minor allele count (MAC < 25), low quality imputation (INFO < 0.8) or with a deviation from the Hardy-Weinberg equilibrium (HWEp < 1e-10) were excluded from further analyses.We conducted two GWAS, on lifetime self-harm ideation or self-harm behaviour using MAGMA41 as implemented on the FUMA web platform42. Briefly, SNPs were mapped to ~20,000 protein coding genes based on their genomic location. Then, the independent SNP association statistics were combined to yield gene-based mean χ2 statistics. Genome-wide significance level was defined as 2.652e-6 .Gene-based association analysis was conducted for 44. Items from the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) assessing self-harm behaviours were included in both cohorts, a detailed description of the items and their application is available in34. Briefly, participants were first asked whether they experienced any suicidal thoughts, then suicide attempt, and finally acts of self-harm not related to suicide attempts. The items used to determine lifetime prevalence of self-harm behaviours were: “Have you ever thought about taking your own life?”; “Have you ever tried to take your own life?” and “(Other than when you tried to take your own life) Did you ever hurt yourself on purpose, for example, by cutting or burning yourself?”.The target sample consisted of individuals from two cohorts of the Queensland Twin Registry. Individuals were recruited and participated in structured telephone or paper interviews assessing psychiatric disorders, substance abuse and living conditions. Detailed information on the cohorts has been published previously45. Briefly, standard protocols for DNA collection and extraction were used. Genotyping was carried out using commercial Illumina SNP arrays. Platform specific QC was performed including: Hardy Weinberg equilibrium deviation, individual SNP call rates, minor allele frequency threshold, and removal of population outliers . Genotype data were imputed using the haplotype reference consortium (HRC) reference panel.The genotyping and quality control (QC) protocol for the target sample have been described previouslysnp2) was calculated using LD-score regression as previously described46. The software ldsc v 1.0.0 was used to calculate both the hsnp2 and the genetic correlation between both GWAS summary statistics. This method relies on the relationship between the non-centrality parameter (NCP or χ2) of GWAS results and LD scores  expected under a polygenic assumption 47. The fact that the expected value of the NCP for a given SNP is a function of the genetic covariance (hg2) of the trait and the LDscore of that SNP, allow us to estimate the SNP based heritability46 (hsnp2) and co-heritability (rg)48 of a set of traits given their summary statistics and known LD patterns. As the population prevalence of broad sense self-harm behaviours on the UK has not been reported, we assumed the population prevalence to be equal to the discovery sample prevalence when transforming to the liability scale using ldsc and thus estimates of hsnp2 should be referred to with caution. Genetic correlations between the traits under study and other traits and diseases (~760 traits) were explored using the LD-Hub web platform49. We used a stringent definition of a significant genetic correlation  imputation quality-variants. The most significant independent SNPs were selected using a conservative clumping procedure 50 to correct for inflation arising from linkage disequilibrium (LD). Eight different PRS were calculated for each individual using different p-value thresholds  as criteria for SNP inclusion on the PRS calculation. PRS were calculated using a dosage assumption, therefore multiplying the effect size of a given SNP by the imputed number of copies (using dosage probabilities) of the effect allele present in an individual. Finally, the SNP dosage effects were summed across all loci per individual.To calculate the genetic predisposition (risk) of our target sample to the traits of interest, each variant’s effect size was obtained from the GWAS summary statistics. Our PRS estimation pipeline excluded self-harm behaviour and self-harm ideation (i.e. the PRS) with actual self-harm phenotypes in the target sample, we employed a linear mixed model regression framework. Briefly, the PRS was added to the model as a predictor variable while accounting for sex, age, age2, sex*age, the first five genetic principal components and imputation run, an in-house set of variables that capture array and cohort differences51, as fixed effects. Correcting for relatedness is crucial when examining family cohorts. Given varying degrees of relatedness in our sample, we employed a linear mixed model using genetic restricted maximal likelihood (GREML) with a random effects variance covariance structure defined by the sample’s genetic relatedness matrix obtained from GCTA 1.91.753. This method has been previously used to deal with related individuals in related target samples of PRS studies54. A partial R2 was used to estimate the variance explained by the PRS using the formula:β represents the PRS fixed effect estimate, 56 to estimate the number of effective variables being tested. The final significance threshold was defined at α < =0.0064To assess the association between the genetic liability to 57 based on available summary statistics leaving out the Australian sample58. The self-harm behaviour (and self-harm ideation) phenotype associations were reproduced with MDD-PRS as a covariate to identify the variance explained by self-harm behaviour (or self-harm ideation) over and above the effect of MDD liability.As sensitivity analysis, we tested whether the liability to major depressive disorder was driving polygenic prediction. To this end, MDD-PRS were calculated using SBayesRself-harm ideation and suicide thoughts were reproduced including NSSH as a covariate. Likewise, the association between PRS for self-harm ideation and NSSH was reproduced with suicide thoughts as a covariate. The same approach was used for the self-harm behaviour PRS but using either suicide thoughts or suicide attempt (the phenotypes with some evidence of association) as a covariate. If the significant PRS-phenotype associations were driven through the same genetic components, adding one of the phenotypes as a covariate should implicitly capture the shared genetic predisposition, thus removing the observed association. Any residual prediction would imply that independent genetic factors, captured by our GWAS, are underlying each phenotype.Further, we were interested on whether the prediction of our PRS on the traits  was due to shared or independent genetic factors. To this end, the significant associations between the PRS for broad sense self-harm ideation and self-harm behaviour were assessed, males and females presented a similar age range, but females showed a higher prevalence of both self-harm ideation and self-harm behaviour. Prevalence of NSSH, suicide thoughts and suicide attempt on the target sample were 3.4, 26.4 and 3.8% respectively. Prevalence of suicide thoughts was slightly lower in the female subgroup while suicide attempt was higher  on the liability scale for both traits was estimated to be 11.1% (SE = 1.7%) for self-harm behaviour and 10.1% (SE = 1.0%) for self-harm ideation. Further, the traits were highly genetically correlated with each other . High genetic correlations with psychiatric disorders such as anxiety, depression and schizophrenia, symptoms such as insomnia, and personality traits such as irritability, miserableness, mood swings, and risk-taking, among others, were identified for both traits  and NSSH   and suicide thoughts (maximum variance explained = 0.13%) . This association was also diminished when correcting for NSSH and further diminished when correcting for suicide attempt , and the associations described above were reproduced but including MDD-PRS as a covariate. The overall variance explained was reduced. Nonetheless, there was still evidence for an association between self-harm ideation significantly predicted suicide thoughts after including NSSH as a covariate; it also predicted NSSH after including suicide thoughts as a covariate, albeit with a smaller proportion of variance explained . The PRS for del Fig. .Figure 4broad sense self-harm by performing GWAS of self-harm ideation and self-harm behaviour in a population-based sample. The identification of only one genome-wide significant locus for each phenotype suggests that better powered genetic studies of self-harm are needed, and indicate that these traits are highly polygenic. Given the self-reported nature of the phenotypes, it is possible that recall bias and differences in subjective understanding of the mental health items could increase the noise to signal ratio in the discovery GWAS. Self-harm is a complex behaviour that encompasses subtypes with varying severities and recurrence rates59, which are not captured by the single item used to ascertain self-harm on the UKB.We explored the genetic architecture of self-harm ideation and four with self-harm behaviour. The gene with the strongest association with self-harm ideation, and the only gene associated with both phenotypes, was DCC. DCC is a gene involved in prefrontal cortex innervation and development. This observation could be consistent with reports of structural abnormalities on the brains of suicidal subjects61. Consistent with our results, DCC has been independently linked to suicidal severity on the UK-B62 and there is evidence of elevated DCC expression in the prefrontal cortex of post-mortem brains of subjects that died by suicide63.We also identified eleven genome wide significant genes using gene-based association tests. Seven