The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins  or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway’s activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development. The Wnt/β-catenin signaling pathway is a highly conserved pathway, which appeared early in phylogenesis and is common to all metazoan life forms. This pathway plays crucial roles during the entire lifespan of all organisms, from early embryogenesis to homeostasis in the adult. Wnt ligands bind to the transmembranal Frizzled receptor family and along with members of the LRP transmembranal co-receptor family, they mediate a large array of cell responses including cell fate speciﬁcation, polarization, migration, and mitogenic stimulation . The firc-fos promoter driving the expression of the luciferase gene (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/). More sensitive TOPflash reporters were generated in Drosophila and zebrafish by increasing the number of TCF/LEF sites to 8, 12, and 16  to generate transgenic or knock-in mouse lines . While tImportantly, recent evidence suggests that mobilization of β-catenin from the cell membrane pool can also trigger the activation of TCF/LEF reporters in a WNT ligand-independent manner . This inDrosophila) to considerably increase reporter protein yields , we increased transcriptional activity further, using 16 TCF/LEF repeats. This was combined with translational enhancers to drive the expression of a nuclear, destabilized, fast maturing Venus  as reporThis resulted in two transgenic lines in which the characterization of the TCF/β-catenin transcriptional activity during embryogenesis is readily observed, including using in vivo imaging. Particularly remarkable is the 16xTF-VNP line, where intense, yet dynamic, reporter activity unveils unexpected features of TCF/β-catenin-responding cells and tissues.This study underlines the importance of developing novel strategies to generate reporters efficient for monitoring of the spatiotemporal dynamics of signaling pathways and it opens new routes of investigation for correlating TCF/β-catenin transcriptional activities with unique cell behaviors during amniote embryonic development.To generate the 12xTF-d2GFP line (TgT2[12TCF/LEF:d2EGFP]), we modified a TCF/β-catenin transcriptional reporter we previously generated, which was intended for in vivo electroporation in chicken embryos  by inserDrosophila myosin heavy chain, which facilitates mRNA export to the cytoplasm . For this, we synthesized and cloned 16 TCF/LEF repeats upstream of the TK minimal promoter, followed by IVS and Syn21 sequences , directlytoplasm . Syn21 iulovirus . A Crystulovirus . FinallyTo validate the efficiency of this construct, we electroporated one-half of the neural tube of HH15 (E2.5) chicken embryos with a DNA mix containing the 16xTF-VNP construct and a TagBFP protein driven by the CAG ubiquitous promoter as a marker of electroporated cells. Twenty-four hours after electroporation, weak expression of 16xTF-VNP was observed in the dorsal neural tube (NT) of the electroporated half, while a stronger expression of the reporter was seen in the migrating neural crest cell population NC; . The co-To test the added value of translation enhancers in a vertebrate environment, we compared the fluorescence levels obtained with the 16xTF-VNP, the 12xTFd2GFP, and a 16xTF-VNP construct lacking the translation enhancers (referred to here as 16xTF-VNP plain). We co-electroporated the right side of the neural tube of E2.5 chicken embryos with the 12xTFd2GFP, 16xTF-VNP plain, or the 16xTF-VNP, together with CAG-TagBFP as internal control . Twenty-We used the same technique as above to generate transgenic quails with this reporter. One female was selected as the transgenic founder using the CrystallGFP marker and crossed with a WT male to expand the transgenic line. The transmission of the transgene to the offspring presented a Mendelian distribution, suggesting a single insertion.To characterize TCF/β-catenin transcriptional activity during embryonic development, we analyzed and compared the activity of the two reporters in the two lines we generated, in whole mount preparations of immunostained embryos, on immunostained sections and in live tissues at different developmental stages. Whole mount preparations of HH21  and HH20  transgenic quail embryos immunostained for GFP or mVenus, Pax7 and Myosin Heavy Chain (MyHC), clarified by the ‘3DISCO’ technique  and imagen route to their sites of differentiation . We observed that the activity of the reporter was not detected in the dorsal neural tube in the 12xTF-d2GFP  and weakXenopus and chicken have led to the premise that these molecules play important functions in the proliferation and/or differentiation of neuronal precursors within the CNS, opposing a Shh gradient from the notochord and floor plate  of the neural tube in mouse and chicken early embryos while their transcripts are absent from migrating NC . Wnt1 anor plate . Howeveror plate . In neuror plate . Wnt sigor plate . The avaor plate .A similar gradient of reporter activity was observed in somites, since it was active in the DML and the transition zone and rapidly decreased in the myotome . As prevThus, the use of destabilized fluorescent reporters to generate these quail lines allows the detection of dynamic changes (increase and decrease) in reporter activity throughout embryonic development that could not be appreciated with stable reporters, such as the BAT-gal mouse line .To characterize TCF/β-catenin activity during early embryonic development, we performed immunostaining on sections and on whole mount embryos, coupled with classical confocal microscopy. We observed that the reporters were first detected at the posterior end of a gastrulating embryo  in the 16xTF-VNP embryos  and two To gain a cellular resolution of the 16xTF-VNP reporter activity, we prepared transverse sections of HH14 (E2.5) embryos at the level of cervical somites, somite I (the newly formed somite) and the PSM, and stained for mVenus, Acetylated Tubulin and DAPI . These sWe also analyzed the activity of the reporters at 9 days of development HH35; . TissuesTCF/β-catenin spatiotemporal activity is highly dynamic, both on a cellular level and throughout development, a feature that has been difficult to study due to lack of reliable destabilized reporters. By using the 12xTF-d2GFP and the 16xTF-VNP transgenic lines, we followed the dynamics of TCF/β-catenin transcriptional activity in vivo. Observation of early somites showed a strong activation of the 12xTF-d2GFP reporter activity in single cells located in the DML . The timA possible limitation of the TCF/β-catenin reporters we designed resides in the half-life of the chromophores we used (i.e. d2EGFP and VNP), which has been estimated to be about 2 hr . This suAn interesting finding of this study was the intense TCF/β-catenin-response in the fore- and hindlimb AER. The AER remained strongly labeled throughout limb growth , embryos were washed in PBS, incubated in 80% glycerol and directly (i.e. less than 24 hr after fixation) examined. To image native fluorescence expression, the detection spectrum for each fluorophore was set, using a Leica sp5 confocal microscope. The imaging parameters were set according to the sample with the strongest fluorescence to avoid over expression, and sample bleaching. These acquisition parameters were then left the same for the rest of the samples in the same experiment. For section preparations, embryos were embedded in 15% sucrose/7.5% gelatine/PBS solution and sectioned with Leica cryostat at 20 µm. Antibody staining was performed as described in Mann–Whitney two-tailed non-parametric tests were applied on the entire population of counted cells to evaluate significance of each treatment. **p-value 0.001–0.01; * p-value ≤ 0.05.Performed as described in http://quailnet.geneticsandbioinformatics.eu/. Embryos were imaged using Leica SP8 confocal microscope on upright DM6000 stand with HCX APO L 20x/1.00 water dipping (WD 2.00 mm). Z-stack images were taken every 15 min for 9 hr  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 Marianne Bronner as the Senior Editor and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Aixa Victoria Morales (Reviewer #1).Thank you for submitting your article "Transgenic quails reveal dynamic TCF/β-catenin signaling during avian embryonic development" 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 found agree that your paper presents a powerful and improved tool to follow Wnt signalling activation in vivo. However, the consensus is that the paper requires extensive revision to demonstrate that the tool is reliable and robust tool. In particular, more transgenic lines are required to demonstrate that this offers a dynamic view of Wnt activation, close to endogenous dynamics. Essential changes are summarized below and elaborated upon in the individual reviews.1. The authors need to better demonstrate the positive effects of the regulatory elements used in the transgenes. Multiple independent transgenic lines are needed in order to draw firm conclusions. In addition, a demonstration of transgene response to small molecules is needed.2. Additional experiments should be added to quantitate the expression levels in vivo. The lifetimes need to be better characterized by analysis of kinetic effects of induction and inhibition of b-catenin signalling .3. As the quail lines may not be generally accessible to many researchers, the authors should provide more data on the expression profiles at different stages of development and better characterize the constructs for use in electroporation.Reviewer #1 (Recommendations for the authors):In spite of the robustness and usefulness of the new Wnt/β-catenin reporter lines, there are some concerns about the results presented:1. The proof of the fine temporal resolution of the dynamics of Wnt signalling using the best of the two constructs (16xTCF-VNP) does not seem strong enough. It is mostly based in a 9 hours recording (Sup. Video 3) of somitic cells. It is unclear how long does it take to the two somitic cells indicated by magenta arrow to change their EGFP levels and also it is unclear if they are more intense at the end of the video because they were out of focus during the first frames of the video. It would be more convenient to explore the property of temporal resolution of 16xTF-VNP in a highly dynamic context such as the segmentation clock in presomitic mesoderm at E2 (similar to Video 4). In that tissue, cyclic expression with a periodicity of around 90 min (chicken) or 120-140 min (mouse) has been shown for several components of the Notch and Wnt signalling pathway . In fact, this recent important work has not been acknowledged by the authors.2. In relation to Supplementary Video 4 (embryos stage E2), the authors speculate that "the reporter-positive cells were found scattered in a wide region of early limb ectoderm, intermingled with reporter negative cells. As the limbs grew, reporter-positive cells migrated towards and coalesced to form the AER, suggesting that they constitute a population of AER progenitors. ". In fact in sections of embryos stage E3, they show how 16xTCF-VNP drives EGFP expression in limb bud mesoderm (besides the expression of AER). In the video at E2 it is unclear if the migrating cells in the hindlimb bud are mesodermal or ectodermal cells. Authors should clarify these aspects  or at least, tone down their interpretations.Reviewer #2 (Recommendations for the authors):Some data are presented to