SPECT-derived dose estimates in tissues of diameter less than 3× system resolution are subject to significant losses due to the limited spatial resolution of the gamma camera. Incorporating resolution modelling (RM) into the SPECT reconstruction has been proposed as a possible solution; however, the images produced are prone to noise amplification and Gibbs artefacts. We propose a novel approach to SPECT reconstruction in a theranostic setting, which we term SPECTRE (single photon emission computed theranostic reconstruction); using a diagnostic PET image, with its superior resolution, to guide the SPECT reconstruction of the therapeutic equivalent. This report demonstrates a proof in principle of this approach.68Ga/177Lu IEC phantom study and a clinical example using 64Cu/67Cu.We have employed the hybrid kernelised expectation maximisation (HKEM) algorithm implemented in STIR, with the aim of producing SPECT images with PET-equivalent resolution. We demonstrate its application in both a dual SPECTRE is shown to produce images comparable in accuracy and recovery to PET with minimal introduction of artefacts and amplification of noise.The SPECTRE approach to image reconstruction shows improved quantitative accuracy with a reduction in noise amplification. SPECTRE shows great promise as a method of improving SPECT radioactivity concentrations, directly leading to more accurate dosimetry estimates in small structures and target lesions. Further investigation and optimisation of the algorithm parameters is needed before this reconstruction method can be utilised in a clinical setting. Nuclear medicine functional imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT) are used extensively for the diagnosis of disease, planning/staging of therapy, radionuclide therapy (RNT) dosimetry calculations and monitoring of response to treatment. More recently, the term “theranostic” has been introduced in nuclear medicine to describe a single molecule, peptide or other targeted agent that is used for both diagnostic imaging and radionuclide therapy labelled with different radionuclides.177Lu) and copper-67 (67Cu) of approximately 20 mm FWHM severely limits the recovery of accurate radioactivity concentrations in vivo due to the PVE MeCOSar-Octreotate (“SARTATE”)  in a trial of subjects who were being treated for unresectable, multi-focal meningioma. The subject presented here received 177 MBq of [64Cu]SARTATE  for treatment planning and was imaged using the same PET/CT scanner as the phantom study. The patient was scanned from vertex of skull to mid-thigh at 1, 4 and 24 h post-injection, with a scan time per bed position of 180 s, 180 s and 300 s in the respective scans. Subsequently, images from cycle 1 of 67Cu therapy with 5.1 GBq of [67Cu]SARTATE  administered 4 weeks after 64Cu imaging were used in the SPECTRE reconstruction. 67Cu SPECT imaging was performed using the same dual-head SPECT/CT system as described previously, with medium-energy-low-penetration (MELP) collimators and an acquisition matrix size of 128 × 128. The 4-h time point data were acquired for 8 s per projection at 3 bed positions covering the vertex of skull to mid-thigh. One hundred twenty projections were acquired over 360° (3° sampling) using continuous rotation mode. Two energy windows were acquired: 67Cu photopeak (163–199 keV) and a lower energy scatter window (143–162 keV). The 4-h post-injection time points for both the PET and SPECT acquisitions were used in the example presented here. An initial OSEM + RM reconstruction of the SPECT data was used to co-register the 4-h 64Cu PET image to the matched time-point 67Cu SPECT data, to be used as the guiding modality in the SPECTRE reconstruction.The SPECTRE reconstruction approach was then applied to a clinical theranostic study using [p = 1, σs = 1, σdp = 5 mm, σds = 5 mm, NN = 3. As in the phantom example, these parameters were chosen based on subjective image quality. Quantitative images with voxel units of kBq/ml were generated for each of the SPECT reconstructions and were co-registered to the matched time-point PET image. VOIs were generated on the PET image using a threshold  and were propagated to each of the SPECT reconstructed images. The SUVmean and SUVmax in each VOI for all reconstructed images was calculated and subsequently used in our analysis.Quantitative SPECT reconstructions used for direct comparisons were the same as those investigated in the IEC phantom study, namely, a conventional OSEM reconstruction with CT-based attenuation and scatter correction (qSPECT) with no post-reconstruction filtering or RM, the STIR-equivalent OSEM implementation with RM (OSEM + RM) and the SPECTRE reconstruction using the HKEM algorithm with RM, with CT-based attenuation correction and window-based scatter correction. No post-reconstruction filter was applied to the OSEM + RM and SPECTRE reconstructions. The number of iterations and subsets for the qSPECT, OSEM + RM and SPECTRE reconstructions were the same as those used in the phantom study, i.e. 4it8s, 40it12s and 40it12s, respectively. The SPECTRE parameters used in the clinical example were σFigure mean and RCmax versus sphere diameter for the 68Ga PET and 177Lu SPECT reconstructions are shown in Fig. The recovery coefficients RCmean) in the SPECTRE reconstruction is higher than the OSEM + RM equivalent in all six spheres. The qSPECT reconstruction has a RCmean of 0.62 in the largest sphere and 0.15 in the smallest sphere, compared to SPECTRE which has a RCmean of 0.82 in the largest sphere and 0.47 in the smallest sphere, showing an improvement in recovery in the largest and smallest sphere by a factor of 30% and > 300% respectively. The maximum recovered voxel values (RCmax) in the four largest spheres of the 68Ga PET series achieves full recovery of the expected radioactivity concentration, whereas the SPECTRE reconstruction only sees full recovery in the two largest spheres. The OSEM + RM reconstruction overestimates the expected concentration in the three largest spheres. Full recovery of the expected concentration in the qSPECT series is only recovered in the largest sphere (RCmax = 1.05). Table mean in the background ROIs for each of the PET and SPECT reconstructions.The mean recovery . Next to this, the same transverse slices for all reconstructions are displayed showing lesions 1, 3 and 4 in a single section through the skull. The same windowing has been applied to each SPECT reconstructed image.Each SPECT reconstruction method using the mean and SUVmax for each VOI are shown in Table It is evident in the qSPECT image that lesions 1, 3 and 4 have poor recovery, as would be expected, compared to PET and both SPECT (OSEM + RM and SPECTRE) reconstructions. The PVE is best illustrated in lesion 4 on the qSPECT reconstructed image; under identical windowing the uptake seen in the VOI is very faint compared to the other reconstructions. The corresponding SUV67Cu SPECT reconstruction without RM (qSPECT) has significantly lower recovery in small VOIs compared to its theranostic 64Cu PET counterpart. The qSPECT 67Cu SUVmean in each lesion is, on average, half that of the 64Cu PET SUVmean estimate. The COV% in the parotids is 16.3%, 25.5%, 31.5% and 26.9% for the PET, qSPECT, OSEM + RM and SPECTRE reconstructions, respectively.Table - emission to deliver the radiation dose also emit photons which can be imaged with conventional gamma cameras. The two radionuclides used in this study both emit photons around 200 keV. The spatial resolution of the gamma camera operating with photons of this energy is poor, measuring around 20 mm FWHM. As many of the metastatic lesions and malignant nodal disease are of this size or smaller it means that we are unable to accurately estimate the amount of radiation delivered to the tissues without addressing the issue of partial volume correction. This, in turn, hampers our ability to better understand the radiobiology of radionuclide therapy, especially in the metastatic setting, and