were associated with self-harm ideation, FAM172A has been previously associated with differential methylation linked to childhood stress in girls64, and is located in a locus recently associated with insomnia65, a phenotype known to be associated with self-harm and suicidality67. Notably, previous studies suggest that the gene SEMA3D, known to be associated with schizophrenia69, could also be associated with suicidality24. A link between SYT14 and bipolar disorder has been reported70. DDX27 has been associated with intelligence72, and a study reporting a relationship between lower IQ and suicide attempt has been published73. Variants near DDX27 and ZNFX1 have been nominally linked to proneness to anger74. No obvious relationship between RPP14 and any psychiatric or behavioural phenotype has been reported in GWAS databases or the literature. Notably, we identified suggestive associations of a cluster of protocadherin genes (PCDH) on chromosome 5. PCDHAC1 is enriched in serotonergic cells in mice75 and PCDH-family differential methylation has been recently associated with early-onset major depression76 and previously associated with schizophrenia, bipolar disorder77 and autism78.Regarding genes associated with LINGO2, FBXO27 and WRB were associated with self-harm behaviour. LINGO2 and its paralog LINGO1 have been linked to neurodegenerative and psychiatric disorders80. Notably, previous results suggest that differential methylation on the promoter of FBXO27 might be linked with childhood physical aggression81, which is known to be highly associated with suicidal behaviours82. While WRB has been linked to cognitive impairment, it is unclear how it relates to self-harm behaviour. Finally, we found evidence for a suggestive association between STK10 and self-harm behaviour. STK10 has been linked with childhood cognitive ability83 and observed to be hyper-hydroxy-methylated upon acute stress84. Altogether these results provide promising candidate genes associated with self-harm ideation and behaviour. Future analyses should focus on replicating these findings and assessing their underlying mechanistic and possible translational roles on self-harm.Three genes: self-harm ideation and behaviour was significant—and some proportion of variance on the studied phenotypes was explained by PRS—, the percentage of variance explained was still far from the heritability estimates for NSSH (h2~37–59%), suicidal ideation (h2~47–66%) and suicide attempt (h2~55%) reported in twin and family studies85. Although the UKB recruitment process does not represent a random sample of the UK population86—and there is evidence of genetic factors associated with completion of the mental health section87— our PRS results provide evidence that the genetic associations discovered have some predictive power  over self-harm related phenotypes on an independent population. The above observations call for novel, well powered genetic studies of self-harm which will be required in order to obtain accurate SNP effect sizes88. A recent study suggest that even after well powered GWA studies have been conducted, most of the missing heritability for a phenotype is tagged by variants with a low MAF that cannot be easily imputed89. Therefore, whole genome sequencing studies of self-harm and suicidality could be paramount to achieve a complete understanding of the genetic architecture underlying self-harm.While the SNP-heritability of 7. Further, the follow up regressions correcting for suicide thoughts when predicting NSSH, and for NSSH when predicting suicide thoughts showed a reduction of the amount of variance explained. This observation is consistent with a partial shared genetic aetiology between them. To the best of our knowledge, this is the first study to date to report on a positive genetic prediction of NSSH. Notably, a previous PRS study assessing depression and self-harm in our sample34 identified an association between the genetic predisposition for depression and suicidal ideation, but no robust association with suicide attempt or non-suicidal self-harm. Thus, two possible explanations exist: (i) the previously stated genetic7 and phenotypic90 correlations between suicide thoughts and NSSH should be explained by depression-independent genetic factors, or (ii) the study by Maciejewski et al. (2017) was underpowered, possibly due to the low accuracy of the available summary statistics at that time and the number of cases in the target population, which limited its ability to detect an association between NSSH and the MDD-PRS.The fact that a PRS for self-harm thoughts was able to explain up to 0.27% of the variance of NSSH is consistent with a previous twin study reporting a significant co-heritability between suicide ideation and NSSHself-harm behaviour and suicide thoughts disappearing after correcting for suicide attempt. Finally, evidence of a shared genetic aetiology between suicide attempt and NSSH did not reach statistical significance, as no PRS simultaneously (robustly) predicted both of them. A possible explanation is that genetic predisposition to NSSH is not associated with suicide attempt predisposition, in spite of the known overlap between NSSH and suicidality91. Another plausible explanation is a lack of power either on the discovery  or target samples to detect an overlap between NSSH and suicide attempt due to a low prevalence of both behaviours.Our results support the existence of a genetic overlap between NSSH and suicide thoughts. We also detected genetic overlap between suicide thoughts and suicide attempt, which was evidenced by the association between the PRS for age at first birth was detected, and this was also observed for the most recent GWAS on depression58. Moreover, another study identified a negative correlation between maternal age at child birth and suicidality. Although the design of this study corrected for genetic confounding92, the association would support the hypothesis that genetic factors predisposing to changes in maternal age could impact on depression and therefore on suicidality and self-harm. The usage of PRS for independent traits to predict self-harm results in a tool of great power to test for pleiotropy and genetic overlap between them. For example, a significant genetic correlation of suicide attempt with insomnia has been reported35, and we have detected insomnia to also correlate with broad sense self-harm. The existence of GWAS summary statistics for insomnia65 makes this hypothesis testable using the approach implemented herein.Self-harm behaviours are likely to share genetic aetiology with several other traits and disorders. We have detected some of these candidate traits, such as neuroticism, nervousness and a risk-taking personality by assessing their genetic correlation with self-harm thoughts and behaviours. Interestingly, a negative genetic correlation of both self-harm phenotypes with 93, comprising a variety of acts such as physical injury and poisoning. Additionally, the self-reported nature of the phenotypes assessed in this study, could be affected by participant specific recall bias, which would bias our results towards the null. In the present study, we modelled a broad self-harm liability regardless of suicidal intent and performed analyses to unveil its underlying aetiology. Several traits, such as depression and other psychiatric disorders are associated with an increased risk for self-harm, as evidenced by the high genetic correlations identified. Our findings might be potentially identifying factors related to the genetic liability to depression and psychopathology in general. Nonetheless, the fact that we identified a positive prediction after accounting for MDD-PRS, and that previous depression PRS were unable to predict NSSH in our target sample34 would suggest our findings to be related to self-harm.Some limitations relevant to this study must be acknowledged. First, this study focused only on a sample of European Ancestry, an approach that allows to avoid biases due to population stratification. However, this hinders our ability to extrapolate to other populations. Furthermore, the samples used in this study were mostly comprised of adults in their late 50 s for the UK-B or in their 40 s for the Australian sample. This limits our ability to understand adolescent and childhood related self-harm. This is important because children and adolescents present a higher prevalence of self-harm compared to adults. Moreover, self-harm is highly complex and heterogeneousself-harm ideation and self-harm behaviour and identified associations with two genetic loci and eleven genes. We characterized the SNP heritability and estimated genetic correlations between the two traits of interest and with a range of other psychiatric, behavioural and physiological traits. Our results suggested an association between the genetic predisposition to broad sense self-harm (both ideation and behaviour) with suicide thoughts. However, no genetic overlap between PRS for self-harm ideation and suicide attempt was detected. The PRS for self-harm behaviour was associated with suicide attempt but the association with NSSH did not reach statistical significance. In addition, our results support a partially common genetic aetiology for NSSH and suicide thoughts and for suicide thoughts and attempt, but no statistically significant evidence for a shared genetic aetiology between NSSH and suicidal attempt. Future studies should leverage novel statistical genetic approaches such as genomic structural equation modelling, to aid in the deconvolution of unique and shared genetic factors between suicidal and non-suicidal self-harm.In summary, we performed GWAS of Supplementary Information.Supplementary Information 2.