suggest that the 16x multimerised line is more sensitive than the 12x multimerised line based on the expression level in neural crest cells in transient expression experiments and in somitic tissue in the transgenic lines. These data are suggestive but not very quantitative. Can this be improved, for instance could embryos/tissues be dissociated and fluorescence intensity be measured in flow cytometry to quantify these effects, preferably at more than one time point?It is also suggested that the 16x reporter is more dynamic since it drives expression of a destabilised GFP variant, however there are no direct measurements of lifetimes to support this. It would be good to try to measure this and demonstrate that this is indeed the case, for instance by treating embryos with a small molecule Wnt/β catenin inhibitor and try to measure the dynamics in the response in both strains. It would also be very useful to have an estimate of the time it takes for the reporter to come on, for instance by locally applying a signal and measuring the kinetics of the response. It is somewhat worrying that in the transfection experiments this appears to take a very long time (up to 24 hours?).It would be really useful to have a stable as well as destabilised reporter and it would be useful to know what these half times are in-vivo and whether they vary development.Drosophila, but it is not clear from the data provided that these help or are necessary in the chick. If there is any information on this it would be helpful to provide this, to guide sensible decisions on future construct complexity.The 16x constructs is quite complex, it contains domains known to enhance translation in An initial characterisation of the reporters showed that at stages E3 and E9 of development activity is seen in places where it is expected. It would however be good to see what the activity looks like at some earlier stages n development (gastrulation/neurulation) when Wnt signalling is known to play a critical role in patterning and differentiation. Since the embryo geometry is simpler at these stages, it will also be easier to document its activity, both in fixed and live preparations. Furthermore since in early development changes in signalling occur fast this will is will also provide better insight in the sensitivity and kinetics of the reporters.Reviewer #3 (Recommendations for the authors):For Figure 1, 2 and 3, the figure legends state that anti-GFP antibodies were used on the embryos and sections before imaging. Does this mean that the endogenous fluorescence was not of sufficient intensity to survive fixation and cryopreservation? What do the fluorescent embryos appear under a stereomicroscope? Can the embryos be live imaged during embryogenesis? The Supplementary Video 3 of a somite is of interest. Could more tissues be shown?Other recommendations:1. Continuation of the regulatory element analysis using electorporation in the neural tube.2. If possible, create independent transgenic lines. Essential revisions:The reviewers found agree that your paper presents a powerful and improved tool to follow Wnt signalling activation in vivo. However, the consensus is that the paper requires extensive revision to demonstrate that the tool is reliable and robust tool. In particular, more transgenic lines are required to demonstrate that this offers a dynamic view of Wnt activation, close to endogenous dynamics. Essential changes are summarized below and elaborated upon in the individual reviews.1. The authors need to better demonstrate the positive effects of the regulatory elements used in the transgenes. Multiple independent transgenic lines are needed in order to draw firm conclusions. In addition, a demonstration of transgene response to small molecules is needed.2. Additional experiments should be added to quantitate the expression levels in vivo. The lifetimes need to be better characterized by analysis of kinetic effects of induction and inhibition of b-catenin signalling .3. As the quail lines may not be generally accessible to many researchers, the authors should provide more data on the expression profiles at different stages of development and better characterize the constructs for use in electroporation.Many questions were raised by reviewers on the reliability of the reporters we have used (12XTFd2EGFP and 16xTF-VNP). As explained in the manuscript, the TCF/LEF binding sites (TCF BS) are well-established tools which have been used for decades in literally hundreds of publications to monitor canonical Wnt signaling, starting with the seminal work of Korinek et al., in 1997. Such constructs were shown to efficiently respond to various members of the pathway, in vivo and in vitrohttp://web.stanford.edu/group/nusselab/cgi-bin/wnt/inhibitors; http://web.stanford.edu/group/nusselab/cgi-bin/wnt/activators_detectors), as well as to small activating and inhibiting pharmacological compounds . Altogether, there is a wealth of data showing that TCF BS reliably respond to Wnt signaling., we have built and extensively characterized the 12XTFd2GFP Wnt reporter used here and compared it to existing constructs containing 3 or 8 TCF-BS.We then showed that an increase in the number of TCF-BS led to a significant enhancement of sensitivity to Wnt signaling that was not detrimental to the reporter accuracy, since the 12XTFd2GFP responded positively to an activated form of b-catenin  and was strongly repressed by the Wnt-inhibiting molecule Dkk1 . As the point of this paper was to combine the Wnt reporter to a destabilized fluorophore, we showed that the expression of the destabilized GFP reporter protein was similar to its mRNA, suggesting that 12XTFd2GFP provides a precise view of cells actively engaged in Wnt signaling in vivo. Those important elements have been added to the text  for more clarity.Drosophila , we were concerned that the basal level of 16xTF-VNP activity may be too high to be useful in our system. In the previous version of the manuscript, we showed that the fluorescent signal of 16xTFVNP was strongly repressed by a dominant form of LEF1 (DN LEF). As using a dominant negative form of LEF1 to repress a TCF/LEF binding site may not be the best of all experiments, we now performed an additional experiment showing that 16xTF-VNP fluorescence is also robustly repressed by the Wnt signaling repressor Axin2 . We believe these explanations and additional data should satisfy the reviewers’ concerns on reliability.The 16xTF-VNP construct builds on the same logic of multimerizing the TCF-BS (up to 16) and therefore it should bear the same reliability to Wnt signaling. An important additional point is the use of translation enhancers, which we show are effective in vertebrates (to a point that we now routinely use them in many constructs we generate in the lab). The point of the destabilized fluorophore is discussed below (response 1 to reviewer 1). Even though a multimer of 16 TCF-BS had already been successfully used in To further characterize the effect of the use of regulatory elements to enhance the activity of the reporter, we have performed additional electroporation experiments that allow quantification of the signals obtained in vivo with (i) the 12XTFd2GFP; (ii) a 16xTF-VNP construct without the translation enhancers (this construct was not used in the previous version of the manuscript) and (iii) a 16xTF-VNP construct with the translation enhancers. These experiments are shown in Sup Figure 2. In addition, additional pictures of side-by-side embryos at the same developmental stage  show that the fluorescent signal is very similar in the two lines, but significantly stronger in the 16xTF-VNP than in the 12XTFd2GFP. This observation also supports the premise that there is no significant positional effect of the transgene insertion into the genome.To address the request for more developmental stages, we now provide 3 novel main figures and 2 Sup Figures that illustrate the expression of the reporters in embryos at E2+ in whole mount  and on sections , at E4 in whole mount , during gastrulation  and at E2- .Reviewer #1 (Recommendations for the authors):In spite of the robustness and usefulness of the new Wnt/β-catenin reporter lines, there are some concerns about the results presented:1. The proof of the fine temporal resolution of the dynamics of Wnt signalling using the best of the two constructs (16xTCF-VNP) does not seem strong enough. It is mostly based in a 9 hours recording (Sup. Video 3) of somitic cells. It is unclear how long does it take to the two somitic cells indicated by magenta arrow to change their EGFP levels and also it is unclear if they are more intense at the end of the video because they were out of focus during the first frames of the video. It would be more convenient to explore the property of temporal resolution of 16xTF-VNP in a highly dynamic context such as the segmentation clock in presomitic mesoderm at E2 (similar to Video 4).The tool we have designed is appropriate to explore dynamic events that have a velocity compatible with the half-life of the Venus-PEST, which is about 90-110 minutes (see below). The changes of fluorescence we observed in Sup video 3 are true changes in Wnt activity. We have now added a timeline to this video, which shows that the increase in activity (from lowest to highest) takes about 5 hours. To address a related comment from this reviewer (“(cells) were out of focus during the first frames of the video), as mentioned in the manuscript, this video was taken with a confocal microscope, which means that all cells are in focus along the entire video. Regarding the segmentation clock, it cycles in chicken PSM with a periodicity of 90’, which we believe is too short to be detected with this tool. Accordingly, in the new Figure 4 and Sup Figure 9, we do not see any sign of periodicity of the reporter fluorescence in the PSM. The paper of Sonnen et al., that is mentioned by reviewer 1 is a KI of Venus-Pest in the axin2 locus. While the Venus-Pest they used should have the same half-life than ours, the periodicity of axin2 expression in mouse was estimated in their work to be 144 minutes, i.e. more compatible with the half-life of VenusPEST.To draw a parallel with the design of Notch reporters that allowed the detection of cyclic Notch signaling in the PSM, Auhlela et al., Delaune et al., Masamizu et al., Sorolodoni et al., resorted to various tricks to further destabilize the fluorescent reporter they used, for instance by adding RNA destabilizing sequences in the 3' UTR. A similar strategy was used by Shimojo et al., to detect cyclic variations of Notch signaling in neurons. The transgenic animals that were used in these studies were designed specifically for that purpose and because the signals they generate is so weak, one can see in the literature that these lines are very seldom used.As mentioned in the manuscript, the signal of a reporter is linearly linked to its stability, and further shortening its half-life will significantly reduce the signal. We have chosen a compromise between high signal and short half-life that may not be suitable for all purposes. We have mentioned the limitation of the reporter lines we designed to detect dynamic changes in Wnt signaling with a short periodicity (2nd§ page 9).2. In relation to Supplementary Video 4 (embryos stage E2) , the authors speculate that "the reporter-positive cells were found scattered in a wide region of early limb ectoderm, intermingled with reporter negative cells. As the limbs grew, reporter-positive cells migrated towards and coalesced