As a large intracellular multiprotein signaling platform, it activates downstream effectors that initiate a rapid necrotic programmed cell death (PCD) termed pyroptosis and activation and secretion of pro-inflammatory cytokines to warn and activate surrounding cells. However, inflammasome activation is difficult to control experimentally on a single-cell level using canonical triggers. We constructed Opto-ASC, a light-responsive form of the inflammasome adaptor protein ASC (Apoptosis-Associated Speck-Like Protein Containing a CARD) which allows tight control of inflammasome formation in vivo. We introduced a cassette of this construct under the control of a heat shock element into zebrafish in which we can now induce ASC inflammasome (speck) formation in individual cells of the skin. We find that cell death resulting from ASC speck formation is morphologically distinct from apoptosis in periderm cells but not in basal cells. ASC-induced PCD can lead to apical or basal extrusion from the periderm. The apical extrusion in periderm cells depends on Caspb and triggers a strong Ca The innate immune system plays a crucial role in the early recognition and eradication of potentially dangerous microorganisms. Inflammasomes are among the best characterized components of innate antimicrobial defense in vertebrates. Inflammasome activation is induced following recognition of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by intracellular pattern-recognition receptors, such as NOD-like receptors (NLRs) and AIM2-like receptors (ALRs). This leads to the formation of large multiprotein platforms, which often contain the adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD), that can recruit and activate proinflammatory caspases . These cInflammasome pathway components are expressed in immune and non-immune cells, but have been studied mostly in cultured cells or bone marrow derived macrophages see e.g. . Recent Epithelia are the first point of contact between the host organism and microbial invaders. Although the role of inflammasome components can be studied in vivo using epithelial infection models, little is known about the direct effects of inflammasome formation on cells and their neighbours in the context of the live tissue. Thus, analysis of this dynamic and rapid process would benefit from precise spatial control of inflammasome formation and live imaging of the ensuing physiological events.Zebrafish are well suited to study innate and adaptive immunity in vivo . Their tIn the zebrafish, ASC is expressed both in epithelial and immune cells, such as macrophages and neutrophils . The larAs in mammals, caspases in the zebrafish are recruited to the ASC speck . The zebUpon cleavage, GSDMs assemble into pores in the plasma membrane and lead to the release of the cell’s cytosol, including inflammatory cytokines , a hallmWe have previously developed optogenetic variants for human and zebrafish caspases which can be activated by light-induced oligomerization of Cry2olig, a photosensitive protein that undergoes rapid homo-oligomerization in response to blue light . This enTo further study inflammasome formation and the resulting cell death in the zebrafish skin upstream of inflammatory caspases, we have now generated an optogenetic variant of zebrafish ASC (Opto-ASC), which efficiently induces speck formation in single cells in both layers of the larval skin. Opto-ASC specks cause inflammatory cell death followed by apical or basal extrusion in periderm cells, which is morphologically distinct from apoptosis. ASC-speck-induced cell death requires Caspa and Caspb but we could not confirm the role of GSDMs in this process; we therefore refrain from calling it pyroptosis. We show that Opto-ASC specks efficiently trigger cell death in the periderm via inflammatory apical or basal extrusion.4  488 nm laser light . After 60 min, we counted the percentage of Opto-ASC-positive epithelial cells in the imaged area and the percentage of speck-forming cells among those Opto-ASC positive cells (2) was sufficient to induce speck formation after several minutes  and expose them to 5% relative 488 nm laser intensity.tg(asc:asc-GFP) line, where the open reading frame of endogenous ASC was tagged with GFP . We injected full-length Opto-ASC, Opto-PYD and Opto-CARD into the ASCΔ2/Δ2 mutant line. We first tested the ability of Asc-mKate2 to function in this mutant and found that it formed specks and induced cell death at the same level as in the wild-type larvae, showing that Asc-mKate2 is fully functional and able to induce cell death ). For all of the constructs, the shrinking of the cell, which we consider an initial sign of cell death upon Opto-ASC activation, was seen as soon as the first oligomerization was detectable.In the wild-type ASC-GFP line, Opto-Asc, Opto-PYD and Opto-CARD induced speck formation within five minutes, whereas in the ASCΔ2/Δ2 knockout line, Opto-ASC, Opto-PYD and Opto-CARD formed multiple smaller aggregates rather than a single large speck, indicating that endogenous ASC may facilitate multiple Opto-ASC aggregates to coalesce to form a single speck. In this mutant, Opto-CARD did not induce cell death, consistent with our earlier finding that inflammatory caspases are recruited via the PYD of ASC  x Tg(Krt4:AKT-PH)) of mixed genetic background . In these two lines, higher percentages of dying cells were basally extruded (38% in WIK and 64% in Tg(Opto-asc)x Tg(Krt4:AKT-PH)).To see if this variation in phenotype was determined by genetic differences, we injected the Opto-ASC construct into different commonly used laboratory strains . In mostTg(UAS:mNG-UtrCH), which we combined with a Krt4:Gal4 driver, and UAS:lyn-tagRFP to label plasma membranes). Larvae carrying these constructs showed stochastic expression of mNeonGreen-UtrCH in periderm cells, which allowed us to follow actin dynamics. The overall response of the actin cytoskeleton was similar in apically -tagged variant of the calponin homology domain of utrophin (UtrCH) expressed under control of the UAS-promoter  with tg and found that the apical actin ridges responded differently. In contrast to the Opto-ASC-activated cells, apoptotic periderm cells retained the original pattern of actin ridges while the cell was shrinking, with a proportional scaling of the pattern until the cell was fully internalized  to a caspa knock-out (KO) line  line , but uneKO) line , as an acaspa deletion had no effect on the morphology or timing of Opto-ASC-induced cell death or the extrusion efficiency . However, periderm cells lacking Caspb were no longer extruded apically  to be extruded from the periderm than during Opto-ASC induced cell death in control larvae  in different F0 KO backgrounds. F0 KO of xtrusion . Double l larvae .We also observed differences in the number and size of cell fragments, as measured by the number and diameter of Akt-PH-GFP labeled membrane vesicles in basally extruded periderm cells, depending on whether death was induced by Opto-ASC with or without Caspb or by Opto-caspase-8 . Again, To test if either caspase-8 or caspase-3 was involved in the execution of ASC-induced cell death we knocked out both genes using the CRISPR/Cas9-based method with four synthetic sgRNAs. However, in both cases the loss of the gene resulted in embryonic lethality so that effects at the larval stage could not be assessed.2+ signaling in the zebrafish skin. We used the Ca2+ signaling reporter GCamp6 to characterize the response of surrounding cells to cell