Indications and diagnostic yield of small-bowel video capsule endoscopy (SB-VCE) are communicated in recent clinical academic guidelines. However, guidelines are based mainly on relatively few, small, selection-biased studies at experienced centers, and thus we lack information on indications and diagnostic yield of SB-VCE in the real-world community setting. The aim of the study was to evaluate indications and diagnostic yield of SB-VCE in the real-world community setting.Our local VCE clinical database was used to identify patients undergoing SB-VCE procedures over a 7-year period (2011–2018). Patients were broadly referred and underwent SB-VCE using PillCam™ SB 2 and SB 3 capsule systems. Procedures were reviewed by local endoscopists, who had undergone similar formal SB-VCE review training. Medical reports of the procedures were composed as such. We retrospectively reviewed all reports and gathered data regarding indications and findings. Diagnostic yield was considered positive if SB-VCE visualized any type of clinically significant pathological finding.536 SB-VCE procedures in 516 patients were included in final assessment. Patient mean (± SD) age was 50 ± 20 years with approximately even female/male ratio (275:241). The overall proportion of positive findings was 42% (225/536). The two main indications were obscure gastrointestinal bleeding  of 46% (246/536) and definite/suspected Crohn’s disease (CD) of 39% (210/536). Positive SB-VCE findings were obtained in 44% (108/246) of procedures with indication of OGIB and in 50% (104/210) of procedures with indication of CD.The indications for SB-VCE are largely consistent with guidelines but with an apparently relatively low diagnostic yield in our real-world community setting. Small-bowel video capsule endoscopy (SB-VCE) has been used as diagnostic tool for a variety of different indications. The importance of clear instructions for optimal clinical use and expectations of SB-VCE has been outlined together with appropriate indications and diagnostic yield in two recent consensus-based guidelines , 2.Guidelines state recommendations about the use of SB-VCE for a few absolute indications: obscure gastrointestinal bleeding (OGIB), known or suspected Crohn’s disease (CD) and small-bowel tumors. Recommendations are mainly based on meta-analyses, systematic reviews and studies conducted at single centers. Some of these studies focus on single indication for SB-VCE with respect to diagnostic yield. Other studies focus on comparison of diagnostic yield of SB-VCE versus other examination techniques such as fiber endoscopic modalities and imaging evaluation of the small-bowel. However, the majority of studies are conducted at experienced centers and with a relatively low number of patients and procedures. As such, they contain the inherent risks of not fully reflecting the real-world community setting –8. This The purpose of the present retrospective observational study was to assess indications and performance expressed in diagnostic yield of SB-VCE in our real-world community setting, using a large unselected number of SB-VCE procedures in an open referral system. The assessment was expected to give an insight into whether real-world community indications and diagnostic yield are in line with clinical guidelines. Accordingly, we believe that the outcomes of this study largely reflect indications for and diagnostic yield of SB-VCE in the real-world community setting.All patients were referred to SB-VCE examination at our institution  following conventional workup including both upper and lower fiber endoscopic examinations without an established diagnosis. Patients could be referred by physicians practicing in any type of health care center . Yet, the majority of patients were referred by gastroenterologists and surgeons practicing at our institution. Patients being controlled and monitored for chronic disease activity (predominately definite CD) were also referred to SB-VCE examination.During a 7-year period (2011–2018), 602 SB-VCE procedures were performed for a variety of indications. The SB-VCE procedures were reviewed by 11 local endoscopists that had undergone the same formal SB-VCE review training. SB-VCE reports were collected consecutively in our local database.SB-VCE reports included the following key parameters for SB-VCE procedure: indication, length, description of bowel visualization, findings and conclusions. Bowel visualization was characterized as being either good or reduced, depending on whether the mucosa was visible for evaluation. If the endoscopist encountered limited visualization and no pathology, the procedure was considered inconclusive and excluded for subsequent analysis. The timing of SB-VCE in the courses of examination for the indications/onset of symptoms varied on a patient case-by-case basis.Data for this study was extracted manually and retrospectively from 536 SB-VCE reports, and pre-SB-VCE data consisted of patient age, gender and date of procedure. No post procedure data were included.Due to the retrospective study design and collection of data, some indications were more frequent than others. As such, the diagnostic yield of the two major absolute indications OGIB and CD were chosen to be addressed in more detail.Medtronic PillCam™ SB2 and SB3 capsule systems were used. The SB2 capsule operating at a fixed picture frequency of 2 frames per second (FPS), while the SB3 capsule operating at a picture frequency of 2 or 6 FPS depending on capsule speed through the gastrointestinal (GI) tract. There was no indication for use of SB-VCE patency capsule in advance of SB-VCE procedures, as MRE scans were performed in selected patients presenting with symptoms, signs or history suggesting mechanical passage issues. The capsule transmitted the acquired images to the data recorder unit (Pillcam™ DR2 and DR3) located outside of the patient’s body. Images were then transmitted from data recorder to workstation (computer). SB-VCE examinations were viewed using the Pillcam™ reader software .SB-VCE procedures were all performed following a standardized protocol. Bowel preparation was initiated the day prior to procedure. Patients were instructed to stay on a clear liquid diet and a bowel lavage was given the evening before and the morning of the procedure. Patients swallowed the capsule and were permitted to go home with a data recorder contained inside a belt. Normal diet was resumed 4 h after capsule ingestion and patients returned with the data recorder the following day.Indications for SB-VCE were categorized in groups in relation to the general underlying issue. OGIB was defined as occult or overt GI