to form the AER, suggesting that they constitute a population of AER progenitors. ". In fact in sections of embryos stage E3, they show how 16xTCF-VNP drives EGFP expression in limb bud mesoderm (besides the expression of AER). In the video at E2 it is unclear if the migrating cells in the hindlimb bud are mesodermal or ectodermal cells. Authors should clarify these aspects  or at least, tone down their interpretations.We now provide sections at different AP levels of a stage HH14 16xTF-VNP embryo . One clearly sees that there are TF-positive cells in the ectoderm in the region of the future limb (see panels I-L). These are likely the same AER progenitors that are observed in Sup Video 4.Reviewer #2 (Recommendations for the authors):Some data are presented to suggest that the 16x multimerised line is more sensitive than the 12x multimerised line based on the expression level in neural crest cells in transient expression experiments and in somitic tissue in the transgenic lines. These data are suggestive but not very quantitative.We have further quantified the fluorescence that was observed with the 12x and the 16x constructs. To do this, we electroporated the neural tube with 3 constructs: (i) the 12XTFd2GFP; (ii) a 16xTF-VNP construct without the translation enhancers (this construct was not used in the previous version of the manuscript) and (iii) a 16xTF-VNP construct with the translation enhancers.The results are shown in Sup Figure 2. They show that the change from 12 TCF/LEF1 binding sites  to 16 BS , together with a fluorophore change , led to a strong increase in the observed fluorescent signal (by about 11 x). The addition of the translation enhancers  further increased the fluorescent signal by about 60%. Altogether, these data demonstrate the utility of the strategy we followed to improve the TOPflash reporter and it shows that translation enhancers which were developed in invertebrates are also active in vertebrates.It is also suggested that the 16x reporter is more dynamic since it drives expression of a destabilised GFP variant, however there are no direct measurements of lifetimes to support this.The half-life of the d2EGFP and the Venus-NLS-PEST are only slightly different. The d2EGFP was designed by Clontech years ago and its half-life is 2 hours. The half-life of Venus-NLS-PEST is reported to be 1.8 hours . Years ago, we tested d2EGFP and Venus-NLSPEST half-life (HL) and confirmed that d2EGFP HL is indeed 2 hours, while Venus-NLS-PEST HL was in our hands more towards 90' (unpublished). Venus is faster-folding than EGFP , which should slightly decrease the reporter response time. All in all, those differences are rather limited and they should not fundamentally change their response to Wnt signaling.It would also be very useful to have an estimate of the time it takes for the reporter to come on, for instance by locally applying a signal and measuring the kinetics of the response. It is somewhat worrying that in the transfection experiments this appears to take a very long time (up to 24 hours?)We have extensively characterized the 12XTFd2GFP construct years ago, showing that the EGFP fluorescence is observed in cells that activate the Wnt response . In the Rios paper, we also compared a stable and a destabilized TF reporter, demonstrating the importance of the latter to obtain a snapshot of the cells actively responding to Wnt. The 16xTF-VNP construct is a variant of the 12x and it reacts similarly. Even though there is likely a delay between the binding of Wnt to its receptor and an observable fluorescence, it is also the case for a transcriptional response to any signaling pathway. Reviewer 2 seems surprised about the 24 hours delay between electroporation and observation: this delay was just out of convenience , but not necessary.Drosophila, but it is not clear from the data provided that these help or are necessary in the chick.The 16x constructs is quite complex, it contains domains known to enhance translation in See above response 1 to the same reviewer 2. Note that we have added a note about an additional IVS cassette upstream of Syn21 in the text (last § page 4).An initial characterisation of the reporters showed that at stages E3 and E9 of development activity is seen in places where it is expected. It would however be good to see what the activity looks like at some earlier stages n development (gastrulation/neurulation) when Wnt signalling is known to play a critical role in patterning and differentiation.We now provide a picture  that shows the fluorescence in the posterior end of a gastrulating 16xTF-VNP embryo.Reviewer #3 (Recommendations for the authors):For Figure 1, 2 and 3, the figure legends state that anti-GFP antibodies were used on the embryos and sections before imaging. Does this mean that the endogenous fluorescence was not of sufficient intensity to survive fixation and cryopreservation? What do the fluorescent embryos appear under a stereomicroscope? Can the embryos be live imaged during embryogenesis? The Supplementary Video 3 of a somite is of interest. Could more tissues be shown?GFP and its variant Venus do not like fixation and their fluorescence tends to fade quite quickly. In contrast, RFP and variants are resistant to fixation. To observe native GFP/Venus after fixation, we image embryos very fast, typically the next day after a step of glycerol-mediated clarification. This point has been clarified in the Materials and methods section . When we need to apply additional antibodies, for instance in Figures 1, 2 and 3, those experiments take days, and we need to restore the GFP/Venus signal with GFP-specific antibodies. While GFP antibody staining can enhance faint signals, it also tends to level down subtle changes in fluorescence intensity. Evidently, those lines were created to be able to perform live imaging, such as those shown in videos 3 and 4 and it is likely that there will be more to come from our and other labs when the new TOPflash line will be up and running.


This cohort study investigates antibody waning after second and third doses of the BNT162b2 (BioNTech/Pfizer) COVID-19 vaccine. What is the durability of the immune response after 3 vaccine doses, and are antibody kinetics associated with SARS-CoV-2 Omicron infection?In this cohort study of 3972 health care workers, reduction in antibody levels 5 months after the third BNT162b2 vaccine dose was slower than after the second, while Omicron's neutralizing response was lower compared with other variants of concern. Peak antibody levels after the third dose were associated with Omicron infection.This study found that the humoral response after the third vaccine dose was sustained for 5 months and that antibody kinetics were associated with Omicron infection. The BNT162b2 two-dose vaccine (BioNTech/Pfizer) has high effectiveness that wanes within several months. The third dose is effective in mounting a significant immune response, but its durability is unknown.To compare antibody waning after second and third doses and estimate the association of antibody kinetics with susceptibility to infection with the Omicron variant of SARS-CoV-2.In a prospective longitudinal cohort study in a tertiary medical center in Israel, health care workers who received the BNT162b2 vaccine were followed up monthly for IgG and neutralizing antibody levels. Linear mixed models were used to compare antibody titer waning of second and third doses and to assess whether antibody dynamics were associated with Omicron transmission. Avidity, T cell activation, and microneutralization of sera against different variants of concern were assessed for a subgroup.Vaccination with a booster dose of the BNT162b2 vaccine.The primary outcome was the rate of antibody titer change over time, and the secondary outcome was SARS-CoV-2 Omicron variant infection, as confirmed by reverse transcriptase–polymerase chain reaction.6 peripheral blood mononuclear cells to 59 (9.3) T cells/106 peripheral blood mononuclear cells.Overall, 4868 health care workers  and 3972 health care workers  were followed up for 5 months after their second and third vaccine doses, respectively. Waning of IgG levels was slower after the third compared with the second dose , as was waning of neutralizing antibody levels . Among 2865 health care workers assessed for Omicron incidence during an additional 2 months of follow-up, lower IgG peak  was associated with Omicron infection, and among participants aged 65 years and older, faster waning of IgG and neutralizing antibodies  were associated with Omicron infection. No waning in IgG avidity was observed 112 days after the third dose. Live neutralization of Omicron was lower compared with previous strains, with a geometric mean titer at the peak of 111 , compared with 942  for WT, 410  for Delta; it demonstrated similar waning to 26  within 4 months. Among 77 participants tested for T cell activity, mean (SD) T cell activity decreased from 98 (5.4) T cells/10This study found that the third vaccine dose was associated with greater durability than the second dose; however, Omicron was associated with greater resistance to neutralization than wild type and Delta variants of concern. Humoral response dynamics were associated with susceptibility to Omicron infection. Furthermore, we investigated the immune response against various VOCs and compared it with the immune response kinetics after the second dose.STROBE) reporting guideline was followed in this study.This cohort study’s protocol was approved by the Institutional Review Board of the Sheba Medical Center, and written informed consent was obtained from all study participants. The Strengthening the Reporting of Observational Studies in Epidemiology . Any HCW who was infected by SARS-CoV-2 during the study was removed from the cohort to a parallel cohort of HCWs who were recovered from COVID-19. Participants were asked to undergo a serology test once every 4 weeks.Data on age and sex were available for all 3972 study participants. Overall, 2723 participants (68.6%) responded to an online questionnaire regarding comorbidities, including immunosuppression and body mass index   with those after the second dose. We included 3972 HCWs who received the third dose and had at least 1 serological test 7 to 140 days after the third dose of vaccine; the number of participants who had tests at different times during the study is presented in To further investigate if the rate of waning was associated with later breakthrough infections, we added 2 months of follow-up to assess the clinical outcome of participants during the Omicron surge, between December 15, 2021, and February 28, 2022. Only participants who were not infected by December 15, 2021, and did not receive a second booster  during the study period were included in this secondary analysis. We compared dynamics of antibody waning after the third dose in individuals infected with COVID-19 during the Omicron surge vs those who remained uninfected during the follow-up period. The study design is depicted in Samples from participants who were vaccinated were tested before receipt of the third dose using the SARS-CoV-2 RBD IgG assay (Beckman-Coulter or after receipt of the third dose using the SARS-CoV-2 IgG II Quant (Abbott) test performed according to the manufacturers’ instructions. All IgG Antibody levels were presented in binding antibody units (BAU) per World Health Organization standard measurements. Antibody detection testing is described in eMethods 3 in the 15 To test the overall neutralizing ability of each serum against