The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists reduce food intake and body weight and are approved for weight loss . Elucidaαs-coupled receptor that signals via protein kinase A  Raptor or Myc-tagged mutant Raptor with alanine replacing serine at the 791st position (Ser791Ala Raptor) in CHO-GLP-1R cells. Using an antibody against the PKA motif RRXS*/T*, immunoblotting of immunoprecipitated Myc-proteins shows that liraglutide and forskolin increase PKA motif phosphorylation on Myc-WT Raptor in a PKA-dependent manner (blocked by H89), but liraglutide fails to increase PKA motif phosphorylation on Myc-Ser791Ala Raptor  and knock-in mice in which endogenous Raptor is replaced by a PKA-resistant Raptor expressing alanine instead of serine at position 791 in all tissues (CMV-Ser791Ala Raptor). WT and CMV-Ser791Ala Raptor knock-in mice lost weight in response to liraglutide treatment, but liraglutide-induced weight loss was attenuated in the CMV-Ser791Ala Raptor knock-in mice  to test whether PKA phosphorylation of Raptor at Ser791 is also required for the weight loss effect of liraglutide in the setting of obesity. Unlike lean mice, obese CMV-Ser791Ala mice were not significantly protected from weight loss induced by liraglutide  where a PKA-mTORC1 interaction mediates the anorectic effect of GLP-1R agonists.In vivo results using novel PKA-resistant Raptor knockin mice suggest that PKA phosphorylation of Raptor contributes to the weight loss effect of liraglutide in lean but not in obese mice. One limitation of these in vivo studies is that PKA-resistant Raptor is expressed in all tissues. Loss of PKA phosphorylation of Raptor in one or multiple tissues may contribute to our observation that CMV-Seraglutide . This coagonists . Regulatagonists , where tagonists . The ARCagonists  and is eagonists . Hindbraagonists . Inhibitagonists . Future 791Ala Raptor knock-in mice were not resistant to the weight loss effect of the MC4R agonist setmelanotide suggests that PKA-mediated phosphorylation of Raptor at Ser791 is not a general mechanism for all Gαs-coupled receptor-targeting weight loss drugs. However, as with our results using liraglutide in HFD-fed mice, a caveat to interpreting our findings with setmelanotide is the use of the whole-body CMV-Ser791Ala mouse model. Since the weight loss effect of setmelanotide is primarily mediated via activation of the MC4R in the PVH, future studies will test the hypothesis that the weight loss effect of setmelanotide will be blunted in PVH MC4R-specific Ser791Ala Raptor knock-in mice. This could establish a role for PKA-mediated regulation of mTORC1 as a key mechanism for weight loss along an ARC (target of GLP-1R agonists)-PVH (target of MC4R agonists) axis.The observation that CMV-SerOur results suggest that liraglutide does not regulate mTORC1 via Akt, as was observed for βAR signaling in adipocytes . InhibitIn summary, we have uncovered a novel signaling mechanism downstream of the GLP-1R characterized by PKA-mediated phosphorylation of Raptor and increased mTORC1 signaling that contributes to the weight-lowering effect of liraglutide. Given the variability in response and adverse effects (e.g. nausea) of current GLP-1-based drugs, the identification of therapeutically relevant signaling profiles downstream of the GLP-1R holds promise for improving efficacy and reducing side effects of these drugs. Furthermore, our findings provide a new framework for future investigations into the significance of the PKA-mTORC1 interaction to other beneficial metabolic effects of GLP-1R agonists. GLP-1R agonists continue to be used as glucose-lowering agents for type 2 diabetes based on their insulinotropic effect via pancreatic β-cells . The PKAChinese Hamster Ovary cells expressing human GLP-1R  were maintained at 37 °C  in Ham’s F12 media containing 10 mM glucose, 1 mM pyruvate, and 1 mM glutamine  supplemented with 10% fetal bovine serum , 10 ml/l penicillin-streptomycin , and 0.8 mg/ml Geneticin G418 Sulfate . β-TC3 cells  were gifg, 10 min, 4 °C), supernatant protein concentration was determined by BCA protein assay . Twenty μg/lane was run  in 4–20% Tris-glycine gels (Invitrogen) and transferred  onto PVDF membranes (Bio-Rad). Blots were blocked in buffer containing 5% BSA  in 1 X Tris-Buffered Saline and 0.1% Tween–20 for 1 hr and incubated in primary antibodies  overnight at 4 °C and secondary antibodies  for 1 hr at room temperature. Blots were imaged with an Odyssey Imaging System and full lanes were analyzed using ImageJ.Cells were washed once with ice-cold 1 X PBS and lysed in RIPA buffer  containing 1 X protease and phosphatase inhibitors. After centrifugation of cell homogenates  Raptor vectors  supernatant protein concentration was determined by BCA protein assay. A total of 200 μg protein was incubated with anti–c-Myc affinity gel  overnight. Beads were washed in lysis buffer and immunoblotting was done as described above using a PKA substrate monoclonal antibody .Myc-tagged wild-type (WT) and PKA-resistant mutant  were crossed to mice expressing the Ingenious construct, and offspring were then crossed to CMV-Cre  mice to generate hemizygous wild-type mice or hemizygous mice with global knock-in of Raptor Ser791Ala. This strategy was chosen because the Ingenious construct in homozygosity prior to Cre recombinase inadvertently created a hairpin loop in the transcript between the two exon 20 regions that was spliced out. Genotyping and cDNA sequencing confirmed expression of Ser791Ala Raptor in place of endogenous Raptor  from weaning and were studied at 12–16 weeks old. Procedures were approved by the Institutional Animal Care and Use Committee at Vanderbilt University (Protocol #M1500158). Sers Raptor . Male miData were analyzed using Prism-9 . One-way or two-way ANOVA or mixed-effects analysis followed by Tukey’s, Holm-Sidak’s, or Brown-Forsythe multiple comparisons was used when appropriate and indicated in the figure legends. Linear regression analysis was also used as indicated in the figure legends. Sample size was determined based on the variability of the primary measurements of interest (phosphorylation of S6 for cell culture studies and body weight for in vivo studies) and the objective of detecting significance (p<0.05) with 90% certainty. Treatments were randomized and blinded to the experimenter when possible. This manuscript provides support for the importance of PKA-dependent mTORC1 activation for the weight-loss effects of Liraglutide, and presumably many GPCR-dependent anorectic agents, while not affecting the food intake-reducing effects of the MC4Ractivator setmelanotide. The weight loss effect of liraglutide however was not impaired after HFD feeding, a model of obesity where GLP1 agonists are being used. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Daniela Cota (Reviewer #2); Hongxia Ren (Reviewer #3).Thank you for submitting your article "Glucagon-Like Peptide-1 Receptor Activation Stimulates PKA-Mediated Phosphorylation of Raptor and this Contributes to the Weight Loss Effect of Liraglutide" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers agreed that the paper has potential, but the authors will need to provide substantial new data to answer the reviewers' concerns.1) Specifically, provide a comprehensive characterization of the mouse model in addition to the studies provided.2) Demonstrate specificity of the weight-loss effects to GPCR-PKA signaling.3) Provide a more comprehensive analysis of the changes in energy expenditure and provide a more comprehensive metabolic phenotyping (insulin levels etc).Reviewer #1 (Recommendations for the authors):1. While I understand that the authors intend to publish a full characterization of the model elsewhere, it is impossible to evaluate the results of this manuscript without some information on the genetics of the model, and some data showing the expression of the mutant, however. The authors could add some more detail about the mouse model to this manuscript and send the manuscript describing the model in more detail for the reviewers to examine.2. The authors should examine the normal regulation of feeding/body weight by non-GPCR-dependent agents . They would want to examine the effects of these on PKA/pS6 in cultured cells, as well.3. The authors should provide direct comparisons of the various conditions, rather than normalizing to individual baseline measurements.Reviewer #2 (Recommendations for the authors):1. The authors claim that the difference in efficacy in weight loss in WT vs raptor mutants treated with Liraglutide is due to a very minor difference in food intake during the second day of treatment. By looking at the data, the sustained weight difference cannot be due to a very transitory difference in food intake, which by the end of the treatment has become comparable to the food intake of vehicle-treated mice. Authors should investigate changes in energy expenditure.2. Related to the above, previously published evidence suggests that central GLP1R agonism stimulates thermogenesis by recruiting the sympathetic nervous system . It would be relevant to assess whether recruitment of the SNS is impaired in the Raptor mutant mice under liraglutide treatment. Authors could assess molecular changes for SNS markers and markers of increased thermogenesis in the brown adipose tissue and in the white adipose tissue. Authors could also assess possible differences in cold-induced SNS activity in their model.3. It is unclear why the authors did not test the relevance of their observations by inducing diet-induced obesity in their mouse model. Does the raptor mutant have a specific phenotype under diet-induced obesity?4. Liraglutide importantly impacts glucose metabolism. Here the authors show that 2-weeks Liraglutide treatment decreases fasting glucose levels independent of genotype. The authors however do not provide any information neither on insulin levels or on insulin sensitivity in their animal model. The mTOR pathway (including raptor) is an important funneling site of the molecular action of insulin. The fact that fasting glucose is decreased similarly in WT and mutant mice, does not preclude possible changes in fasting insulin or during dynamic tests (insulin tolerance tests).5. Statistical analyses carried out in figure 3 do not consider as independent factors the treatment and the genotype. Authors need to carry out 2-way anovas. Only if there is an interaction, the authors can conclude that for instance the total body mass  or fat mass  is different between liraglutide-treated WT and mutant mice.eLife. Your revised article has been evaluated by Mone Zaidi (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Glucagon-Like Peptide-1 Receptor Activation Stimulates PKA-Mediated Phosphorylation of Raptor and this Contributes to the Weight Loss Effect of Liraglutide" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:All three reviewers and the reviewing editor feel that the revision only partly addressed the concerns raised. Specifically, the work lacks studies in diet-induced obesity which are required to explain the therapeutic mechanisms of GLP1 agonists, the failure to examine the anorectic response to other GPCR agonists to establish the specificity of the phosphorylation site for GLP1's signaling/effects versus that of other signaling pathways as the latter is what the authors imply.Reviewer #1 (Recommendations for the authors):The authors have made several useful changes to this revised manuscript, including adding additional information about the genesis of their mouse model. The validation remains a bit less complete than one would like, however- in the methods the authors state that they have sequenced the cDNA from these mice and refer to Supplemental Figure 1B, but the data are not provided here (or elsewhere).I am also disappointed that the authors have not examined the anorectic response to other agents, as this would provide a useful and important control for the specificity of the phosphorylation site for Lira (or other GPCR agonists).Reviewer #2 (Recommendations for the authors):The authors have only partly addressed the concerns of this reviewer.In particular, although already pointed out by this reviewer, the authors have decided to not carry out studies under diet-induced obesity. This reduces the interest for the data in their present form, which remain preliminary.Liraglutide is used for weight loss in patients suffering from