death induced by Opto-ASC specks or Opto-caspase-8. We crossed the Tg(Opto-asc) line to Tg(ß-actin:GCamp6), a line expressing GCamp6 under the ubiquitously active ß-actin promoter  within an imaging area of around 24 cells corresponding to 13 mm2  appear as strong lines following the appearance of the speck (yellow arrow), and the sporadic Ca2+ signals of surrounding cells appear as the weaker lines. The speck is visible following its appearance as a magenta line, until it disappears following cell lysis. The extruded cell appears as a cyan shadow once extruded, and remains visible until lysis.To analyze these observations in a more quantitative manner, we used a two-dimensional representation of time and space  showed only sporadic Ca2+ signaling. We counted the number of responding cells in the ring of cells directly adjacent to the dying cell, and the next ring beyond those. We detected on average 21 basal cells and 12 periderm cells responding to Caspase-8-induced cell death with increased Ca2+ levels within the first 15 min after the cell started to shrink   and duriThese findings indicate that different types of cell death are not entirely separable, but that they represent different manifestations of parallel and interconnected signaling pathways, the overall balance of which defines the final outcomes. This is also evident from other studies in the past, where disrupting one pathway directs the cell towards a different pathway that may have a different phenotypic outcome . PerhapsIn the case of zebrafish periderm, we identified Caspb as essential for apical extrusion. In the absence of Caspb, cells die with an apoptotic morphology in response to speck formation. The requirement for Caspb for apical extrusion and lysis might also explain the difference in the response to ASC-speck formation between the periderm and the underlying basal cells. According to cell-type-specific RNA sequencing data , basal cAlthough Caspa localizes to the ASC speck  and is nZebrafish Caspa and Caspb are thus redundant in their role as rapid cell death inducers, but the downstream mechanisms that lead to either extrusion or apoptosis of periderm cells are yet to be identified. These mechanisms may be deployed differently in different situations. For example, in Caco-2 cell monolayers caspase-1 causes pyroptosis with rapid apical extrusion and a lamellipodial response in neighbouring cells which close the wound within approximately 70 min . Wound c2+ response in the case of ASC-induced death, but not apoptosis, indicates signalling from the dying cell to the surrounding epithelium, and this signal differs between cells dying apoptotically or by ASC-induced death. Caspase-8-induced apical extrusion of MDCK cells is preceded by a strong calcium wave in surrounding cells . Human GsdmE is activated downstream of caspase-3 during apoptosis  and it pin cells  and is nin cells . The relin cells .Danio rerio) were cared for using standard procedures as described previously (Tg(HSE:mCherry-Cry2olig-asc) line was generated by co-injecting embryos at the one-cell stage with the HSE:mCherry-Cry2olig-asc plasmid with transposase mRNA (100 ng/μl). To follow the actin dynamics in skin cells, we used the Tg line (Tg(6xUAS:mNeonGreen-UtrCH_6xUAS:lyn-tagRFP) generated in the lab of D.Gilmour by Jonas Hartmann. Actin dynamics in basal skin cells were visualized in the Tg line (Tg(6xUAS:mNeonGreen-UtrCH_6xUAS:lyn-tagRFP). To induce specks and follow endogenous ASC we used Tg(HSE:asc-mKate2) and the endogenously tagged asc:asc-GFP line  line  crossed l4) line  crossed GFP line . To induasc, asc(4xmut), PYD and CARD published in HSE:mCherry-Cy2olig-asc plasmid as background and were modified by seamless cloning using the NEB Assembly Kit, after SmaI/EcoRV or XmaI/EcoRV digestion of the plasmid (replacing asc). The following constructs were generated and used for transient expression: HSE:mCherry-Cry2oligR489E, HSE:mCherry-Cry2olig-PYD andHSE:mCherry-Cry2olig-CARD. All inserts were verified by sequencing, and constructs are deposited in the European Plasmid Repository (https://www.plasmids.eu) For expression of transient constructs, we co-injected expression plasmids with Tol2 transposase mRNA (100 ng/µl) in embryos at the one-cell stage.The Cry2olig-mCherry construct (plasmid #60032) was obtained from Addgene, and plasmids containing Larvae were screened at 2.5 dpf for expression of tag:RFP in the heart as described in asc were used with the following sequences: 5′-GGTGGAGATCGAAGATCAAG-3′ and 5′-GCAGCTGCAGGAGGCTTTTG-3′ respectively. Guide RNAs were synthesized using MEGA shortscriptTM Kit (Applied Biosystems), according to the manufacturer’s protocol, and were purified using RNeasy Mini Kit . Cas9 mRNA was synthesized using mMESSAGE mMACHINE SP6 Transcription Kit . 2 nL of a mixture containing 250 ng/μL gRNA and 0.1 mg/mL Cas9 protein was injected into the yolk of one-cell AB zebrafish embryos.We designed a custom gBLOCK (Integrated DNA Technologies) incorporating a guide RNA-targeting sequence preceded by a T7 promoter sequence. Guide RNAs against exon 1 of Genotyping was performed by extracting genomic DNA from fin clips or larvae using the QuickExtract DNA extraction solution (Epicentre) and amplifying loci using EMBL in-house Phusion polymerase, primers are listed in the Key Resource Table.caspa, caspb, gsdmea, gsdmeb and caspase-8a. sgRNA target sites were selected for high predicted cleavage efficiency within various exons of candidate genes using CCTop (The F0 CRISPR screen was performed as described in ng CCTop .Sequences of synthetic sgRNAs and gene identifiers are listed in the key resource table. We used 3–4 synthetic sgRNAs (Sigma) per gene, which were diluted in water to a concentration of 100 µM. Cas9 protein was produced by the EMBL PEPCore facility and stored in a concentration of 62.5 µM in Cas9 buffer . For each experiment, we freshly mixed equal volumes of sgRNA and protein solution (1 µl sgRNA per 1 µl Cas9) and injected the mixture into early embryos within the first 15 min after fertilization. All CRISPR KOs were confirmed by PCR and sequencing around target sites.2+ reporter, we used the 488 nm multiline Argon laser (Lasos Laser GmbH). To image mCherry and lyn-tagRFP, we used a HeNe laser at 561 nm. For detection of the far-red dye DRAQ7 we used the HeNe 681 laser.Zebrafish larvae were anaesthetized using 4 mg/ml tricaine pH 7 and mounted on their right side in 0.75% low melting agarose on MaTec glass bottom petri dishes . Live imaging was performed at 20 °C using a Zeiss LSM780 NLO confocal microscope (Carl Zeiss) and a 40×C-Apochromat (NA 1.20) water immersion objective (Carl Zeiss). To image GFP, mNeonGreen and the GCamp6 Ca2) to stimulate oligomerization of optogenetic constructs.We determined the laser intensity for the 488 nm laser at regular intervals using a chroma slide at a fixed laser power of 5%. The total laser power was measured using a laser power meter (THORLABS GmbH Dachau Germany SN: P0023941) equipped with the sensor S121C 400–1100 nm with a capability of measuring 500 mW. Whenever not stated otherwise, we used 5% 488 argon laser power  of the LSM780 NLO and operated the microscope using the Zen Black Software  together with the Pipeline Constructor Macro .For overnight imaging of whole larvae, we used a 20 x air objective (Carl Zeiss) and acquired multiple stacks (2x7) to cover the entire larva. Stacks were then stitched together using the Zen Black software. Stacks were acquired every 15 