Supplemental Digital Content is available in the text. TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20.TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell–derived cardiomyocytes.MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20.Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell–derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.We demonstrate that circRNAs formed from the  TTN (Titin) transcript and forms a functional motif when both ends of the RNA join on circularization.We uncovered the large, heart-specific circular RNA cTTN1, which is derived from the I-band region of the cTTN1 is necessary for normal cardiomyocyte organization, and we show that the motif that this and other RBM20-dependent circular RNAs form by circularization is crucial to allow binding of SRSF10 to exert its function.TTN transcript forms functionally important circular RNAs, and together with our finding that genomic regions corresponding to motif formation are less tolerant to sequence variation this suggests that variants that affect formation and function of these circRNAs might relate to cardiac pathology.This reveals that the RBM20, mutations in the I-band region of the TTN gene also may have as-yet unsuspected clinical relevance by hindering formation or function of these circular RNAs.Besides mutations in In particular, variants in this region may have more clinical relevance than was assumed on the basis of the role of this region in the formation of the TTN protein.TTN) gene consists of 363 exons to produce the largest human protein. It is regarded as a bidirectional spring that provides mechanical support to the sarcomere but also receives incoming signals.1 With the advent of next-generation sequencing, it became clear that truncating variants in TTN are found in 20% to 30% of patients with a dilated cardiomyopathy (DCM).2 While the role of TTN in cardiomyocyte function and structure is well established, far less is known about the possible regulatory roles of the RNA transcript (>100 kb) that TTN produces.The human titin , which results in the formation of single-stranded circRNA molecules. Depending on the linear splicing in the template pre-mRNA, this circRNA might contain all or only a few of the exons between the acceptor and donor exons. Although for most of the circRNAs the function is still elusive, several functions have been described, and no general function seems applicable to all of them. The described functions range from microRNA sponging5 to facilitation of transcription by direct association with RNA polymerase II,6 to interaction with proteins,7 to acting as a template for protein synthesis.8It was long assumed that in pre-mRNAs exons are always spliced to an upstream exon to generate a linear mRNA, which then serves to translate to protein. However, with the advent of next-generation sequencing, it became increasingly clear that many genes can also form circular RNAs (circRNAs) by so-called back-splicing.RYR2 and TTN produce large numbers of circRNAs.12 In our study, we also showed that the splice regulator RBM20 is necessary to produce a large proportion of these TTN-derived circRNAs.9 This class of RBM20-dependent circRNAs is derived mainly from the I-band region of TTN, which is the region that is alternatively spliced in RBM20 mutation carriers.14 This suggests that loss of these circRNAs in RBM20 mutation carriers might contribute to the development of the severe form of DCM combined with lethal arrhythmias, but also that TTN mutation carriers in the I-band region could develop similar phenotypes as a result of the distortion of formation or function of these circRNAs. Therefore, we asked if this specific class of RBM20-dependent circRNAs formed from the I-band region of the TTN transcript have important functions in cardiomyocytes.We and others have shown the expression of thousands of circRNAs in the human and rodent heart, where especially the mRNA transcripts of Excel Files I through VI in the Data Supplement. Data of RNAseR-treated human heart tissue RNA sequencing (RNAseq) were used under license for the current study; data are available via the corresponding authors. CircRNAprofiler is available on github page: https://github.com/Aufiero/circRNAprofiler.15 The code for the genetic constraint analysis is available at https://github.com/ImperialCardioGenetics/cTTN.Raw sequencing data for circRNA detection in human heart and cTTN1 inhibition in human induced pluripotent stem cell (hiPSC)–derived cardiomyocytes (hiPSC-CMs) are available via NCBI accession numbers PRJNA5333243 and PRJNA630157, respectively. All data with regard to bioinformatic analysis are included in Data Supplement, which also includes primer and shRNA sequences and antibody details in Tables I, II, and III in the Data Supplement, respectively.Extended methods are described in the 16 The dermal fibroblasts were obtained with informed consent after approval by the Institutional Review Board committee of Rambam Medical Center. Colonies of these hiPSCs were cultured in mTeSR-1 on plates coated with growth factor–reduced Matrigel. Cardiac differentiation was performed following a previously published protocol17 with the slight adaptation that cells were cultured in RPMI/B27 medium from days 7 to 10. Cells were metabolically selected for at least 2 weeks by culturing in CDM3 medium without glucose supplemented with 20 mmol/L sodium lactate.18 All experiments were conducted on hiPSC-CMs 40 to 60 days after the start of differentiation, and each observation was replicated in 2 to 5 independent experiments with hiPSC-CMs from different differentiations.The fully characterized hiPSC line derived from a healthy man was previously published.We cloned shRNAs targeting the back-splice junction of cTTN1 and a negative control shRNA (shSCR) into the pLKO.1 vector and produced third-generation lentivirus in RPMI/B27 or CDM3 medium, depending on the experiment. hiPSC-CMs were dissociated with the use of TrypLE express and replated 2 to 4 days before lentiviral transduction to ensure homogeneous cell populations between conditions. The hiPSC-CMs were transduced either with freshly produced lentivirus or with a specific amount of transducing units, depending on the density of the cells. Experiments were performed on cells between 4 and 8 days after transduction.RNA was isolated with TriReagent. RNAseq was performed on a Illumina NextSeq 500 platform in paired-end mode with a read length of 150 bp and analyzed by use of the R Bioconductor packages DEseq2 and DEXseq for differential gene expression and exon usage, respectively. For detection of most circRNAs, we used end-point polymerase chain reaction (PCR) with divergent primers to amplify the back-splice region, and for detection of cTTN1, we used a TaqMan-based quantitative PCR system with similar divergent primers and a probe targeting the back-splice junction. To visually detect cTTN1 in fixed cells, we used a FAM-labeled probe directed against the back-splice junction, where the signal was amplified by incubation with an anti-FITC antibody followed by incubation with a TSA Plus fluorescence kit. RNA immunoprecipitation was performed with the MagnaRIP RNA binding protein immunoprecipitation kit. RBM20 knockout (KO) mice hearts for RNAseq were derived from studies approved by the Institutional Animal Care and Use Committee of the University of Amsterdam and performed in accordance with the guidelines of this institution.19 and in a second step, the expected numbers were corrected for sequencing depth at low-coverage sites. The model was validated using TTN synonymous, missense, and truncating variants; the first 2 are assumed to be under minimal selection, and truncating variants are known to cause DCM.We compared the observed number of rare variants  in the gnomAD reference population (version 2.1.1) exome data sets  with the number of variants that we would expect to see under neutral variation. The expected number of variants