obesity and not to induce weight loss in lean individuals. The observations provided are made in non-obese mice. Whether what the authors observe still stands true in obesity and helps explain the therapeutic action of liraglutide remains to be demonstrated.It is possible that mutant mice may have per se a phenotype in DIO, and this information would be relevant. Whether under DIO, mutant mice would respond similarly or differently from WT mice to Liraglutide would also be relevant information, even if this is a whole-body and not a cell-specific mutant, a limitation that could then be discussed by the authors.Reviewer #3 (Recommendations for the authors):The authors have made an effort to address the reviewers' comments. As a result, the quality of the manuscript was improved. However, the reviewer has the following questions to be further clarified.1. Appropriate method of statistical analysis needs to be used for results presented in Figure 1 and the accompanying supplemental figure. The reviewer suggests to use 2-way ANOVA instead.2. For signaling experiments using chemical probes, it is necessary to show the time course of the signaling dynamics and dose-dependent effect (as opposed to demonstrate the result using single dose and single time point). Essential revisions:The reviewers agreed that the paper has potential, but the authors will need to provide substantial new data to answer the reviewers' concerns.Reviewer #1 (Recommendations for the authors):1. While I understand that the authors intend to publish a full characterization of the model elsewhere, it is impossible to evaluate the results of this manuscript without some information on the genetics of the model, and some data showing the expression of the mutant, however. The authors could add some more detail about the mouse model to this manuscript and send the manuscript describing the model in more detail for the reviewers to examine.We gladly agree to this request and now provide detailed information about the Raptor knock-in mutant model in the Methods section (lines 206-215) and in Figure 3—figure supplement 2.2. The authors should examine the normal regulation of feeding/body weight by non-GPCR-dependent agents . They would want to examine the effects of these on PKA/pS6 in cultured cells, as well.We agree with the reviewer that this is an important question that will be more appropriately addressed once we have identified the specific tissues/cell types in which Raptor signaling mediates the weight loss effect of GLP-1R agonists and generate the appropriate tissue/cell-specific Raptor mutant mouse line.3. The authors should provide direct comparisons of the various conditions, rather than normalizing to individual baseline measurements.We thank the reviewer for this comment. We now provide direct comparisons of the various conditions used in the cell culture experiments in Figure 1  and in vivo experiments .Reviewer #2 (Recommendations for the authors):1. The authors claim that the difference in efficacy in weight loss in WT vs raptor mutants treated with Liraglutide is due to a very minor difference in food intake during the second day of treatment. By looking at the data, the sustained weight difference cannot be due to a very transitory difference in food intake, which by the end of the treatment has become comparable to the food intake of vehicle-treated mice. Authors should investigate changes in energy expenditure.791Ala Raptor mutant mice during liraglutide treatment, these mutants also display reduced energy expenditure relative to vehicle-treated mice that lasts for a longer duration during the liraglutide treatment period than in wild-type mice. We hypothesize that both of these phenotypes contribute to the relative resistance to liraglutide-induced weight loss in the Raptor mutant mice.we agree with the Reviewer and have now conducted an experiment in metabolic cages. As now shown in Figure 3K-N, along with a more rapid rebound in food intake in the Ser2. Related to the above, previously published evidence suggests that central GLP1R agonism stimulates thermogenesis by recruiting the sympathetic nervous system . It would be relevant to assess whether recruitment of the SNS is impaired in the Raptor mutant mice under liraglutide treatment. Authors could assess molecular changes for SNS markers and markers of increased thermogenesis in the brown adipose tissue and in the white adipose tissue. Authors could also assess possible differences in cold-induced SNS activity in their model.Gastroenterology 2004; Knauf C et al. Endocrionology 2008) GLP-1R agonist administration actually lowers energy expenditure even in wild-type mice. However, our data do suggest that the duration of liraglutide-mediated reduction of energy expenditure is greater in the Raptor mutant knock-in mice compared to control mice, even though there is no significant difference in absolute energy expenditure between genotypes. One potential interpretation of this is that mechanisms that could contribute to restoring energy expenditure (e.g. thermogenesis) are activated in control mice, but this is impaired in the Raptor mutant knock-in mice. This is a hypothesis that would be better tested in tissue-specific Raptor mutant knock-in mice since this would avoid confounding effects of impaired PKA-mediated regulation of Raptor in multiple tissues .Although GLP-1R agonists have been shown to increase sympathetic tone and stimulate BAT thermogenesis in rodents, it does not appear that these phenotypes contribute to parameters that regulate body weight . As shown by our results in Figure 3 and studies conducted by other groups  is an important funneling site of the molecular action of insulin. The fact that fasting glucose is decreased similarly in WT and mutant mice, does not preclude possible changes in fasting insulin or during dynamic tests (insulin tolerance tests).We agree with the reviewer and now include data showing that in addition to no effect of liraglutide on glucose levels in the Raptor mutant mice, there is also no difference between wild-type and Raptor mutant mice on fasting insulin levels . We also provide data for the Reviewer in 5. Statistical analyses carried out in figure 3 do not consider as independent factors the treatment and the genotype. Authors need to carry out 2-way anovas. Only if there is an interaction, the authors can conclude that for instance the total body mass  or fat mass  is different between liraglutide-treated WT and mutant mice.We thank the Reviewer for this suggestion and have re-analyzed these results using 2-way ANOVA tests. There is a significant genotype and treatment interaction with time for the various comparisons in Figure 3 that show significant differences.[Editors’ note: what follows is the authors’ response to the second round of review.]Reviewer #1 (Recommendations for the authors):The authors have made several useful changes to this revised manuscript, including adding additional information about the genesis of their mouse model. The validation remains a bit less complete than one would like, however- in the methods the authors state that they have sequenced the cDNA from these mice and refer to Supplemental Figure 1B, but the data are not provided here (or elsewhere).We apologize for the confusion. The results of the sequencing validation are shown in new Figure 3—figure supplement 2. We also provide additional details for the design and creation of this mouse model in the Methods section (lines 280-283).I am also disappointed that the authors have not examined the anorectic response to other agents, as this would provide a useful and important control for the specificity of the phosphorylation site for Lira (or other GPCR agonists).791Ala Raptor knockin mice. Like the Glp1r, the MC4R is a Gαs-coupled receptor, so this provides a valuable comparison to the Glp1r. As we now show in the new Figure 5, unlike what we observed with liraglutide, CMV-Ser791Ala Raptor knockin mice are not resistant to the weight loss effect of setmelanotide. This suggests that the phosphorylation of Raptor by PKA at Ser-791 does not contribute to the weight loss effect of all Gαs -coupled receptor agonists equally. These results are also described in the Results section (lines 141-152) and in the Discussion (lines 193-202).Following this suggestion from the reviewer, we have now conducted additional experiments in which we assessed the weight loss effect of the melanocortin-4 receptor (MC4R) agonist setmelanotide in wild-type and CMV-SerReviewer #2 (Recommendations for the authors):The authors have only partly addressed the concerns of this reviewer.In particular, although already pointed out by this reviewer, the authors have decided to not carry out studies under diet-induced obesity. This reduces the interest for the data in their present form, which remain preliminary.Liraglutide is used for weight loss in patients suffering from obesity and not to induce weight loss in lean individuals. The observations provided are made in non-obese mice. Whether what the authors observe still stands true in obesity and helps explain the therapeutic action of liraglutide remains to be demonstrated.It is possible that mutant mice may have per se a phenotype in DIO, and this information would be relevant. Whether under DIO, mutant mice would respond similarly or differently from WT mice to Liraglutide would also be relevant information, even if this is a whole-body and not a cell-specific mutant, a limitation that could then be discussed by the authors.791Ala Raptor knockin mice with liraglutide after having been rendered obese following feeding with a 60% high fat diet (HFD). As shown in the new Figure 4, unlike the results obtained in lean mice, obese CMV-Ser791Ala Raptor knockin mice were not resistant to the weight loss effect of liraglutide. However, it must be noted that the CMVSer791Ala Raptor knockin mice had significantly higher body weight and adiposity following the HFD exposure prior to any treatment . It has been our experience that mice with higher starting body weight and adiposity tend to lose more weight in response to liraglutide. We support this with additional data in new Figure 4—figure supplement 1 showing a positive correlation between initial body weight and liraglutide-induced weight loss. This could offset any attenuation of the liraglutide effect in the CMV-Ser791Ala Raptor knockin mice. Therefore, we determined the average baseline body weight of all mice prior to liraglutide treatments (43.4g) and divided results into those from mice with baseline body weights above 43.4g and those with baseline body weights below 43.4g. We show that there is no difference in the weight loss effect of liraglutide between wild-type and CMV-Ser791Ala Raptor knockin mice that have and average baseline body weight above 43.4 g ; however, there is a tendency for the CMV-Ser791Ala Raptor knockin mice to be resistant to the weight loss effect of liraglutide when looking at mice whose baseline body weights are below 43.4g. These findings suggest that PKA phosphorylation of Raptor at Ser-791 may contribute to the weight loss effect of liraglutide in obese mice below a specific weight threshold. Results are described in the Results section (lines 121-140) and in the Discussion (lines 171-183).Following this suggestion from the reviewer, we have now conducted experiments treating wild-type and CMV-SerReviewer #3 (Recommendations for the authors):The authors have made an effort to address the reviewers' comments. As a result, the quality of the manuscript was improved. However, the reviewer has the following questions to be further clarified.1. Appropriate method of statistical analysis needs to be used for results presented in Figure 1 and the accompanying supplemental figure. The reviewer suggests to use 2-way ANOVA instead.Following this recommendation from the reviewer, we have now re-analyzed these data using 2-way ANOVA.2. For signaling experiments using chemical probes, it is necessary to show the time course of the signaling dynamics and dose-dependent effect (as opposed to demonstrate the result using single dose and single time point).Following the recommendation from the reviewer, we show both dose response and time course experiments that were conducted in order to select the proper dose and time exposure of liraglutide for the signaling experiments . Different doses of the inhibitors are already shown in Figure 1.