min for 12–16 hr.For high-resolution time-lapse imaging of the Opto-ASC speck, Opto-PYD and Opto-CARD we used a Zeiss 880 Airyscan and imaged in Airy fast mode with a C-apochromat 40 x/1.2 W Korr FCS M27 objective. For activation of opto-constructs and detection of GFP we used 0.2% 488 nm and for detection of mCherry 1.5% 561 nm. To detect the GCamp6 signal, we optimized imaging conditions to reach a frame rate below 5 sec. The stack size was 8 µm (1 µm z resolution).We used Fiji ImageJ for image analysis . Images Line profiles for high resolution images of specks were normalized by dividing by the average gray value and subtraction of the lowest gray value. For measuring speck size, a line was drawn through the longest diameter of the speck; the diameter was determined by measuring the distance between the two points of maximum slope of the plot profile along the line.To determine the growth of specks, we marked the outlines of the speck in each time frame for Opto-ASC and ASC-GFP separately and measured the area. We started the measurements at the first time point where mCherry/GFP visibly aggregated. We calculated the time difference starting from the appearance of the first mCherry/mKate2 aggregates to the time point where total intensity (intensity * area) of the speck reached a constant value.To determine if a periderm cell was extruded apically, basally or in both directions we used bright field imaging. Anything that was retained below the periderm layer was considered as basally extruded, anything that left the tissue and dissociated from the epithelial layer into the surrounding water was considered apically extruded.2+ signaling in bystander cells in response to cell death, we generated kymographs for a circular area around the dying cells using the Radial Reslice Fiji plugin . P-value ranges are included in figures as follows: * is p<0.05, ** is p<0.01 *** is p<0.001 and **** is p<0.0001. For comparison of different heat shock conditions  and the For the detection of cell lysis, we used Draq7 (Invitrogen), a far-red dye that stains nuclear DNA only if membrane integrity is lost. We preincubated fish in 0.3 µM (1:1000) Draq7 in E3 fish medium for 1 hr and added the dye directly to the low-melting agarose and the fish medium in which larvae were imaged, to a concentration of 0.3. µM.To inhibit GSDMN pore formation, we treated 3 dpf larvae for with 10 µM Disulfiram (Merck) or 25 and 100 µM dimethyl fumarate for 1 hour before and during imaging. Exposure for 2 hr or longer (e.g. over night) was lethal for zebrafish larvae at a dose as low as 1 µM of Disulfiram and 10 µM for dimethyl fumarate (DMF). All inhibitors were dissolved in DMSO.We also treated 2 dpf larvae over night with 10 µM to 100 µM Gasdermin D inhibitor LDC7559 (MedChemExpress). The inhibitor was added directly to the fish water as described in  This important study describes a novel optogenetic approach to study the responses to inflammasome activation and cell death in zebrafish. The validation of the model is compelling, and its application reveals novel insights in the response to cell death. This new model should help further advance future efforts centered on cell death responses in vivo. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  General Statements [optional]We are obviously very pleased with the general support expressed by the referees, and appreciate their critical comments. We detail below how we propose to respond to their suggestions and queries.In view of the fact that my lab is no longer in existence, I will have to rely on the kind generosity of my colleagues at EMBL to host former team members (the two first authors) for a limited period to come back to Heidelberg to carry out any further experimental work that may be needed. This means we will have to limit the work we can do to those experiments with the highest priority. However, we are optimistic that we will be able to obtain indicative results.We will also follow most of the referees’ other suggestions and requests for additional data and quantifications, as outlined  below.Description of the planned revisionsReviewer #1 ):Minor comments1. Specific experimental issues that are easily addressable:There's a significant concern with the use of LDC7559 (line 387) as a putative small molecule inhibitor of gasdermin D function to test roles (or lack thereof) of the zebrafish gasdermins in the ASC-triggered lytic cell death responses. A recent study  has reported that LDC7559 does not inhibit gasdermin D (and possibly other gasdermins) but rather acts as an allosteric activator of PFKL (phosphofructosekinase-1 liver type) in neutrophils and thereby suppress generation of the NADPH required for the phagocytic oxidative burst and consequent NETosis. Thus, use of LDC7559 as a presumed gasdermin inhibitor in the current manuscript is problematic and should be deleted. As an alternative pharmacological approach to suppress gasdermin function, the authors might consider the use of either disulfiram  and/or dimethylfumarate . These would be straightforward additional experiments.We have ordered the reagents to do these experiments. We are optimistic that we will obtain data that will strengthen this part of the ms and be a pointer for future studies by others.We propose to keep the information on LDC7559 included, but to discuss the reservations the referee lists above – otherwise, others might ask why we did not even try this inhibitor.2. Are prior studies referenced appropriately? there are some problems; see below.2a. One paper is cited twice in lines 724-726 and 727-729.2b. Another paper is cited twice in lines 790-792 and 793-795.2c. No journal is included for the referenced study by Shkarina et al. in lines 827-828.2d. No journal is included for the referenced study by Stein et al. in lines 831-832.2e. No journal is included for the referenced study by Masumoto et al. in lines 793-795.2f. No journal is included for the referenced study by Kuri et al. in lines 774-775.We are embarrassed about these omissions and mistakes and have corrected them..3. Are the text and figures clear and accurate? Generally, yes but with a few exceptions noted below:3a. line 28: "morphological distinct" should read "morphologically distinct".3b. line 161: this sentence contains in parentheses "for how long?" I think this was a comment by one author that wasn't removed from the final submitted manuscript.3c. line 945: spelling "balck" > "black".3d. line 268: "whereas showed a delayed speck formation": the authors need to specify what model/ condition showed a delayed speck formation.3e. line 262: spelling "egnerated" > "generated".Thank you, all corrected.Reviewer #2 ):1. In Figure 3 and Figure 4, the majority of Opto-Asc localizes to the plasma membrane but not endogenous Asc. It seems that tagging affects its localization, which could potentially explain its slow kinetics in oligomerization.