bleeding. Anemia was considered a reliable indication for occult OGIB since occult bleeding would intuitively be the only reason for SB-VCE examination of anemia. CD was categorized as patients either having definite or suspected CD. Defecation disorders consisted of patients suffering from symptoms or signs of predominately diarrhea, alternatively alternating defecation pattern or mucus in stool. Furthermore, indications that were vague or not frequent enough for categorization into respective groups were assembled into a common group named miscellaneous.Findings were in some instances categorized primarily if they included non-specific findings, such as mucosal changes (erythema and edema) or mucosal lesions .The definition of clinically significant findings was defined as any type of pathological finding and not necessarily having a direct relation to the indication. These findings were classified as being contributive to diagnostic yield (i.e. positive findings).As a measurement of consistency and quality control of SB-VCE examinations, the median small-bowel transit time (SBTT) was calculated and compared with historical data reported from healthy individuals.Out of the 602 SB-VCE reports, a total of 66 reports were either not available (6 reports) or evaluated as inconclusive (60 reports) due to external factors . Specific causes for inconclusive reports are presented in Table Patients were referred across all age groups with increased frequency of patients in their twenties and sixties. Patient mean age was 50 ± 20  with approximately even female/male ratio (275/241). A majority of procedures  had good visualization of the small bowel, while a minority of procedures  were described as having reduced visualization albeit sufficient for diagnostic intend. SBTT was available for a majority of procedures . Overall median SBTT was estimated to 4 h and 18 mins (range 1 h to 14 h and 37 mins). No complications in relation to the procedures was registered and no capsule retention was reported.The major indications for SB-VCE were OGIB 46% 246/536) and CD 39% (210/536). Only a minority of CD patients, 15% (31/210), were diagnosed with CD before undergoing SB-VCE. Defecation disorder 5% (28/536), miscellaneous 4% (20/536), abdominal pain 3% (15/536), suspicion of tumor 2% (10/536) and suspicion of celiac disease 1% (7/536) were minor indications  for SB-VCE examinations was 42% 225/536). For the two main indications, OGIB and CD, the proportion of positive findings was 44% (108/246) and 50% (104/210), respectively. Specific findings for main indications OGIB and CD are depicted in Figs. 6. For thThe objective of this retrospective study was to assess indications and diagnostic yield of SB-VCE in a real-world community setting. We believe that the performance of SB-VCE observed in the present study reflects the real-world community setting in our health system in Denmark and maybe also in other similar health systems, since the assessment was based on a broad non-selective referral system and large number of SB-VCE procedures, outnumbering subjacent studies in recent guidelines , 2.The main indications were OGIB and CD, which is in line with guidelines , 2. HoweFurthermore, patients were not always recommended to undergo SB-VCE examination according to a distinct protocol, which is a consequence of the open referral system we have at our institution. This could explain some of the unconventional and uncommon relative indications provided by the referring physician. Finally, the studies referred to in the guidelines are characterized by generally including SB-VCE procedures with distinct indications and conducted at experienced centers , 2. ThusThe overall diagnostic yield observed in the present study was 42%, which is in line with the estimate of 48% observed in a recent Italian prospective multicenter study by Soncini et al.  DissectiWe need to acknowledge that comparing indications, and especially diagnostic yield, carries considerable risks of reaching inappropriate conclusions due to differences in study designs, including definitions and other confounders. The definition of what contributes to diagnostic yield is identical to that which is considered of clinical significance, and varies among studies. Thus, as stated in Enns et al. , standarThe variation in the distinction between significant and non-significant finding is also associated with the inherent intra- and interobserver bias of the present study. Having 11 different endoscopists review procedures could be a potential source of interobserver bias. On the other hand, this also reflects real-world community setting, where several physicians practice at the same institution. In a few instances, doubtful findings were conferred with colleagues (i.e. second opinion). Yet, most of the evaluations were concluded by a single reviewer and, in our opinion, likely reflect real-world practice.The evaluation of the SB-VCE procedures in the present study had a limited degree of standardization and nomenclature, which could contribute to increased interobserver bias, as outlined in other studies , 12. TheA weakness of the present study was the retrospective design and the inherent confounders and bias. Accordingly, SB-VCE procedure data were not uniformly registered. Furthermore, 11% of procedures had to be excluded, as they were not fit-for-purpose due to mainly impaired visualization of mucosa. In addition, given the different level of experience of the endoscopists, a procedure could generate different conclusions (interobserver variability) and thereby affect study findings. However, as SBTT  was in the anticipated range  and the One could argue that another potential weakness of the present study was the use of both SB2 and SB3 VCE systems, as this could be accompanied by differences in diagnostic yield depending on the system used. However, a recent study comparing these two systems did not support this claim, as only minor differences in diagnostic yield were recorded between the two capsule systems. A more detailed description of mucosal villi alterations is the main advantage of using the newer SB3 system .Finally, the time from the onset of symptoms to SB-VCE examination has been shown to influence the diagnostic yield in patients with indication of OGIB , 16. In We conclude that indications for SB-VCE in our real-world community setting are largely consistent with guidelines, but with an apparently relatively low diagnostic yield. It is evident that the VCE field needs further attention for standardization and refinement of definitions. Finally, adoption of a standardized referral protocol could also increase SB-VCE outcomes.