Cadmium-zinc-telluride (CZT)-based detectors exhibit higher diagnostic sensitivity in myocardial perfusion imaging (MPI) than conventional Anger-MPI for detection of coronary artery disease (CAD); however, reduced specificity and diagnostic accuracy of CZT-MPI were observed. This study aims to compare these different camera systems and to examine the degree of inter-rater reproducibility among readers with varying experience in MPI.83 patients who underwent double stress/rest examinations using both a CZT and conventional SPECT cameras within one visit were included. Anonymized and randomized MPI-images were distributed to 15 international readers using a standardized questionnaire. Subsequent coronary angiography findings of ten patients served as a reference for analysis of sensitivity and specificity.P < 0.05). CZT-MPI exhibited higher sensitivity than Anger-MPI (87.5% vs. 62.5%) and significantly reduced specificity (40% vs. 100%). Readers experienced with both camera systems had the highest inter-rater agreement indicating higher reproducibility .Image quality was significantly better in CZT-MPI with significantly lower breast attenuation (Higher diagnostic sensitivity of CZT-MPI offers advantages in detection of CAD yet potentially of at the cost of reduced specificity, therefore it requires special training and a differentiated evaluation approach, especially for non-experienced readers with such camera systems.The online version contains supplementary material available at 10.1007/s12350-022-03054-w. However, this comes potentially at the cost of reduced specificity in obese patients examined in this position, as argued in multiple studies.9Myocardial perfusion imaging (MPI) is one of the most often used techniques worldwide for the detection and assessment of coronary artery disease (CAD). The diagnostic performance of conventional single-photon emission computed tomography (Anger-SPECT) is well established, with current data yielding estimates for sensitivity ranging between 0.82 and 0.91, and for specificity between 0.70 to 0.90, depending on clinical history and the cutoff chosen for significant CAD.9 Furthermore, most dedicated CZT cameras for MPI are not equipped with an integrated computed tomography (CT) component with most studies performed without attenuation correction (AC), harboring the risk of attenuation artifacts being interpreted as perfusion abnormalities.10In contrary to Anger-MPI, the ability to image upright and therefore the tendency for imaging of a mainly obese patients may result in reduced diagnostic specificity with inferolateral segments still suffering marked soft tissue attenuation on CZT cameras.13 In a meta-analysis performed by Nudi et al.8, CZT-MPI presented a relatively high rate of false positive results and reduced specificity particularly in imaging of obese patients, respectively. In the majority of the studies included, patient cohorts were paired based on specific clinical characteristics.With the introduction of high speed solid-state cardiac cameras first studies comparing these technologies suggested high correlation in image quality compared to conventional SPECT, with an equivalent level of diagnostic confidence.The objective of this study was to examine inter-rater reproducibility, diagnostic certainty, reader experience effects between CZT-MPI and conventional Anger-MPI in the same patient cohort in a multi-center analysis with readers of varying experience in the assessment of SPECT-MPI. In addition, we delineated whether and to what extent the two camera systems, differ in terms of image quality and susceptibility to artifacts.Retrospective multicentric reading study with 15 international readers from the field of nuclear cardiology with varying experience in MPI. The readers evaluated anonymized and randomized MPI findings of 83 patients acquired with a CZT-based as well as with one of two conventional Anger SPECT cameras . Informed consent was waived due to the retrospective character of the study.14 In a proportion of patients, prior coronary angiographic data indicated the extent of vessel involvement (N = 41). Other patients received a subsequent coronary angiography depending on symptoms and the results of the non-invasive diagnostic work-up. Relevant coronary angiography results, defined as results acquired no later than 90 days after MPI, were available in ten patients. As part of the clinical routine, cardiovascular risk factors  were present in 64 patients.Of the 361 patients who underwent myocardial perfusion imaging (MPI) in the year 2013 as part of the clinical routine in the Klinikum rechts der Isar, Munich, Germany, we identified 83 patients who were sequentially examined with a CZT  and with one of the two available conventional SPECT cameras  within one visit both after stress and at rest. Data were originally collected in routine clinical practice at our institute when the CZT camera was newly introduced and were used for comparison with the conventional SPECTs to familiarize the readers with this new technology. The pretest probability for the presence or absence of significant obstructive CAD, defined as a ≥ 70% stenosis of at least one coronary artery was calculated using a consortium score to validate the indication for non-invasive diagnostics.15 with an encrypted invitation letter containing a Uniform Resource Locator (URL) address with an individual transaction number (TAN) password for each reader. A standardized questionnaire broadly used in the interpretation of MPI with questions regarding image quality, artifacts, final diagnosis, diagnostic certainty and the 17-segment model of the American Heart Association (AHA)16 was used. This standardized questionnaire is used for purely image-based evaluation of data, which make up the main building blocks of MPI findings. No clinical information was provided, and all readers were blinded to the modality of examination .15 international readers from the field of nuclear cardiology with varying experience in the interpretation of SPECT-MPI were recruited. They were divided based on underlying experience into three groups of five participants each: Five readers with experience with both conventional SPECT cameras and a CZT-SPECT camera (group 1); five readers who only have experience with conventional SPECT cameras (group 2) and five readers with little or no experience in MPI interpretation (group 3). The anonymized and randomized MPI images were distributed in the form of static images of the short axes and horizontal/vertical long axes as well as the polar maps by means of an online study. The online study was carried out using Evasys Survey Grid17 This system uses pixilated CZT crystal detector columns mounted in nine vertical columns that are arranged in a 90 ° geometry. No attenuation correction (AC) was applied in this study, since the D-SPECT camera used at the time of the study did not support it.The recorded light pulses are amplified with photoelectron multipliers (PMT) that deliver electrical pulses proportional to photon energy. In this way, the activity of the radiotracer in the examined organ is visualized as a projection onto a plane. The gamma camera, which rotates around the patient and depicts isotope activity in multiple angle projections, enables the three-dimensional reconstruction of isotope activity and distribution in the body. Two conventional cameras were used in this study . A D-SPECT system  was used for CZT-MPI.79 patients were examined via one-day protocol and four patients via two-day protocol. The patients with the one-day protocol were examined sequentially with both camera technologies, first in stress and then in rest . Mean time interval between the stress examinations was approximately 33 (± 31) minutes when CZT-MPI was performed as first procedure vs. 58 (± 15) minutes when Anger-MPI was performed as first procedure.18 resulting in mean dose of 414 (± 221) MBq for the stress study and 838 (± 282) MBq for the rest study in the one-day protocol. Using the two-day-protocol injected activities were 518 (± 255) MBq (stress) and 548 (± 230) MBq (rest), respectively. On conventional SPECT cameras, all patients were imaged in a supine position and on the CZT-SPECT system all patients were imaged using the standard semi-supine position.Injected activities of the perfusion tracer Tc-99m sestamibi were body-weight adjusted according to current guidelinesScan durations were 20 min fixed per scan in the conventional cameras and on average 12 (stress) resp. 5 (rest) minutes the D-SPECT using count limits.19 This quantitative analysis software enables the quantification and thus the objectification of tracer accumulation in the heart both at rest and under stress. It has been validated in multiple phantom studies and in several data sets of healthy patients and patients with CAD of various degrees of severity.20 After volumetric data analysis and image reconstruction, radiotracer activity is assessed in sections along the short and long axes of the left ventricle. Furthermore, a polar map of the radiotracer distribution throughout the left ventricle is generated.Analysis of the volumetric data on the regional tracer distribution resulting from MPI was performed with a dedicated software (MunichHeart).16. As displayed in Figure A standardized myocardial scoring system for the resulting polar maps is available for quantification of results (summed stress score (SSS), summed rest score (SRS), and summed difference score (SDS) as described by Berman et al.21 When individual values occurred very frequently, the sign test was calculated.22 Statistical significance was denoted by P values < .05. Inter-rater agreement was analyzed with the Fleiss' Kappa statistics.23 The analysis of the Inter-rater agreement was divided in two categories, the first regarding all answers for diagnosis , and the second regarding abnormality in comparison of normal versus abnormal findings . Sensitivity, specificity, and accuracy were calculated using the angiography group (N = 10) and a negative control group which did not undergo angiography after non-invasive exclusion of CAD (N = 8). A positive finding was defined at a cutoff of ≥ 70% luminal diameter narrowing of at least one coronary vessel. Eight patients of the angiography group displayed coronary artery stenosis with the cutoff as defined above and two patients had normal angiograms. A total of ten patients were defined as a negative group consisting of patients with no relevant coronary artery stenosis in coronary angiography (N = 2) as well as patients who had intermediate pretest probability for CAD, yet no indication for a coronary angiography after revision of all non invasive examinations and available data and exclusion of CAD in interdisciplinary consensus (N = 8).Statistical analysis was performed using R statistical software (R version 3.6.3). The Wilcoxon test was used to test differences between the two camera technologies or between two groups of readers.The individual coronary territories were compared in accordance with the 17-segment model of the AHA. A positive MPI finding was defined as an SDS of at least 2 and/or an SSS of at least 4. The SRS was assessed analogously to the SSS.2. Cardiovascular risk factors were documented only in 64 patients and 41 patients had pre-existing CAD as summarized in Table 83 patients aged between 38 and 91 years  were enrolled. 