-1 and LRRFIP2 OPEN; https://doi.org/10.1038/srep29834).That is an interesting suggestion. The membrane enrichment is indeed reproducible and we have no full explanation for it. However, ASC itself seems to have some affinity for the cell cortex as seen by its association with the apical actin ridges in keratinocytes in the resting state (see e.g. figure 3A). Affinity of ASC for actin is also documented in the literature:):Main points:1. So far, it remains a bit unclear how the authors define precisely speck versus any aggregate and the light induced clusters of Cry2 olig. Is it related to the timescale of formation and/or the lifetime of the aggregates? Is it related to their size?There Is no ‘formal’ definition of an inflammatory speck apart from it being the unusually large aggregates that ASC forms once it is activated. Light-induced clusters of Cry2Olig alone, or of Cry2olig fusions with proteins that do not normally oligomerize are much smaller (extensive documentation in the literature).A speck is thus a stable aggregate of ASC which is usually around 1 µm in size and is able to activate downstream caspases. But neither of these aspects alone are unique to ASC: prion-like structures can also be large aggregates (indeed ASC-specks have been compared to prions), and much smaller molecular assemblies can activate caspases. Thus ‘speck’ is more an operational definition, and ‘natural’ specks do have both of these properties, but as our experiments show, the properties can actually be separated. I would rather not try to narrow or change the definition, but leave any further discussion to the experts in the field.Figure 4E shows a number of variants of ‘speck’-like and other multimers: ASC-mKate and Opto-ASC form large single specks in the presence of endogenous ASC. Opto-ASC specks are only slightly smaller than those formed by endogenously tagged ASC-GFP . Opto-PYD recruits endogenous ASC and becomes incorporated into a speck of approximately the same size, while Opto-CARD does so less efficiently. All of these kill cells. In the absence of endogenous ASC, Opto-ASC forms much smaller specks, and very many in each cell, but these are still functional as seen by the fact that they still kill cells . Both Opto-PYD and Opto-CARD also form only the small aggregates (quantification will be included), with Opto-PYD still killing the cell by virtue of its ability to recruit caspases via their PYD, whereas Opto-CARD does not.Since the authors use most of the time constant blue light illumination, could they also assess how long the speck remains after stopping blue light exposure and quantify their lifetime (relative to the CRY2olig cluster lifetime)?Briefly, any speck that contains a functional ASC moiety remains stable and does not disassemble once the blue light is turned off. In skin cells it is not possible to make quantitative measurements because they are killed by the speck. Opto-ASC specks remain stable until they are taken up by macrophages, as originally reported for ASC-GFP specks in Kuri et al. 2017.Stability can best be assessed in muscle cells, which do not die upon speck formation. The figure below shows that specks begin to form within minutes of a short pulse of illumination and remain stable (and indeed grow further) for at least 60 min.Similarly could they provide some comparison of the size and localisation of CRY2 olig clusters compared to the speck.For size, see above. In addition, the size of the Cry2 oligomers as well as of Opto-ASC specks can vary with expression levels.For location, Cry2olig clusters are usually distributed throughout the cell, as seen in most of the right panels in Figure 4E, and in earlier work in cultured cells . ASC specks can form anywhere in the cell, while Cry2olig-ASC has a preference for the cell cortex, but this is not absolute. In keratinocytes, but not in basal cells, the speck usually forms close to the lateral membrane. In the absence of endogenous ASC no real speck is formed but Opto-ASC in this case shows no clear localisation of Opto-ASC to the membrane.In view of the variation we see, a strict quantification is difficult: what would be the ‘correct’ definition of classes to look at? To make statistically significant statements, we would need an enormous number of examples in which we could control for all of the variation of expression levels, cell size, day to day variation etc, and we currently don’t have these. We hope the qualitative evidence in the micrographs we show represents the differences well, and we will be happy to provide a larger number of images, if the referees feel this would be helpful.With the non functional CRY2olig Asc fusion (Cter fusion), do they still see transient olig2 clustering which then reverse when blue light illumination is gone? I think it might be useful to clarify these points in the main text since most of the quantifications are based on speck localisation/numbering, so their characteristics have to be very well defined.That would be interesting to work out, but after our initial experiments with this construct, we did not pursue this further, since it was not a pressing issue at the time. If we can fit this into our planned experimental time table, we will re-assess it. However, while of interest, we feel these data would not add substantially to what we know at this point.2. In all the snapshots of speck formation, there seems to be a relative enrichment of the ASC signal at the cytoplasmic membrane (relative to the cytoplasm) prior to strong speck formation. This seems specific of optoASC as it does not seem to happen for the endogeneous ASC or upon overexpression of ASC-mKate (both in this study and in the previous study published by the same group). Is this apparent membrane enrichment something reproducible? (I see that on pretty much every example of this manuscript). If so what could be the explanation? Is there an actual recruitment at the membrane or is it because the membrane/cortical pool takes longer to be recruited in the speck (hence looking relatively more enriched at intermediate time points).See our speculations in response to point 1 of the first referee.We too would really like to understand this, but see no easy and efficient way of testing it at this point.3. There is also a very distinctive ring accumulation that seems to match with apical constriction and/or a putative actomyosin ring  . Is it something already known? Could the authors comment a bit more on this? This could suggest that ASC accumulates in actomyosin cortex, which would be a very interesting property.We see that we had failed to be clear about this.There are two types of actin-labelled rings that appear around dying cells. One is formed by the epithelial cells that surround the dying cell. This structure becomes visible as soon as the cell begins to shrink. That it is formed by the surrounding cells is clear from mosaics where the dying cell does not express the actin marker  and the parts of the ring are seen only in the subset of surrounding cells that do express the marker. This ring is also not circular, but follows the polygonal shape of the shrinking cell. We believe that this is the contractile structure that closes the wound, as observed in many other cases of wound healing.The other is the one the referee describes here. It is formed within the dying cell, as shown by the fact that it is visible in labelled cells when all the surrounding cells are negative for the marker. The other difference is that it appears only once the dying cell has already contracted considerably and begins to round up and be extruded . The third referee had raised a similar point in relation to the same structure seen in Figure 6C, and we provide below the requested analysis. It relies on resolution in the y-axis, which is unsatisfactory, but nevertheless, it is clear that this ring is in a plane above the apical surface of the epithelium . It may well simply be actin appearing in the entire cortex of the cell as it rounds up and looking like a ring when seen from above. A completely different method for