under neutral selection was predicted based on sequence context and methylation level,9 Here, we used the paired-end RNAseq reads of 3 human control hearts, 3 DCM hearts, and 3 hypertrophic cardiomyopathy hearts and aligned them against the human genome reference hg19 using MapSplice, CircMarker, and NCLscan. We predicted both the RNA-binding protein and miRNA binding using our in-house previously published R-based computation framework, circRNAprofiler.15For prediction of RNA-binding protein and microRNA (miRNA) binding, we used the raw sequencing data of our previously published study.TTN.For bioinformatic prediction of exons included in cTTN1, we used RNAseq data of RNAseR-treated RNA samples isolated from left ventricular tissue and the computational tool circAST, which determines the exons derived from aligning of these sequencing data to the human genome and thus derived from circRNAs in a specific region of 20 As a consequence, the data shown for continuous variables are a mean±SEM of n=6 to 15 biological replicates derived from 2 to 5 differentiations. For categorical data, the percentage of cells in all groups is depicted per condition. To compare continuous variables between 2 groups, we used the Mann-Whitney U test; to compare continuous variables between 3 groups, we used the Kruskal-Wallis test combined with the Dunn post hoc test. To compare the distribution of cells in different categories between 2 groups, we used the χ2 test. To compare the effect of loss of RBM20 over time (days 1–8), we made use of a 2-way ANOVA to test the effect of loss of RBM20, the day effect, and the interaction term between RBM20 and day. In case the interaction term was significant, we determined the differences between the shRNA against RBM20 and the negative control shRNA by pairwise comparison, in which we used Bonferroni correction for multiple testing. All performed tests were 2 sided, and a value of P<0.05 was considered significant.Data obtained from hiPSC-CMs are a combination of 2 to 5 independent experiments on cells from independent differentiations, with at least n=2 biological replicates per independent experiment. Data from these independent experiments are combined by use of Factor Correction.Figure Ia and Ib in the Data Supplement). This motif occurs almost exclusively in the back-splice junction of RBM20-dependent circRNAs derived from TTN; it is found in only 3 other non-TTN-derived circRNAs, whereas it is found in 44 TTN-derived circRNAs. The biological importance of this motif is further suggested by the trend for selective constraint we found within the human population .21 We clearly found no selective constraint when the above-described motif was formed by linear TTN splicing independently of whether this splicing occurred in exons included or always excluded from circRNAs. However, we did detect a trend toward reduced selective constraint within this motif when it is both formed by back-splicing and present within an exon . Loss of SRSF10 was shown previously to result in embryonic lethality attributable to an underdeveloped myocardium and distorted cardiomyocyte calcium handling.23 A second bioinformatic comparison using all annotated SRSF10 binding motifs in ATtRACT revealed an enrichment in the back-splice junction and the full sequence of TTN- versus non–TTN-derived circRNAs, in the full sequence of TTN-derived circRNAs versus the linear TTN mRNA, and in the exons included versus excluded from the circRNAs . To test this predicted binding, we performed RNA immunoprecipitation of SRSF10 in hiPSC-CMs. Indeed, all tested circRNAs in which the back-splice junction forms the motif strongly bind SRSF10. In addition, circRNAs that contain the motif in their full sequence  also bind SRSF10. In contrast, circRNAs without the motif do not bind SRSF10 . In addition, in a RNA immunoprecipitation in which we overexpressed SRSF10 and an artificial circRNA construct containing the motif, mutation of the motif impaired this binding . This suggests that cTTN1 is very large and could potentially span 35 to 58 exons in total. We used shRNAs that target the back-splice junction of cTTN1 to selectively knock down cTTN1 without affecting its linear counterpart . Loss of cTTN1 caused missplicing of SRSF10 targets such as CASQ2 and MEF2A in the same way that loss of SRSF10 does, without affecting SRSF10 expression levels . Furthermore, contraction analysis of engineered heart tissues after selective loss of cTTN1 demonstrated markedly reduced contraction amplitudes 7 and 14 days after generation of the engineered heart tissues . These contraction amplitudes obtained by the MUSCLEMOTION algorithm highly correlate with absolute force.25 However, despite these severe derangements, expression of NPPA and NPPB was not increased after loss of cTTN1 .In addition, selective loss of cTTN1 caused gross abnormalities in hiPSC-CMs, where it disrupted sarcomere structure and induced cell death  showed ample putative miRNA binding sites, and this was confirmed by finding 6 miRNAs upregulated after inhibition of cTTN1 in hiPSC-CMs . We then compared our RNAseq after loss of cTTN1 . Together, these findings indicate that downregulation of MYBPHL could also affect the observed phenotype. However, inhibition of miR-34a-5p, the miRNA with 8 binding sites in the MYBPHL 3′ untranslated region, in hiPSC-CMs did not relieve MYBPHL repression .We further showed by fluorescent in situ hybridization and cell fractionation experiments that cTTN1 localizes to both the nucleus and cytoplasm. This broad distribution, combined with its very large predicted size, indicates that cTTN1 might have different functions according to its location Figure G and 2H.9 and there already hypothesized that the loss of these RBM20-dependent circRNAs might play a role in the adverse effects caused by loss of RBM20. Indeed, even the selective loss of cTTN1 recapitulates not only the structural derangements seen after loss of RBM2027 but also the splice abnormalities caused by loss of RBM20 . Strikingly, this also involves missplicing of TTN itself, which shows that cTTN1 is a crucial regulator of TTN splicing, the transcript from which it derives.We showed earlier that cTTN1 is an RBM20-dependent circRNARBM20 itself . However, we find by immunofluorescent in situ hybridization that cTTN1 colocalizes with RBM20 in distinct nuclear foci . Together, these results suggest a role for cTTN1 in keeping RBM20 localized to the nucleus to perform its splicing function.Because cTTN1 is formed only in the presence of RBM20 and cTTN1 seems to also regulate the same splicing effects, we asked how cTTN1 is involved in the proper function of RBM20. Loss of cTTN1 does not decrease the levels of i Figure D. These 29 We speculated that after loss of RBM20 by shRNA inhibition, it would take longer for cTTN1 to also disappear. We would thus create a situation in which cTTN1 is still present while RBM20 is already absent. Indeed, on knock-down of RBM20 in hiPSC-CMs, RBM20 itself is already lost 1 day after transduction, whereas cTTN1 starts to diminish only from day 2 onward  primary cells or in vivo. HiPSC-CMs are known to be immature fetal-like cardiomyocytes30 with a higher expression of the fetal TTN isoform, which includes the I-band region in the transcript. This limits the RNA template for circRNA production of circRNAs derived from the I-band region. As a consequence, cTTN1 had relatively lower expression in these cells compared with the human heart. This suggests that cTTN1 has a more important role in the adult heart and that loss of cTTN1 in the adult heart would result in a more tremendous phenotype.We studied cTTN1 because it is the highest expressed circRNA in the human heart and is downregulated in DCM.31 revealed that the formation of the SRSF10 binding motif in the back-splice junction of RBM20-dependent circRNAs is conserved to mice. The difference between mouse and human is the exons used for back-splicing to create this motif