Anxiety and depression are common in the modern world, and there is growing demand for alternative therapies such as meditation. Meditation can decrease perceived stress and increase general well-being, although the physiological mechanism is not well-characterized. Endocannabinoids (eCBs), lipid mediators associated with enhanced mood and reduced anxiety/depression, have not been previously studied as biomarkers of meditation effects. Our aim was to assess biomarkers (eCBs and brain-derived neurotrophic factor [BDNF]) and psychological parameters after a meditation retreat.  This was an observational pilot study of adults before and after the 4-day Isha Yoga Bhava Spandana Program retreat. Participants completed online surveys  to assess anxiety, depression, focus, well-being, and happiness through validated psychological scales. Voluntary blood sampling for biomarker studies was done before and within a day after the retreat. The biomarkers anandamide, 2-arachidonoylglycerol (2-AG), 1-arachidonoylglycerol (1-AG), docosatetraenoylethanolamide (DEA), oleoylethanolamide (OLA), and BDNF were evaluated. Primary outcomes were changes in psychological scales, as well as changes in eCBs and BDNF. P < 0.001). All improvements were sustained 1 month after BSP. All major eCBs including anandamide, 2-AG, 1-AG, DEA, and BDNF increased after meditation by > 70% (P < 0.001). Increases of ≥20% in anandamide, 2-AG, 1-AG, and total AG levels after meditation from the baseline had weak correlations with changes in happiness and well-being.  Depression and anxiety scores decreased while focus, happiness, and positive well-being scores increased immediately after retreat from their baseline values ( A short meditation experience improved focus, happiness, and positive well-being and reduced depression and anxiety in participants for at least 1 month. Participants had increased blood eCBs and BDNF, suggesting a role for these biomarkers in the underlying mechanism of meditation. Meditation is a simple, organic, and effective way to improve well-being and reduce depression and anxiety. In the modern world, stress and anxiety have become synonymous with success and productivity. Over the course of a lifetime, almost half of Americans are estimated to have a mental disorder such as anxiety (>25% incidence) or mood disorder (>20% incidence) , 2. Therα, and increases in β-endorphins have also been shown with various meditation/yoga practices [Although certain benefits of meditation have been widely acknowledged, the physiological basis and underlying mechanisms have not been well-characterized. Cortisol levels may be reduced –7, and tractices .9 -tetrahydrocannabinol (THC) [Endocannabinoids (eCBs) are lipid mediators found in the brain and peripheral tissues that mimic the action of Δol (THC) . The eCBol (THC) . Serum lol (THC) . We hypoTo gain greater insights into the psychological and physiological effects of meditation, we evaluated participants at Bhava Spandana Program (BSP), an experiential program leading to deep states of meditativeness in just a short period of time. In Sanskrit, “Bhava” means “sensation” and “Spandana” can be loosely translated as “resonance.” Essentially, it means “resonance with life.” The program  is a 4-dThe BSP is a 4-day, 3-night, residential program offered to those who have been initiated into Shambhavi Mahamudra. This advanced meditation program is designed to provide the opportunity to go beyond the limitations of body and mind and experience higher levels of consciousness. Bhava Spandana offers the experience of a world of unbounded love and joy.Through powerful processes and meditations, BSP creates an intensely energetic situation, where individuality and the limitations of the5 sense organs can be transcended, creating an experience of oneness and resonance with the rest of existence. Humans have lived within the limitations of human senses. Bhava Spandana is like giving one a lift or jump over the wall to have a peep at life beyond the limitations of the five sense perceptions. Past BSP participants have described feelings of blissfulness, ecstasy, intense happiness, inclusive perception, and higher states of consciousness during and after the program.Our hypothesis was that BSP meditation would significantly reduce depression and anxiety and improve happiness and positive well-being in the short-term as well as in the long-term. Furthermore, these changes in psychological well-being are associated with objective changes in blood eCB and BDNF levels. This study specifically aimed (a) to assess the impact of this 4-day guided, experiential meditation retreat on mental health and well-being , (b) to correlate reported psychological changes with objective blood biomarkers (eCBs and BDNF) immediately after the retreat, and (c) to assess any persistent impact on participants' psychological well-being one month after the retreat.In October 2017, adult participants (≥18 years) were registered to participate in the 4-day BSP meditation program at Isha Institute of Inner-Sciences, McMinnville, Tennessee, USA. All registrants received an e-mail which invited them to participate in an online survey 2 weeks before the meditation program. Participation in the study was voluntary. Individuals who were unable to read and/or comprehend the consent forms were excluded. For the blood biomarker subset of the study, individuals were excluded if they reported use of active marijuana, opioid, or other illegal drugs, or if they were taking antidepressant medication. The protocol was reviewed and approved by Institutional Review Board of Indiana University School of Medicine. All participants gave electronic informed consent.The BSP is a 4-day advanced yoga and meditation program designed to enhance participants' perception and sensitivity to life by going beyond the limitations of body and mind and experiencing higher levels of consciousness . ParticiParticipants completed the preprogram (“baseline”) survey within 2 weeks before the retreat and postprogram (“immediately after retreat”) survey within 2 weeks after the retreat, and a 1-month follow-up survey. These surveys included scales that have well-established reliability and validity in the literature. Depression is measured by the 20-item Center for Epidemiologic Studies Depression Scale  (CES-D).Anxiety is measured by the 8-item Patient-Reported Outcomes Measurement Information System (PROMIS) Emotional Distress-Anxiety (short form) . The scaMindfulness is measured by the 15-item Mindful Attention Awareness-Trait Scale (MAAS) . The MAAWell-being is measured by Ryff's 42-item Psychological Well-Being (PWB) Scale . Ryff's Only those who completed the first survey were asked to complete the postintervention surveys. Those who did not complete the survey within a week of the first invitation were sent e-mail reminders.Survey participants were given the option for blood biomarker evaluation. Informed consent for blood draws was obtained, and the participant's samples were collected at the following time points: (1) preprogram (up to 2 days before program began) and (2) postprogram (within 2 days after program ended). Standard sterile venipuncture technique was used to collect 10 mL of blood divided into different sampling tubes for measuring anandamide, 1-AG, 2-AG, DEA, OLA, and BDNF.For participant confidentiality, samples were deidentified with a code for sharing with individuals outside of the study team. The biomarker samples were sent immediately to the analysis lab in Denver, Colorado. As anandamide is quickly catabolized by Fatty Acid Amide Hydrolase (FAAH), the fresh blood samples were centrifuged and extracted plasma was frozen immediately to preserve and accurately measure this biomarker.t-tests were used to compare biomarkers for significant change from before meditation to after meditation.Repeated measures analysis of variance (ANOVA) was used to compare preprogram (baseline), immediately after program, and 1-month follow-up survey responses; an unstructured variance-covariance matrix was used. In the subset of subjects with biomarkers assessed, paired Pearson correlation coefficients were used to evaluate the associations between the changes in biomarkers and changes in psychological states. In addition, the change in biomarkers was categorized into two groups, including one group where there was at least a 20% increase and another where there was less than 20% increase. The responses to the psychological scale were separately analyzed for both of these groups. A 5% significance level was used for all tests.Three hundred forty-eight participants completed the preprogram