49 patients (59%) were male. The body mass index (BMI) was between 16.5 and 55.4 kg/mP < 0.05, Figure P < 0.05), regardless of reader experience . Higher breast attenuation was observed in females when examined with Anger-MPI , yet this difference was not significant (P=0.052). A significant difference in breast attenuation between female and male patients was observed only in MPI findings of the Anger cameras and only by readers who only had experience with conventional SPECT cameras . Another possible source of artifacts is liver and bowel activity. The CZT camera exhibited slightly higher liver and bowel activity, but no significant difference for any reader group was observed . This observation was made by the experienced readers (groups 1 and 2), but not by the inexperienced readers . Although the stress MPI was performed as the first procedure in 80% of the patients on the CZT camera, and a significant negative correlation between time and liver and bowel artifacts was confirmed, this potential artifact was not significantly higher for the CZT camera compared to conventional SPECT . This correlation was also apparent with significantly higher mean SSS and SDS scores in all groups on CZT-MPI . Mean SRS scores were not significantly different . In contrary, inexperienced readers (group 3) reached the highest SSS .Regarding the final diagnosis, all reader groups observed a significantly higher rate of abnormalities in CZT-MPI . Group 2 showed significantly lower inter-rater agreement in CZT-MPI in comparison to Anger-MPI . Group 3, with little or no experience in MPI, achieved a significantly higher degree of agreement in CZT-MPI than with the Anger-MPI , respectively. Regardless of the modality, the inter-rater agreement of group 3 was always lower compared to group 1  reached a significantly higher degree of inter-rater agreement in comparison to reader groups 2 and 3 . Mean overall diagnostic certainty was comparable for both camera systems .For the assessment of interpretative certainty, the answers were divided into three columns: equivocal, probable , and definite . Higher values indicated greater interpretative certainty regarding final diagnosis based purely on image interpretation. Experienced readers (groups 1 and 2) had significantly higher interpretative certainty than readers with little or no experience in MPI interpretation (group 3), . Specificity of the CZT camera was significantly lower than of the conventional cameras . Higher rates of false positive evaluations resulted in overall reduced accuracy for CZT MPI, as shown in Table P <0.05).In patients undergoing coronary angiography, eight patients had relevant coronary artery stenosis, defined as a ≥ 70% stenosis of at least one coronary artery, and two patients had normal angiograms. A negative control group (N = 10) was defined as patients with no relevant coronary artery stenosis in coronary angiography N = 2) as well as patients who had intermediate pretest probability for CAD, yet no indication for a coronary angiography after revision of all non invasive examinations and available data and exclusion of CAD in interdisciplinary consensus (N = 8). Overall, the D-SPECT showed a higher sensitivity compared to conventional SPECT cameras  and significantly lower specificity for the CZT camera were observed (40% vs 100%). In the meta-analysis by Nudi et al.8 comparing CZT-MPI and conventional Anger-MPI, breast attenuation and obesity were discussed as possible causes for the reduced specificity of CZT technology as extra-cardiac soft tissue activity can still hamper image quality and contrast-to-noise ratio possibly generating false positive results arising especially in inferior and inferolateral segments.10 Some studies suggest greater diagnostic accuracy in combined supine and prone CZT MPI without adversely affecting its sensitivity.25 However, this comes at the cost of extended examination time and is therefore recommended in the case of inferolateral ischemia in patients with a history of previous inferolateral MI.26 Therefore, routine prone imaging in CZT-MPI and adequate training and learning curve of the technologists, especially with patient positioning, are key in yielding the best images this technology can provide. Lack of experience of the technologists at the time of introduction of the D-SPECT could have contributed to the low specificity of this camera in our study.Better image quality of the CZT camera was confirmed and is technically due to its significantly enhanced sensitivity of count detection compared to conventional Anger cameras as previously confirmed in phantom models by Zoccarato et al.6 discussed that due to the improved spatial resolution of the CZT detectors, better visualization of non-serious perfusion defects in the absence of significant CAD can also impair the specificity of CZT-based camera systems as also observed in this study. A large multicenter study evaluating fractional flow reserve (FFR) in coronary lesions found that the majority of coronary lesions with a narrowing of 50–70% have no hemodynamic relevance.27 Although the degree of agreement between conventional Anger-MPI and FFR measurements has been well investigated, to our knowledge, this is not the case for CZT-MPI.28 Therefore, further investigation of CZT-MPI in correlation with the FFR will certainly be required in the future.Although the stress MPI with the CZT camera was performed as first procedure in 80% of the patients, artifacts due to liver and bowel activity were not considered significantly more serious for the CZT camera. It is noticeable that the experience of the reader plays a significant role in the interpretation of artifacts as such. All inexperienced readers (group 3) achieved a significantly lower specificity with the CZT camera (mean specificity of 20%). Overall, they observed significantly more abnormalities in the CZT-MPI findings than in conventional Anger-MPI findings. The tendency for overestimation of the disease was shown repeatedly in questions about diagnosis and in assessments with significantly higher SSS and SDS reached by this group Figure . Fiechte29Significantly higher Inter-rater agreement in the reader group with experience with CZT based cameras (group 1) compared to the rest of the groups (2 and 3) with no significant experience with CZT-MPI interpretation illustrates that regardless of the underlying experience, dedicated training with such systems is required to avoid over- or underestimation of results. Importantly, for optimal image acquisition technologists should also undergo training and acquire experience regarding optimal patient positioning in CZT-MPI.It is important to emphasize that a purely image-based, visual evaluation of the myocardial perfusion scintigraphy can be considered as a study limitation. This does not reflect the clinical reality, as various clinical parameters play a role in the diagnostic work-up of CAD and everyday decision-making. However, as we were interested in a direct head-to-head comparison of imaging systems, we considered this unavoidable. The use of a standardized reading template, the anonymization and randomization of the modality of the examination (CZT- vs. Anger-MPI) and the multicentric approach helped increase the power of this study and reduce familiarity bias. Another limitation in the use of a single cohort and sequential double examination is the extra cardiac clearance pattern and the limited but nevertheless existing decay of radiotracer with time between the examinations. However, a study with separate examination protocols for the same patient cohort on different days is not justifiable due to the unnecessary radiation exposure. In the present study, no attenuation corrected images  were provided to the readers. This came at the risk of attenuation artifacts being misinterpreted as perfusion abnormalities However, routine attenuation correction  is not performed in our clinic, so that attenuation-corrected images would only be available for a small number of patients. Therefore, we decided to compare the images primarily created in the clinic with standard positioning of the patient, also in order to better capture the different image properties of the respective camera systems.CZT-MPI delivers higher diagnostic sensitivity compared to conventional Angel-MPI whilst exhibiting significantly lower specificity. Our investigation has shown that the reduced accuracy of CZT-MPI due to low specificity compared to Anger-MPI—especially achieved by nuclear cardiologist with no previous experience in CZT-MPI—illustrates the necessity of a dedicated training. The importance of a differentiated evaluation and interpretation of the results of non-invasive diagnostics in everyday decision-making and clinical practice was demonstrated.CZT-MPI displays superior image quality and lower susceptibility for artifacts. Reader experience plays a major role in the interpretation of SPECT images, especially when acquired by camera systems with high diagnostic sensitivity. Therefore, when introducing a CZT-detector-based camera, dedicated training of the medical staff is recommended.Supplementary file1 (PPTX 8306 KB)Supplementary file2 (MP3 7589 KB)Below is the link to the electronic supplementary material.


With advances in organ matching and preventing acute graft-versus-host-disease (aGvHD), chronic graft-versus-host disease (cGvHD) following allogeneic hematopoietic stem cell transplantation (HSCT) has become a focus of transplant-related morbidity and mortality. Given that cGvHD often presents years following a transplant, our objective was to estimate its burden of cost resulting from allogeneic HSCT based on published estimates of incidence, morbidity, the value of lost work time and survivorship. Our choice of a ten-year time horizon is novel to the field of rare disease and was determined to be meaningful after consultations with present co-authors, including five physicians, one of whom is a transplant surgeon. A total of 44 450 cGvHD patients in the United States were estimated to require treatment over the next decade (from 2015 to 2025). This estimate is based on the last 5 years of trends reported in the transplant registries. What is not reported in any registry is that these patients will accrue a total of 605 631 years of lost wages, a collective lost productivity that will cost society over $27 Billion in the decade ahead: more than five times ($27B vs. $5.2B) the estimated ten-year cost of treating the condition.