imaging would have to be set up to document this reliably, but we hope that these explanations help to clarify the confusion we may have created.We do not see this accumulation in cells that leave the epithelium towards the interior (see figure in the response to ‘minor points’ below).4. In the end, since cell death can also occur without visible speck formation, I am wondering if they are eventually the most relevant structure to be quantified. Is it because speck can be dissolved upon caspase activation and could it relates to the speed at which caspase are activated (which may not leave enough time for strong aggregation and visible speck formation)? I believe it would help to get more explanation/discussion on this point.As already mentioned above, it is indeed not obvious what the significance of the large speck is . We agree that it is not necessarily functionally relevant for the signalling outcome to quantify this property – but nor was this the purpose of this work. Regardless of what kind of aggregate is formed, the optogenetic tool allows the induction of ASC-dependent cell death, and therefore the study of the ensuing cellular events.5. The compensatory mechanisms that lead to cell death/extrusion despite depletion of caspb is very interesting. Could the authors use some pan caspase inhibitor (like zvad FMK) to confirm that this block opto-ASC cell death also in this context? Alternatively could they check the status of effector caspase activation using live probe (nucview) or immunostaining in the context of caspb depletion?Those would be interesting avenues to pursue. However, for the reason stated above , we are forced to restrict ourselves to the most important experiments, and think we should prioritize the ones mentioned above.6. If I understand well, Figure 7C on the right side suggest that the double KO cells don't extrude . I don't think these results are mentioned at any point in the main text, but it would be important to include them (since this is an important control).This interpretation is in fact correct, and we have changed the labelling in the figure to ‘no immediate death’7. Waves of calcium following cell death and cell extrusion have been previously characterised . Interestingly, in this previous article they observed waves of calcium near Caspase8 induced death (in MDCK) as well as near laser induced death in zebrafish, while apparently the authors don't see such Calcium waves upon Caspase8 activation in the zebrafish here. I think it would be important to include a comparison of the authors results with this previous paper in the discussionWe have included this in our discussion.8. There is also a previous study which characterised the impact of caspase1 on cell extrusion  which promotes apical extrusion in Caco2 cells. I think it would also be important to include this work in the discussion and to compare with the results obtain here in vivo.We have included this in our discussion.Other minor points:1. Line 439: are the numbers given in percentage? if these are absolute numbers, it is out of how many cells ? Same remark line 445: what are the number of cases representing? (percentage?)We have rephrased this to make it unambiguous.2. Figure 5: could the authors show periderm and basal cell extrusion with the same type of markers? (membrane or actin or ZO1)? This would help to really compare accurately the morphology and the remodellings associated.We used Utr-mNeonGreen to lable actin both in periderm and basal cells. Actin labeling of extruded periderm cells is shown in figure 6C, actin labeling of a dying basal cells and the overlying periderm cells is shown in supplementary figure 5A.3. Is there any obvious differences in cell size or characteristic cell shape between the classic lab strains  and the WIK and experiment strain used here? I do acknowledge that this is clearly not the focus of this study, but given this striking difference (which is related to an important question in the field of extrusion), it would interesting to mention this if there is anything obvious.We will make these measurements and include the data.4. Figure 6C: what is exactly the localisation in Z of this strong actin accumulation observed during apical extrusion? Is it apical or is it rather on the basal side of the cell? A lateral view of actin could be useful in this figure for all the different conditions described.See response to ‘main point 3’ above.The images that show this are below. However, even from these images it is hard to appreciate the locations. They are in fact much easier to see by following the movies over time, and through the z-sections at any given time point. We will of course submit the movies with the manuscript.5. Figure S3B: could the authors show the utrophin-neonGreen channel separatly? Is there a ring of actin in the dying cell? Also are the membrane protrusion formed more basally? .6. Figure 4A legend: I guess the authors meant red arrowheads rather than frame ?This has been corrected7. I list below a number of typos I could find in the main textThanks for noticing these, we have corrected all of these, as well as further typos we found.Line 29: in.Line 30: but.Line 151 : from the …[…] (tissue ?)Line 161: there is most likely a text commenting that was not removed (for how long?)Line 262: generated (egnrtd).Line 268: whereas showed a delay (the subject is missing).Line 269: a point is missing.Line 362: which the lack.Line 368: a point is missing.Line 400: a space is lacking "cellsdepending".Line 438: shrinkwe (space).Line 459 : or I infections.Line 525: there is a point missing.Description of the revisions that have already been incorporated in the transferred manuscriptWe have thoroughly re-edited the text, not only for the typos etc. mentioned by the referees, but also for other aspects of grammar, ambiguity etc. We also added text on the additional points in the main text and discussion as suggested by the referees. We will obviously have to do a final edit once we have carried out the additional experiments.Description of analyses that authors prefer not to carry outAs indicated in the introductory note, we would love to continue this project, including in the directions the referees propose, but we will have to limit ourselves to those that are most important to strengthen the points we make. The experiments mentioned by referee 3 under point 5 would take some testing of conditions and reagents, and while we will give it a try while the two first authors will have the opportunity to return to EMBL for additional experimental work, we can only do this if the experiments with higher priority will work as smoothly and efficiently as we are hoping they will. And we agree with the referee that this is an interesting avenue to pursue, but feel it is not essential to support the validation of the tools we describe or support the other results we present.5. The compensatory mechanisms that lead to cell death/extrusion despite depletion of caspb is very interesting. Could the authors use some pan caspase inhibitor (like zvad FMK) to confirm that this block opto-ASC cell death also in this context? Alternatively could they check the status of effector caspase activation using live probe (nucview) or immunostaining in the context of caspb depletion?Those would be interesting avenues to pursue. However, for the reason stated above , we are forced to restrict ourselves to the most important experiments, and think we should prioritize the ones mentioned above.


Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders  and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants. Dysglycaemia refers to an abnormality in blood glucose level, which can include hypoglycaemia (low blood glucose level) or hyperglycaemia (high blood glucose level). Monogenic diabetes is a rare form of inherited diabetes mellitus caused by heterozygous or homozygous defects in a single gene involved in β-cell development or function. It accounts for approximately 1% to 6% of pediatric diabetes patients . The mosGenetic testing is strongly recommended for a rapid and accurate diagnosis of monogenic diabetes or CHI in order to allow the clinician to make a correct diagnosis.In the past 10 years, the next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research ,9. NGS eIn this manuscript, we aim to describe the strategy for the validation of a home-made NGS “dysglycaemia panel” consisting of 44 candidate genes related to glucose metabolism disorders  and FRG, using Ion AmpliSeq technology combined with Ion-PGM. We also reported the results of the first genetic analyses.All the procedures adopted for the validation of the home-made NGS “dysglycaemia panel” follow the guidelines reported in ACMG Standards for clinical next-generation sequencing .Thirty-two anonymized DNA samples of patients, previously genotyped by Sanger sequencing, were used to optimize the methodology and perform the panel validation. Different types of mutations such as missense, nonsense, frameshift A localizGenomic DNA was extracted from EDTA whole blood using QIAamp DNA Blood Midi kit  and purified with Amicon Ultra 0.5 mL  according to standard procedures. Eluted DNA was quantified with Nanodrop Spectrometer .www.ampliseq.com (accessed on 30 March 2023)) according to hg19. The list of the genes included in the NGS panel is reported in Primers for 44 genes causative of dysglycaemia and its complications were designed using the Thermo Fisher Scientific Ion AmpliSeq Designer platform  and then quantified following the Qubit 2.0 fluorimeter  instructions using HS dsDNA kit. The quantified libraries were pooled and diluted once to 100 pM.The library was amplified by emulsion PCR on Ion sphere particles (ISPs) using the Ion PGM Hi-Q OT2 Kit according to standard protocol . Ion 316 chips were used to sequence eight samples simultaneously. Sequencing was performed on an Ion PGM System  using the Ion PGM Hi-Q Sequencing kit according to the manufacturer’s instructions. Sequencing data were analyzed with Coverage Analysis and Variant Caller plugins available within the Ion Torrent Suite software TS 5.18 and contextually with Ion Reporter. All the pathogenic or likely pathogenic variants detected by Ion PGM were confirmed using specific couples of primers by direct sequencing of the PCR products.In all the runs, the mean coverage was over 200×, and the mean length of the amplicons was about 200 bp. All data of the variants selected for the evaluation of the “dysglycaemia panel” performance are reported in In this study, we report the successful validation of a custom-designed targeted Ampliseq panel for the detection of variants causative of dysglycaemia and its complications. Recently, in the literature, several reports on the application of NGS approach for genetic diagnosis of monogenic diabetes have been published ,18,19. AIon torrent sequencing system performed by PGM measures the hydrogen ions released during the incorporation of dNTP in the amplification of the DNA target template. The protons release causes a decrease of the pH level in the solution present inside the chip. Ph variation is directly proportional to the number of bases incorporated in the template. When target region is rich of homopolymer repeats, the multiple incorporation of dNTP may lead to a high number of hydrogens released, and a high electronic signal was detected by the sensor. Consequently, variants occurring in this genomic region may be missed In our study, all the four missed frameshift mutations are not located in the genomic region rich of homopolymers on repeat regions. Therefore, we may suppose that the failure of detection was related to the primer design used for the library amplification, even if the theoretical coverage of HNF1a, GCK, and SLC5a2 genes was predicted as 100% by the Ampliseq primer design tools. As a consequence of this fact, in order to detect these variants, we decided to include in our protocol the Sanger sequencing of GCK exon 10, HNF1a exon 4 and 7, and SLC5a2 exon 11 using specific pairs of primers (available on request).MODY is defined as an autosomal inherited form of diabetes mellitus caused by mutations in a single gene. In 2013, Lopez et al. described the co-inheritance of HNF1a and GCK mutation in one MODY patient . In the In conclusion, our panel based on the Ampliseq approach can be useful for identifying pathogenic variants in multiple genes in a short time. This could help to detect several defects in children and young adults who need to receive a genetic diagnosis necessary for optimal treatment. Sanger sequencing will be included in our analytical protocol to avoid missing frameshift variants due to dysglycaemia.


A Bonferroni correction was applied, resulting in an alpha level of 0.005. A total of 279 pharmacists participated in this study. After the CE session, mean knowledge scores increased . After the CE session, there was no significant increase in pharmacists’ knowledge about the approach that is not recommended when discussing vaccination beliefs with a patient , determinants of vaccine uptake , and social determinants of health that can influence vaccination rates . There was a significant change in pre- and posttest knowledge for the remaining seven items.Pharmacists promote vaccinations and challenge misconceptions about vaccine hesitancy, yet pharmacists’ knowledge of vaccine confidence has not been assessed. The objective of this study was to compare pharmacists’ knowledge of coronavirus disease 2019 (COVID-19) vaccine confidence before and after a live continuing education (CE) session. This pretest–posttest study evaluated the differences before and after a live CE session on COVID-19 vaccine confidence provided to pharmacists at a nationwide health technology company. Participants’ total pretest and posttest scores were compared using paired  The Centers for Disease Control and Prevention (CDC) define vaccine confidence as the conviction that vaccines are effective, safe, and part of a trustworthy medical system . BuildinThe strategies to influence people’s confidence in COVID-19 vaccines include improving healthcare professionals’ confidence in vaccines, encouraging healthcare professionals to get vaccinated, and healthcare professionals recommending vaccines to patients . A coordp < 0.001) [p < 0.001) [p < 0.001) [Previous work has explored vaccine hesitancy among healthcare professionals, and assessed pharmacists’ perceptions and acceptance of COVID-19 vaccines ,15,16. F< 0.001) . Another< 0.001) . FurtherThe purpose of this study was to compare the knowledge of COVID-19 vaccine confidence before and after a CE session on this topic among pharmacists at a Nationwide Health Technology Company in the US.This study used a pretest–posttest study design. Pharmacists employed at a Nationwide Health Technology Company in the US, who participated in a company-sponsored CE session designed to provide information on cultivating confidence on vaccines (described below), were eligible to participate in the study.CE sessions are an internationally recommended lifelong learning approach for healthcare professionals to equip them with the necessary knowledge, skills, and ethical attitudes, while keeping them current and competent in their practice ,21. The The pretest–posttest questionnaire consisted of 10 knowledge-based items based on the content of the CE presentation. The questionnaire was developed in an iterative fashion by the research team, with regular input from the clinical pharmacists. The 10 questionnaire items included: (1) what percent of COVID-19 cases in the US are due to the Delta variant as of September 2021 ; (2) the percentage