hence lead to more personalised treatments.The field of theranostics in nuclear medicine has enjoyed a great deal of expansion over the past decade. Importantly, this is translating into improved outcomes for patients . However68Ga PET/177Lu SPECT, the SPECT reconstructions that incorporated RM showed improved recovery of the radioactivity concentration in all six of the spheres. The SPECTRE reconstruction showed better recovery of the mean activity concentration in all six of the IEC phantom spheres compared to OSEM + RM for the same number of iterations, with a more noticeable improvement in recovery in the two smallest spheres. Recovery of the concentration in the background (BKG RCmean), that is quantitative accuracy, was similar for all PET and SPECT reconstructions and was within ± 5% of the known background concentration. The SPECTRE reconstruction (for the chosen HKEM parameters) and the 68Ga PET image have similar noise properties in the background compartment, with a COV of 4.3% and 4.1% respectively, compared to the OSEM + RM reconstruction which has a COV of 20.0%, indicative of higher noise in the background. The OSEM + RM (40it12s) reconstruction has a “blobby” noise texture in the background, a result likely due to the reconstruction algorithm fitting the noise in the data too closely, resulting in a modified noise texture that has supressed high-frequency noise features at the cost of amplifying mid-frequency noise correlation with a larger number of iterations [In our phantom study using a combination of erations . This sterations –36.Figure 68Ga point source placed in the centre of the FOV of our PET/CT system has a transverse spatial resolution of approx. 5.8 mm FWHM [68Ga PET IEC acquisition was 7.5 mm FWHM, which is consistent with our previous report of the intrinsic spatial resolution of 68Ga PET imaging: intrinsic reconstructed resolution of 5.8 mm FWHM with a post-reconstruction Gaussian filter of 5 mm FWHM results in a resolution of approx. 177Lu IEC qSPECT reconstructed image was found to be 18.2 mm FWHM compared to the estimated SPECTRE resolution of 8.8 mm FWHM, suggesting an improvement in resolution by a factor of ~2. Improving spatial resolution by a factor of two leads to an expected improvement of volume resolution of a factor of 8 (i.e. 23). For comparison, the OSEM+RM reconstructed SPECT image was found to have a global resolution of 10 mm, demonstrating that conventional PVC methods such as RM do well in recovering radioactivity concentrations and improving resolution, but noise amplification, introduction of Gibbs artefacts, and the overestimation of the true radioactivity concentration in the spheres support the fact that more sophisticated approaches to partial volume correction are needed. The NRMSE results reported in Table 177Lu IEC phantom data demonstrates that a PET image can be used to guide the SPECT reconstruction, producing images with comparable resolution, and hence recovery of true radiopharmaceutical uptake, to PET.We have previously reported that a  mm FWHM . The estmentclass2pt{minimmean and SUVmax in small structures, where the proposed SPECTRE method saw an average increase by a factor of approx. 2.7 in the mean radioactivity concentration in small lesions when compared to the standard qSPECT reconstruction. In Fig. mean by a factor of 3.3. All VOIs in the SPECTRE and OSEM + RM reconstructions have a higher SUVmean and SUVmax than the respective PET measurement, except for the pituitary gland which is significantly higher in PET. This may be attributed to the different physiological conditions between diagnostic imaging and therapy where an amino acid infusion is administered for nephroprotection purposes during PRRT. The amino acid infusion could have a pharmacokinetic effect on the pituitary uptake and retention, causing the radioligand to washout more quickly. The co-administration of amino acids during 67Cu therapy makes it difficult to directly compare uptake in the PET and SPECT images as the biodistribution may be affected. The parotids, being the largest volume investigated in this study, are suitable for comparing the noise characteristics in each of the reconstructed images. For this VOI, the SPECTRE reconstruction had similar noise properties to the qSPECT reconstruction, which was very blurred and hence relatively low in noise level. In contrast, the OSEM + RM reconstruction had a significantly higher COV%, indicative of a higher level of noise.The reconstruction methods that address the collimator-detector response resulted in a higher SUVOur investigations into use of the HKEM algorithm for theranostic PET-guided SPECT image reconstruction have identified a number of issues that need to be considered and investigated further. The HKEM algorithm was originally implemented for reconstructing emission data (PET) using anatomical image data (CT or MRI) as the guiding modality and thus has not yet been optimised for the use of functional data as the guiding modality. Further investigation into how to best implement functional data as the prior needs to be explored. Additionally, accurate spatial co-registration between the PET and SPECT data is a requirement and is affected by issues such as different bed shapes between the PET and SPECT cameras, the position of the subject’s arms (i.e. above their head or by their side) in each modality, and the presence of physiological motion or variability . As we chose to use the head for our clinical study, which is relatively immune to spatial variations in features, the dependence of the SPECTRE algorithm on these spatial misalignments has not been tested as yet and will be the subject of future work.The superior noise properties of the SPECTRE reconstructions seen in this work suggests the possibility that this approach to SPECT image reconstruction will be useful in the reconstruction of low-count SPECT data and this will also be the subject of future work. Optimisation of the SPECTRE reconstruction approach in a theranostic setting will likely involve joint optimisation of the PET image reconstruction, as the guiding modality, combined with a range of HKEM parameters in order to achieve an optimal SPECTRE reconstruction result. Robustness to accurate registration and sensitivity to the differences in the PET and SPECT pharmacokinetics need to be considered.SPECT imaging plays an important role in monitoring organ and lesion absorbed dose but is severely limited by its poor spatial resolution, particularly for medium energy gamma photon imaging. Mitigation of PVEs in order to obtain accurate lesion and normal organ dosimetry is a much-needed step towards personalised RNT, where individualised doses, patient-specific treatment planning, and investigation into radiobiological effects and response to treatment are all directly linked to our ability to make accurate image-based dosimetry measurements. We have demonstrated that the theranostic paradigm can be exploited in SPECT image reconstruction to improve the poor spatial resolution of gamma camera imaging of therapeutic radionuclides when the corresponding PET image is used as guiding information incorporated into the reconstruction process. This novel approach to SPECT image reconstruction, using matched pairs of diagnostic (PET) and therapeutic (SPECT) imaging, is a promising method for improving SPECT image quality and thus our ability to accurately monitor treatment, leading to better quantification of the dose delivered to small structures such as nodal and metastatic disease and target lesions. The SPECTRE approach to image reconstruction shows clear improvements over conventional reconstructions incorporating resolution modelling due to its better noise characteristics and lack of Gibbs and other resolution modelling artefacts. Further investigation and optimisation of the algorithm parameters is needed and should be investigated for a number of theranostic oncological applications that use radionuclides for therapy.