More than 4 billion doses of the Coronavirus disease (COVID-19) vaccine have been administered worldwide but the relationship between the different vaccines and the development of renal disease is unknown. We present a case of tubulointerstitial nephritis following administration of the Oxford-AstraZeneca COVID-19 vaccine.A previously fit and well 51-year-old female presented on 27th May 2021 with a one-month history of weight loss, fatigue, nausea, and a metallic taste. She had an acute kidney injury with a creatinine of 484 umol/L. She was on no regular medications and denied taking any over-the-counter or alternative medicines. She had received her first dose of the Oxford-AstraZeneca vaccine on 23rd March 2021 and her second dose on 20th May 2021. A renal biopsy was performed the following day. The 19 glomeruli appeared normal to light microscopy but the tubulointerstitial compartment contained a dense inflammatory infiltrate including many eosinophils. There was widespread acute tubular injury with tubulitis, but no established or longstanding atrophy. A diagnosis was made of an acute tubulointerstitial nephritis. She was commenced on oral prednisolone and her renal function improved. She did not require renal replacement therapy at any time.To our knowledge, this was the first described case of acute tubulointerstitial nephritis following administration of the Oxford-AstraZeneca COVID-19 vaccine, although a number of cases have emerged more recently. In our case the patient was very fit and well, had no previous past medical history and had not taken any recent prescribed, over-the-counter or alternative medications. The absence of these provoking factors in our case makes the vaccine the most likely explanation for the development of tubulointerstitial nephritis although the pathophysiology behind this remains unknown. Given the unprecedented number of vaccinations being delivered around the world, nephrologists should be aware of this possible link although more research into the topic is required. More than 4 billion doses of the Coronavirus disease COVID-19) vaccine have been administered worldwide since the beginning of the vaccination programme. The relationship between the different COVID-19 vaccines and the development of renal disease is currently unknown. However, there are emerging reports of the vaccines being linked with the development of de novo and recurrent glomerular disease. A recent review article summarised several cases where patients had developed new or relapsing glomerulonephritis following administration of the COVID-19 vaccine [9 vaccineThe incidence of tubulointerstitial nephritis following administration of the COVID-19 vaccine is less widely reported although several cases have started to emerge within the literature –6.Acute tubulointerstitial nephritis is typified by the presence of inflammation and oedema within the renal interstitium and is associated with acute renal injury. The clinical features are non-specific and include arthralgias, fever, a maculopapular rash and eosinophilia. Many cases present with no symptoms at all and therefore the condition is thought to be under-diagnosed by clinicians . Drug-inWe present a case of tubulointerstitial nephritis in a patient with no previous history of renal disease following the second dose of the Oxford-AstraZeneca COVID-19 vaccine. Importantly, she had a very minimal past medical history and had taken no recent prescribed or over-the-counter medications.A previously fit and well 51-year-old female was referred to our renal team on 27th May 2021 with a presumed acute kidney injury (creatinine of 484 umol/L). She had never had her renal function checked previously. This was on a background of a one-month history of feeling non-specifically unwell. She gave a history of weight loss (6 kg), fatigue, nausea and a metallic taste. She was on no regular medications and denied taking any over-the-counter or alternative medicines. She denied any illicit drug use. She had received her first dose of the Oxford-AstraZeneca vaccine on 23rd March 2021 and her second dose on 20th May 2021. Her physical examination was unremarkable. Specifically, she had no signs or symptoms of uveitis although a formal ophthalmological examination was not sought.Her blood pressure at presentation was 145/81. Her urinalysis showed 1+ protein and was positive for blood. The urine protein:creatinine ratio was 50 mg/mmol. Her CRP was 77, albumin 34 g/l and eosinophils were within the normal range at 0.16 10*9/L. Her acute renal screen was unremarkable; ANCA and Anti-nuclear antibody (ANA) were negative and her complement screen showed a mildly raised C3 of 1.89 g/L. She underwent a renal biopsy the following day that showed a total of 19 glomeruli, none were sclerosed. The glomeruli appeared normal to light microscopy. The tubulointerstitial compartment contained a dense inflammatory infiltrate including many eosinophils (Fig. She was commenced on 40 mg of oral prednisolone which is the standard starting dose in our local renal unit. Her renal function continued to deteriorate for 3 days to a maximum creatinine of 626 umol/L before starting to improve. She did not require renal replacement therapy at any time.Her renal function continued to improve on a 3-month weaning course of oral prednisolone, which was stopped at the end of August 2021. Her creatinine improved to 104 umol/L on 16th September 2021 and her latest creatinine was 78 on 17th March 2022.To our knowledge, this was the first described case of acute tubulointerstitial nephritis following administration of the Oxford-AstraZeneca COVID-19 vaccine. One case of tubulointerstitial nephritis was reported after the administration of the Pfizer-BioNTech vaccine in a patient with pre-existing chronic kidney disease [A further case of tubulointerstitial nephritis was reported following administration of the first dose of the BioNTech vaccine . A 44-yeA number of case reports of tubulointerstitial nephritis have emerged more recently –6. They Interestingly, acute tubulointerstitial nephritis has also been associated with the influenza vaccination in one case study . HoweverThere have also been various case reports linking the vaccine with the development of de novo and relapsing glomerulonephritis .Given that billions of people are being vaccinated around the world, it is plausible that the development of rare kidney disease is temporally associated with the COVID-19 vaccination but may be unrelated to the vaccine itself. It is very difficult to prove a causal relationship in all the above case studies. It is also conceivable that another ingredient or adjuvant within the vaccine preparation could cause a tubulointerstitial nephritis.In our case the patient was very fit and well, had no previous past medical history and had not taken any recent prescribed, over-the-counter or alternative medications. The absence of these other provoking factors in our case makes the vaccine the most likely explanation for the development of tubulointerstitial nephritis. The timescale between receiving the vaccination and presenting with acute kidney injury also supports this theory although the pathophysiology behind this remains unknown. Given the unprecedented number of vaccinations being delivered around the world, nephrologists should be aware of this possible link although more research into the topic is required.


Editorial on the Research TopicCardio-oncology: mechanisms and therapeuticsCancer and cardiovascular disease share similar risk factors and are both prevalent among aging populations. Individuals with a history of cancer are exposed to a 2–3 times higher chance of getting acute coronary syndrome (ACS), which can persist for up to 10 years after a cancer diagnosis , 5. In tIn addition, novel cancer therapies have tremendously improved the survival of cancer patients but also increased treatment-related side effects , 7. CardThe purpose of this research topic is to bring together a collection of works that provide novel insights into interactions between cancer and cardiovascular disease as well as mechanisms and therapeutics of anticancer treatment-induced cardiotoxicity. All contributions to this research topic concentrate on one or more of the above-mentioned study topics and several studies referenced below are representative.6-Methyladenosine (m6A) are increasingly recognized to play important roles in physiology and disease  levels. The proper structure and function of junctophilin-2 (JPH2) are recognized to be indispensable for proper excitation-contraction coupling in cardiomyocytes -induced cardiotoxicity in vitro and in vivo by upregulating PAK2 and XBP1s. Overexpression of PAK2 or XBP1s reduced miR-194-5p- exacerbated cardiomyocyte apoptosis. This work was the first to identify a novel pathogenic miR-194-5p/PAK2/XBP1s axis in DOX-induced cardiotoxicity, hence proposing a potential target for the prevention and treatment of DOX-induced cardiotoxicity.Doxorubicin is a popular anticancer agent but is well-known for its cardiotoxicity in many patients. The mechanisms underlying doxorubicin (DOX)-induced cardiotoxicity remain not fully understood. miRNAs are widely involved in the progression of various cardiovascular diseases . Fa et aPeng et al. suggested that NT-proBNP could predict cardiovascular symptoms in individuals with myocardial damage following PD-1 inhibitor therapy, while highly sensitive troponin T (hsTnT) is the best cardiac biomarker for mortality prediction in symptomatic patients. This study may help medics to perform risk stratification for patients at an earlier time and to implement effective interventions at the early stage of PD-1 inhibitor-related myocarditis.In recent years, immunotherapy has achieved great success in cancer treatment. Unfortunately, cardiotoxicity appears to have emerged as an unneglectable issue recently . The worCosyns et al. The most common complications were acute renal failure (25.9%), embolic events (21.7%), and congestive heart failure (18.1%). This is a sizable observational cohort of IE patients with cancer. It sheds light on current IE cancer patient profiles, treatment, and outcomes. Considering the lack of randomized and large-scale observational data on IE cancer patients, this registry provides a unique viewpoint on IE management in cancer patients.Infective endocarditis (IE) occurs more frequently in cancer patients as compared with the general population . IE was Oikawa et al. consecutively enrolled 169 patients who planned to receive cardiotoxic chemotherapy for 12 months of follow-up and found that the incidence of CTRCD was greater in the high D-dimer group than in the low D-dimer group . High D-dimer levels at baseline were an independent predictor of the development of CTRCD, according to multivariable logistic regression analysis . It is suggested that D-dimer may be a potential predictor of CTRCD and has clinical practical value.Improved early detection methods have allowed a larger number of cancer patients with cancer therapeutics-related cardiac dysfunction (CTRCD) to live longer . Oikawa Varkoly et al. performed high-throughput sequencing analysis of RNA obtained from blood samples of 28 patients with hematological malignancies who had undergone chemotherapy. The result suggested that patients with low LVEF had influenza orthomyxovirus, avian paramyxovirus, and retrovirus sequences present. This is the first study to use high-throughput, blinded, unbiased sequencing to test for RNA viruses in circulating blood and associate those findings with abnormalities in heart function in patients who have recently finished chemotherapy. This study raises attention to RNA virus infections in individuals with chemotherapy-related cardiomyopathy.It has been hypothesized that immunosuppression after chemotherapy increases opportunistic viral infections . VarkolyHang et al. followed up 197, 699 colorectal cancer patients for 37 months and examined the risks of cardiovascular death (CVD) in patients with CRC. They revealed that CVD ranked first and accounted for 41.69% of the major cause of non-cancer deaths. In addition, the nomogram for CVD prediction in CRC patients was created. This nomogram performed quite well and might assist physicians in providing customized care in clinical settings.Colorectal cancer (CRC) patients are potentially at high cardiovascular risk . Hang etWith the generous support from all editors, publishers, reviewers, and authors involved in this research topic, we have successfully finalized this wonderful collection focusing on mechanisms and therapeutic in Cardio-Oncology. Future studies on the mechanism and management of cardio-oncology are expected to continually improve the survival and life quality of cancer survivors. The enormous issues posed by tumor-cardiovascular comorbidity, however, deserve more attention given its rising incidence and the continuously aging population. There is substantial opportunity for the collaboration between oncologists and cardiologists to work together to improve the outcome of cancer patients with cardiovascular comorbidity.