survey and 323 participants completed the postprogram survey, resulting in a 93% response rate. The 1-month follow-up survey was completed by 308 participants. One hundred and forty-two participants volunteered for blood sampling for biomarker assays before and after meditation retreat. Subject characteristics are shown in P < 0.001; Scores for depression (CES-D) and anxiety significantly decreased from baseline to immediately after retreat , happiness, and psychological well-being (PWB) increased significantly from baseline to immediately after retreat  by 2.0 ng/mL (SD = 2.8) with an effect size of 0.71, and 71% of participants had an increase of at least 20%. Similarly, BDNF increased (P < 0.001) by 5945 pg/mL (SD = 8414) with an effect size of 0.71, and 53% of participants had an increase of at least 20%.Endocannabinoids  and BDNF were analyzed in 142 participants. Compared with baseline levels, all immediately after retreat biomarker levels were higher . Some inParticipants with increases in 2-AG levels larger than 20% after meditation from the baseline had larger increases in mindfulness, happiness, and positive well-being  scores (Supplementary Table ). SimilaIn this large, prospective study, we showed that a short, intense guided experiential meditation (BSP) significantly decreases anxiety and depression and improves psychological well-being, happiness, and mindfulness and that these improvements sustain for at least a month. For the first time, we showed that objective blood biomarkers, eCBs  and BDNF, all increased following an advanced meditation, BSP, suggesting a role for these mediators in the underlying mechanisms of meditation. The specific mechanisms linking meditation to positive psychological effects have not been well-characterized in the literature, and this research provides early evidence that the eCB system and BDNF are significantly increased after advanced meditation.Low blood levels of anandamide and 2-AG have been reported in patients with depression . DeficieThe level of increase in eCBs following meditation was large enough to be deemed clinically significant, as effect sizes ranged from 0.77 to 0.90, and some participants had 2-3-fold increases. This is more than reported eCB increases associated with moderate intensity aerobic physical exercise  and sexuActivation of CB1 receptors has been shown to increase BDNF expression . We demoThe sustained positive impacts from meditation, even one month after BSP, are encouraging. The BSP participants experienced persistent positive benefits from the short and intensive BSP meditation, extending beyond an immediate “high” or bliss feeling from increased eCBs. An intense short-term negative experience can lead to diseases like posttraumatic stress disorder; thus an intense short-term positive experience might similarly lead to lasting positive psychological consequences. Completion of Inner Engineering program (level 1) and daily practice of Shambhavi kriya is a requirement for BSP, and participants were encouraged to continue this daily practice after the BSP to maximize potential benefits. Continued daily meditation may have contributed to some of the sustained positive results at 1 month after BSP in our study. Given certain logistical constraints (some BSP participants were from different states or other countries), we did not collect blood samples to reassess biomarkers at 1 month. This could be evaluated in future studies.Weak but positive correlations between biomarker changes and changes in psychological measures, including some interindividual variations, were observed in this study. The findings suggest that there are interindividual variations in objective biomarkers, similar to variation in experiences and happiness levels. Because of the disparity in subjective experience and physiological responses, the benefits and expression of biomarkers from meditation may vary between individuals. This needs further study to explore the interindividual variations in subjective and biomarker responses in people simultaneously participating in a meditation program. Factors such as genetics/epigenetics, gene expression, and corresponding psychological health may explain these interindividual variations.Strengths of this study are prospective design, relatively large number of meditators participating simultaneously in validated psychological surveys and blood biomarkers before and after meditation, hypothesis-driven approach to mechanistically associate the beneficial effects of meditation with simultaneous objective biomarker assay associations, use of validated scales for sustained psychological benefits of meditation for up to 1 month, and generalizability of the findings as the study meditators had diversity in terms of age, sex, race, and ethnicity. Limitations included lack of 1-month postretreat biomarker assessment, as discussed above. Additionally, the 4-day BSP retreat had multiple daily meditation sessions, and the “immediately-after-meditation” blood samples were actually collected after the entire program. Because eCBs (particularly anandamide) are highly labile, biomarker expression would likely have been even higher if samples had been collected during the retreat. The study population had low baseline levels of anxiety and depression; therefore, differing results might be obtained by analyzing the effects of meditation on patients with psychological disorders. Finally, participants were not surveyed on postretreat meditation or other activities that may have contributed to the sustained long-term benefits.Since the meditators in this study were prospectively and objectively followed over 3 time points, each individual essentially served as his/her own control. To rule out a placebo effect, we analyzed objective biomarkers in addition to self-reported psychological changes. The lack of a separate, matched control group may be viewed as a study limitation. However, the significant changes in objective biomarkers and the sustained psychological benefits (despite only a brief meditation retreat) strongly support that this is a true effect rather than a placebo. This pilot study provides evidence to support future larger studies (including a control group) with longer-term follow-up to assess the persistent benefits of meditation.More research is needed to understand the role of the eCB system in the mechanisms of meditation. In future, the effects of meditation may be replicated through pharmacologic intervention, as FAAH inhibitors can reduce anandamide degradation. Inhibition of FAAH has been considered as a treatment option for anxiety and other psychological disorders , 51–54; An intense 4-day guided Isha meditation retreat significantly decreased depression and anxiety while improving happiness, mindfulness, and psychological well-being. Increased blood endocannabinoids and BDNF were also observed immediately after the intense meditation, potentially explaining the underlying mechanism behind the improvements in happiness and other psychological benefits. The psychological effects of the meditation retreat were sustained for at least 1 month. Meditation might serve as a simple, organic, nonpharmacological, and effective low-risk therapy or a prevention strategy for depression and anxiety, while improving happiness and well-being. Future studies are needed to investigate the role of the eCB system as mediators of the positive effects of meditation, the sustained benefits of meditation over longer-term, and reasons for interindividual variability in meditation response.