and thereby the exact composition of the full circRNA sequence. The conservation of the motif formation, together with the genetic constraint of the motif in human circRNAs, implies that this mechanism is of biological importance.One could argue that the fact that cTTN1 is not conserved to rodents implies that the mechanisms we describe are less important. However, scrutinizing our previously published RNAseq data of RBM20 KO mice24 This tool reports which exons within a certain region are detected in RNAseq data. We investigated the region from which cTTN1 is derived (TTN exons 79–145) in RNAseR-treated RNAseq data, in which all noncircRNAs are degraded before sequencing. This means that all reported exons within this region are potentially derived from cTTN1. However, cTTN1 is derived from the I-band region of TTN, from which the majority of circRNAs within TTN are derived.9 As a consequence, the reported exons in the circAST analysis could also be derived from other circRNAs stemming from this region. In addition, the exons detected in the RNAseq data showed variability between the 3 RNAseq samples that we analyzed (Excel File III in the Data Supplement), which indicates variability in which exons are included in the circRNAs. This could be caused by differences in back-splicing and thus which circRNAs are formed, but it could also be caused by alternative linear splicing that occurred within the formed circRNAs. We defined cTTN1 as the circRNA formed by back-splicing of exon 79 and 145, but based on the circAST analysis, we cannot exclude that several isoforms of cTTN1 exist. This implies that different cTTN1 isoforms could underlie the different mechanisms we describe. Unfortunately, the possible large size of cTTN1 and the uncertainty about its composition mean that overexpression as a possible rescue for RBM20 mutation carriers would be a very uncertain path to take.We describe that cTTN1 is potentially a very large circRNA that could span 35 to 58 exons (6.6–10 kb). This prediction is based on our analysis using the circAST tool.28 In these foci, the mRNA of the RBM20 splicing targets is produced from their respective DNA templates and immediately spliced by RBM20.28 It has also been shown that most RBM20 mutations occur in the RSRSP domain,32 which is necessary for nuclear localization of RBM20. Therefore, RBM20 mutations in this domain lead to translocation of RBM20 out of the nucleus and, not surprisingly, to defects in splicing of the RBM20 targets.33 Furthermore, in homozygous genome-edited pigs with a mutation in this RSRSP domain, ribonucleoprotein granules accumulated abnormally in the cytoplasm.34 These dysregulated granules were linked to myocardial cellular pathobiology and heart failure and therefore were introduced as a radical new concept underlying DCM in RBM20 mutation carriers.34 We show that selective loss of cTTN1 also leads to mislocalization of RBM20 from the nucleus to cytoplasm, which thus could lead to the observed phenotype via the loss of splicing in the nuclear foci but probably also via dysregulation of ribonucleoprotein granules.RBM20 has been shown to exert its splicing function in nuclear foci.28 and that these factors are able to splice mRNA targets at similar positions as RBM20. This hypothesis is supported by the 4-nt long recognition motif of RBM20 (UCUU),36 which is also part of the recognition motif of other splice factors. Some of these splice factors are, according to our RNA-binding protein analysis using circProfiler (Excel File II in the Data Supplement), also able to bind cTTN1 in the full circRNA sequence. This would mean that these splice factors splice the same mRNA targets in the nuclear foci as long as cTTN1 is present and recruiting them to these foci.The experiment in which we detected splicing of RBM20 and SRSF10 targets at several time points after inhibition of RBM20 suggests that cTTN1 affects splicing directly, because in this experiment splicing of these targets is largely preserved as long as cTTN1 is still present while RBM20 is already absent. We therefore hypothesize that cTTN1 is able to recruit other splice factors to the nuclear foci where normally RBM20 is present and exerting its functionTTN transcript in that it produces important regulatory RNA molecules, of which one eventually also regulates splicing of the TTN transcript. These circRNAs stem from the I-band region of the TTN transcript that hitherto received less attention because it is spliced out in the healthy heart. However, these data suggest that this region is an important source of muscle-specific regulatory RNAs and could potentially also be a source of disease-causing mutations. Therefore, we believe that variants in the I-band region should also be considered when patients are checked for disease-causing mutations. This is even more important when the phenotype resembles the phenotype of DCM with arrhythmias as seen in RBM20 mutation carriers, whereas no RBM20 mutation is detected.We find a novel role for the This study provides a first example of a functional RNAsequence formed by circularization that cannot be readily recognized in the human genome and illustrates how circularized RNA contributes to sequence diversity in a functional manner.The authors thank Malou van den Boogaard, Dylan K. de Vries, Guillermo Griffith, and Corrie de Gier-de Vries for experimental assistance; Esther E. Creemers for scientific discussions; and Cris dos Remedios, Albert Suurmeijer, Marijke Wasielewski, Jan Jongbloed, Jolanda van der Velden, and Torsten Rasmussen for human tissue samples.This work was supported by grants from ZonMw to Dr Tijsen (Veni 91616150), the Netherlands Cardiovascular Research Initiative to Dr Pinto (CVON-ARENA-PRIME), the Wellcome Trust (107469/Z/15/Z), Medical Research Council (UK), British Heart Foundation (RE/18/4/34215), and the NIHR Imperial College Biomedical Research Centre to Dr Ware.None.Expanded MethodsData Supplement Figures I–IXData Supplement Tables I–IIIData Supplement Movies I–IIData Supplement Excel Files I–VIReferences 37–44


More than 88 million Americans are at risk of developing type 2 diabetes mellitus (T2DM). The National Diabetes Prevention Program’s Lifestyle Change Program (DPP LCP) has been shown to be effective in reducing the risk of progressing from prediabetes to T2DM. However, most individuals who could benefit from the program do not enroll.The aim of this trial is to test the real-world efficacy of 3 mobile phone–based approaches to increasing enrollment in the DPP LCP including a best-practice condition and 2 novel approaches.We will conduct a 3-armed randomized clinical trial comparing enrollment and 1-month engagement in the DPP LCP among adults with prediabetes from 2 health care settings. Participants in the best-practice condition will receive SMS-based notifications that they have prediabetes and a link to a website that explains prediabetes, T2DM, and the DPP LCP. This will be followed by a single question survey, “Would you like the DPP LCP to call you to enroll?” Participants in the 2 intervention arms will receive the same best-practice intervention plus either 2 mobile 360° videos or up to 5 brief phone calls from a health coach trained in a motivational coaching approach known as Motivation and Problem Solving (MAPS). We will collect measures of diabetes-related knowledge, beliefs in the controllability of risk for T2DM, risk perceptions for T2DM, and self-efficacy for lifestyle change pre-intervention and 4 weeks later. The primary outcomes of the study are enrollment in the DPP LCP and 4-week engagement in the DPP LCP. In addition, data on the person-hours needed to deliver the interventions as well as participant feedback about the interventions and their acceptability will be collected. Our primary hypotheses are that the 2 novel interventions will lead to higher enrollment and engagement in the DPP LCP than the best-practice intervention. Secondary hypotheses concern the mechanisms