Infrared neuromodulation (INM) is a promising neuromodulation tool that utilizes pulsed or continuous-wave near-infrared (NIR) laser light to produce an elevation of the background temperature of the neural tissue. The INM-based cortical heating has been proven as an effective modality to induce changes in neuronal activities. In this paper, we investigate the effect of INM-based cortical heating on the characteristics of interictal epileptiform discharges (IEDs) induced by penicillin in anesthetized rats. Cortical heating was conducted using a NIR laser light guided through a needle-like silicon-based waveguide probe. We detected penicillin-induced cortical IEDs from preprocessed micro-electrocorticography ( Highly synchronized electrical discharges known as interictal epileptic discharges (IEDs) appear between seizure periods2. IEDs occur due to the simultaneous discharge of a group of neurons in an area referred to as the epileptic focus3. Based on underlying neural activities, IEDs are classified into two main types5. Type-1 (IED with a slow-wave component) expresses the IEDs that correspond to the recruitment of large populations of excitatory and inhibitory cells. Type-1 is described as a spike with fast negative change and a slow positive component. Type-2 (IED without a slow-wave component) reflects the high synchrony in the underlying neuronal network and recruiting of excitatory cells in a more local area resulting in a higher negative peak amplitude. Type-2 is associated with fast ripples and results from an enhancement of synaptic excitation due to the weakening of inhibition. Moreover, type-2 IED reflects a dynamic increase in excitatory activity and the small width of this IED expresses the increased synchronization in the neural networks that are mainly supported by glutamatergic mechanisms4.Epilepsy is a neurological disease that can be described by sudden changes in the brain’s electrical activity that cause symptoms in brain functionality8. Thermal-based modulation of neural activity depends on the temperature-induced changes in the transmembrane capacitance and non-uniform conductance changes within the dynamics of various ion channels10. The high-resolution spatial induction of a temperature gradient using near-infrared illumination14 has been proven as an effective neuromodulation approach15. Infrared neuromodulation (INM) is a promising thermal-based modulation tool relying on the use of pulsed or continuous-wave near-infrared (NIR) laser light (wavelength range between 1400 and 2100 nm) to produce an elevation of the background temperature of the tissue and thus altering neuronal activities16. Xia and Nyberg illustrated that the firing rate of neurons can be reduced by 80% in a cell culture of rat cortical neurons expressing drug-induced epileptiform activities using continuous NIR light (2)12. Furthermore, during INM experiments with continuous NIR light (2), Furuyama and colleagues reported a clear suppression in the normalized spike counts during epileptiform activity induced with bicuculline methiodide in the CA1 hippocampal region of Mongolian gerbils13.Neuromodulation tools and modalities such as electrical, thermal and transcranial magnetic stimulation had been developed and invested in for years to mitigate or modulate epileptic seizures and IEDs17. In addition, the underlying neural mechanism of the abnormal neuronal hyperexcitability, seizures and epilepsy belongs to the extreme effect of 19. Moreover, hyperthermia increases membrane sodium and/or calcium conductance, tissue metabolic rate and the intrinsic excitability of pyramidal cells and interneurons20. Accordingly, the previous findings of INM and hyperthermia give the following hypotheses. (1) Continuous wave NIR light illumination plays an influential role in suppressing epileptic discharges in terms of IED frequency13 and peak-to-peak amplitude of epileptiform discharges12. (2) In contrast, hyperthermia has an inhibitory effect on 17. (3) Finally, hyperthermia leads to the excitability of both excitatory and inhibitory neurons in both CA1 and CA3 pyramidal cells due to changes in the intrinsic membrane properties20. Based on above, this research investigates further on the thermal effect of INM by addressing the following questions:Can the thermal effect of NIR illumination in a drug-induced acute epilepsy model be interpreted using quantitative features extracted from epileptiform discharges?How much the pulsed NIR illumination at different frequencies may affect the investigated features of IEDs and thus the underlying neuronal activities?In this study, cortical heating is carried out using an optrode probe, which is a multimodal photonic device that uses pulsed and continuous NIR light illumination to conduct a controlled cortical heating16. Horváth et al. have proved that this optrode device can be efficiently, repeatedly and safely used to perform deep tissue INM in anesthetized rats21. Here, infrared-based neuromodulation was performed in nine anesthetized rats. The recordings contain micro-electrocorticography . The dura mater was left intact during the whole experiment except in a few cases (n = 3) where the bone could not be removed from the craniotomy without tearing the dura, as they were tightly fused.All experiments were performed according to the EC Council Directive of September 22, 2010 (2010/63/EU), were in compliance with the ARRIVE guidelines, and all procedures were reviewed and approved by the Animal Care Committee of the Research Centre for Natural Sciences and by the National Food Chain Safety Office of Hungary (license number: PE/EA/672-6/2021). Acute in vivo experiments were carried out on Wistar rats . A mixture of intramuscularly administered ketamine (75 mg/kg) and xylazine (10 mg/kg) was used to induce anesthesia. During the electrophysiological recordings, supplementary doses of ketamine/xylazine were injected intramuscularly to maintain the anesthesia. A homeothermic heating pad connected to a temperature controller  was used to keep the physiological body temperature of the animals. After reaching the appropriate anesthetic depth for surgery, the head of the rat was fixed in a stereotaxic frame , then the skin and the connective tissue was removed from the top of the skull. Next, a cranial window with a size of about 10 mm 22 with 32 platinum recording sites (150 24) designed for infrared stimulation (see “25). A stainless-steel needle inserted in the nuchal muscle of the animal served as the reference and ground electrode during recordings.A flexible, polyimide-based mentclass2pt{minimmentclass2pt{minimCortical electrical activity was collected using an Intan RHD2000 electrophysiological recording system  equipped with a 32-channel headstage  was injected into the brain using a 34-gauge needle. The needle attached to a Hamilton syringe was mounted on a micromanipulator, then slowly  using a 10-second-long sliding window with 0.25-sec of overlap between windows, we calculate the square signal of the window to differentiate between IED and background activity easier, then we compute the local-maxima peaks within the squared signal; (3) an adaptive threshold is computed which equals the average of detected local-maxima peaks within the squared signal and the average of the squared signal set; (4) the adaptive threshold is used to detect IEDs. In summary, the IED detection method depends on calculating the detection threshold adaptively in a sliding 10-sec window. This method showed an accuracy of 95Interictal discharges were detected using a custom Matlab script. Our IED detection approach had the following steps: (1) filtering the recording site’s signal in the 30–300 Hz range using a second-order Butterworth band-pass filter because this frequency range represents the most dominant band that contains the pure interictal discharges28 that expresses the synchrony state of the underlying neuronal network4; (2) the ratio of the negative-peak  to the peak-to-peak amplitude28 which is positively correlated to the recruitment rate of excitatory cells5; (3) the ratio of delta and theta (2–8 Hz) band power to the (2–300 Hz) band power30 obtained using power spectral density (PSD), where increased delta and theta synchronization refers to cortical dysfunction thus the decreased ratio can be interpreted as partial normalization of cortical activity32; (4) the ratio of gamma (28–80 Hz) band power to the 2–300 Hz band power using PSD, where increased gamma results from the dysfunctional state of the underlying multiple ion channels or neurotransmitter receptors in awake subjects31; (5) IED frequency that is calculated from the total count of detected IEDs for every 40 seconds. In this study, the clustered IED frequency during each 40-sec time-frame was sufficient for expressing the IED occurrence of each INM phase  signal using a 300 ms-long segment. The center of the thresholded part of square signal  of each pairwise X combination expresses the reliability of that correlation and its importance. All statistical processes were computed using descriptive statistics functions in MATLAB. The null hypothesis shows that there is no relationship and there is no similar change of feature during laser ON and OFF phases among rats and trials with the same protocol. Changes with p value < 0.05 are considered statistically significant.The values of each extracted feature per trial/rat/frequency/site group are formulated as change rate per 40 seconds during INM phases , then normalized to the change rate of the last 40 seconds of PCtrl in array form called X with  length. To measure the similarity of each X array among trials/rats, Pearson correlation was implemented between the average of all X arrays  and the average of X arrays . The final correlation  about 20-30% amplitude suppression compared to the baseline but the reduction is smaller at the furthest recording sites  about 0–15% of suppression  but that increase does not exceed the values of PCtrl phase. Using 1 kHz pulsed NIR light, negative-peak feature tends to slightly decrease  during laser ON phase. During 1 Hz pulsed NIR light, negative-peak feature shows a constant rate of change beside optrode  and a clear decrease in the furthest channel . With 1 kHz pulsed NIR light, the effect on the 2–8 Hz frequency band becomes weaker , and with 1 Hz of NIR light there are narrow changes (more stable) in 2–8 Hz band power  and this effect becomes dominant with 4 mins NIR ON. Beside NIR laser illumination, there is a moderate positive correlation . This elevated state of gamma activity is maintained at 1 kHz pulsed NIR light  and far from optrode . At pulsed illumination, a moderate increase in bandpower can be observed during the stimulus onset, which is more dominant as the stimulus onset time is increased. Heating phase-related changes: the induction of gamma oscillation is obvious during heating up phase and this elevated state is maintained at the maximal steady cortical temperature  where the correlation and p value show instability in IED frequency among rats and trials so there is no clear evidence about the dominant change in IED frequency. With 1 kHz pulsed NIR light  and with 1–100 Hz frequency of pulsed NIR light there is no clear evidence about the dominant change in IED frequency. At 1–100 Hz illumination, in addition to the long-term maximal temperature  or boosting the temperature of the tissueFrequency-related changes: the normalized changes of the peak-to-peak amplitude of IEDs show an apparent reduction in the amplitude 37. The mild increase in peak-to-peak amplitude  band power clearly increases during the P2ON and P4ON phases, where both INM protocols produce an induction in gamma rhythm  band power to the total band (2–300 Hz) power shows a proportional increase during the heating-up phase, particularly when pulsed NIR light is used. However, during the steady-state maximum temperature, the relative power of delta and theta bands decreases proportionally.The increase in the ratio of gamma (28–80 Hz) band power is entirely correlated with increasing cortical temperature.While in the immediate vicinity of the stimulation site there are no clear changes in IED frequency associated with temperature. Additionally, steady-state maximum temperature during trials does not illustrate any consistent changes in IED frequency.The effects of INM on IED frequency and peak-to-peak amplitude exhibit great variability between animals. These effects are reversed depending on the frequency of pulsed-IR illumination.This effect of INM could potentially restrict its use in therapeutic applications related to epilepsy. However, the thermal effect of INM on cortical neurons induces changes in other characteristics of IED, which could prove beneficial for future applications.Finally, the research outcomes highlight the following new hypotheses:There is a distinct effect of the heating-up phase and maximal cortical temperature on IED characteristics.There is reverse relation between the amplitude and frequency of IEDs and based on the frequency of NIR light.A low rate of heating up the cortex has a more inhibitory impact on the sharp negative-polarity of IEDs.There is no clear evidence that the INM procedure can suppress the IED frequency in anesthetized rodents.This paper investigates the INM-based cortical heating procedure and its effect on interictal discharges in a penicillin-induced acute model. With the use of the multimodal photonic device we successfully achieved precisely controlled cortical heating experiments using a laser diode source. By characterizing the detected IEDs in Supplementary Figures.Supplementary Figures.