HIV-TRACE that uses a single-linkage algorithm identified more nodes in the same networks that were connected by sparse links than Cluster Picker. This suggests that the choice of the program used for identifying networks should depend on the study aims, the characteristics of the population being investigated, dynamics of the epidemic, sampling design, and the nature of research questions being addressed for optimum results. HIV-TRACE could be more applicable with larger data sets where the aim is to identify larger clusters that represent distinct transmission chains and in more diverse populations where infection has occurred over a period of time. In contrast, Cluster Picker is applicable in situations where more closely connected clusters are expected in the studied populations.HIV-TRAnsmission Cluster Engine (HIV-TRACE) and Cluster Picker are some of the most widely used programs for identifying HIV-1 transmission networks from nucleotide sequences. However, choosing between these tools is subjective and often a matter of personal preference. Because these software use different algorithms to detect HIV-1 transmission networks, their optimal use is better suited with different sequence data sets and under different scenarios. The performance of these tools has previously been evaluated across a range of genetic distance thresholds without an assessment of the differences in the structure of networks identified. In this study, we tested both programs on the same HIV-1  In this study, we expound on a previous study9 that evaluated the performance of these tools by looking at difference risk groups and analyzing the structure of HIV-1 transmission networks identified by both programs. A total of 2,017 HIV-1 pol sequences from cross-sectional surveys conducted in Ugandan fisherfolk communities (FFCs) (n = 728) of Lake Victoria, female sex workers (FSWs) (n = 592), and general population (GP) (n = 697) groups were analyzed for a 7-year period (2009–2016). These populations were surveyed under the Medical Research Council (MRC)/Uganda Virus Research Institute (UVRI) and London School of Hygiene and Tropical Medicine (LSHTM) Uganda Research Unit's Molecular Epidemiology Study that aimed to determine HIV-1 transmission networks in high-risk and GP groups in Uganda. The term fisherfolk was used to refer to persons living in villages that are located along the shores of Lake Victoria, whereas the GP refers to neighboring inland or mainland communities that are mostly agrarian or involved in trade. The term FSWs includes persons involved in either commercial or transactional sex. In this study, transmission networks referred to genetically closely related HIV-1 sequences based on a predefined GD threshold and/or a monophyletic group on a phylogenetic tree with high bootstrap support (>0.95) where two highly similar sequences are known as pairs and more than two sequences as clusters.4 In identifying networks, HIV-TRACE calculates the pairwise GDs between HIV-1 sequences, whereas Cluster Picker relies on maximum GD but also considers a high bootstrap support at the common node for closely related sequences on a phylogenetic tree. A GD of 1.5% was chosen as the optimum threshold for use in this study based on previous evaluations done across different GD thresholds in similar populations.4 In our previous study, we analyzed HIV-1 sequences with epidemiological linkage data across a range of GD thresholds (0.5%–5%) and considered a maximum GD of 1.5% as an ideal cutoff to identify viral transmission networks in the studied populations.1Transmission network analyses are critical for making inferences about HIV-1 spread and patterns of mixing between different populations, important for the design of effective interventions.n = 91) and clusters  were detected at the same GD threshold in HIV-TRACE. This difference in results was attributed to the different algorithms implemented in each program for network identification. HIV-TRACE uses a single-linkage algorithm,7 which means that the networks it identifies can be connected by just a few individuals with sparse links, whereas Cluster Picker searches for more closely connected clusters  that comprised transmission pairs were detected by Cluster Picker, whereas additional nodes  that linked to other external or sparse nodes were detected by HIV-TRACE. This finding suggests that Cluster Picker may be more preferable for use in more homogenous populations such as the FFCs where recent ongoing HIV-1 transmissions are common and where HIV-1 sequences are likely to cluster more densely. In contrast, HIV-TRACE may be more suited for use in populations that are more diverse and where the sampling could span for a longer period of viral transmission. Nonetheless, in both programs, a greater proportion of HIV-1 transmission networks consisted of pairs with fewer larger clusters, which would be expected in a mature and generalized HIV epidemic similar to that in Uganda.7 As both programs use different algorithms to identify linked sequences, HIV-TRACE detected more and larger clusters compared with Cluster Picker at a GD of 1.5%. However, in a separate study9 that evaluated the performance of both programs across a range of GD thresholds (1%–5.3%) using a mixed data set that included next-generation sequence data, HIV-TRACE tended to detect fewer clusters than Cluster Picker at lower GD thresholds (<3%). In this study, the performance of both programs was evaluated at a single GD cutoff determined as ideal for use in our study setting.4We identified more and larger clusters with HIV-TRACE than Cluster Picker at a maximum GD of 1.5%. HIV-TRACE and Cluster Picker both exploit the GD between HIV-1 sequences to detect transmission networks although Cluster Picker additionally uses branch support values on phylogenetic trees to confirm linkages. However, bootstrap values can change as additional sequences are added to a tree, which could cause clusters to diminish when revisiting a data set upon including new sequences. This feature is absent in HIV-TRACE, which makes it a more suitable program for tracking clusters over time. Also, because HIV-TRACE uses a single linkage approach and rapidly computes pairwise GDs between individual sequences, it does not have to store large matrices and can quickly process very large data sets.9 were based on Ugandan HIV-1 sequences that may not be representative of other populations elsewhere in the world.A limitation of this study is that the comparison between HIV-TRACE and Cluster Picker was performed on sequence data where the clusters were not already known. Additional comparison using data with known clusters would further inform the network analysis and is a consideration for our future analyses. It is also important to note that the results presented in this study and those from a previous investigation2 Therefore, choosing the right program to use for network identification should depend on several factors that include the aims of the study, the dynamics of the disease epidemic in a particular population, and the type of sampling design.10In conclusion, HIV-TRACE was generally found to detect more and larger clusters than Cluster Picker at a GD of 1.5%. Furthermore, HIV-TRACE detected clusters that were connected by sparse links in the transmission chains. This implies that the choice of the GD threshold applied should put into consideration the characteristics of the population being investigated.


Xenopus laevis retinotectal circuit. We show that microglia regulate pruning of retinal ganglion cell axons and are important for proper behavioral response to dark and bright looming stimuli. Using bioinformatics, we identify amphibian regulator of complement activation 3, a homolog of human CD46, as a neuronally expressed synapse-associated complement inhibitory molecule that inhibits trogocytosis and axonal pruning. Using a membrane-bound complement C3 fusion protein, we demonstrate that enhancing complement activity enhances axonal pruning. Our results support the model that microglia remodel axons via trogocytosis and that neurons can control this process through expression of complement inhibitory proteins.Partial phagocytosis—called trogocytosis—of axons by microglia has been documented in ex vivo preparations but has not been directly observed in vivo. The mechanisms that modulate microglial trogocytosis of axons and its function in neural circuit development remain poorly understood. Here, we directly observe axon trogocytosis by microglia in vivo in the developing  Microglia, the immune cells of the CNS, are vital for the maintenance and development of a healthy brain. Constantly surveilling the brain , these hStudies depleting microglia or disrupting microglial function provide indirect evidence to support the hypothesis that microglia remodel synapses through phagocytic mechanisms. An inherent weakness of indirect approaches is that the source of the synaptic material within microglia is unknown. For example, it is possible that synaptic components may be found within microglia due to clearance of apoptotic neurons rather than synaptic pruning. More direct approaches are required to verify whether microglia collect synaptic material from living neurons. Currently, direct evidence of complete elimination of synapses by microglial engulfment remains elusive. However, instead of removing entire synapses, microglia have been documented engaging in trogocytosis, or partial elimination, of axons and presynaptic boutons in ex vivo organotypic cultures and in fixed brain tissue using electron microscopy , althougEven if we accept the hypothesis that microglia trogocytose the axonal compartment, many questions remain. What impact does partial elimination of presynaptic structures have on circuit remodeling? It is unclear if this phenomenon is required for proper wiring of neuronal circuits. Does microglial trogocytosis of axons affect axon morphology? While disrupting microglial function enhances axon tract outgrowth , it is uXenopus laevis retinotectal circuit. We then developed an assay to monitor microglial trogocytosis of axons among the population of microglia in the optic tectum. To investigate the functional role of microglial trogocytosis, we depleted microglial cells and found