of action of the 2 intervention arms: (1) whether changes in risk perception are associated with program enrollment among participants in the mobile 360° video group and (2) whether changes in self-efficacy for lifestyle change are associated with program enrollment among participants in the MAPS coaching group. Finally, exploratory analyses will examine the cost effectiveness and acceptability of the interventions.The project was funded in September 2020; enrollment began in February 2021 and is expected to continue through July 2022.We are conducting a test of 2 novel, scalable, mobile phone–based interventions to increase enrollment in the DPP LCP. If effective, they have tremendous potential to be scaled up to help prevent T2DM nationwide.ClinicalTrials.gov NCT04746781; https://clinicaltrials.gov/ct2/show/NCT04746781DERR1-10.2196/28884 Among US adults, 34.5% have prediabetes, placing them at increased risk of type 2 diabetes mellitus (T2DM) . ExtensiThere are several reasons for low enrollment and engagement in the DPP LCP. First, many people with prediabetes are unaware of their risk for T2DM or do not believe that they are at risk of developing T2DM or its associated complications. Second, many individuals are not aware of the appropriate lifestyle changes that can prevent progression to T2DM -8. This Most prior research on DPP LCP enrollment interventions has tested the effectiveness of medical providers notifying their patients of their prediabetes, counseling them, and referring them to the DPP LCP. These studies have reported DPP LCP enrollment rates of 8%-11% ,13. We bIn this project, we propose to address these issues by directly connecting with individuals with prediabetes through mobile phone–based interventions in a 3-armed randomized controlled trial. We will compare a best practice intervention with 2 novel interventions our research group has developed and pilot tested: mobile 360° videos and Motivation and Problem Solving (MAPS)–based phone counseling.Aim 1 is to conduct a 3-armed randomized clinical trial comparing enrollment and 1-month engagement in the DPP LCP among adults with prediabetes receiving risk notification and education alone, risk notification and education plus the mobile 360° video, and risk notification and education plus MAPS.Aim 1.1 is to examine the mechanisms underlying the mobile 360° video by comparing changes in deliberative, experiential, and affective risk perceptions across study arms.Aim 1.2 is to examine the mechanisms underlying MAPS by comparing changes in DPP LCP–related self-efficacy across study arms.As noted in the previous section, whether an individual enrolls in the DPP LCP is a multifactorial process that includes their awareness, knowledge, risk perception, and unique barriers. Our intervention is designed to address each of the factors. First, to address low awareness of prediabetes, individuals with a diagnosis of prediabetes within the past 5 years documented in their electronic health record (EHR) will be informed via text message that they have prediabetes and sent a link to the website, Do I Have Prediabetes , that indeliberative risk perceptions , affective risk perceptions , and experiential risk perceptions . The videos are hypothesized to increase participants' affective  and experiential (gist-based) risk perceptions and the likelihood of enrolling in the DPP LCP. The proposed mechanisms of action for MAPS counseling include increasing both motivation and self-efficacy .Individuals will then be randomized to receive only the initial best practice intervention, the best practice plus the mobile 360° videos, or the best practice plus MAPS counseling. The proposed mechanism of action for the mobile 360° videos divides risk perceptions into The DPP LCP is an evidence-based program that is available at hundreds of locations throughout the United States as well as online . It is aIn the best practice condition, we will send a text message to individuals who, according to their health records, have prediabetes. This message will notify them that they have prediabetes and refer them to a website that provides information about prediabetes and the effectiveness of the DPP LCP. This component of the intervention will increase participants’ awareness of the condition and the DPP LCP without adding to the workload of primary care clinicians. Additionally, this is a process that can be automated to regularly reach new individuals with prediabetes within a health system. While it is not yet standard clinical practice to notify individuals with prediabetes of their condition and offer the DPP LCP to them, there is evidence that individuals with prediabetes who are notified about their status are more likely to engage in self-directed healthy lifestyle changes . AdditioThe mobile 360° videos (in which the viewer moves their phone to “look around” the world of the video) are intended to increase individuals’ risk perceptions regarding the potential adverse outcomes that might occur should they develop T2DM. Our team has designed and pilot tested two 3-minute videos with accompanying voiceover (in either English or Spanish) and soundtrack. In the first video A, the naMAPS is a hybrid counseling technique that combines motivational interviewing and social cognitive, practical problem-solving to enhance motivation and self-efficacy for behavior change. MAPS has been shown to help people effectively participate in evidence-based programs and change their health behaviors ,24. AsidA standardized MAPS training and treatment manual has been developed and used in pilot work and will be utilized in this project. Coach training will include both online “classroom” instruction and practice coaching sessions with feedback from a MAPS expert (KL).The MAPS counseling itself will focus on promoting enrollment and engagement in the DPP LCP by addressing each participant’s unique values and barriers. MAPS uses a combination of motivational enhancement and social cognitive approaches based on motivational interviewing and practical problem-solving approaches. All participants will receive up to 5 telephone counseling calls lasting approximately 10 minutes during the 4 weeks following study enrollment. The timing of the MAPS counseling calls will be negotiated between the participant and health coach, as has been done for other MAPS trials. The MAPS counselor will also help each participant develop an individualized “wellness plan” that may not only include goals related to DPP LCP enrollment and engagement but also other potential stressors and concerns . Thus, MAPS will assist individuals with various life stressors that may ultimately affect their DPP LCP engagement. MAPS counseling will be conducted via telephone in Spanish or English, and information from each session will be documented in a secure REDCap database.All MAPS counseling sessions will be audio-recorded, and the recordings will be uploaded into the study database (in REDCap). Ongoing training and monitoring of recorded calls will ensure that the delivered MAPS follows the protocol precisely. A MAPS expert will review at least one recording per coach per week and provide detailed feedback and guidance as necessary to the health coach.Study participants will be recruited from 2 sites: University of Utah Health and the Midvale Community Building Community Clinic (CBC). University of Utah Health is the Mountain West’s only academic health care system and includes 5 hospitals, 12 community clinics, and several specialty centers. The Midvale CBC is an outpatient community health clinic providing medical, dental, physical therapy, and mental health services to low-income and uninsured families, with a predominantly Spanish-speaking clientele.Spanish- and English-speaking patients ages 18-89 years from University of Utah Health and the Midvale CBC will be invited to participate in the study.Patients will be considered eligible for the proposed trial if (1) they are aged 18-89 