the wild type (WT) virus and specifically to compare with neutralizing levels of Sheba HCWs after 2 and 3 vaccine doses, we used pseudovirus neutralization as previously described.20 Inclusion criteria for selecting the neutralizing antibody group are described in eMethods 2 in the 22 Further details can be found in eMethods 4 in the 14To measure the quality of IgG antibodies, we used urea as a chaotropic reagent and tested the strength of interaction between the IgG and the viral antigen (the RBD) as was recently described.To identify infection during the study period, participants were asked to undergo a SARS-CoV-2 test, either RT-PCR (Seegene) or a rapid antigen test in case of exposure to an individual with a detected SARS-CoV-2 infection or the development of potential COVID-19 symptoms (eMethods 1 in the R squared = 0.92)  for second and third vaccine doses. Abbott IgG levels after the second dose were imputed from Beckman-Coulter IgG levels using data on 215 selected serum samples not included in the HCW cohort using a cubic polynomial equation in log Beckman-Coulter level , followed by a fixed effect linear decline from 30 days onward. Rates of decrease in antibody levels were analyzed using a linear mixed model in which log antibody level was the dependent variable and each individual’s peak level was modeled as a random effect. Separate models were run for second and third vaccine doses. For neutralizing antibody levels, the decrease starting at 70 days after the second vaccine dose but not the third dose was found to be slower than the initial rate of decline and was modeled accordingly; rates of decline reported are initial rates, and all rates are expressed on the original scale as percent decreases per day. Age  and sex were included as fixed-effect–adjusting covariates, and their interactions with peak level and rate of decline were included. For comparisons between doses, estimated mean peak levels, rates of decrease, and the level at day 140 after the third vaccine dose were standardized to the distribution of age and sex of individuals in the second dose cohort. Standard errors of estimates were model based for neutralizing antibodies, but for IgG, a bootstrap procedure was used to account for the extra uncertainty from imputing Abbott IgG levels for the second dose cohort (eMethods 6 and 7 in the Graphs were created using GraphPad Prism software version 9.0 (GraphPad Software). Correlations between IgG and neutralizing antibody levels for each period were assessed by Spearman rank correlation. Paired pre- and post-third vaccine dose avidity, neutralization, and T cell activation were compared using the Wilcoxon signed-rank test. Statistical analysis was performed using SAS statistical software version 9.4 (SAS Institute). We report all parameters with 95% CIs, with the interpretation as per the model fit.In total, 8092 samples from 3972 HCWs who received 3 vaccine doses  years; 996 [74.9%] women) were collected from August 5, 2021, until December 29, 2021. Of these, clinical follow-up data were available for 2865 HCW, who were followed during the Omicron surge, between December 15, 2021, and February 28, 2022. . SamplesIgG waning was slower after the third dose of the vaccine  vs after the second dose  . NeutralA correlation and constant regression association in all study periods were observed between IgG and neutralizing antibodies (Spearman rank correlation between 0.59 and 0.74) after the third dose . A random sample of 150 sequenced samples from this period consisted of 100% Omicron variant. A total of 2865 of 3972 HCWs who were not infected before the beginning of follow-up and did not receive the fourth dose were followed up during this period B. OveralDemographics of individuals who were infected vs those not infected are described in eTable 8 in the 6 PBMCs and decreased to 59 (9.3) activated T cells/106 PBMCs within 3 to 5 months. During this time, the percentage of participants with no T cell response (no reaction on the Elispot) increased from 7 participants (9.1%) to 16 participants (20.8%). The dynamics of IgG and neutralizing antibodies tested in this group were similar to those of the full cohort  and 85 to 112 days after the third dose (at trough). Baseline characteristics of this population are described in eTable 3 in the Serum samples from 25 randomly selected individuals with 4 consecutive monthly samples were tested. Baseline characteristics of this population are described in eTable 5 in the In this cohort study, we found a significantly slower waning of humoral response after the BNT162b2 third dose vaccine compared with after the second dose; however, a high rate of infection (40%) was seen during 2 months of the Omicron surge among HCWs vaccinated with a booster dose 5 to 6 months earlier. In addition, lower peak IgG levels were associated with susceptibility to Omicron infection, as was a faster decay of IgG levels, particularly among individuals aged 65 years or older. Finally, we observed that the waning of neutralization against the Omicron variant was similar to that against other strains and was consistently lower than against Delta and WT during 4 months of follow-up.20 followed by significant antibody waning,23 which was associated with approximately 30-fold and 6-fold lower IgG and neutralizing antibody levels, respectively, 6 months after the second dose.9 The superior humoral response after the third compared with the second dose was associated with in an increase in not only the quantity (IgG levels), but also the quality (avidity) of IgG antibodies.15 The durability of neutralizing titers after the third vaccine dose may, therefore, be associated with the persistence of high-quality IgG antibodies observed in our study for at least 4 months. With time and possible additional vaccine doses, future studies should continue to examine the interplay between quantity and quality of IgG antibodies given that these are crucial factors associated with vaccine efficacy.Two doses of the BNT162b2 vaccine elicit a rapid induction of humoral response25 yet the threshold for protection is unknown because defining it would require measuring antibody levels just before infection. Alternatively, the rate of waning is more obtainable, and thus we examined whether this could be a factor associated with susceptibility to infection. Our results showed that at least in individuals aged 65 years or older, the rate of immune response waning was associated with Omicron transmission. Those aged 65 years or older who were eventually infected by Omicron had a steeper decrease in neutralizing and IgG antibody titers. Factors associated with variation in the rate of decrease among individuals of the same age group have yet to be studied. A future model estimating the risk of an individual being infected based on peak antibody levels and the rate of their decline could potentially estimate the individuals who should receive additional boosters and when they should receive them.Previous studies have shown that antibody levels are correlated with protection from infection,26 Here, we reported a similar waning of direct microneutralization against different VOCs within 4 months after the third dose. This suggests that within a few months of waning, the neutralization efficacy of Omicron may be insufficient to prevent infections. Indeed, 2 studies28 found that protection against Omicron infections after a third mRNA vaccine dose waned over time. During the Omicron surge in Israel, which began 4 months after third dose vaccine rollout, a high rate of Omicron infections was reported,29 raising suspicion that immunogenicity toward this variant was decreased despite relatively high IgG and neutralizing antibody levels. Overall, these results suggest that the relative resistance of the Omicron variant to the humoral response induced even by a full vaccine series may be the major factor associated with a high transmission rate.We and others have previously found that neutralizing antibody levels after 3 vaccine doses were approximately 4-fold to 10-fold lower against the Omicron compared with WT and Delta VOCs.30 that a second booster (fourth dose) was associated with IgG and neutralizing antibody levels similar to those induced by the first booster.30 However, we found that it was not associated with effective prevention of Omicron mild and asymptomatic infections among HCWs.30 However, a second booster was efficacious in protecting against severe disease and death compared with 1 booster dose.31 Despite the relatively slow waning of the immune response observed in this study, the lack of protection from infection by emerging SARS-CoV-2 variants suggests that repeated boosters of currently available vaccines may have reached a limit of protective outcomes in young and healthy populations. These results additionally suggest that the timing of second and third boosters in such populations should be further considered and studied. Our results in this study suggest that some populations may be more prone to Omicron infections and that, therefore, an updated Omicron vaccine could be advantageous. Indeed, preliminary data suggested that a bivalent Omicron BA.1 and prototype booster vaccine were associated with higher Omicron BA.1 and BA.4 and 5 neutralizing titers compared with the prototype vaccine alone.33 This result suggests that vaccine strain updates may be associated with protection against infection. Future studies should assess the effectiveness of a modified vaccine against infection of Omicron and other new emerging variants.We have recently foundThis study has several limitations. First, given that all participants were HCWs, they were primarily women and relatively younger and healthier than the general population, thus potentially limiting the study’s generalizability. Second, despite being validated on 215 samples before comparison, tests after the second dose were performed using a different assay and were thus imputed to be comparable with tests after the third dose. Although unlikely, this may potentially be a source of bias. Third, we have investigated the response only to the BNT162b2 vaccine, while the durability after other vaccines may be different. Fourth, the study period after waning was relatively short. Further studies to assess waning after longer periods are needed. Furthermore, given that participants were not blinded to their serology testing, those who had lower serological markers could have potentially had a lower threshold for RT-PCR testing, and this may have created bias. However, due to the outstanding surge in infections, all HCWs, were encouraged to obtain a weekly RT-PCR SARS-COV test during the study period in addition to testing after exposure or due to symptoms, regardless of their serological tests.Numerous studies have found that mRNA vaccines played a major role in protecting the world population against COVID-19. This cohort study found that the humoral response after a third dose was sustained for months with a minor decrease in antibody levels and that antibody levels were associated with infection with the Omicron variant, and thus, infection may potentially be estimated. Nevertheless, our results suggest that the humoral response generated by vaccination may not be enough to protect against Omicron infection. BNT162b2 booster doses have been found to be protective against severe disease and mortality, yet if reducing transmissibility and achieving herd immunity is the goal, our results suggest that a different vaccination strategy may be required.