that this enhanced axon arborization and reversed the behavioral responses to dark and bright looming stimuli. We identified amphibian regulator of complement activation 3 (aRCA3)  axons by individual microglial cells in real-time using the developing  (aRCA3) , a homolelopment . In thisXenopus laevis tadpoles were labeled with IB4-isolectin conjugated fluorophores for in vivo imaging. IB4-isolectin binds the RET receptor tyrosine kinase on microglial cells . SYP is a presynaptic vesicle protein , and SYPTo assess the functional roles of microglial trogocytosis, we first depleted microglia using PLX5622. PLX5622 is an inhibitor of colony stimulating factor one receptor (CSF1R), which is a tyrosine kinase receptor essential for microglia survival . AnimalsNext, we interrogated the effect of microglial depletion on the morphology of single RGC axons. Axons were followed for several days in control and microglia-depleted animals . MicroglXenopus laevis and in zebrafish have shown that the retinotectal circuit is a vital processing and decision-making center for the visual detection of looming objects   has six CCP or CCP-like domains (Danio rerio (rca2.1) , and Coturnix japonica (MCP-like) each have five CCP domains.Using NCBI protein-protein BLAST, the  (aRCA3) . aRCA3 iopicalis . Like hu protein . As a coman CD46 . Despiteman CD46  and rodeman CD46 , the Gal domains , and hom(rca2.1) , Equus cTo investigate whether RGC neurons endogenously express aRCA3, retinal sections were probed by fluorescence RNAscope in situ hybridization . Probes It is unknown whether aRCA3 localizes to the correct cellular compartments to protect axons from complement attack. To investigate this, we examined the localization of aRCA3 by tagging it with mCherry . This aRNext, we examined the effect of aRCA3 overexpression on microglial trogocytosis. pHtdGFP was expressed in RGC neurons with or without overexpression of aRCA3, and microglial trogocytosis was quantified by measuring microglial green fluorescence on day 4 and day 5 post-labeling. Expression of pHtdGFP in RGC neurons resulted in a significant correlation between microglial green fluorescence and the number of pHtdGFP-labeled axons on day 5 post-labeling , a relatWe then examined the effect of aRCA3 overexpression on axon morphology in single RGC axons over several days . OverexpWhile CD46 is best known for its ability to inactivate complement C3 and complement C4, CD46 can also signal through intracellular tyrosine kinase activity under certain conditions . This raXenopus laevis complement C3 and fused the N-terminus to the extracellular C-terminus of Xenopus laevis VAMP2. This design was chosen in favor of a GPI anchor design that modifies the C-terminus C345C domain of complement C3 as this domain undergoes major rearrangement during activation and proteolysis   . In contslation) .VAMP2-C3 was co-expressed with pHtdGFP in RGC neurons, and axons were monitored over several days . To contPrevious real-time imaging experiments in ex vivo slice culture have shown that microglia trogocytose presynaptic elements . Our reaNumerous past studies have shown that during development microglia have synaptic components within phagocytic compartments . HoweverActivation of complement C3 exposes a reactive thioester bond that covalently attaches to amine or carbohydrate groups on cell surfaces . MicroglWhile VAMP2-C3 clearly produced effects on axon morphology, we did not rule out the possibility that the expression of VAMP2-C3 in RGC neurons may be producing effects on axons that are not mediated by the complement system. Additionally, we were unable to determine whether VAMP2-C3 expression enhanced microglial trogocytosis as electroporation of RGC neurons with VAMP2-C3 rarely yielded labeled axons. We speculate that expression of VAMP2-C3 in RGC neurons reduces viability, as previous studies have shown that the complement cascade mediates phagocytosis of RGC neurons during development . FurtherXenopus laevis aRCA3, an endogenously expressed, synaptic vesicle-associated, complement inhibitory molecule. aRCA3 is the most similar amphibian homolog of human CD46. CD46 is a membrane-bound complement inhibitory molecule that cleaves activated complement C3 and C4 .Xenopus laevis frogs (RRID:XEP_Xla200) were maintained and bred at 18°C. Female frogs were primed by injection of 50 IU pregnant mare serum gonadotropin . After 3 days, male and primed female frogs were injected with 150 IU and 400 IU of human chorionic gonadotropin  into the dorsal lymph sac, respectively. The injected male and female frogs were placed in isolated tanks for mating. Embryos were collected the following day and maintained in Modified Barth’s Saline with HEPES (MBSH) in an incubator at 20°C with LED illumination set to a 12 hr/12 hr day-night cycle and staged according to Nieuwkoop and Faber (NF) developmental stages  injection to the third ventricle of 1 mg/ml Alexa 594 conjugated IB4-isolectin. A minimum of 48 hr was allowed to pass before live imaging studies were commenced to allow for binding and update of IB4-isolectin by microglial cells. For concurrent labeling of brain structures, 1 mM CellTracker Green BODIPY in 10% DMSO was injected icv after isolectin injection. For labeling of RGC axons, electroporation of plasmid DNA into RGC cells was performed as described previously . In briePLX5622 was dissolved in DMSO at 20 mM, aliquoted, and stored at −20°C. Thawed aliquots of PLX5622 were sonicated briefly for 1 min before dilution in polyethylene glycol (PEG) 400. PEG 400 solution was then further diluted in a solution of 0.1X MBSH containing non-ionic surfactants poloxamer 407 and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) to form a mixed micelle drug delivery vehicle . Final vTwo-photon live imaging of axons and microglia cells was performed as described previously . TadpoleFor real-time imaging studies, stage 46–48 tadpoles were immobilized by brief (2–8 min) immersion in freshly thawed 2 mM pancuronium bromide in 0.1X MBSH. Animals were then maintained in 0.1X MBSH for imaging. Z-stacks were collected at 6 min intervals. For daily imaging studies, stage 43–47 tadpoles were anesthetized by immersion in 0.02% MS-222. Z-stacks were collected, and animals were returned to 0.1X MBSH rearing solution.When imaging microglia (Alexa 594 conjugated IB4-isolectin) and RGC axons (eGFP or pHtdGFP) concurrently, excitation wavelength was set at 830 nm and a 565 nm emission dichroic was used in conjunction with green (500–550 nm) and red (584–676 nm) filters for fluorescence emission detection on separate photomultiplier tubes. When imaging microglia (Alexa 594 conjugated IB4-isolectin) and brain structures (CellTracker Green BODIPY), imaging was done sequentially. Alexa 594 was first imaged with excitation wavelength set to 810 nm and fluorescence emission detection through the red filter. Imaging of CellTracker Green BODIPY was then performed at excitation wavelength 710 nm and the fluorescence emission detected through the green filter. When imaging RGC axons alone (pHtdGFP), excitation wavelength was set to 910 nm and fluorescence emission detected through the green filter. When imaging aRCA3-mCherry-Myc and SYP-pHtdGFP, images were captured concurrently at wavelength 990 nm through the red and green filters, respectively.Laser irradiation injury was carried out by repeatedly scanning an approximately 10 × 10 μm region under high laser power at wavelength 710 nm for 10 s.For visualization purposes, the 3D object counter  and 3D RTwo-photon z-stacks were captured daily from 2 to 5 days post-electroporation. Z-stacks were denoised with CANDLE , and werThe number of microglia and microglia process numbers were counted manually within 150-μm-thick z-stacks of the optic tectum.For microglial mobility experiments, microglia in the optic tectum were imaged over 2 hr at 6 min intervals and manual tracking of microglia was carried out using the TrackMate plugin .Collected z-stacks were denoised with the 3D hybrid median filter plugin. Time series were registered by descriptor based series registration . MicroglMicroglia were labeled with Alexa 594-conjugated isolectin and axons with pHtdGFP or SYP-pHtdGFP as described above. On day 4 and day 5 post-labeling, 150-µM-thick z-stacks were collected from the optic tectum. Laser power and photomultiplier tube voltage was kept constant on both days. The number of labeled axons was counted manually. In studies when axons were labeled with pHtdGFP, data were excluded if axonal blebbing was detected, or if the number of labeled axons fell from day 4 to day 5. This data exclusion step was not performed when axons were labeled with SYP-pHtdGFP. Using the red microglia channel, microglia 3D ROIs were automatically generated using the 3D object counter plugin . MicroglTwo-photon z-stacks were captured on day three post-electroporation. aRCA3-mCherry-Myc and SYP-pHtdGFP channels were denoised using the 3D hybrid median filter ImageJ plugin. A 3D mask of the axon was generated by summing the red and green channels and using an intensity threshold for segmentation of the axon. Pearson’s correlation coefficient  of the mStage 47 tadpoles were placed within a closed 60 mm petri dish and allowed to swim freely. The petri dish was placed on the bottom of a large shallow  glass culture dish filled completely with 0.1X MBSH. The large shallow glass culture dish was placed on a purple 3D printed stage to allow for automated segmentation of albino tadpoles. A webcam was placed above the tadpole to record tadpole behavior, while a 2000 lumens projector customized by 3D printing to shorten the focal distance, was used to project visual stimuli onto a piece of 96 white 20 lb bond copy paper taped to the side of the large glass culture dish.2) expanding circle was shown over a white (208 cd/m2) background. For bright looming stimuli, a white expanding circle was shown over a black background. During looming, the diameter of the circle expanded exponentially at 10% per frame at 60 frames per second, from a diameter of 54 pixels (7 mm) until it encompassed the entire screen 0.5 s later. After another 0.8 s, the screen was reset and a 10 s refractory period commenced. In parallel to stimulus presentation, 480 p video was recorded by webcam