years, (2) have a diagnosis of prediabetes within the past 5 years documented in the EHR (ICD-10 code R73.03), and (3) have an email or mailing address and a mobile telephone number on record with the health system.Patients will be excluded from the study if they have any of the following diagnoses: T2DM (ICD-10-CM E11), type 1 diabetes mellitus (ICD-10-CM Diagnosis E10), diabetes mellitus due to underlying condition (ICD-10 E08), drug or chemical induced diabetes mellitus (ICD-10 E09); gestational diabetes (ICD-10 024.4), neonatal diabetes mellitus (ICD-10 P70.2), or post-pancreatectomy diabetes mellitus (ICD-10 E13). Individuals who are currently pregnant will be excluded from the trial. The rationale for this exclusion is that these women would be excluded from participating in the National DPP LCP. In addition, individuals who do not have an email address or mailing address or who do not own a smartphone will be excluded from the trial, simply because they could not complete the trial.This is a 3-arm stratified randomized clinical trial comparing enrollment and 1-month engagement in the DPP LCP among adults with prediabetes receiving risk notification and education alone vs risk notification and education plus mobile 360° video vs risk notification and education plus MAPS. It should be noted that this study is not powered nor designed to compare the effectiveness of the mobile 360° videos vs MAPS nor does the design allow for testing of the additive value of mobile 360° videos plus MAPS.Potential participants who do not opt-out will then be sent an SMS message with a link to a survey. The survey will begin with a short eligibility screening section. Individuals who are screened out will be thanked for their time and informed that they do not meet the criteria for participating in the study.Individuals who are not screened out will be directed to the consent cover letter and the remainder of the baseline survey. The baseline survey will collect information about demographics , diabetes-related knowledge (11 items), and beliefs about the controllability of risk for type 2 diabetes (4 items) using 2 subscales on the risk perception for developing diabetes scale . It willAfter completing the baseline survey, individuals will be allocated to the condition according to randomization tables generated by the projects statistician (YZ). These tables will be based on permuted block randomization, stratified by health system and sex, with block sizes randomly generated as 5, 10, or 15. In accordance with our planned sample sizes in each arm, each block will maintain a 1:2:2 ratio in sample size across the treatment groups .Two days after completing the baseline survey, all participants will be sent a hyperlink to the education website, Do I Have Prediabetes? . This wiFour days after completing the baseline survey, participants randomized to the mobile 360° video arm will be sent the first video; 2 days later, they will be sent the second video. Up to 2 reminder texts 1 day apart will be sent to individuals who do not access the links for the videos. Similar to the educational website, the videos will be delivered as an iframe within a web-based survey. This will allow us to assess the duration of time spent watching the video and to collect immediate feedback on participants’ impressions of each video.A health coach trained in MAPS will call the participants randomized to that group; the first call may be used to initiate MAPS coaching or to simply schedule the upcoming MAPS counseling sessions. Participants will be offered up to 5 coaching calls over 4 weeks, each lasting approximately 10 minutes. During these sessions, participants will explore their motivation and goals and how enrollment in the DPP LCP may fit with these goals. Additionally, MAPS coaches will help participants identify barriers to achieving their goals and troubleshoot ways to address these barriers. If a participant enrolls in the DPP LCP, any remaining MAPS calls will serve to reinforce the positive change the individual has made, provide accountability, address ongoing challenges and barriers, and encourage continued engagement with the DPP LCP. All MAPS sessions will be audio-recorded, and at least 1 session per week per coach will be reviewed by a MAPS expert. Feedback will be provided to coaches as needed. This process will insure the fidelity of the MAPS intervention. Participants will have the option to tell the MAPS coaches that they would like to stop receiving calls at any point.Ten days after study enrollment, all participants will be sent a single question survey: “Would you like the DPP LCP to call you to help you enroll?” The contact information for participants who respond “yes” will be sent to the referral coordinator at their respective health system's DPP LCP, who will then call them to enroll. The rationale for this piece of the intervention is to mimic the current workflow for individuals to enroll — in most cases, a health care provider places a referral to the program via the EHR, and the program contacts the potential participant to enroll them. Due to the COVID-19 pandemic, both study sites have been offering the DPP LCP virtually, either as an asynchronous online program or through synchronous group sessions. During the study, participants will have the ability to select whatever DPP LCP format they prefer, including in-person sessions, if available.The follow-up survey will be sent via text message to all participants 4 weeks after the baseline survey is completed. The survey will include all of the items described in the baseline survey . The survey will conclude with a 9-item questionnaire about the practical barriers and facilitators that were relevant to choosing whether to enroll in the DPP LCP such as perceptions of the need for counseling to change one's lifestyle  ; accessiWe will use Qualtrics survey software for all questionnaires used in this study . This plEach participant will be sent US $20 electronic gift cards upon completion of the baseline survey and follow-up survey . To motivate completion of all study procedures, participants who complete both the baseline and follow-up questionnaires will be placed into a lottery for 1 of 5 US $100 electronic gift cards.The primary outcomes of the study are DPP LCP enrollment and 4-week engagement, as defined by the CDC . These dThe DPP LCP at University of Utah Health is covered by some insurance plans and requires an out-of-pocket fee for those without coverage. The cost for the online program is currently US $504, and the cost for the in-person program is US $425. Some scholarships are available for those who meet income guidelines. These costs may change during the course of the study. The Midvale CBC’s program is offered free of charge and is currently funded by a grant. To minimize the problem of cost as a barrier to enrollment, University of Utah Health participants who are uninsured or underinsured may be invited to participate in the Midvale CBC program. Otherwise, all other participants will only be invited to attend the program offered by their health system.If the mobile 360° videos and MAPS are successful in increasing enrollment and early engagement with the DPP LCP as they have been in our pilot work , they have tremendous potential to be scaled up to help prevent T2DM nationwide. More than 80% of adults in the United States own a smartphone . TherefoStatistical power for the primary analysis in Aim 1 was calculated using the statistical power calculation software PASS 14 . Based oF tests for continuous variables. If participants’ demographics and baseline measures of risk perception are not balanced across the 3 treatment groups at baseline, we will use inverse probability of treatment weighting with propensity scores to create a synthetic sample and compare the difference in the causal average treatment effects across the 3 treatment groups [We will use logistic regression to compare the effects of the 3 treatments on