Remote patient monitoring (RPM) has demonstrated value as a tool to aid patients in management of their chronic illness in the home. Although the Mississippi Diabetes Telehealth Network Study (MSDTNS) was successful in reducing HbgA1c levels for patients participating in RPM in the Mississippi Delta, the long-term effect of RPM on patients and how to support patients to maintain the treatment effect after discharge remain unclear.This study evaluated the long-term effectiveness of an RPM program after the intervention was withdrawn.A retrospective review of medical records of patients who completed all phases of the MSDTNS from 2014 to 2016 was performed over a period of 6 months. Data collected included HbgA1c values, demographics, and changes in social determinants of health.Of the 31 participants, African Americans displayed a significant difference in HbgA1c values compared with Caucasians since the end of the MSDTNS. No significant effect of other variables, such as income, marital status, insurance coverage, or age, on the change in HbgA1c values was detected since the end of the original study.This limited study implies that African Americans are at higher risk for an increase in hemoglobin A1C after the program is completed. More investigation is needed to identify ways to reduce their risk and equalize the long-term effects of RPM on clinical outcomes of patients in rural or underserved communities. Using remote patient monitoring (RPM), a key component of telehealth, to aid in managing chronic diseases such as diabetes improves clinical and economic outcomes, especially for patients living in rural areas that lack access to health care services.5Setting appropriate goals is the initial step in managing diabetes. With the exception of pregnant women, HbgA1c levels should be <7%. Lowering the HbgA1c levels to <7% reduces symptoms of diabetes and its long-term complications such as kidney dysfunction, visual impairment, cerebrovascular events, and heart disease.6 A nurse will then provide individualized diabetes education based on the nurse–patient discussion of mutually set goals.Identifying barriers to suboptimal glucose control, including lifestyle (diet and exercise), nonadherence to medication/monitoring practices, and lack of knowledge of the disease process, is pertinent in education disbursement and goal development for patients on RPM.7 Diabetes management information can be provided to the patient using routine telephone calls, by direct messaging, or during alert situations (notification of elevated glucose) identified by the RPM nurse.8Mutual goal setting allows the patient to become more engaged in their care, increasing their ability to self-manage their skills related to monitoring their diabetes.Over the enrollment period, patients learn to appropriately monitor glucose, dietary intake, exercise options, and medication management. The goal is to provide lasting education to carry the patient through their lifetime with the chronic disease state.Outcomes reported upon completion of the RPM program have typically shown improvements in HbgA1c without showing sustained or at goal HbgA1c levels over time. Other than the expected HbgA1c levels reported, researchers have also investigated reasons behind potential successes, identifying barriers and groups with potential for disparity that would need additional support to improve success rates of RPM for diabetes management.6 In the study led by Michaud et al., RPM for diabetes management was generally found to show improvement in HbgA1c numbers. However, while women were found to be more successful in completion of the study, they had less improvement of HbgA1c at 0.4% when compared with men at 0.8% reduction.8In a review completed by Andrès et al., RPM was found to show improvements in HbgA1c for participants in addition to reducing instances of hypoglycemia when compared with standard care practices.9 Randall et al. found that while enrolled in RPM for diabetes management, patients were attending the same number of in-office primary care visits. This information is attributed to RPM nurses encouraging patients to seek follow-up for trending elevations in glucose, which possibly would not have happened otherwise, thereby mitigating further complications or hospitalizations.10Relatedly, Michaud et al. found that women with public insurance (Medicare and Medicaid), higher baseline HbgA1c values, and reduced engagement in participation had less success with RPM.7 Although a multitude of factors may affect the perceived success of RPM in diabetes management, studies by Michaud et al. and Su et al. suggest that patient engagement could be the driving force between marginal and significant improvement of HbgA1c levels.9Su et al. found that patients who were more engaged in RPM were more successful in reaching HbgA1c goals in a 6-month time period. They defined better engagement as patients who monitored and uploaded their bio-clinical data more often, usually daily, than those who did not.11 While RPM has been shown to improve clinical aspects of diabetes such as improved HbgA1c, long-term diabetes maintenance data are lacking and should be explored further.5Based on the lack of information regarding follow-up post-RPM completion, more thorough studies should be completed looking at long-term sustainability of HbgA1c levels over a longer duration of time and with larger sample sizes. In their meta-analysis of systemic reviews of randomized controlled trials, Lee et al. recognize the significance that RPM plays in reducing HbgA1c levels, specifically with patients whose HbgA1c level is >8% before implementation.12 With the rural health clinic as the center of primary care and the tertiary center as the provider of specialty services, telehealth was used to connect patients living with uncontrolled diabetes to members of the care team not readily available in their communities.Recognizing the importance of effective management of chronic diseases for the reduction of overall health care costs, the University of Mississippi Medical Center and its partners initiated the Mississippi Diabetes Telehealth Network Study (MSDTNS) to implement RPM with a collaborative model for managing diabetes.Patients in the study were educated on managing diabetes as well as required to complete the daily health session of measuring their blood glucose at home. The nurses who coached the patients through their experience while on the program then reviewed these measurements. The findings of this program contributed to expansion of the RPM program. Of 171 patients originally enrolled in the MSDTNS, 115 completed all phases of the study.There was a significant difference in the HgbA1c values from baseline to 3-, 6-, 9-, and 12-month values. Maximum benefit was achieved after 3–4 months on the program and was maintained over the 12-month period. It was concluded that RPM and telehealth could be effective tools for assisting home-based patients in self-management of diabetes in rural areas.Even with the perceived success of this initial study, the long-term effect of RPM on patients and how to support patients to maintain the treatment effect after discharge remain unclear. To explore this, we conducted a retrospective study on patients who completed all phases of the MSDTNS.This retrospective study evaluates the long-term effectiveness of an educational intervention and chronic disease management program powered by telehealth after the intervention was withdrawn by comparing participants' most current HbgA1c levels with their levels upon completion of the original study that concluded in 2016. The time from withdrawal of the remote monitoring intervention to attempt to follow-up was ∼4 years.1.Were the outcomes from the MSDTNS, specifically the HbgA1c level reductions, sustained after withdrawal of the technological intervention?2.What is the participant's current financial or social status since completion of the study, which may have affected their health?The research questions were as follows:The study period for this exploration was 6 months. The original study site served as the site of consent as the providers there had established relationships with the participants. Many of the patients continued care at this site after the original study concluded.The inclusion criteria for this retrospective study were as follows:Completion of the MSDTNS18 Years of age or olderNot pregnantInstitutional review board approval was secured through the University of Mississippi Medical Center as per policy. Clinic staff who were trained on the research procedures contacted prior study participants by phone and invited them to participate in the follow-up study. Calls were made over a 6-month period in an effort to reach as many participants as possible. After informed consent was obtained, participants were administered a short survey  asking qThe responses to the survey were deidentified and coded. The researcher at the site also logged the participants' current HbgA1c values and any results that were available in their electronic health record since the conclusion of the original study. If the participant did not have an HbgA1c test within the past 4 months or was not scheduled for one within the next 3 months, a level was drawn at that time. Other information collected included name, age, race, medical history, marital status, and insurance status. Investigators did not ask questions regarding new health problems since the withdrawal of the remote monitoring program.Descriptive statistics, mean and standard deviation (SD) for continuous variables and frequency and percentage for categorical variables, were used to summarize end-of-intervention and last HbgA1c measures, the outcome variable, and baseline patient characteristics. The Wilcoxon signed-rank test was conducted to examine the change in HbgA1c from the end of the intervention to follow-up.Linear regression models were performed on available cases to evaluate the effect of each socioeconomic variable on the change in HbgA1c, adjusting end-of-intervention measures of HbgA1c. If significance was detected in regression at the 0.1 level, we further evaluated the change in HbgA1c for the socioeconomic variable. Then, we conducted the Wilcoxon signed-rank test again to examine the change in HbgA1c for each race group.In addition, missing values occurred in end-of-intervention HbgA1c, age, and marital status. To maximize the use of available information and avoid bias from missing data, we conducted multiple imputations, a well-established strategy that replaces missing data with several sets of plausible values and aims to provide statistically valid inferences. We conducted 10 imputations using fully conditional specification with the Markov chain Monte Carlo procedure.13Specifically, linear regression is used to impute end-of-intervention HbgA1c and age, and logistic regression is used to impute marital status (married yes/no). All