To validate the specimen-pooling strategy for real-time reverse transcription PCR detection of severe acute respiratory syndrome coronavirus 2, we generated different pools including positive specimens, reflecting the distribution of cycle threshold values at initial diagnosis. Cumulative sensitivities of tested pool sizes suggest pooling of <6 specimens for surveillance by this method. After the first report of the coronavirus disease (COVID-19) outbreak in Wuhan, China . Therefore, we evaluated the pooling strategy for SARS-CoV-2 testing using clinical specimens from 3 hospitals in South Korea: Seoul Medical Center and National Medical Center, both in Seoul, and Jeonbuk National University Hospital in Jeonju. The Institutional Review Boards of the hospitals approved this study. Written consent from participants was waived.Testing pooled specimens is a well-known method and has been used in blood banks worldwide to screen for infectious disease; however, only a few studies have evaluated specimen pooling for SARS-CoV-2 (https://www.copangroup.com). Laboratory diagnosis of SARS-CoV-2 infection was performed with all specimens using the following rRT-PCR kits targeting the E and RdRp genes: STANDARD M nCoV Real-time Detection  or PowerCheck 2019-nCoV Real-Time Detection .Pooled upper respiratory specimens were prepared from 50 individual SARS-CoV-2–positive specimens and 300 individual SARS-CoV-2–negative specimens. Either a single nasopharyngeal swab (NPS) or a nasopharyngeal and an oropharyngeal swab (NPS/OPS) were collected in an eNAT tube  values of r RT-PCR for patients confirmed positive during January 20–March 2, 2020 , Real-prep , and eMAG . We followed the extraction protocol provided by each manufacturer with an input volume of 200 μL and elution volume of 50 μL.The following 3 automated RNA extraction systems were used: MagNa Pure 96 . The distribution of Ct values in individual specimens (t>30, which was near the cutoff value. We selected positive samples for pooling according to this distribution pattern.We performed all statistical analyses with MedCalc version 19.2.1 (MedCalc Software Ltd, pecimens  showed nt values increased for both the E and RdRp genes as the pool size increased (The pooled positive specimens had 100% sensitivity in pool sizes 2, 4, and 6 and 97%–99% sensitivity in pool sizes 8, 10, and 16 . To ensuWe evaluated the clinical sensitivity and specificity of SARS-CoV-2 rRT-PCR using pooled upper respiratory specimens from confirmed cases. Because pooled specimens are expected to be used as a screening tool, the clinical sensitivity of pooled specimens at a given pool size is especially important.t values of positive specimens from patients at the time of diagnosis were not considered in the study design. The Ct values of specimens in previous studies were relatively low (t values, meaning low virus titers, are expected to be vulnerable to pooling, the distribution of Ct values in the actual population should be reflected when determining the pool size. We analyzed the actual distribution of Ct values from 4,364 initially confirmed cases, and the distribution showed skewness with regard to the PCR cutoff value.A limitation of previous studies is that the C<64; however, we do not recommend increasing the pool size to 64, corresponding to a theoretical increase in Ct values of 6, given the associated loss in sensitivity; doing so may cause false negative results.Yelin et al. (<6 specimens in clinical practice. Pooling >6 specimens might cause false-negative results, considering the observed abundance of specimens with high Ct values in the population.The pooling strategy showed efficiency when the positive rates in the population were low (t value <35 within 40 amplification cycles. Therefore, this study did not include individual specimens with a Ct value >35, which is interpreted as an inconclusive result by this kit. Third, we did not evaluate cost-effectiveness on the basis of the hypothesized prevalence. Last, we did not evaluate the effect of specimen volume in the pools; increasing the input volume from each specimen may improve the sensitivity of the pooling test.This study has some limitations. First, the analytical performance of the PCR kit used has not been evaluated fully because it is one of the earliest available commercial PCR kits that received the Emergency Use Authorization in Korea. Second, the positive cutoff in the kit was a COur protocol will be helpful for screening persons in groups at high risk for COVID-19 infection quickly and quarantining those confirmed positive, even in situations with limited time and test resources. Epidemiologic factors should be considered when choosing an adequate pooling number. Symptomatic case-patients should be tested individually without pooling to enable effective and timely action. We have included practical guidelines for specimen-pooling procedures in the Appendix.Additional information about sampling from pooled specimens for rapid mass screening for coronavirus disease.


Since December 2019, SARS-CoV-2 has infected millions of people worldwide, causing excess deaths and a surge in demand for ICU beds. With no effective therapies against SARS-CoV-2, randomized trials of several potential therapeutic agents, including steroids, have been conducted. Although use of steroids in patients with ARDS  has beenThe RECOVERY trial compared administration of 6 mg/day dexamethasone for 10 days to usual care in 6425 hospitalized patients with SARS-CoV-2 infection. Survival was significantly higher in the dexamethasone-treated patients, especially in the subgroup of 1007 patients receiving invasive mechanical ventilation , 299 [9]Most multicenter randomized trials stratify by center to minimize potential center bias, but this was not done in the RECOVERY trial . AlthougMoreover, several factors known to contribute to mortality in COVID-19 were not measured, including ethnicity and obesity. For patients receiving mechanical ventilation, severity of hypoxemia, ventilator settings and other types of organ support were not reported, yet are associated with outcome. For those factors that were measured, their frequencies in the two study groups were not reported for the subgroups. The possibility of an imbalance between the groups therefore cannot be excluded.p = 0.0084), even though the RECOVERY trial contributed 57% of the weight of the meta-analysis, and the trial results seemed to show a greater benefit in ventilated than in non-ventilated patients.The strength of this meta-analysis  is the iMoreover, from the three trials stopped prematurely –10, the Assessing outcome at 28 days may not be optimal for several reasons. In a randomized trial of corticosteroids in patients with ARDS, short-term variables improved with steroids, but there was no decrease in ventilator-free days at day 180 and even an increase in 90-day mortality in some subgroups . In the The RECOVERY trial results suggest that corticosteroids may be beneficial in patients with COVID-19,especially when mechanical ventilation is required. However, the absence of stratification and incomplete information about some factors associated with outcome may have resulted in imbalance between the treated and control groups. While the meta-analysis points toward a beneficial effect of corticosteroids, it also has limitations, precluding definitive conclusions. Finally, beneficial effects at the relatively short day-28 mortality endpoint may not translate into longer-term benefit. Studies to assess longer term physical and functional outcomes in critically ill COVID-19 patients are warranted.


The tumour microenvironment contains a unique population of cells, of complex origin and diverse functionality, known as Cancer associated fibroblasts (CAFs). In recent years there has been a growing interest in targeting CAFs to aid cancer diagnosis and therapy. Amongst the approaches utilized so far, targeting Fibroblast activation protein (FAP) has shown a lot of promise. In this review, we will focus on our current knowledge of the biology of CAFs as well as theranostic applications that will enhance cancer diagnosis and therapy in cancers carrying a distinct CAF signature.Cancer Associated Fibroblasts (CAFs) form a major component of the tumour microenvironment, they have a complex origin and execute diverse functions in tumour development and progression. As such, CAFs constitute an attractive target for novel therapeutic interventions that will aid both diagnosis and treatment of various cancers. There are, however, a few limitations in reaching successful translation of CAF targeted interventions from bench to bedside. Several approaches targeting CAFs have been investigated so far and a few CAF-targeting tracers have successfully been developed and applied. This includes tracers targeting Fibroblast Activation Protein (FAP) on CAFs. A number of FAP-targeting tracers have shown great promise in the clinic. In this review, we summarize our current knowledge of the functional heterogeneity and biology of CAFs in cancer. Moreover, we highlight the latest developments towards theranostic applications that will help tumour characterization, radioligand therapy and staging in cancers with a distinct CAF population. Cancer remains the second leading cause of death in the world , despiteThe TME may consist of more than 50% of the tumour mass and together with tumour cells it coordinates rapid proliferation, local invasion and eventually metastasis to the surrounding tissues . In addiTME-specific markers, found during tumour development, can be useful not only to understand the composition of TME, but also to aid diagnosis and therapy strategies . It has Growing evidence proves that CAFs can stimulate