the likelihood of enrollment and 1-month engagement in the DPP LCP. The distributions of participants’ demographics and baseline measures of risk perception and self-efficacy will be summarized and compared by treatment groups using chi-squared tests for categorical variables and t groups . We willTo examine the mechanisms underlying the mobile 360° video by comparing changes in deliberative, experiential, and affective risk perceptions across study arms, we will calculate the changes in risk perception scores across all 3 treatment groups using the scoring procedures described by Ferrer et al . We will, we will calculate the changes in self-efficacy across all 3 treatment groups using the scoring procedures described by Wilson et al [To examine the mechanism of the MAPS intervention by comparing changes in DPP LCP–related self-efficacy across study armson et al . We willon et al . Our hypIn addition to the efficacy data collected in the trial, the study team will collect a series of process measures . We willThe project was funded in September 2020. The institutional review board approved the study on January 13, 2021. Enrollment began on March 10, 2021 and is expected to continue through July 31, 2022. As of April 27, 2021, 39 individuals had begun the study.In this project, we will conduct a 3-arm randomized controlled trial intended to compare the efficacy of a best-practice approach and 2 novel interventions on enrollment and engagement in the DPP LCP. These interventions will be delivered directly to individuals with prediabetes through their mobile phones. To standardize the offer of referral to the DPP LCP, all participants will receive an offer to be referred to the National DPP LCP 10 days after completing the baseline survey. Our primary outcome will be participants' enrollment and 1-month engagement in the National DPP LCP.This study is a randomized trial targeting patients with prediabetes within 2 health systems, including a community clinic serving primarily low-income, Spanish-speaking patients and a large academic medical center. The interventions will be delivered by mobile phones, making them scalable to large, diverse populations if found to be effective. The interventions will be delivered in English and Spanish, expanding the generalizability of study findings. The use of a theoretical framework for the study and validated questionnaires to elucidate the mechanism of action of the 2 novel interventions is a strength, as is the collection of process measures.This study is not powered nor designed to compare the effectiveness of the mobile 360° video vs MAPS nor does the design allow for testing of the additive value of mobile 360° video plus MAPS. Because each of these interventions is quite novel, we felt that first they should have demonstrated incremental efficacy over the best-practice condition. If these interventions are found to be incrementally effective, future trials will be designed to compare the effectiveness of these interventions head-to-head as well as to evaluate a combination of the interventions . In addition, the differences in number of contacts between the best-practice condition, the video condition, and the MAPS condition may present a confound, making it unclear if outcomes differ due to number of contacts or intervention content. Finally, in this trial, we are only able to assess 1-month DPP LCP engagement. Future work should explore whether these interventions impact longer-term (12 months) engagement and health outcomes.Potential challenges we may encounter include difficulties with recruitment and retention. If we have difficulties with recruitment, we will expand our recruitment population to individuals with A1C levels between 5.7% and 6.4% rather than just those with a documented prediabetes ICD-9 code. If we have challenges with recruitment, we will revisit the frequency and timing of contact with participants. If many returned surveys have significant amounts of missing data, we will follow up with participants and ask them to respond to missing items. Additionally, if we are unable to meet our recruitment goal at the smaller Midvale CBC, we will enroll uninsured or underinsured Spanish-speaking patients from the University of Utah to supplement the Midvale sample.1c and weight. In this future trial, we will also compare the cost effectiveness of these interventions.All of the data will be used to inform the design of a large, SMART in which individuals will first receive risk notification and education; then, nonresponders will be randomized to mobile 360° video or nothing, and subsequent nonresponders will be randomized to MAPS phone counseling. Primary outcomes will be enrollment and 1-year engagement in the DPP LCP. Secondary outcomes will include changes in an objective measure of risk for T2DM including hemoglobin A


We thank the author of the letter for the interest in our article.We agree with the author that the comparison of vaccine efficacy must be based on good clinical data. As vaccine efficacy could only be established in a controlled environment such as a clinical trial, we could not do so in our study. We also acknowledged in the paper the lack of detailed information regarding vaccine recipients who remained healthy and/or those who did not succumb to the disease, thereby limiting our ability to infer vaccine effectiveness in the Malaysian populations. However, inferences on vaccine effectiveness in Malaysia were reported by Suah et al. using datasets with relatively more data points concurred with the findings in our study.,The COVID-19 vaccination programme in Malaysia was implemented in phase 1 (frontline workers), phase 2 , and phase 3 (other adults aged 18 and above). BNT162b2 was the first vaccine to be initiated in Malaysia and was the predominant vaccine used for phase 1 and the early half of phase 2 between February 2021 and May 2021.In our dataset, the median time from being fully vaccinated (2 weeks after the second dose) to death were 10 weeks for the recipients of BNT162b2 and 11 weeks for the recipients of the inactivated vaccine. While we did not analyse immunological data, the higher mortality rates among recipients of inactivated vaccines also coincided with the World Health Organization's recommendation to prioritise an additional dose of COVID-19 vaccine for the recipients of inactivated vaccines aged above 60 at the time of data analysis and reporting. Despite the reduction in vaccine effectiveness over time and their protection against emerging variants of SARS-CoV-2, encouraging reports on the protective effects of a 3-doses homologous/heterologous booster regimen and among convalescents who were fully vaccinated further support the booster practice adopted by many countries worldwide.We observed higher mortality rates among those fully vaccinated with inactivated vaccines than those who were partially vaccinated after accounting for age between 25 August and 14 September 2021. While the observations were atypical, the difference in the age-standardised mortality rates (ASMR) between the 2 groups was not statistically significant. As we further stratified the analysis by the presence of comorbidities, higher ASMR was observed among those with comorbidities in the partially and fully vaccinated groups, respectively.,14Finally, the author concluded that in actual community use, the total COVID-19 deaths per million was lower in Lao People's Democratic Republic (hereinafter Laos), which mainly administered inactivated vaccines compared to Singapore that rolled out predominantly mRNA vaccines. Nonetheless, cross-country comparisons of vital COVID-19 indicators are complex due to the multifarious factors that must be considered. Using case fatality rate (CFR) as a case in point, COVID-19’s CFR in Laos when 49% of the total population was fully vaccinated, was approximately 0.4%. When 49% of the total population in Singapore was fully vaccinated, COVID-19 CFR in Singapore was 0.06%.None.