using the OpenCV library (Dark or bright looming stimuli were presented using custom code written in Python 3. An expanding circle was projected onto the culture dish (800 × 600 pixels or 10.6 × 8 cm) using the PsychoPy library . For dar library . Ten looCustom computer vision tadpole tracking code was written using Python three and OpenCV . FeatureAn automated python script was used to randomize tadpole videos and a user blinded to treatment categorized tadpole responses to looming stimuli as defensive escape behavior (positive response), absence of defensive escape behavior (negative response), or undeterminable . Tadpole responses were categorized as undeterminable if the tadpole was moving quickly when the looming stimulus was presented. The response rate was calculated as the number of positive responses divided by the combined number of positive and negative responses.Positive escape responses were further analyzed to compare motor data. Escape distance and maximum velocity over 2 s from the onset of the loom was calculated. Escape angle was also measured from heading data. In dark looming stimuli trials, the escape angle was defined as the absolute value of the change in heading from 0 to 0.6 s. For bright looming stimuli trials, the escape angle was defined as the absolute value of the change in heading from 0 to 1.2 s. This difference is because dark looming stimuli evoked escape movement beginning approximately 0.5 s after looming onset, whereas bright looming stimuli evoked escape movement beginning approximately 1.0 s after looming onset.To identify candidate complement inhibitory proteins, the STRING database  was usedThe Allen Brain Map Human Transcriptomics Cell Types Database  was usedXenopus laevis genome with NCBI protein-protein BLAST  was queried in the in BLAST . The topThe SMART protein domain research tool  was usedXenopus laevis aRCA3 were modeled separately by trRosetta and joined using pyMOL  and homogenized by sonication and processed according to manufacturer’s protocols to isolate mRNA. Superscript IV reverse transcriptase (Invitrogen) was then used according to manufacturer’s protocol to generate whole tadpole cDNA.Xenopus laevis cDNA was carried out using primers flanking genes of interest. Primers were generated according to mRNA sequences for aRCA3 (XM_018246573.1), VAMP2.S (NM_001087474.1), and c3.L (XM_018253729.1) predicted by NCBI (Cloning of  by NCBI  and Xenb by NCBI . Synapto by NCBI . pHtdGFP by NCBI . PCR wasThe SYP-pHtdGFP fusion protein was created by in-frame fusion of pHtdGFP to the C-terminus of SYN between a three amino acid linker (QGT). The aRCA3-mCherry-Myc fusion protein was created by an in-frame fusion of mCherry-Myc to the C-terminus of aRCA3 between a two amino acid linker (AC). The Myc-VAMP2-C3 fusion protein was created by an in-frame fusion of Myc to the N-terminus of VAMP2 between a four amino acid linker (PGKI) and an in-frame fusion of C3 to the C-terminus of VAMP2 between an 18 amino acid linker (ASIKSPVQPLSAHSPVCI). The long linker for VAMP2-C3 was chosen to ensure that the C-terminus transmembrane domain of VAMP2 would not sterically hinder proper folding of C3.Statistical analysis was performed using Graphpad Prism nine software. All data is presented as mean ± SEM, unless otherwise noted. Data was assessed for normality by D’Agostino-Pearson omnibus test and examination of quantile-quantile plots and assessed for homoscedasticity by examination of residual plots. All statistical tests were two-sided, and differences were considered significant for p<0.05.When normality and homoscedasticity assumptions were met, differences between two groups were tested by unpaired t-test. Welch’s t-test was used if the assumption of homogeneity of variance was not met. Non-normally distributed pairwise comparisons were tested by Mann-Whitney test.If no datapoints were missing, two-way repeated measures ANOVA was used to test group data. If an interaction was found, the interaction was reported and Sidak’s multiple comparison test was used to test between groups or time points as appropriate. If no interaction was found, then the main effect was reported. Conversely, if the data contained missing values , the mixed effect restricted maximum likelihood model was used.Correlations were calculated using Pearson’s correlation coefficient. Trendlines were plotted using simple linear regression. Significance testing of the difference between two correlation coefficients was carried out using Fisher’s z transformation.Log-transformed histograms were fit by Poisson regression with no weighting. The means of the best-fit curves were compared by likelihood-ratio test.https://doi.org/10.6084/m9.figshare.12895172.v1. The documented source code and user guide for the looming stimulus presentation and tadpole tracking software module, XenLoom (Beta), developed in this study are available at https://github.com/tonykylim/XenLoom_beta.The quantitative data used for analyses and figure generation in this paper are publicly available at  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Xenopus laevis tadpoles as a model system, you have used molecular manipulations, live imaging and behavior, to provide compelling in vivo evidence that microglia prune axons via trogocytosis – a partial engulfment and breakdown of axon terminals, identifying this process as an important regulator of axonal pruning to promote correct neural wiring during development.Your study on microglial pruning of axon endings is excellent and well documented. Using the developing retinotectal projection of Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Carlos Aizenman (Reviewer #1); Cornelius T Gross (Reviewer #2); Amanda Sierra (Reviewer #3).Thank you for submitting your article "Microglial trogocytosis and the complement system regulate axonal pruning in vivo" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Xenopus retinotectal system was in general praised by three reviewers. The longitudinal in vivo fluorescent imaging to simultaneously monitor microglia motility and retinal ganglion cell axon terminal branch dynamics in the optic tectum are superb and informative. Examining the functional consequences of this process, as well as relating the process of trogocytosis to the complement system, are solid additions to your study.Your study linking microglial trogocytosis, or partial engulfment of axon endings, to the maturation of axonal terminal arborizations in the developing The reviewers had some issues, nevertheless, on controls for autofluorescence and for the complement experiments, literature citation and discussion, and in some cases, overinterpretation of your data. Most of their comments can be readily amended with your existing data. Below, is a summary of their comments and suggested revisions, taken from the appended reviews and from the reviewer discussion, which was more interactive and thorough than usual with the reviewers ultimately agreeing with each other, all to your benefit.Revisions:1) Measuring changes in branching versus axon length: Reviewer 2 questioned whether microglia depletion results only in a change of branching, but not overall length of axons. During the reviewer discussion, reviewer 1 pointed out that the changes observed occur over a short time course, of small lengths of an otherwise large arbor and so changes in axon length would not be a useful measure. I agree. This point should be taken up in the Discussion.2) Controls for autofluorescence, to demonstrate true trogocytosis signal vs autofluorescence:a) As suggested by reviewer 3, is it possible to quantify with appropriate negative controls  to demonstrate the specificity of the quantification?b) Reviewer 2 suggests that traces for all individual cells in the validation experiments  should be shown to understand the variability in the persistent change in GFP signal that remains after contacts are completed. In addition, a spectral analysis should be done to help rule out autofluorescence.c) Correlative EM, also suggested, would be a labor-intensive addition, and is not deemed necessary if one of the former suggestions are taken.3) Complement experimentsa) Reviewer 2 thought that the proper control for the VAMP2-C3 overexpression is VAMP2 itself, is not ideal, citing that VAMP2-C3 fusion might have dominant negative effects that could have little to do with C3; also, that a better control would be VAMP2-C3* where C3* is non-cleavable or even a VAMP2-GFP fusion. He also wondered why you did not use a GPI anchor if the idea was to present C3 on the membrane, and whether you had evidence that C3 in this fusion form is functional. In the Consultation session, reviewer 1 did not think that the VAMP-GFP fusion or uncleaveable C3 would be better, and surmised that you used VAMP rather than GPI anchors to limit expression to presynaptic terminals. Reviewer 2 accepts this point but please discuss the inherent weaknesses in using the VAMP2-C3 control, either in the Materials and methods and/or Results.b) Reviewer 3 thought that for the aRCA3 overexpression and VAMP2-C3 expression experiments , you should show direct evidence that trogocytosis is altered by quantifying the axonal fluorescent signal in microglia , as proof that engulfment is potentiated or decreased (instead of showing only the downstream effect in axons). Reviewer 1 rebutted this comment by indicating that he did see a difference in axon phenotypes for depletion and CD46 homolog or C3 overexpression. He also acknowledges that you do discuss and invoke compensations due to the cell autonomous nature of the CD46 homolog and C3 over-expression. However, he did find it disappointing that you did not follow up on a single axon level with longitudinal in vivo live imaging what the impact of microglia contacts is in these circumstances. Please address the shortcomings of these experiments in the Discussion.c) Reviewer 2, # 6 comment on why depletion of microglia does not alter the number of axons, only their branching, but manipulations of complement seem to alter total numbers of axons. Please cite the possibility that C3 has an impact on microglia-mediated neuron elimination via apoptosis . Reviewer 1 thought that both microglia depletion and complement manipulation altered number of axonal branches, not