a malignant tumour phenotype due to their heterogeneous nature and functional diversity. Others suggest that a distinct CAF subpopulation, known as myofibroblasts (myCAFs), can have a more tumour-restraining role . It is tUnder normal conditions, fibroblasts are quiescent spindle shaped cells located in the connective tissue of most organs. They regulate the polarity and function of normal epithelium by generating the basement membrane. During tissue fibrosis and wound healing, these same fibroblasts are transformed into smooth muscle reactive fibroblasts and contribute to tissue repair . The assStudies have shown that CAFs are responsible for the synthesis and remodelling of the ECM and play various roles in cancer metastasis, angiogenesis, and drug resistance. Even though extensively studied in vitro, the CAF population remains largely uncharacterized in regards to its origin, biology, and subtypes , presentA number of different tissue types are responsible for the origin of CAFs , highligThe best-studied cell population of CAF origin consist of mesenchymal stem cells (MSCs). In vitro and in vivo studies have provided evidence that MSCs from bone marrow (BM-MSCs) can differentiate into a CAF subtype in tumours such as gliomas, breast, and pancreatic cancers ,17.In addition to the adjacent sources, a number of CAFs can be identified from a non-fibroblastic origin. Such cells can be epithelial or endothelial cells, through epithelial-to-mesenchymal transition (EMT) or endothelial to mesenchymal transition (EndMT), respectively. The latter transition, was first observed during heart formation where an exchange in TGF-β signalling from endothelial to fibroblast-like cells was identified in cardiac tissues, suggesting that a similar phenomenon occurs in cancer tissues . During Adipocytes can also transform into fibroblasts through expression of mesenchymal bone marrow lineage-committed markers , peroxisome proliferator activated receptor-γ (PPARγ)) . AdipocyThe difficulty of recognizing CAFs and their origin, lies in the absence of specific markers, although markers that are not expressed in normal cells can be used as an indicator, such as the aforementioned α-SMA . AnotherSeveral studies utilizing cell culture as well as in vivo experiments in mouse models, have suggested a diverse number of functions that CAFs might employ. In their study, Olumi et al. were among the first to show that CAFs promote tumour growth in a prostate cancer mouse xenograft model while normal fibroblasts have more inhibitory properties . In addiIt is well known that tumour growth depends on the uncontrollable proliferation of tumour cells . This ra2O2) by cancer cells initiates oxidative stress in CAFs, followed by reduction of mitochondrial function, increase of glucose uptake and ROS levels, and finally leads to CAF differentiation. As a result, a reactive TME is generated by ROS, where CAFs sustain the energy needed for cancer cell proliferation Ga-FAPI-2 and [68Ga]Ga-FAPI-4 in two patients, whereas further PET/CT scans were acquired of 25 patients. Similar to literature values for [18F]FDG  ,109,110.ax 7.41, ), [68Ga]3 ± 7.5, ) 200 MBql et al.  quantifi68Ga]Ga-FAPI-46 showed a favourable dosimetry profile with an estimated whole-body dose of 5.3 mSv for an administration of 200 MBq. The biodistribution study showed high TBRs increasing over time, making this an interesting tracer for future theranostic applications Tc-FAPIs were developed as well and showed specific binding to recombinant FAP-expressing cells with high affinity [99mTc]Tc-FAPI-34 was applied for diagnostic scintigraphy and SPECT of patients with metastasized ovarian and pancreatic cancer for follow-up to therapy with [90Y]Y-FAPI-46. [99mTc]Tc-FAPI-34 accumulated in the tumour lesions also shown in PET/CT imaging using [68Ga]Ga-FAPI-46, making this a powerful tracer for diagnostic scintigraphy, especially in cases where PET imaging is not available. Additionally, the chelator used in this compound allows labelling with the therapeutic radionuclide 188Re. Watabe et al. applied radionuclides with relatively long half-lives, 64Cu and 225Ac to label FAPIs for studies in mice with human pancreatic cancer xenografts. Accumulation levels in the tumour and most normal organs were significantly higher for [64Cu]Cu-FAPI-4 than for [68Ga]Ga-FAPI-4. [225Ac]Ac-FAPI-4 injection showed significant tumour growth suppression in PANC-1 xenograft mice indicating that [64Cu]Cu-FAPI-4 and [225Ac]Ac-FAPI-4 could be used in theranostics for the treatment of FAP-expressing pancreatic cancer [Loktev et al.  developeaffinity . The leac cancer .18F-labelled FAPI ([18F]FGlc-FAPI) and concluded from a preclinical study it is an interesting candidate for translation to the clinic, taking advantage of the longer half-life and physical imaging properties of 18F. Giesel et al. [18F]F and 68Ga. In ten patients with lung cancer PET-scans were acquired after administration of [18F]F-FAPI-74, with the highest contrast achieved 1 h p.i. in primary tumours, lymph node and distant metastases with SUVmax > 10, respectively. The radiation burden of a diagnostic [18F]F-FAPI-74 PET-scan was lower than that of PET-scans with [18F]F-FDG and other [18F]F-tracers, whereas [68Ga]Ga-FAPI-74 was comparable to other 68Ga-ligands. The high contrast and low radiation burden of FAPI-74 PET/CT favours multiple clinical applications. So, tumour-to-background contrast ratios of different FAPIs were equal to or even better than those of [18F]FDG [18F]FDG, no diet or fasting in preparation for the examination is necessary, and image acquisition can potentially start a few minutes after tracer application. Despite their advantages, utilization of FAPIs in the clinic may face some limitations as there is still not enough information on its specificity, suggesting a decreased performance in patients who might have other diseases that are characterized by high levels of fibrosis.Toms et al.  synthesil et al.  describe[18F]FDG ,114. In 90Y]Y-FAPI-4 was chosen for a proof of principle approach in a final stage breast cancer patient with bone metastases [The efficacy of clinical radioligand therapy via crossfire effects is not yet known. [tastases . This waThe results of the first FAPI tracers are very encouraging for theranostic applications for a wide variety of tumours. Here, we highlight several opportunities for future FAP tracers.2O2 and glutathione. These tumour specific properties are currently evaluated in pro-drug strategies [As most clinically valuable tracers belong to one family of small molecule tracers, based on FAP inhibitor UAMC-1110, it is currently the only available FAP inhibitor with low nanomolar activity and high selectivity. This calls for the development of novel small molecule FAP inhibitors, of which the scaffolds can be used for the design of new FAP theranostics with new and potentially improved properties. Another strategy, adopted by Clovis Oncology, is based on a peptidomimetic FAP-2286) to target FAP  and the 6 to targrategies ,120,121 Despite the increasing progress in cancer research and the development of novel therapeutics, conventional therapeutics are not adequate enough. The majority of efforts so far were only focused on targeting the cancer cells and even though highly abundant, CAFs have long been ignored in these efforts. The emerging role of CAFs is slowly appearing to be of great importance and their functions in tumour progression and metastasis have become an attractive target for imaging and therapy. However, a number of challenges need to be addressed in order to reach translation from bench to bedside. Looking at their structural and functional complexity, diagnostic markers that can specifically recognize CAFs are still limited. This complexity is also outlined by the various CAF cell origins. In the TME, CAFs co-exist with tumour cells providing them the paracrine niche and nutrients that are needed for tumours to grow. Breaking this contact and pinpointing the role of each of the microenvironment compartments, may appear pivotal for discoveries of novel therapeutic interventions. Fortunately, technological advances over the past years are opening new opportunities in understanding the complex biology of CAFs in cancer and will pave the way for new targets that can be used in future developments, one of those targets being FAP. The high and rather selective tumour uptake of very promising FAP-targeting tracers as described opens up applications for non-invasive tumour characterization, staging examinations, and radioligand therapy in many different cancers with a high content of activated fibroblasts.