variables are included in the imputation model. Then, a linear regression model was performed on each of the 10 sets to estimate socioeconomic effects on change in HbgA1c, and results were combined using Rubin's rules.All data analyses were conducted using the SAS statistical software .Thirty-one of 115 (27%) completers from the original study consented to participate in this study. The remaining 84 completers were lost to care and could not be found to offer participation in this follow-up study. Basic characteristics of participants are presented in Sixteen participants were married. Among the 26 participants who had hypertension, 1 also had heart failure. Sixteen participants used multiple insurance providers. Medicare was the most frequently used insurance among participants and 14 (45%) participants had it. Thirteen (42%) participants used the Magnolia Health Plan or Medicaid. Other insurance was used by 65% of participants.p = 0.33).The HbgA1c value was recorded at baseline for 30 participants with a mean of 7.76 (SD = 1.26). At the end of the study, the HbgA1c value was increased by 0.27 (SD = 1.77), on average, from the baseline. No significant change was detected at the 0.05 level through the Wilcoxon signed-rank test (p = 0.07). Compared with Caucasians, African Americans are expected to have a higher increase of 1.16  in the outcome at the end. Other socioeconomic variables do not show a significant effect on the change in HbgA1c.The estimates of linear regression models are displayed in p = 0.01) in the HbgA1c value, from 7.63 (SD = 1.31) to 8.39 (SD = 1.82), while Caucasians had an average decrease of 0.64  in the HbgA1c value, from 8.03 (SD = 1.18) to 7.39 (SD = 1.15). The change in HbgA1c was further investigated for each race group. African Americans had an average increase of 0.72  in the change in HbgA1c compared with Caucasians.After multiple imputation on baseline HbgA1c, age, and marital status (married yes/no), results of the regression analysis show that African Americans, on average, have a higher increase of 1.19  of the 115 participants who completed the original study were African American and 18 (15.7%) were White.Although the limited retrospective study does imply that African Americans are at higher risk for having an increase in HbgA1c after the program is completed, more work is needed to investigate the specific reason for this increased risk. Once the reasons for the increased risk are identified, interventions can be tested in this population to reduce their risk and equalize the long-term effects of RPM on clinical outcomes of patients in rural or underserved communities.Through investigating the actual real-world situation of rural patients, what this study does illuminate is the possibility that RPM programs may prevent patients with chronic conditions from becoming lost to follow-up. While intense RPM programs are not the only solution to prevent gaps in follow-up care, the findings suggest that patients with chronic conditions could benefit from additional support after participating in RPM programs.


Novel interventions should be developed for people who have undergone psychological trauma. In a previous case study, we found that the number of intrusive memories of trauma could be reduced with a novel intervention. The intervention included a brief memory reminder, a visuospatial task and mental rotation, and targeted trauma memory hotspots one at a time in separate sessions.This case series (N=3) extended the first case study with 3 new cases to determine whether a similar pattern of beneficial results is observed. We explored whether the brief intervention would result in reduced numbers of intrusive memories and whether it would impact symptoms of posttraumatic stress, depression and anxiety, and general functioning. Acceptability of the intervention was also explored.A was a preintervention baseline phase and B intervention phase). Intrusions were targeted individually, creating repetitions of an AB design.A total of 3 women completed the study: 2 with posttraumatic stress disorder and other comorbidities and 1 with subthreshold posttraumatic stress disorder. The primary outcome was the change in the number of intrusive memories from the baseline phase to the intervention phase and at the 1-month follow-up, with an assessment of the intrusion frequency at 3 months. Participants monitored the number of intrusive memories in a daily diary for 1 week at baseline, for maximum of 6 weeks during the intervention phase and for 1 week at the 1-month and 3-month follow-ups. The intervention was delivered in person or digitally, with guidance from a clinical psychologist. A repeated AB design was used  reduced from 38.8 per week during the baseline phase to 18.0 per week in the intervention phase. It was 13 at the 3-month follow-up. The total number for P4 reduced from 10.8 per week at baseline to 4.7 per week in the intervention phase. It was 0 at the 3-month follow-up. The total number for P5 was reduced from 33.7 at baseline to 20.7 per week in the intervention phase. It was 8 at the 3-month follow-up. All participants reported reduction in posttraumatic stress symptoms in the postintervention phase. Depression and anxiety symptoms reduced in 2 of the 3 participants in the postintervention phase. Acceptability was favorable.We observed good compliance with the intervention and intrusive memory diary in all 3 cases. The number of intrusive memories was reduced for all participants during the intervention phase and at the 1-month follow-up, with some improvement in other symptoms and functioning. Further research should explore the remote delivery of the intervention and whether nonspecialists can deliver the intervention effectively. Most people experience psychological trauma  in their lives ,2, and mAs noted previously , althougbrief memory reminder cue for one specific intrusive memory of trauma, followed by a 25-minute Tetris gameplay with mental rotation . The ine lines; -18. It hproaches ,19-21. Tbriefly bring the visual content of the memory to mind without becoming emotionally overwhelmed by a method agreed with the participant . Next, the participant was taught to use mental rotation. She then played Tetris for 25 minutes using mental rotation. She monitored her specific intrusive memories in a daily diary so that the impact of the intervention on a distinct intrusive memory could be easily observed. The total number of intrusive memories decreased from 12.6 per week at baseline to 6.1 per week in the postintervention phase. Furthermore, the number of intrusive memories continued to reduce to only 1 memory per week at the 3-month follow-up. Symptoms of posttraumatic stress and depression and anxiety reduced in the postintervention phase, whereas functioning improved. The participants considered the intervention to be an acceptable way to reduce the number of intrusive memories. The next step in exploring the effects of the intervention involves examining if they extend to other cases of women after trauma and whether remote (rather than in person) delivery is a feasible delivery method given restrictions occurring during the COVID-19 pandemic  [We adapted the intervention for women in Iceland who experienced intrusive memories of trauma, as reported in a recent case study . Some ofolation) .repeated AB design in which each specific memory is targeted in separate sessions, so that we can consider the effects of an individual intervention session on each specific memory over time [In this case series, we aimed to extend our previous case study to a short case series of trauma-exposed women in Iceland, drawn from an epidemiological study of trauma experienced by women in Iceland. The intervention sessions took place either in person in a university setting or remotely using a web-based platform. We examined whether the novel intervention approach could reduce the number of intrusive memories of trauma (primary outcome) and whether reductions were maintained at follow-up (1 and 3 months). As before, the brief intervention was guided by a clinical psychologist, targeting one distinct intrusive memory at a time per session. The acceptability of the intervention was also explored along with adaptions in intervention delivery. Again, we explored whether having fewer intrusive traumatic memories would also be associated with improvements in general functioning, posttraumatic stress, and depression and anxiety symptoms (secondary outcomes). The design adopted here can be described as a within-subject ver time ,20.As in our previous study , we predMeasures section) or not likely PTSD , using clinical interviews. In all, 2 semistructured interviews were administered in the substudy .Participants were drawn from an epidemiological study of trauma experienced by women in Iceland (as in our previous case study ). As desWomen who took part in the Social Trauma Project substudy (both likely PTSD group and not likely PTSD group) were screened for the presence of intrusive memories of trauma, as in our previous case study . As befoIn this study, 72 women from the substudy, who provided their consent to be contacted for further research were assessed for inclusion in this study .Inclusion criteria were 1) having experienced criterion A trauma as defined by DSM-5 [ having eIn all, 5 women who met the inclusion criteria were included, ranging in age from 39 to 66 years . Participants are referred to as P1, P2, P3, P4, and P5. The initial 2 participants recruited did not complete the intervention sessions due to nonsuitability of the study timing in their lives . Both P1 and P2 were excluded from the data analyses, although data from the baseline were collected. After P1 and P2 wished to cease their participation, a question was added to the recruitment process asking if the participants foresaw any obstacles to their participation in the study. P1 did not meet any diagnostic criteria for psychological disorders according to the MINI. P2 met the diagnostic criteria for bipolar disorder and reported subthreshold PTSD symptoms.A total of 3 patients completed the study, 2 (67%) with PTSD and other comorbidities and 1 (33%) with subthreshold PTSD. P3, woman aged ≥40 years, met the criteria for major depressive disorder and PTSD. P4, a woman aged ≥60 years, did not meet any diagnostic criteria but reported subthreshold PTSD according to the CAPS-5. P5, a woman aged ≥50 years, met the diagnostic criteria for major depressive disorder, social anxiety disorder, and PTSD. P3 reported having 10 different intrusive memories of physical violence that occurred in childhood. P4 reported having 3 different intrusive memories from childhood sexual abuse, and P5 reported 6 different intrusive memories from childhood sexual abuse.The case series used a single-symptom approach, in which each intrusive memory was targeted in different sessions, that is, one at a time ,19. Specrepeated AB design whereby the length