number of axons. Please address this point in the Discussion.4) RGC subtype specificity; pruning in areas outside the tectum:a) As per reviewer 1's comments, that because there is a bigger effect on dark looming detectors than light looming detectors, different types of axons (from different RGCs having different response selectivity) could be differently selective to microglial pruning.b) Inhibition of microglial activation by PLX5622 might also affect remodeling in visual circuits, and could account for the behavioral difference.Please discuss.5) Unease with the behavioral results: Reviewer 2, comment #4 and Figure 9: the "behavioral part of the manuscript is only weakly linked to the main story". Reviewer 1 somewhat concurs, especially since this behavior is something that is known to require intact tectal circuitry. To this end, reviewer 3 proposes that to ensure that microglia outside the optic tectum, including the retina, regions involved in locomotion, as well as peripheral macrophages are not affected, evidence should be provided that the depleted animals do not have impairments in their vision, locomotor activity, and overall health. Reviewer 2 subsequently agreed to include the behavior, provided that you explicitly state that the mechanism that links depletion to behavior is far from understood, and remove Figure 9 summary, as it now stands, "to offset the abundance of correlative findings and hype in this field and reinforcement of (such) data". Reviewer 3 adds that you could modify Figure 9 to summarize what is known and what is not, and put question marks whenever appropriate. The latter would be a compromise position.6) Literature citation: All three reviewers ask for a more critical appraisal of the literature regarding microglial synaptic pruning, to highlight what you have done so well and how it is novel : Although previous and widely cited studies argue for microglial pruning, based on KO and depletion models, there is little direct evidence that microglia actively select the synapses to be removed . Other live imaging studies have failed to show that microglia grasp and digest synapses from living neurons; this includes the Weinhard and Gross study using organotypic cultures. They instead report axonal trogocytosis, for the first time, in vivo. Yours is the first study very nicely demonstrating trogocytosis by live imaging.Reviewer #1:This is a beautiful study, and probably one of the best work I've seen from this group, already known for excellent, high-quality papers in developmental neuroscience.The authors use an in vivo system to examine the hypothesis, based on prior in vitro data, that microglia prune axons via troglocytosis – a partial engulfment of an axon, basically by engulfing and breaking down axon terminals. The advantages of this in vivo system is that they can also push this hypothesis much further by examining the functional implications of microglial troglocytosis, and directly link the role of the complement system in synaptic pruning to microglial engulfment of axon terminals.Xenopus laevis tadpoles as a model system, the authors present a compelling set of experiments, that include molecular manipulations, live imaging and behavior, to provide compelling in vivo evidence that microglia troglocytose presynaptic axon terminals, and that this is an important regulator of axonal pruning to promote correct neural wiring during development.Using the developing retinotectal projection of Xenopus microglia are identified by labelling with IB4-isolectin, and using in vivo imaging are found to me highly motile and responsive to injury, consistent with known MG functions. Remarkably MG enter the neuropil and are shown to associate with a green FP labelled retinal axon and depart with little bits of green fluorescence in their bellies. Moreover, using labelled synaptophysin (a synaptic vesicle protein) they show that MG are actually engulfing synaptic terminals. This increase in green fluorescent in the MG was shown to be highly significant as shown as an increase over a day. These experiments convincingly show troglocytosis by MG of synaptic terminals in vivo and in real time. The provided videos are incredible.Their experiments are as follows: Next they look at the developmental consequences of MG pruning. In the first experiment they deplete MG using an inhibitor of a kinase necessary for MG survival. Depletion of MG results in increased branching of RGC axon terminals, suggesting that MG are negatively regulating axon branching over development. To test the consequences of this, they use a behavioral response to either dimming or brightening visual stimuli. Remarkably, MG depletion induced remodeling suppresses responses to dimming stimuli and enhances responses to brightening stimuli. While this is a puzzling finding, it does show that disrupted remodeling does cause abnormal behavior.To find whether the complement system is important for regulating MG pruning, they first look for complement regulating proteins known to be associated in the brain, and identify human CD46 as a top candidate. The closes protein to this was the frog aRCA3 protein, a transmembrane protein with several complement control protein domains (CCCPs). Using RNAscope in situ hybridization, they find that aRCA3 is expressed in the RGC layer in the retina, and that it co-localizes with RGC axon terminals. By overexpressing aRCA2 (and thus inhibiting complement) they see decreased MG troglocytosis and enhanced axonal branching, whereas increasing complement expression in axons  they saw increased pruning.Together these data show compelling evidence that the complement system regulates activation of microglia, and thus axonal pruning, which results in normal sculpting of axonal projections in the developing brain.I really struggled to find something I didn't like about this study. The data is high-quality, the logic is clear, the findings are incredibly cool and compelling. This paper will be of interest to a wide range of scientists, including developmental neuroscientists, systems neuroscientists, those interested in the role of the immune system in the brain, etc. What I especially like is how it brings together a bunch of disparate ideas and creates a sense of clarity that makes it one of those papers that read as an instant classic.Reviewer #2:Xenopus vertebrate model. They take advantage of the unique power of this system to perform longitudinal in vivo fluorescent imaging to simultaneously monitor microglia motility and retinal ganglion cell axon terminal extensions in the optic tectum. Their major novel observation is that microglia appear to persistently retain cytoplasmic GFP signal expressed in axons following their contacts with axons. They invoke microglia-neuron trogocytosis as a possible mechanism to explain this phenomenon. They then go on to show that pharmacological depletion of microglia results in an increase in axonal branching and conjecture that trogocytosis is involved in reducing axonal branching by a trogocytosis-dependent pruning phenomenon. In a digression they also show that the same pharmacological treatment results in changes in behavioral responses to dark and light looming stimuli. In the second half of the work they overexpress a Xenopus CD46 homolog and show that it reduces GFP retention and increases axonal outgrowth/branching and overexpress C3 and show that it decreases GFP retention and decreases axonal outgrowth/branching. The authors conclude that microglia restrict axon terminal outgrowth by trogocytosing axon material via a complement-dependent mechanism.This manuscript by Lim et al. takes important steps to link microglia to the maturation of axonal terminal arborization in the The work makes several important observations and has the potential to link microglia function to the pruning of axon terminals. In particular, several observations are important for the field in my opinion and could be elaborated more, including the finding that microglia depletion results in a change of branching, but not overall length of axons, and the report of frequent switching back and forth between ramified and amoeboid morphologies and entry and exit from the neuropil layer. However, several data are overinterpreted in my view and the authors need to hone closer to the findings so as to emphasize their most important contributions.Issues that should to be addressed:1) The GFP retention assay needs controls in order to rule out confounds due to the background autofluorescence mentioned by the authors. Traces for all individual cells in the validation experiments  should be shown for the reader to understand the variability in the persistent change in GFP signal that remains after contacts are completed. In addition, a spectral analysis should be done to help rule out autofluorescence.2) To causally link GFP retention to axonal remodeling it would be helpful to follow the axonal arborization of neurons that have been contact by microglia. Do they change in a manner consistent with the microglia depletion experiments (changes in branching but not axon length)? Without this kind of data it is hard to understand the impact of microglia contacts on axons.3) Figure 3EF: it seems odd that Syp-GFP is taken up by microglia as it should be primarily localized to vesicles. Does this suggest that microglia engulf axons with their vesicles inside?4) The behavioral part of the manuscript is only weakly linked to the main story. Microglia depletion will clearly have multiple effects on development beyond the remodeling of axons in the optic tectum and without a more restricted depletion or manipulation of microglia and documented interpretable changes in the responsible circuitry, the link to changed behavior is tenuous. Given this weak mechanistic link, Figure 9 is premature and misleading and should be eliminated.5) It is not clear that the proper control for the VAMP2-C3 overexpression is VAMP2 itself. I'd be concerned that a VAMP2-C3 fusion might have dominant negative effects that could have nothing to do with C3 and could explain the difference with VAMP2. The better control would be VAMP2-C3* where C3* is non-cleavable or even a VAMP2-GFP fusion. Also, if the idea was to present C3 on the membrane then why didn't the authors use