To evaluate the impact of positive end-expiratory pressure (PEEP) on intracranial pressure (ICP) in animals with different respiratory mechanics, baseline ICP and volume status.2O from 5 to 25 cm H2O. Measurements were taken in pigs with normal ICP and normovolemia (Series I), or with intracranial hypertension  and normovolemia (Series II), or with intracranial hypertension and hypovolemia (via exsanguination) (Series III). Pigs randomized to the control group received only hydrochloride instillation while the intervention group received additional chest wall strapping. Common carotid arterial blood flow before and after exsanguination at each PEEP level was measured in pigs with intracranial hypertension and chest wall strapping (Series IV).A total of 50 male adult Bama miniature pigs were involved in four different protocols . Under the monitoring of ICP, brain tissue oxygen tension and hemodynamical parameters, PEEP was applied in increments of 5 cm HICP was elevated by increased PEEP in both normal ICP and intracranial hypertension conditions in animals with normal blood volume, while resulted in decreased ICP with PEEP increments in animals with hypovolemia. Increasing PEEP resulted in a decrease in brain tissue oxygen tension in both normovolemic and hypovolemic conditions. The impacts of PEEP on hemodynamical parameters, ICP and brain tissue oxygen tension became more evident with increased chest wall elastance. Compare to normovolemic condition, common carotid arterial blood flow was further lowered when PEEP was raised in the condition of hypovolemia.The impacts of PEEP on ICP and cerebral oxygenation are determined by both volume status and respiratory mechanics. Potential conditions that may increase chest wall elastance should also be ruled out to avoid the deleterious effects of PEEP.The online version contains supplementary material available at 10.1186/s12868-021-00674-9. In other words, an increased ERS may attribute to either the increase in EL due to pulmonary diseases such as acute respiratory distress syndrome (ARDS), or the rise in ECW due to chest wall impairment such as intra-abdominal hypertension, or both [L and ECW were not differentiated in previous studies [A few studies investigated the potential role of ERS , 11, 14.RS –17. CereRS . Here, tpressure . It is w or both . EL and  studies , 11, 14.RS, which attribute to high EL, ECW, or both [CW. However, no inter-group  difference was observed in pigs with elevated baseline ICP. In addition, because of the severe hypotension induced by high PEEP, ICP decreased (rather than increased) in the intracranial hypertension conditions. Due to the nature of a preliminary study, several important factors were not controlled in that study . In a series of randomized-controlled experimental studies, we re-evaluate the impact of PEEP on ICP in animals with different respiratory mechanics, baseline ICP and volume status.Our group explored the impact of PEEP on ICP under different conditions of elevated E or both . We founComprehensive methods are available  monitoring. An esophageal catheter was inserted to measure esophageal pressure . ARDS  was induced via instillation of 0.1 mol/L hydrochloride down the endotracheal tube. ECW was raised by chest wall strapping [−1; FiO2 1.0; PEEP 5 cmH2O; the rate was initially set to 20 min−1 and adjusted to maintain a PaCO2 between 35-45 mmHg) and randomized to chest wall strapping or control group (10 per group). PEEP was gradually increased from 5 to 25 cmH2O in a 5 cmH2O interval. Hemodynamical parameters, respiratory mechanics, ICP, and PtiO2 were measured in each PEEP setting.Central venous and femoral arterial lines were placed for hemodynamical monitoring and fluid administration. Animals received a tracheotomy and continuous sedation and paralysis. One burr hole was created for ICP and brain tissue oxygen tension , and the same data were collected as in Series I.A second burr hole was created, and a Foley catheter was inserted in this series. ICP was increased by inflating the balloon with saline at a rate of 0.5 mL/min. The target was a stable ICP level between 25 and 30 cmHSeries II, except that animals were exsanguinated before randomization. The target of exsanguination was a decrease in cardiac output (CO) by ≥ 20%. Animals were then also randomized (6 per group), and the same data were collected as in Series I and II.The preparation was as in Series I. ARDS was induced, and the chest wall was strapped in all animals . ICP monitor and Foley catheters were inserted to produce intracranial hypertension as in Series II. Ultrasonography was used to measure common carotid arterial blood flow: an averaged velocity (V) was measured over an entire respiratory cycle. Vessel diameter (D) was measured at the locations where the flow velocity was measured in M-mode. The blood flow passing the measurement location was calculated as 0.25*V*π*D2. PEEP was stepwise increased and measurements were taken. Animals were then exsanguinated as in Series III. PEEP was increased again, and the blood flow measurements were repeated.In this before-after comparison, hemodynamical monitoring and ventilation settings were as in t-test was used as appropriate. Significance was established at p < 0.05. Analyses were performed with SPSS Statistics software and GraphPad Prism.All continuous data were tested for normality of distribution (Shapiro-Wilk) and equal variance, as appropriate. Groups were compared using Mann-Whitney (Rank Sum) tests, and changes over time were examined using Friedman RM-ANOVA on ranks where group distribution was not normal. Where distribution was normal and variance equal, two-way ANOVA or Student’s CW was significantly increased by chest wall strapping, while no difference in EL was observed between the control and chest wall strapping groups , and the magnitude was significantly higher in the chest wall strapping group , but the magnitude was similar between groups , while no difference was observed between groups , but no difference was observed between the two groups , while no inter-group difference was observed . A significantly greater decrease was observed in the chest wall strapping group , and the magnitude was greater in the chest wall strapping group . There was a significantly greater drop in CO in the chest wall strapping group ; (2) Increasing PEEP resulted in decreased ICP if the animals were volume-depleted (Series III); (3) Increasing PEEP resulted in decrease in PtiO2 in either normovolemic or hypovolemic conditions (Series I–III); (4) The impact of PEEP on hemodynamical parameters, ICP and PtiO2 became more evident when ECW was increased (Series I–III); (5) Compare to normovolemic condition, common carotid arterial blood flow was further lowered when PEEP was raised in the condition of hypovolemia, which