of baseline  and intervention (B) phases varied for each distinct intrusion, according to which intervention session the intrusive memory was targeted in. Thus, the baseline phases for each distinct intrusive memory were used as control periods in the comparison with the intervention, that is, the number before and number after being targeted.Thus, the design for each participant was a A daily diary was used to monitor the number of each intrusive memory over time. That is, for 1 week before intervention, then over 6 weeks (maximum) of intervention; then for 1 week at the 1- and 3-month follow-ups. Intrusive memories were targeted individually in up to 6 intervention sessions. These sessions were guided by a clinical psychologist, who was a specialist in trauma-focused cognitive behavior therapy. After the first session, participants were able to self-administer the intervention if they wished for memories that they had already targeted in the session. P3 received 2 repetitions of an AB design, P4 only targeted 1 memory (and thus no repetitions of an AB design), and P5 received 4 repetitions of an AB design.The primary outcome was the change in the number of intrusive memories per week from baseline to the intervention phase and to the long-term follow-ups (1 and 3 months). Participants further completed secondary outcomes—self-report measures for posttraumatic stress, depression and anxiety symptoms, and functional impairment—at baseline, the last intervention session, and 1- and 3-month follow-ups.in real time support from a clinical supervisor via telephone directly after participant sessions regarding any case specific adaptions needed . Twice a month, the researchers joined remote  peer-group training meetings with other international researchers about the intervention (convened by EAH and LS).Researchers delivering the intervention  underwent training and received clinical supervision, promoting adequate intervention delivery and protocol adherence. Training involved 2 in vivo workshops for 3 days and then again approximately 6 months later for 2 days, delivered by psychologists with expertise in developing the intervention and delivering it in other settings (MK and EAH). The theoretical background and practical aspects of the intervention  were covered in the workshops, as well as role-playing with trainers and feedback until sufficient performance was reached. While data collection was ongoing, the researchers received supervision. Such supervision included weekly supervision meetings, as well as more hotspots sheet in a way that the participant could also see it. Participants then labeled each intrusive memory with a symbol  and were instructed on how to monitor their frequency each day in a pen-and-paper diary (primary outcome measure). To indicate when a certain memory was experienced, the participants recorded the symbol corresponding to a given memory for each time frame of that day. Each diary was divided into 7 days and each day, into 4 periods (see Measures section). The participants also completed baseline questionnaires (secondary outcomes) in the baseline session.A similar procedure was followed as in our previous case study ; in the In each intervention session maximum 6 sessions), the participant chose 1 intrusive memory to target (by looking through their diary entries) and completed the intervention procedure (guided by the researcher). The memory chosen could be the most distressing or frequent or one chosen to be targeted by the participant for other reasons. As in our previous case study  sessions or KanstMarathon mode with the ghost piece off. When an intervention session took place remotely with a video call, Tetris gameplay was performed on the participants’ own computer with a shared screen so that the researcher could monitor the gameplay, especially regarding mental rotation. Only one distinct intrusion was targeted for each session. To select which intrusion was targeted, the participant (not the researchers) selected which intrusive memory it was. At the end of the last intervention session, secondary outcome measures were completed again.After the memory reminder procedure, participants were trained on how to use the Tetris game and practiced using mental rotation. They then played Tetris using mental rotation for 25 minutes . For in-Participants were invited to self-administer the intervention for memories already targeted in a session using a mobile version of the Tetris created by Electronic Arts . For exaWhen the COVID-19 pandemic started , researchers switched to remote (rather than in person) delivery through Kara Connect, which is a General Data Protection Regulation–compliant web-based platform certified by the Icelandic Directorate of Health. The last intervention session for P3 was performed remotely, intervention sessions 2 and onward were performed remotely for P4, and all sessions were remotely delivered for P5. Tetris was played on the web on the participants’ computers (ghost piece off and sound set to 0%) with a shared screen so that the researcher could monitor participants’ gameplay via Kara Connect, which increased the likelihood of instruction adherence, especially regarding the use of mental rotation.At both the 1- and 3-month follow-up time points, participants recorded the number of intrusions in their diary daily again for 1 week and completed the secondary outcome measures. Data were entered via laptop into a REDCap  database (REDCap), an encrypted electronic software stored on a secure server .Please note that the measures described here have already been described in our previous case study  and are The Clinician-Administered PTSD Scale (CAPS-5) is a 30-item semistructured interview used to assess symptoms of PTSD from an index of trauma and symptom severity (in the past month) according to DSM-5 [α=.88), test-retest reliability (α=.83), and good convergent validity (α=.83) [to DSM-5 . Items a (α=.83) The MINI for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, assesses Axis I psychiatric disorders according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, using a structured diagnostic interview. For most diagnoses, the MINI has good sensitivity and specificity . InterraThe intrusive memory diary, a pen-and-paper diary similar to that used in Thorarinsdottir et al [ir et al , was adair et al ,20. It iThe primary outcome was the change in the number of intrusive memories from the baseline to the intervention phase and at the 1-month follow-up. The original CTR additionally prespecified a measure of intrusive memories at the 3-month follow-up (“Change in the number of intrusive memories of trauma from baseline to 3 month follow-up”) but incorrectly stated that the measure was “Questions about the frequency of intrusive memories for the past day or week ,” specifically “How often did this memory come up yesterday?” and “How often did this memory come up per day in the past week?” However, this was incorrect, as we had changed this measure at the study start to use the same diary as for the earlier time points , that is the “Number of intrusive memories of traumatic event recorded by participants in a diary daily  per week over the baseline phase and during one week at three month follow-up.” The measure has been updated retrospectively in the CTR for this 3-month period and should be interpreted with caution.PTSD symptoms in the past month were evaluated using the PCL-5, a self-report scale with 20 items. Each symptom is rated on a 5-point Likert scale , with higher scores indicating greater severity. The PCL-5 evaluates the severity of PTSD symptoms according to DSM-5 criteria. It has strong internal and test-retest reliability and good convergent and discriminant validity [α=.95).validity . Criterivalidity . The IceDepression symptoms in the past 2 weeks were evaluated using the Patient Health Questionnaire-9 (PHQ-9), a self-report measure with 9 items rated on a 4-point Likert scale  [α=.89). A 5-point change in the total PHQ-9 score was considered clinically significant [ery day) . The PHQery day) . The Icenificant .Anxiety symptoms in the past 2 weeks were evaluated using the Generalized Anxiety Disorder-7 scale (GAD-7). In this self-report questionnaire, each item is rated on a 4-point Likert scale . The GAD-7 assesses symptoms of general anxiety disorder and its severity and has great internal reliability and good test-retest reliability [α=.90). A 4-point change on the GAD-7 is considered clinically significant [iability . In geneiability . The Icenificant .Functional impairment in the previous week was evaluated using the Sheehan Disability Scale (SDS). This self-report measure has three domains: (1) work or school, (2) social, and (3) family life, assessing functional impairment using a 11-point scale  [α=.84) [tremely) . A 3-poitremely) . To assetremely) . The Ice [α=.84) .Self-guided adherence for daily life using the gameplay part of the intervention was rated “How often did you manage to play Tetris after you experienced an intrusive memory?” .Feasibility and acceptability ratings for the intervention were assessed with 2 ratings “Would you recommend playing Tetris to a friend?” and “Do you consider gameplay to be an acceptable way to reduce the daily frequency of intrusive memories?” The scores ranged from 0 to 10. Higher scores indicated greater acceptability. Open-ended questions included (1) “How did you feel about playing Tetris after you had an intrusive memory?” and (2) “Did you find the intervention helpful? If yes, how?”The impact of intrusive memories on concentration, sleep, and stress in the past week was evaluated using 6 self-rated items: a total of 2 items assessed general concentration difficulties and difficulties in concentration due to intrusive memories , 1 item assessed concentration disruption in minutes in the past week, and 2 items assessed the impact of intrusive memories on sleep . An item assessed the impact of intrusive memories on stress levels .General impact of intrusive memories was assessed using 2 ratings of intensity and vividness of the intrusions on a 11-point scale .Intrusion diary adherence item was assessed using the rating “How accurately did you fill out the diary?” .Impact of intrusive memories on daily functioning was evaluated via an open-ended question: “How have the intrusive memories affected your ability to function in your daily life in the past week?” and a self-rated question, “Have the intrusive memories affected your ability to function in your daily life?” .per week was calculated for comparability. Missing data were dealt with by excluding these time points from the calculations and using available data (see Results section). For example, a participant had a baseline period of 14 days, but data were present for only 6.5 days; thus, the total number of intrusions per week was calculated as 10 intrusions / 6.5 days × 7 = 10.8 intrusive memories per week during baseline.The