a GPI anchor? Moreover, it would be good to have evidence that the C3 in this fusion form is in fact functional.6) If depletion of microglia does not alter the number of axons, but just their branching, why do manipulations of complement seem to alter total numbers of axons? The argument made in the Discussion that in this case there are compensations is possible, but other mechanisms should be considered, in particular the possibility that C3 has an impact on microglia-mediated neuron elimination via apoptosis .7) The authors should present the criteria that define trogocytosis in the manner used in this manuscript, especially as there is no EM data to support its morphological assessment.8) The images in Figure 7 do not appear to be representative of the data in the accompanying graph for branch numbers.9) Several citations are referred to incorrectly: Weinhard et al., 2018 presented evidence for trogocytosis in fixed brain tissue using electron microscopy; Paolicelli et al., 2011 did not investigate complement factors.Reviewer #3:Xenopus tadpole retinotectal circuit as a model.The manuscript "Microglial trogocytosis and the complement system regulate axonal prunning in vivo" by Lim and Ruthazer is a very elegantly designed and written paper showing for the first time in vivo evidence of microglial trogocytosis, using the The paper is very solid and I only have a few comments.1) The authors seem to consider that microglial synaptic pruning is well-established whereas axonal trogocytosis is not but this is not exactly the case. The evidences for microglial pruning synapses come from indirect studies using KO and depletion models – these studies demonstrate a role for microglia but do not provide direct evidence that microglia actively selects the synapses to be removed , although this is widely stated in the literature. Live imaging studies have failed to show that microglia grasp and digest synapses from living neurons. In fact, the Weinhard and Gross paper using organotypic cultures was unable to observe this phenomenon and instead reported axonal trogocytosis.The authors should make a more critical appraisal of the literature regarding microglial synaptic pruning, as in fact it further highlights the relevance of their in vivo model for live imaging studies of microglial interaction with spines and axons.2) This reviewer is a bit skeptic about the basal green autofluorescence in microglial cells and the ability of the authors to discriminate it from true "trogocytosis" signal. Would it be possible to perform some sort of quantification with appropriate negative controls  to demonstrate the specificity of their quantification?Additional evidence of the specificity of their analysis could be provided by doing correlational EM analysis of the microglia imaged by 2p.Finally, the results will be more solid if the authors could quantify the length of the axonal arbor before and after microglial contact – to estimate the size of the arbor removed.3) In the microglial depletion experiments the authors should more explicitly discuss that their manipulation is likely to affect microglia outside the optic tectum, including the retina, regions involved in locomotion, as well as peripheral macrophages. They should thoroughly discuss and provide as many evidences as possible that the depleted animals do not have impairments in their health status, vision, or locomotor activity (this is slightly touched upon in the Discussion), or any other factor that may affect their performance in the behavioral tests.4) In all the aRCA3 overexpression and VAMP2-C3 expression experiments , the authors should show the direct evidence that trogocytosis is altered by quantifying the axonal fluorescent signal in microglia , as proof that their manipulation had the predicted effect of potentiating or decreasing engulfment (instead of showing only the downstream effect in axons).eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Carlos Aizenman (Reviewer #1); Amanda Sierra (Reviewer #3).Thank you for submitting your article "Microglial trogocytosis and the complement system regulate axonal pruning in vivo" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Xenopus laevis tadpoles as a model system, you have presented a varied set of experiments that include molecular manipulations, live imaging and behavior, to provide compelling in vivo evidence that microglia prune axons via trogocytosis – a partial engulfment of an axon and breakdown of axon terminals, identifying this process as an important regulator of axonal pruning to promote correct neural wiring during development.Your study on microglial pruning of axon endings through partial engulfment is excellent and well documented. Using the developing retinotectal projection of Especially pointed is the experiment to overexpress aRCA2  and thus inhibiting complement, when you see decreased microglial trogocytosis and enhanced axonal branching, whereas increasing complement expression in axons  you see increased pruning. Together with the functional experiments, these data show compelling evidence that the complement system regulates activation of microglia, and thus axonal pruning, which results in normal sculpting of axonal projections in the developing brain.Your revisions are apt and thorough, and address the concerns of the reviewers. Reviewer 1 in fact said, "(this is) probably one of the best work I've seen from this group, already known for excellent, high-quality papers in developmental neuroscience."Revisions:Reviewer 3 had two caveats and you can address these by textual revision:1) Refrain from using the term "activated microglia" or "microglial activation" since there is no unique state of activation. Rather, microglia show different responses to different stimuli. As you show, in physiological conditions, they are far from "resting" and play critical roles by responding to neuronal cues.2) This reviewer had initial concern about the specificity of your novel imaging method, which you largely addressed with new figures. However, you have not provided additional evidence of engulfment with an independent method, such as correlational EM analysis, as this reviewer recommended. You lightly touched on this issue by mentioning the EM study performed by Weinhard et al. , but a more explicit acknowledgement of the need of independent corroboration of their findings is still needed. Revisions:1) Measuring changes in branching versus axon length: Reviewer 2 questioned whether microglia depletion results only in a change of branching, but not overall length of axons. During the reviewer discussion, reviewer 1 pointed out that the changes observed occur over a short time course, of small lengths of an otherwise large arbor and so changes in axon length would not be a useful measure. I agree. This point should be taken up in the Discussion.The following text has been added to the Discussion:“Another interesting possibility is that microglia preferentially engage in trogocytosis at short, dynamic, branches over longer, established, mature branches. In this case, microglial depletion results in a greater number of short branches, which over the short time course in this study, do not significantly impact axon branch length.”2) Controls for autofluorescence, to demonstrate true trogocytosis signal vs autofluorescence:a) As suggested by reviewer 3, is it possible to quantify with appropriate negative controls  to demonstrate the specificity of the quantification?b) Reviewer 2 suggests that traces for all individual cells in the validation experiments  should be shown to understand the variability in the persistent change in GFP signal that remains after contacts are completed. In addition, a spectral analysis should be done to help rule out autofluorescence.c) Correlative EM, also suggested, would be a labor-intensive addition, and is not deemed necessary if one of the former suggestions are taken.The important negative control experiment to address the question of autofluorescence, in which the experiment is performed with unlabeled axons, was actually integrated into our original experimental design in Figure 3C and D . No significant increase in green fluorescence was detected in this condition.We have also added a supplemental figure  which follows over time the changes in intensity of green fluorescence associated with each microglial cell that contacts (or fails to contact) a GFP-expressing axon. The stable levels of green fluorescence in non-interacting microglia lends further support to our finding that green fluorescence in microglia increases specifically as a result of axon-microglia contact.In addition, we have added a new quantitative analysis  which examines green fluorescence across all individual cells in the trogocytosis assay validation experiment.3) Complement experimentsa) Reviewer 2 thought that the proper control for the VAMP2-C3 overexpression is VAMP2 itself, is not ideal, citing that VAMP2-C3 fusion might have dominant negative effects that could have little to do with C3; also, that a better control would be VAMP2-C3* where C3* is non-cleavable or even a VAMP2-GFP fusion. He also wondered why you did not use a GPI anchor if the idea was to present C3 on the membrane, and whether you had evidence that C3 in this fusion form is functional. In the Consultation session, reviewer 1 did not think that the VAMP-GFP fusion or uncleaveable C3 would be better, and surmised that you used VAMP rather than GPI anchors to limit expression to presynaptic terminals. Reviewer 2 accepts this point but please discuss the inherent weaknesses in using the VAMP2-C3 control, either in the Materials and methods and/or Results.We added to the Results to explain why we didn’t use a GPI anchor design:"This design was chosen in favor of a GPI anchor design that modifies the C-terminus C345C domain of complement C3 as this domain undergoes major rearrangement during activation and proteolysis  . In contrast, the N-terminus of complement C3 is exposed on the surface of the protein and located on the MG1 domain, a domain that does not undergo marked confirmational changes upon complement C3 activation and proteolysis .”Also added to the Discussion to discuss the shortcomings of the VAMP2 control:“While VAMP2-C3 clearly produced effects