was probably the cause of lowered ICP and PtiO2 (Series IV).The main findings of our study were: (1) ICP was elevated by increased PEEP in both normal ICP and intracranial hypertension conditions in animals with normal blood volume (L and decreased ECW [L) is less likely to be transmitted into the pleural space, and thus has less impact on central venous pressure and cerebral venous return. In other words, although a high PEEP is usually applied in the treatment of ARDS, the pulmonary disease per se might be a “protective” factor against the deleterious effects of PEEP to the brain. However, EL is not routinely measured in clinical settings; instead, ERS was often considered synonymous of EL. One should keep in mind that ERS and EL are not interchangeable because ECW is also a key determinant of ERS. Our data from Series I to III clearly demonstrated that an increased ECW resulted in a more considerable influence of PEEP on ICP. Our finding suggests that the possible underlining conditions which can increase ECW  must be ruled out before a high PEEP is applied in brain-injured patients.As shown by Chapin et al., the transmission of airway pressure to pleural pressure depends on the relative compliance of the lung and the chest wall. The transmission is less “efficient” with increased Eased ECW . This meSeries I and II), raising PEEP resulted in decreased ICP if the animals were volume-depleted (Series III). This can be explained by the influence of PEEP on CO and thus cerebral perfusion. As proposed in a conceptual framework that the two important determinants of the impact of elevating PEEP on ICP are the intracranial compliance and the “net” change of cerebral blood volume (CBV); the latter is determined by the balance between the arterial inflow  and venous outflow . Increased PEEP and subsequent increased pleural pressure impede the global venous return to the heart, lead to a decrease in CO and cerebral perfusion [Series III. In this case, the influence of decreased CO  exceeded the influence of decreased venous return (which reduces venous outflow and increases CBV), which resulted in a negative “net” change of CBV and eventually a decreased ICP.In contrast to the normovolemic conditions . Therefore, the most possible cause of lowered ICP and PtiO2 was decreased cerebral perfusion. More data was obtained to support the concept in Series IV. We measured the blood flow in the common carotid artery and a significantly greater decrease of blood flow was observed after exsanguination when the same PEEP was applied. Take all data together, we can conclude that PEEP can affect ICP predominantly via its hemodynamical effects, which always functions on both the arterial (perfusion) and venous (returning) sides simultaneously. And here, increased ECW amplifies the effects of PEEP.Mean arterial pressure (MAP) and CPP were also lower when high PEEP levels were applied. In animals with intact cerebral autoregulation, vessels in the brain can maintain a constant cerebral blood flow via regulating the vascular tone throughout a wide range of MAP or CPP , 26. In tiO2 was also measured in the present study, and we found that increasing PEEP resulted in decreased PtiO2 in either normovolemic or hypovolemic conditions. The impact of PEEP on cerebral oxygenation was not well understood yet. Results from previous studies are variable [2O) improved PtiO2 [tiO2 was observed. The conflicts of our data to previous studies can be explained as follows: assuming a relatively constant O2 consumption, the change of PtiO2 should be the consequence of changed O2 delivery. Thus, the two key determinants of O2 delivery to the brain were CO and arterial O2 saturation. Unlike Nemer’s study where increasing PEEP resulted in a considerable improvement in P/F ratio (from 108.5 to 203.6 mmHg), the improvement in P/F ratio was less obvious  but with depleted blood volume (like in Series III). In fact, we did perform some pilot experiments to exam this. Briefly, a crossover rather than a randomized-control experiment was completed in four pilot animals with normal ICP and depleted blood volume . ICP decreased with the increment of PEEP in all animals, but the magnitude was small  to exam the impact of hypovolemia and speculated that the conclusion might also be true in animals with normal ICP, although without additional experiments.It is also interesting to know the influence of increased PEEP on ICP and other parameters in animals with normal ICP (like in Series I vs. Series II). In other words, the four components of ICP were not investigated in isolation and the quantitative way in the present study and thus may limit our interpretation of the effects of a specific factor. Moreover, several other factors that may have an impact on these four components, such as pulse amplitude, respiratory wave amplitude and pressure reactive index  were not considered in the present study. (5) The present study investigates only two different volume statuses (normovolemia vs. massive blood loss) while intermediate states, for example, mild hypovolemia due to diuretics or hyperosmolar agents, or small amount of blood loss, are more likely to occur in the clinical setting. Further studies are necessary to confirm the effects of PEEP in these situations.Our study has some limitations: (1) The data were obtained with experiments of short duration; therefore, the long term effects of PEEP were still unclear; (2) Blood flow was measured at the common carotid artery rather than the internal carotid artery (which perfuse the brain). In the present study, we used a Vevo 3100 ultrasonic system with an ultra-high-frequency probe designed for small animal research. The ultra-high-frequency probe provided high resolution while the maximal scan depth was limited. The internal carotid artery was too deep to display by using the current device, so we chose common carotid artery instead; (3) Although a decreased cerebral venous return was proposed as the main reason for increased ICP, the outflow was not measured in this study. We measured arterial blood flow via Doppler ultrasound; however, using the same methodology to assess cerebral venous return was suggested to be inaccurate ; (4) IndCW is high. In brain-injured patients, PEEP should be applied under the monitoring of MAP, ICP, and CPP; meanwhile, potential conditions that may increase ECW should be ruled out to avoid the deleterious effects of PEEP.Both volume status and respiratory mechanics determine the impacts of PEEP on ICP and cerebral oxygenation. PEEP increases ICP in stable hemodynamic conditions while decreases ICP in hypovolemia. The effects are more profound when EAdditional file 1. Comprehensive experimental methods.Additional file 2. Details of respiratory mechanics, hemodynamical parameters and blood gas analysis.