Persecutory delusions are strong threat beliefs about others’ negative intentions. They can have a major impact on patients’ day-to-day life. The Feeling Safe Programme is a new translational cognitive-behaviour therapy that helps patients modify threat beliefs and relearn safety by targeting key psychological causal factors. A different intervention approach, with growing international interest, is peer counselling to facilitate personal recovery. Combining these two approaches is a potential avenue to maximize patient outcomes. This combination of two different treatments will be tested as the Feeling Safe-NL Programme, which aims to promote psychological wellbeing. We will test whether Feeling Safe-NL is more effective and more cost-effective in improving mental wellbeing and reducing persecutory delusions than the current guideline intervention of formulation-based CBT for psychosis (CBTp).A single-blind parallel-group randomized controlled trial for 190 out-patients who experience persecutory delusions and low mental wellbeing. Patients will be randomized (1:1) to Feeling Safe-NL (Feeling Safe and peer counselling) or to formulation-based CBTp, both provided over a period of 6 months. Participants in both conditions are offered the possibility to self-monitor their recovery process. Blinded assessments will be conducted at 0, 6 (post-treatment), 12, and 18 months. The primary outcome is mental wellbeing. The overall effect over time (baseline to 18-month follow-up) and the effects at each timepoint will be determined. Secondary outcomes include the severity of the persecutory delusion, general paranoid ideation, patient-chosen therapy outcomes, and activity. Service use data and quality of life data will be collected for the health-economic evaluation.The Feeling Safe-NL Trial is the first to evaluate a treatment for people with persecutory delusions, while using mental wellbeing as the primary outcome. It will also provide the first evaluation of the combination of a peer counselling intervention and a CBT-based program for recovery from persecutory delusions.Current Controlled Trials ISRCTN25766661 (retrospectively registered 7 July 2022). The order of the items has been modified to group similar items , the largest treatment effects for persecutory delusions to date [The new translational Feeling Safe Programme, a targeted psychological treatment addressing key contributory causal factors, has demonstrated large treatment effects for persecutory delusions  using brief CBT modules and supporting patients to relearn safety. At the same time, professional peer counsellors focus on addressing personal recovery by sharing of experiences, identifying meaning in past and current experiences, and helping people to build experiential knowledge. Besides the close collaboration between therapists and peer counsellors, Feeling Safe-NL offers a trauma-imagery module based on the evidence for trauma imagery as a treatable psychological causal factor of threat beliefs , 24. TheThis randomized controlled trial will recruit outpatients with persecutory delusions, independent of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-classification. The primary hypothesis is that the Feeling Safe-NL Programme will lead to greater improvement in mental wellbeing over time (from baseline to 18-month follow-up) than formulation-based CBTp. The secondary hypotheses are that the Feeling Safe-NL Programme will be more effective than formulation-based CBTp over time (from baseline to 18-month follow-up) in 1) reducing the severity of the persecutory delusion, 2) reducing general paranoid ideation, 3) improving patient-chosen outcomes of therapy and 4) improving activity levels. We also hypothesize that the Feeling Safe-NL Programme will be more cost-effective than formulation-based CBTp (from baseline to 18-month follow-up). The trial will also include an explanatory component. We hypothesize that changes in the key maintenance factors , resilience, and personal recovery will mediate changes in mental wellbeing and persecutory delusions. We will also record all service use, and other relevant health economic data, for the health-economic evaluation.The primary objective is to test whether the Feeling Safe-NL Programme is more effective in improving mental wellbeing over time than formulation-based CBTp. The overall effect over time (baseline to 18-month follow-up) and the effects at each timepoint will be determined .The secondary objectives are to test whether the Feeling Safe-NL Programme is more effective than formulation-based CBTp over time in (1) reducing the severity of the persecutory delusion, (2) reducing general paranoid ideation, (3) improving patient chosen outcomes of therapy, and (4) improving activity levels. Additionally, we investigate the mediators of improved mental wellbeing and reduced persecutory delusions and whether the Feeling Safe-NL Programme is more cost-effective than formulation-based CBTp.A pragmatic single-blind superiority randomized controlled trial with two parallel groups. Patients will be randomized 1:1) to the Feeling Safe-NL Programme or formulation-based CBTp. Measurements are conducted at baseline, 6-, 12-, and 18-month follow-up  that offer care to people with persecutory delusions.We will recruit outpatients with persecutory delusions, independent of DSM classification. The inclusion criteria are main persecutory belief held with at least 60% conviction; low mental wellbeing . Therapists will receive weekly supervision from a clinical psychologist and CBT supervisor. Therapists start seeing participants for the trial after four recorded therapy sessions (two for Feeling Safe-NL and two for formulation-based CBTp) that have been rated and scored ≥ 4 on each relevant item of the Cognitive Therapy Rating Scale-Revised for Psychosis. Professional peer counsellors meet the standards of the Dutch professional competence profile , 19 and Interventions will be discontinued when a participant no longer wants to continue the intervention or when a participant is an early therapy completer. The interventions that are tested are found to be feasible and safe in this population and the study is situated in routine clinical practice.Both formulation-based CBTp and Feeling Safe-NL involve shared decision-making and tailoring of CBT-techniques to each individual patient to promote adherence and outcomes. Therapists and peer counsellors generally see participants at their local community mental health team, but home visits are possible to improve adherence to the interventions.Standard care will continue as usual and will be monitored by the research team.There are no provisions for post-trial care since the interventions are found to be feasible and safe in this population and the study is situated in routine clinical practice. Usual care will continue when the trial ends.28 over time  [The primary outcome will be psychological wellbeing mepoint) .The secondary outcomes include persecutory delusion severity  , generalin prep), reasoning biases  [We will also assess several mediators: trauma imagery  –45, insoMADS-PM) –52, persMADS-PM) , and resMADS-PM) .Other study parameters include the therapeutic relationship   and workFollowing enrolment, participants will be allocated to formulation-based CBTp or the Feeling Safe-NL Programme. Assessments will be carried out at baseline and after 6 (post-intervention) 12 and 18 months. A schematic diagram is provided in Table The sample size for longitudinal intention-to-treat (ITT) analyses with linear mixed models (LMM) was calculated according to the method presented by Liu and Liang (1997) and based on data from previous RCTs –15, 24. Our strategy for achieving adequate participant enrolment to reach the target sample size is to recruit at several large mental healthcare institutions that collaboratively offer care to more than 200,000 patients. Additionally, we work with a committed group of peer counsellors and therapists and trained trial assessors who are involved in informing patients and community health team staff.http://www.randomizer.org). The allocation sequence ensures an equal assignment to both interventions for each site.Patients will be randomized by our independent randomization bureau using a scientific randomization program on the internet 42 consists of six items which assess the following dimensions of delusions: frequency of preoccupation with delusions, duration of preoccupation with delusions, conviction, frequency of distress, intensity of distress and disruption to life caused by beliefs. Test–retest reliability has been found to be high [The Psychotic Symptom Rating Scales-Delusions Subscale (PSYRATS-DS) be high . Scores  be high .The Revised-Green et al., Paranoid Thoughts Scales (R-GPTS) is an 18-item self-report questionnaire that assesses ideas of persecution (10-item) and social reference (8-item) and was found to have excellent psychometric properties . A hieraThe time budget measure is used to assess  activity. The measure is specifically designed for people experiencing psychosis .The CHoice of Outcome In Cbt for psychosEs (CHOICE) short form  is a 12-Resource use will be measured with the Trimbos Institute and iMTA Cost Questionnaire for Psychiatry (TIC-P), short version. Using the TIC-P, we will measure healthcare usage, patients’ and their family’s out-of-pocket costs, and productivity losses owing to absenteeism and presenteeism. The EuroQol 5-dimension 5-level questionnaire (EQ-5D-5L) will be used to measure health-related quality of life (HRQoL). The EQ-5D-5L describes health states over five domains of mobility, self-care, usual activities, pain/discomfort, and anxiety/ depression. Dutch tariffs  will be The Trauma And Life Events checklist (TALE) is a 21-item checklist for traumatic experiences and shocking life events in people with psychotic disorders that was recently developed . It coveThe Counsellor Rating Form – Short (CRF-S)  is a 12-The Working Alliance Inventory – Short Form Revised  (WAI-SR)The Trauma Screening Questionnaire (TSQ) is a 10-item self-report questionnaire that assesses post-traumatic stress symptoms . The scaThe PTSD Checklist for the DSM-5 (PCL-5) is a 20-item self-report questionnaire assessing PTSD symptoms. The questionnaire has been validated in many different types of samples . The DSMThe Insomnia Severity Index (ISI) is a 7-item self-report questionnaire that assesses the severity of both night-time and daytime components of insomnia .The Brief Core Schema Scales (BCSS) is a 24-item self-report questionnaire that assesses schemata concerning self and other people . The queThe Dunn Worry Questionnaire (DWQ) is a 10-item questionnaire that assesses general problematic worry . The scaThe Voice Impact Scale (VIS) is a 24-item self-report questionnaire that assesses the impact of voices. It contains three 8-item subscales: negative impact of voices, positive impact of voices, and living well with voices. The psychometric qualities of the VIS are good. A publication about the psychometrics is underway.49 is an 8-item questionnaire assessing avoidance and associated distress. The scale