number of intrusive memories of trauma was recorded by participants in the diary daily  during the baseline phase and each week during the intervention phase (weeks 1-6) and during 1 week at the 1-month follow-up. The primary outcome was the change in the number of intrusive memories of the trauma. The timeframe was baseline week to the intervention phase (weeks 1-6) and follow-up (1 month). In practice, owing to scheduling reasons, the baseline phase was longer than 1 week, and as anticipated, the number of intervention weeks varied. Therefore, because these periods had different time lengths, the mean number When examining change over time, the percentage reduction in total intrusions per week was calculated from the baseline phase to the intervention phase to other periods as follows: 1– (mean number per week during intervention phase / mean number per week during baseline) × 100. For example, for the same participant there were 4.7 intrusions per week in the intervention phase, which was calculated as 1 – (4.7 / 10.8) ×100 = 57% reduction in the intervention phase compared with the baseline.Please note that at the 3 month-follow-up, we did not use the telephone questions on the CTR (NCT04209283) about the frequency of intrusive memories for the past day or week  but instead replaced this with the same 1 week diary used at earlier time points. We noted the use of a diary at 3 months in our ethics submission, but we incorrectly specified it in our CTR and did not update the CTR on this point until the submission of this paper.We examined the number per week of targeted memories in comparison with nontargeted memories. This was done by calculating the number in the same way as described above for targeted memories . However, standard baseline and intervention periods were established for nontargeted memories , as they were not targeted by the intervention. The baseline for each nontargeted memory was 1 week , and the intervention phase was determined as the period from when any memory was targeted with the intervention.A descriptive approach was used to explore whether clinically meaningful changes were observed in the overall symptoms of posttraumatic stress, depression, anxiety, and functional impairment.This study was approved by the National Bioethics Committee of Iceland (VSNb2017110046/03.01). The participants provided written informed consent before the start of the study. All the sessions followed a written protocol. No adverse events related to the intervention were reported. Participants were asked to briefly consider the visual content of the traumatic memories they selected, which might have resulted in some distress. Previous research has indicated that this intervention approach is well tolerated, including in inpatients with complex PTSD . HoweverProcedure, Training section).The study was registered before the start of the study on ClinicalTrials.gov (NCT04209283) on December 24, 2019. The manuscript contains anonymized summary-level data. The study materials may be made available upon reasonable request with an appropriate material transfer agreement with the University of Iceland or Uppsala. We note that delivery of this intervention at present requires prerequisite training and supervision by psychologists with experience of developing it  was targeted 5 times and the other (Memory B) was targeted once. P3 monitored the memories quite accurately, but data were missing for days 15 to 22 during the intervention phase. P4 reported having 3 intrusive memories of childhood sexual abuse, of which only 1 (Memory A) was targeted with the intervention 5 times. P4 monitored the memories accurately, and data were missing for half of day 1 and for days 8 to 15 during the baseline phase. P5 reported having 6 intrusive memories of childhood sexual abuse, 4 of which were targeted. One of the memories (Memory A) was targeted 3 times, whereas the others  were targeted once. P5 accurately monitored their memories with no missing data. No attempts were made to retrieve the missing data. On average, 90% of the diary data were completed for all 3 participants. No data were missing for the secondary outcome measures.The total number of intrusive memories per day throughout all phases for each participant  is shown in We now consider each of the participant graphs shown in Change in the Total Number of Intrusive Memories section; however, please see earlier notes about the change in measures at this time point in contrast to the original CTR.P3 had 13 intrusive memories in their diary at the 3-month follow-up (67% reduction from baseline). P4 had 0 intrusive memories (100% reduction from baseline) at the 3-month follow-up, whereas P5 had 8 in the week of the 3-month follow-up , and P4 rated the item at 3 (unlikely to recommend to a friend). They also rated if they considered gameplay to be an acceptable way to reduce intrusive memories. P3 and P5 rated the item at 8 (high acceptability), and P4 rated it at 3 (low acceptability). When participants were asked to rate how they felt about playing Tetris after having an intrusion, P3 noted that “It reduced the emotion, sometimes I was able to concentrate and think my way through it. Sometimes I experienced a kind of peace within.There was a clear reduction in posttraumatic stress symptoms (PCL-5) from baseline to the postintervention phase, which tended to continue to drop further during follow-up for all 3 participants who completed the intervention, suggesting clinical improvement . DepressThe ratings of the impact of intrusive memories on concentration, stress, and sleep are included in ” and P5 said, “I experience distress and nightmares.” At the 1-month follow-up, P3 disclosed, “When I am under a lot of stress, they disturb me more.” P5 reported, “The images no longer have color, cause less disruption and are less frequent.” At the 3-month follow-up, P3 said, “I am under a lot of stress and dealing with a certain communication problem which triggers my PTSD and the intrusions” and that “I can now comprehend that this is only a memory, and I don’t feel as distressed.” P5 reported, “They mostly impact my anxiety” and “I am not a person that easily believes in things, but this intervention works.” For all phases following the baseline, P4 reported, “The memories have no impact anymore.”The reported impact of intrusive memories on the ability to function in daily life over the intervention and follow-up periods showed improvement . At baseline, the participants were asked to respond to an open question about how their intrusive memories impacted their ability to function in daily life. At baseline, P3 said, “When I have this overwhelming feeling, I find it difficult to be around other people, the worst thing is how it affects my ability to stay present for my daughters.” P4 said, “I have anger inside me, I constantly think back to how no-one noticed what was done to me.” P5 reported, “I get very stressed and anxious; it takes a lot of energy to get out of the emotion...” In the last intervention session, P3 reported, “The intrusions disrupt my concentration,The aim of this case series was to extend our previous case study by evaluating a novel visuospatial intervention designed to reduce the number of intrusive memories of trauma ,14,37. TNot only did the targeted intrusions reduce in this case series but nontargeted intrusions were also reduced in the intervention phase. Although this appears relatively more so than the targeted ones, results must be treated with caution because of the potential floor effects on the low baseline number of nontargeted intrusions rendering comparisons misleading. Kessler et al  found thWe examined whether a reduction in the number of intrusions would have an impact on the symptoms of posttraumatic stress, depression and anxiety symptoms, and general functioning (secondary outcomes). The general pattern was that posttraumatic stress symptoms were reduced for all participants in the postintervention phase . Depression and anxiety symptoms were reduced at times suggestive of a clinically significant change for 2 (P3 and P5) of the 3 participants in the postintervention phase ,33, wherP3 and P5 rated the intervention as acceptable; using Tetris gameplay was an acceptable method to reduce the frequency of their intrusive memories and noted that they would recommend the intervention to a friend. This is similar to what Holmes et al  found amTwo of the participants ceased participation in the study after the baseline phase; the reasons were unrelated to the intervention but were related to scheduling issues in their daily life. However, it is important to determine who is most likely to benefit from the intervention and how to educate individuals about it in a way that increases the chance of people making an informed choice of whether they are able to try it or have the time to take part in a course of treatment. It could also be explored whether the intervention could have an impact on the frequency of intrusive memories with fewer guided intervention sessions, which would reduce the participation load.Owing to the COVID-19 pandemic, the delivery of the intervention was changed from face to face to remote . Originally, our plan was to gradually move toward remote delivery in future studies. When the pandemic struck, it forced the change to occur more quickly during the intervention phase of the study. This turned out to be a positive development, as the data did not suggest that the intervention became less effective by being delivered remotely. Recent research by Singh et al  indicateTargeting established intrusive memories of trauma that participants had been experiencing for some years  with a brief visuospatial intervention, involving a brief memory reminder and Tetris gameplay with mental rotation, seems to show promise for further exploration as a method to reduce their frequency. These early data suggest that the intervention might also result in symptom reduction related to posttraumatic stress, anxiety and depression, and improved functioning; however, further studies are needed. Because of its simplicity, this intervention might be capable of removing common barriers to existing treatment options after trauma, such as for PTSD, including some patients’ reluctance to talk about and describe their trauma in detail to a therapist, high costs, and a limited number of qualified therapists . The intThe results of this study are encouraging, and the effects of the intervention on the number of intrusive memories need to be further explored. Continuing to develop this kind of scalable intervention is crucial to reach a large number of people in need of treatment after experiencing trauma. Future research should further examine the feasibility and acceptability of remote delivery by nonexperts in mental health  and whether fewer intervention sessions can yield similar results. Randomized controlled trials are required to assess the intervention.