of US adults (>18 years old) who are fully vaccinated (as of September 2021); (3) the approaches that should not be taken when discussing vaccination beliefs with a patient; (4) the recommended order in which a healthcare provider should have a conversation with a patient about vaccines, according to the WHO; (5) the most effective vaccine after >120 days of full vaccination (as of August 2021); (6) the health outcomes of myocarditis for adolescents (12–17-year-olds) who are vaccinated versus those who are unvaccinated (for every million second dose of vaccination over 120 days); (7) determinants of vaccination uptake; (8) social determinants of health that can influence vaccination rates; (9) the percentage of adults who stated that they will not get the vaccine according to the national immunization survey (as of September 2021); (10) assessment tools that can be used to identify and assess an individual’s social determinants of health. Each item had five response options. Participants were asked to select the one best response option provided for each item. Items were selected to represent the specific content covered in the CE presentation. The content validity of the questionnaire was reviewed and approved by a team consisting of experienced clinical and research pharmacists. The items had an internal consistency (Cronbach’s α) of 0.65, which indicated that the items would form an acceptable scale of moderate internal consistency .t-test, where an a priori alpha level of 0.05 indicated significance. Pretest and posttest scores for each of the ten items were compared using chi-squared tests. A Bonferroni correction was applied to account for the multiple comparisons, resulting in an a priori alpha level of 0.005 (0.05/10 = 0.005) for the categorical analysis. All analyses were conducted using SAS on Demand for Academics .Pharmacists were invited to voluntarily participate in this study via email two weeks before the CE session. Pretest data were collected up to two weeks before the CE presentation, and posttest data were collected immediately after the CE presentation using the proprietary learning software provided by the health technology company. The pretest and posttest responses were matched using a unique identifier within the software. Each item was scored 1 or 0 depending on whether it was correct (1) or incorrect (0), and scores were summed for each subject. Scores therefore ranged from 0–10 for each subject. The mean pretest and posttest scores were compared using a paired p < 0.0001). The percentage of correct answers for each item asked during the pretest and posttest is shown in A total of 279 pharmacists from the health technology company participated in this study. The mean knowledge scores of cultivating confidence in vaccines significantly increased from 5.2 ± 1.5 before the CE session, to 7.4 ± 1.35 after the CE session (p < 0.005). However, there was no change in the proportion of correct answers pre- and posttest for the remaining items regarding the options that describe the approach not recommended when discussing vaccination beliefs with a patient , determinants of vaccine uptake , and the social determinants of health that can influence vaccination rates .After the CE session, there was a significant increase in pharmacists’ knowledge about the percentage of COVID-19 cases due to the Delta variant, the percentage of US adults (≥18 years old) who are fully vaccinated (as of September 2021), the recommended order in which a healthcare provider should have a conversation with a patient about vaccines according to the WHO, the vaccine that is most effective 120 days after full vaccination, the health outcomes of myocarditis for adolescents who are vaccinated versus those who are unvaccinated, the percentage of US adults that have stated they will not get the vaccine according to the National Immunization Survey (as of September 2021), and the assessment tool that can be used to identify and assess an individual’s social determinants of health , a positive attitude (mean 8.43), and good practice  in regards to COVID-19 [In the pretest, there were three items for which the proportion of participants who correctly answered the item exceeded 70%, and no significant improvement was seen for the posttest . This indicated that pharmacists already had knowledge of these aspects of vaccine confidence. A cross-sectional study undertaken in Pakistan to identify the status of knowledge, attitude, and practice regarding COVID-19 among healthcare workers reported that the majority of healthcare workers have good knowledge  of their respondents correctly indicated the items addressing specific vaccinations for newborns , and nearly all of their respondents (>90%) recognized the need for further education in this field . FurtherIn light of the evidence of vaccination knowledge gaps among pharmacists, the spread of vaccine hesitancy among healthcare professionals, and low participation rates in CE sessions despite their benefits, the findings from this study will contribute to the literature in various ways. First, to our knowledge, this is the first study that assessed a CE session for improving pharmacists’ knowledge of COVID-19 vaccine confidence. In response to the COVID-19 public health crisis, the results from the current study demonstrated that by allowing pharmacists to obtain an educational intervention, such as a continuing education session on COVID-19 vaccine confidence, is important in enhancing pharmacists’ knowledge to provide appropriate counseling for their patients and supporting vaccination uptake. Second, the study findings provided additional evidence to the literature of the role of CE sessions in improving knowledge among pharmacists. This has been demonstrated by the significant increase in pharmacists’ knowledge of COVID-19 vaccine confidence after the CE session. Finally, the study identified various COVID-19 knowledge gaps among pharmacists before the CE session. This finding adds more to the literature regarding COVID-19 knowledge gaps among pharmacists. As pharmacists have an important role in promoting and supporting the uptake of vaccination, the need for lifelong learning for pharmacists on this topic and others is important.Limitations of this study include the nature of the pre- and posttest research design. It is common that a pretest will likely get participants to be more aware of the test itself, and alert participants to the limited material required to score better on a posttest rather than acquiring adequate general knowledge for the subject of interest. In addition, the intervention was limited to one continuing education session, and the posttest was administrated immediately after the end of the session, which is primarily a measure of short-term memory retention. However, previous research has demonstrated that short-term knowledge gains are associated with long-term retention; this does not necessarily reflect the magnitude of knowledge acquired over the long term ,31. DemoFuture studies may include addressing items where pharmacists’ knowledge remained low, and evaluating the effect of the CE program among pharmacists at other organizations or to other healthcare professionals. In addition, pharmacists’ knowledge should be assessed over a longer time period  to measure longer-term knowledge retention. The relationship between knowledge about vaccines and their sociodemographic variables should be considered in future research as well. Continued work to raise awareness among pharmacy professionals regarding their role in educating citizens about health aspects, such as the importance of vaccination, is recommended. In addition, further assessment is needed regarding pharmacist’s skills in communication and identification of false beliefs among the population.This pretest–posttest study showed that pharmacists’ mean knowledge scores of COVID-19 vaccine confidence significantly improved after attending a CE session on the topic. Further work is required to address knowledge gaps that remained after the CE session, and to expand this CE opportunity to other healthcare professionals in different settings.