has demonstrated good internal reliability, test–retest reliability, and validity.The Oxford Agoraphobic Avoidance Scale (O-AS)in prep) assessing avoidance in the past two weeks  and in situ-defences  which are strategies people use to keep themselves safe.The Oxford Paranoia Defence Behaviours Questionnaire (OPDBQ) is a newly developed questionnaire  Interview  is a briThe Fast and Slow Thinking (FaST) Questionnaire  is a 10-The Maudsley Assessment of Delusions Schedule – Probability of Being Mistaken (MADS-PM) is a commonly used ‘Yes’ or ‘No’ item assessing whether people consider the possibility that they are being mistaken about their persecutory delusion .The 15-item questionnaire about the process of recovery (QPR) assesses personal recovery . The QPRThe Brief Resilience Scale (BRS) is a 6-item questionnaire of resilience that was found to have good psychometric properties .Semi-structured qualitative interviews will be conducted with participants regarding their experiences with both therapies. The interviews that are conducted post-therapy will mainly focus on patients’ experiences with (1) collaborating with their therapist and peer counsellor (if Feeling Safe-NL) and effect on their recovery and (2) self-monitoring and visual feedback using the m-Path application. Interviews conducted 1-year after therapy completion will mainly focus on participants’ experiences with long-term recovery. Therapists and peer counsellors will also be interviewed after seeing several participants. The interviews will mainly focus on (1) their collaboration in the Feeling Safe-NL Programme and (2) using the m-Path application. Participants will be interviewed until saturation is achieved on all topics.This project team is committed to a recovery-oriented approach, and everyone working on the trial will be trained to ensure respectful and meaningful interactions with patients and community mental health staff. Trial assessors can visit participants at their local community mental health team or at their homes. Participants are also compensated for their time and travel. We also know from previous trials that patients remain motivated and generally like to contribute to this type of study . Additiowww.m-path.io). Audio recordings of the therapy sessions (for supervision and fidelity ratings) and qualitative interviews will also be stored in agreement with the VU University Amsterdam guidelines using the program Research Drive.Questionnaire data  will be collected in the Data Manager module of Research Manager in agreement with local mental healthcare institutions and VU University Amsterdam guidelines. Data from self-monitoring measures will be collected using the GDPR compliant platform m-Path of the KU Leuven (Participants are given a research number (before randomization) and a randomization number (after randomization). No participant information is stored in the Research Manager. Participant information is kept on a separate and secured drive in a recruitment file with a password. This data is only accessible by the principal investigator, the project coordinator and trial assessors. In line with WMO guidelines, data will be stored for 15 years after the last intervention has been provided. We will save the key file about the participants for the entire period of 15 years for safety reasons.N/a. The trial involves an investigation of two psychological interventions using questionnaires and interviews.The primary continuous outcomes for mental wellbeing will be analyzed with linear mixed models (LMM) with outcome measurement (at the three follow-up timepoints) as the dependent variable and based on the intention-to-treat (ITT) principle. Baseline values will be added as covariates, time as a categorical variable, and treatment condition as a fixed effect. The regression coefficient of the LMM will be used as the between-group effect size (Feeling Safe-NL vs formulation-based CBTp) since this is the estimated difference in results between the groups.The secondary continuous outcomes for the persecutory delusion, general paranoid ideation, patient-chosen therapy outcomes and activity will also be analyzed with LMM with outcome measurements (at the three follow-up timepoints) as the dependent variable and based on the intention-to-treat (ITT) principle. Baseline values will be added as covariates, time as a categorical variable, and treatment condition as a fixed effect. Between-group effect sizes (Feeling Safe-NL vs formulation-based CBTp) will be derived from the regression coefficient of the LMM. Outcomes for conviction of the main persecutory delusion (PSYRATS-DS) will also be dichotomized with recovery being defined as conviction falling below 50%. Logistic generalized estimating equations (GEE) with exchangeable correlation structure ITT analysis will be used to examine the effects on dichotomous outcomes, since GEE is reported to be a better estimator of effects in dichotomous outcomes than LMM . EffectsWe will also carry out parallel mediation analysis using the PROCESS macro for SPSS  to asses2) = 5) of 5 points in WEMWBS mental wellbeing, based on studies on the WEMWBS and the Feeling Safe Trial [Both (1) cost-effectiveness analysis (CEA) and (2) cost-utility analysis (CUA) will be conducted alongside the randomized trial. The outcome is Treatment Response (TR) defined as a clinically relevant change, in the CEA. In the CUA the outcome will be QALYs as estimated by the EQ-5D-5L using Dutch tariffs. Since we need to assign costs to meaningful outcomes, we use both QALYs and clinically relevant change defined using the primary outcome of mental wellbeing. For a clinically relevant change, we use Jacobson & Truax reliable change. For mental wellbeing measured with the WEMWBS, this corresponds to an improvement of 5 points. We calculated this as follows: SE means = 8.2 * square root (10.83) = 3.38; reliable change index: (2 × (3.38)fe Trial , 68. Foufe Trial , 70. Sinfe Trial . To visufe Trial .Inductive Thematic Analysis , 74 willN/a. Analyses are conducted at the end of the trial.N/a. There is no plan for any additional analyses.Missing outcome data in the clinical effectiveness analyses will be handled by the LMM analysis (i.e. given its robustness to missing data) . In the Data will be made accessible after an embargo period (completion of PhD project), for verification purposes, safety monitoring, and future research with some restrictions: (1) the approval of the participants as indicated in the consent form, (2) conditions related to data security, (3) quality assessment of the proposed research, (4) collaboration in using the data set. Potential restrictions for data access are evaluated with the privacy experts at the various partner organizations to ensure the protection of the study population, and even if the data are not made available following request, we will make the metadata describing our research publicly available through registration in PURE and any other outputs that do not involve data collected about the participants.The trial is run by the principal investigator, project coordinator (PhD) and trial assessors who meet on a (bi-) weekly basis. There is also a meeting with the trial steering committee every three months including members of the Oxford Cognitive Approaches to Psychosis (O-CAP) research group, trial supervisors, peer counsellors, local principal investigators of the participating mental healthcare institutions, and the randomization bureau.N/a. The interventions are proven to be acceptable and are used in clinical practice.The interventions  are found to be acceptable and are used in clinical practice. Therefore, no major adverse events related to the interventions are expected. Nor is there any expectation that combining Feeling Safe and peer counselling would result in adverse outcomes. Adverse events reported spontaneously by the participant or observed by the investigators or staff will be recorded. If a serious adverse event occurs, the responsible therapist (and peer counsellor) takes action. The principal investigator will report the serious adverse event through the web portal to the accredited medical ethics committee that approved the protocol within 7 days of first knowledge for serious adverse events that result in death or are life-threatening followed by a period of a maximum of 8 days to complete the initial preliminary report. All other serious adverse events will be reported within a period of a maximum of 15 days after the investigator has first knowledge of the serious adverse events. An elective hospital admission, suicidal thoughts, self-harm, and crisis contacts with mental healthcare professionals will not be considered as a serious adverse event as this regularly occurs within this population. Somatic illnesses will also not be considered as a serious adverse event as this is unrelated to participation in the study.N/a. A trial steering group was established instead of a data monitoring committee.Important protocol modifications  will be submitted to the relevant medical ethics committee for review and communicated during the meeting with the trial steering committee and during assessments with participants, if applicable.https://www.gedachtenuitpluizen.nl). This organization is dedicated to disseminating psychological therapies for people with threat beliefs and/or hallucinations. It is also the central training institute in The Netherlands for psychological therapies in psychosis. Materials will be made available to a wide group of people using the organization’s website and newsletter. With the dissemination funding from the sponsor, we can also develop a training course and associated materials.Results will be communicated through publications in journals and presentations at established conferences. Participants who have agreed to receiving the results will be sent a newsletter. Broader dissemination of the findings will occur through the organization Cognition & Psychosis  combining Feeling Safe with peer counselling; (2) by introducing trauma imagery as a treatable maintenance factor; and (3) introducing visual feedback of self-monitoring to empower and support people in shared decision-making and tracking of progress. Feeling Safe-NL is the first program for patients with persecutory delusions in which therapists and professional peer counsellors collaborate to improve mental wellbeing and feelings of safety. Therefore, the program moves towards a dual approach to therapy involving systematically removing the factors that hamper recovery while synergistically promoting personal recovery. The Feeling Safe-NL Programme addresses the key problems people with persecutory delusions are seeking help for regardless of diagnostic category. The current trial aims to determine the effectiveness and cost-effectiveness of the Feeling Safe-NL Programme above that of the guideline intervention formulation-based CBTp. The potential is a therapy that may be more effective and cost-effective on the long-term. Outcomes are expected in 2026.Protocol version 2 (21–12-2022). Recruitment started on the 14th of March 2022 and is expected to be completed in March 2025.