Timely assessment and treatment of patients with neovascular AMD (nAMD) are crucial to preservation of vision. Loss to follow up (LTFU) in these patients is a problem but this has not been systematically investigated.A retrospective review of electronic medical records of patients with nAMD first treated with anti-VEGF therapy from 1st Jan 2014 to 31st Dec 2018, was conducted in January 2021. Any patient not seen for more than 12 months was classed as no longer attending.Of the 1328 patients who attended between 2014 and 2018, 348 had failed to attend and were eligible for inclusion in this study. Reasons noted for discontinuation of care: discharged by clinician (33.3%), died (20.7%), moved to another unit outside of area (17.5%), stopped attending due to ill-health (13.5%), discharged due to failure to attend (5.6%) and patient choice to no longer attend (4.6%). There were 16 (4.6%) who did not receive any further appointments despite clinician request for follow-up. After 5 years, 50.5% of patients were no longer attending for treatment. Age was a factor in failure to attend, with 7 out of 12 patients aged >100 years no longer being followed up, compared to 1 out of 11 of 50–59 year-olds.When analysing visual outcomes in an AMD service it is important to characterise the patients who are lost to follow up. The outcomes for this group may be avoidably poor and understanding the factors influencing LTFU rate is crucial to addressing shortcomings in a hospital AMD service. Age-related macular degeneration (AMD) is the second most common cause of visual impairment in the developed world. The prevalence of cases is increasing due to the aging population . In the In the UK, the cost of anti-VEGF treatment is covered by the National Health Service and is free-of-charge for the patient. Our tertiary referral centre follows the Treat and Extend regimen for treatment, with patients receiving intravitreal injections at intervals based on their disease activity . If the This study aimed to identify patients with neovascular AMD (nAMD) who were no longer attending regular hospital follow-up and the rate at which they dropped out of treatment. Our aim was to investigate factors influencing discontinuation of anti-VEGF therapy.A retrospective electronic case notes review was undertaken of patients who received an initial intravitreal injection of anti-VEGF  during the period 1st January 2014 to 31st December 2018 at Gloucestershire Hospitals NHS Foundation Trust. These data were taken from electronic medical records (EMR) kept on Medisoft or Intersystems TrakCare. Those over 50 years of age with a diagnosis of neovascular AMD and who received an initial injection of aflibercept, ranibizumab or bevacizumab in either their first or second eye during this time period were included in analysis.The following data were recorded for each patient: age at time of data collection (or at death), gender, first and last visit dates, LogMAR best-corrected visual acuity (BCVA) at baseline and at last follow-up visit in the treatment and fellow eye , number of injections in treatment eye, whether on regular treatment or post-treatment observation, and presence of active or inactive disease in the fellow eye.Patients who did not have a follow up appointment for greater than 12 months were recorded as no longer attending, and therefore included in our study. For these patients, EMR entries recording reasons for ceasing to attend were noted. This data had been accurately recorded over the 5-year period by the AMD coordinators with entries made onto the electronic health record if a patient phoned up or cancelled an appointment and we have reported the data that they recorded. If patients failed to attend a minimum of three booked appointments without contacting to rearrange, this led to an automatic discharge from follow up. The reasons provided for discontinuation of treatment included: follow-up cancelled by patient, patient deceased, discharged by clinician, multiple failures to attend leading to automatic discharge, ill-health, or transfer to another unit. Patients discharged were divided into two groups: those in whom further treatment was felt to be futile (below treatment criteria = BTC), either due to failure to improve or progressive decline in visual acuity despite treatment, or those discharged after a period of disease inactivity on OCT.A total of 1328 patient received an initial anti-VEGF injection for nAMD in the study period, January 2014 to December 2018. The mean age was 84 years (range: 53–103 years) at death or last visit. There were 348 who had not had a follow-up appointment after 1 year from last appointment, and were therefore assumed to have discontinued care: 223 women (64.1%) and 125 men (35.9%). The mean duration of follow-up for these patients was 26.3 months (±2.1), with on average 6.8 (±5.0) injections received.n = 71), or their BCVA was consistently below treatment criteria (LogMAR 1.20) and further anti-VEGF injections were thought to be futile (n = 45). Table p = 0.006, paired sample t-test). The average age in this group was 87 years (range: 62–103 years), and 61.2% were women.In 116 patients (33.3%), the primary reason for stopping follow-up was discharge from hospital eye services. All of these patients had ceased anti-VEGF therapy. They had inactive disease with stable visual acuity and OCT appearance  had died. A further 47 (13.5%) patients were unable to attend further follow up due to ill-health. Sixty-one patients (17.5%) moved out of the Gloucestershire area to seek further follow-up in another NHS Trust.n = 16, 4.6%), those who did not attend on more than three occasions and were automatically discharged , and finally those who did not receive any further appointments despite clinician request for follow-up . Further detail about these patients is provided in Table The remaining LTFU patients whose anti-VEGF therapy abruptly stopped were those who declined any further follow up  and Gillies (53% at 5 years) and comparable to those reported by Pushpoth in another UK-based study (49% at 4 years) and by Falk (47% at 4 years) . Vaze reWe found that few patients cancelled appointments due to anxiety around injections or found injections too painful to continue. Of our cohort, this was only the case for four patients, who were all LTFU in the first 2 years of treatment. These patients had six injections before cancelling follow up, demonstrating a window of opportunity to counsel patients or discuss options for anaesthesia to prevent drop out.Comparable studies have shown that issues with transport are a major burden to patients who have to attend regularly . In DroeIt is of concern that 16 people were not sent a follow up appointment. Of these 16 patients, 15 were not receiving regular treatment because the choroidal neovascular membrane had stabilised. It is possible that these patients had therefore been discharged, but without the decision being formally documented in their medical records. The final patient who was not sent an appointment had initially cancelled due to travelling and later died. These patients who are lost to follow up due to administrative errors, rather than clinician or patient choice, are extremely important to identify to prevent avoidable harm. As reported by Davis, vulnerable patients are at higher risk of harm from LTFU, and AMD patients are in this category, as many are elderly or visually impaired .The limitations of this study include its retrospective design, relying on the accuracy of clinical documentation. Using this method of patient identification for LTFU may also miss some patients, as some may not have been seen for more than 12 months and then been re-referred to our service, and, therefore, not been included in this dataset. Greater than 12 months since last visit was deemed to be a sufficient time period to class as LTFU, as it was felt unlikely that patients would have an interval greater than one year between appointments. This is also the consensus from other authors , 19. It This study provides reassuring evidence that when the decision is made to permanently stop treatment in nAMD, it is a decision that is usually made by the lead clinician rather than the patient due to pain, anxiety, transport issues or unsatisfaction with visual outcomes. This appreciation of the causes for loss to follow up, and those at higher risk, allows us to better personalise services in the future to improve patient outcomes. Further analyses of the reasons for LTFU from larger datasets, such as the National Ophthalmology AMD dataset, will allow greater planning of service provision.In 2014 the Health Quality Improvement Programme commissioned the Royal College of Ophthalmologists to undertake a feasibility audit in the treatment of “wet” Neovascular Age Related Macular Degeneration.In the feasibility audit data were not available to explain why patients were lost to follow-up and only six of 32 centres had any patients reported as having died.This study provides a detailed account of the reasons for loss to follow up, which should provide a benchmark for services to compare their loss to follow up results with in the newly commissioned National Audit of treatment of “wet” Neovascular Age Related Macular Degeneration.