on axon morphology, we did not rule out the possibility that the expression of VAMP2-C3 in RGC neurons may be producing effects on axons that are not mediated by the complement system.”b) Reviewer 3 thought that for the aRCA3 overexpression and VAMP2-C3 expression experiments , you should show direct evidence that trogocytosis is altered by quantifying the axonal fluorescent signal in microglia , as proof that engulfment is potentiated or decreased (instead of showing only the downstream effect in axons). Reviewer 1 rebutted this comment by indicating that he did see a difference in axon phenotypes for depletion and CD46 homolog or C3 overexpression. He also acknowledges that you do discuss and invoke compensations due to the cell autonomous nature of the CD46 homolog and C3 over-expression. However, he did find it disappointing that you did not follow up on a single axon level with longitudinal in vivo live imaging what the impact of microglia contacts is in these circumstances. Please address the shortcomings of these experiments in the Discussion.Added to the Discussion:"Additionally, we were unable to determine whether VAMP2-C3 expression enhanced microglial trogocytosis as electroporation of RGC neurons with VAMP2-C3 rarely yielded labeled axons. We speculate that expression of VAMP2-C3 in RGC neurons reduces viability, as previous studies have shown that the complement cascade mediates phagocytosis of RGC neurons during development . Furthermore, our study indirectly examined the effects of VAMP2-C3 and aRCA3 on microglial trogocytosis. Future experiments could directly examine the effects of VAMP2-C3 expression and aRCA3 overexpression on trogocytosis rates by real-time imaging.”c) Reviewer 2, # 6 comment on why depletion of microglia does not alter the number of axons, only their branching, but manipulations of complement seem to alter total numbers of axons. Please cite the possibility that C3 has an impact on microglia-mediated neuron elimination via apoptosis . Reviewer 1 thought that both microglia depletion and complement manipulation altered number of axonal branches, not number of axons. Please address this point in the Discussion.We changed the Discussion to be clearer that the number of axons was not examined in this study. Our experiments select for labeling of isolated pHtdGFP-expressing single axons that can be reconstructed for morphometric analysis. This experimental design therefore does not provide direct or indirect information about the total number of retinal axons.4) RGC subtype specificity; pruning in areas outside the tectum:a) As per reviewer 1's comments, that because there is a bigger effect on dark looming detectors than light looming detectors, different types of axons (from different RGCs having different response selectivity) could be differently selective to microglial pruning.b) Inhibition of microglial activation by PLX5622 might also affect remodeling in visual circuits, and could account for the behavioral difference.Please discuss.Added to the Discussion:“Conversely, it is possible that dark and bright looming-sensitive RGC axons are differentially affected by microglial depletion or the circuitry of the retina upstream of RGCs is remodeled following microglial depletion, both of which could produce the abnormal behavioral outcomes we observed.”5) Unease with the behavioral results: Reviewer 2, comment #4 and Figure 9: the "behavioral part of the manuscript is only weakly linked to the main story". Reviewer 1 somewhat concurs, especially since this behavior is something that is known to require intact tectal circuitry. To this end, reviewer 3 proposes that to ensure that microglia outside the optic tectum, including the retina, regions involved in locomotion, as well as peripheral macrophages are not affected, evidence should be provided that the depleted animals do not have impairments in their vision, locomotor activity, and overall health. Reviewer 2 subsequently agreed to include the behavior, provided that you explicitly state that the mechanism that links depletion to behavior is far from understood, and remove Figure 9 summary, as it now stands, "to offset the abundance of correlative findings and hype in this field and reinforcement of (such) data". Reviewer 3 adds that you could modify Figure 9 to summarize what is known and what is not, and put question marks whenever appropriate. The latter would be a compromise position.We concur and have removed Figure 9C which relied too heavily on correlative findings and added to the Discussion:"Indeed, the mechanism linking microglial depletion to improper response to looming stimuli is unclear and warrants future investigation.”6) Literature citation: All three reviewers ask for a more critical appraisal of the literature regarding microglial synaptic pruning, to highlight what you have done so well and how it is novel : Although previous and widely cited studies argue for microglial pruning, based on KO and depletion models, there is little direct evidence that microglia actively select the synapses to be removed . Other live imaging studies have failed to show that microglia grasp and digest synapses from living neurons; this includes the Weinhard and Gross study using organotypic cultures. They instead report axonal trogocytosis, for the first time, in vivo. Yours is the first study very nicely demonstrating trogocytosis by live imaging.We added to the Introduction:“Studies depleting microglia or disrupting microglial function provide indirect evidence to support the hypothesis that microglia remodel synapses through phagocytic mechanisms. An inherent weakness of indirect approaches is that the source of the synaptic material within microglia is unknown. For example, it is possible that synaptic components may be found within microglia due to clearance of apoptotic neurons rather than synaptic pruning. More direct approaches are required to verify whether microglia collect synaptic material from living neurons.”Modified the main text to be more assertive about trogocytosis results.Modified the Introduction to include the mention of electron micrographic data in the Weinhard et al., 2018 study.Removed incorrect references attributing complement studies to Paolicelli et al.Revisions:Reviewer 3 had two caveats and you can address these by textual revision:1) Refrain from using the term "activated microglia" or "microglial activation" since there is no unique state of activation. Rather, microglia show different responses to different stimuli. As you show, in physiological conditions, they are far from "resting" and play critical roles by responding to neuronal cues.We have removed both of these references to microglial activation from the manuscript.2) This reviewer had initial concern about the specificity of your novel imaging method, which you largely addressed with new figures. However, you have not provided additional evidence of engulfment with an independent method, such as correlational EM analysis, as this reviewer recommended. You lightly touched on this issue by mentioning the EM study performed by Weinhard et al., but a more explicit acknowledgement of the need of independent corroboration of their findings is still needed.The first paragraph of the Discussion section, summarizing our results now explicitly acknowledges that our finding was not confirmed using correlative EM:“As SYP-pHtdGFP is primarily localized to synaptic vesicles, this suggests that the axonal material that microglia engulf contains presynaptic vesicles and is consistent with correlative electron microscopy studies that have demonstrated putative presynaptic vesicles within microglia , although we did not use correlative electron microscopy to confirm this in our study.”


To the Editor—Pandemics have occurred throughout history and have generally led to catastrophic aftermaths across all levels of society. Notable examples include the black death in the 14th century and the 1918 Spanish flu; both names strike like a universal apocalyptic chorus.1 Although the understanding of microbes, the emergence of epidemiology, and rapid advancement of science and medicine over the 19th and 20th centuries have kept many old pathogens at bay and have led to an unprecedented quality of life,2 the modern world has already faced 2 dangerous new pandemics (2009 H1N1 influenza and COVID-19) in the 21st century.These recent pandemics are of particular concern not only because the medical and scientific communities were already aware of (and quite familiar with) both families of viruses but also because we currently live in an era that boasts stellar healthcare quality compared to just a century ago. For these reasons, the ongoing COVID-19 pandemic merits closer examination as a case study for learning new approaches to restraining future outbreaks.3 It began as an epidemic but has since spread to >150 countries and sovereignties.4 Although some countries and sovereignties  have managed to contain the spread of SARS-CoV-2, others  have experienced an unexpected number of COVID-19 cases and are struggling with overburdened healthcare facilities despite the fact that all 6 of these countries (except Spain) quickly implemented travel restrictions to mainland China within the first week5 and executed domestic lockdowns to restrict community spread. However, a major difference that helps explain the disparity in the severity of the pandemic is evident in these countries’ initial responses to COVID-19.The COVID-19 pandemic originated in Wuhan, China, in December 2019.Three countries and sovereignties that have managed COVID-19 remarkably well  happen to be densely populated regions whose citizens frequently travel to and from mainland China, which makes their achievement noteworthy. Since the emergence of SARS-CoV-2, these countries and sovereignties have made testing widely available and accessible and have carried out rigorous contact tracing, thus facilitating the diagnosis and treatment of every case. Ministers in these countries have also been warning about the impending outbreak and relaying accurate information around the clock. Although they initially created panic, these efforts resulted in a perpetual minimal relative number of new cases over the past few months ,7 upon observing a surge in new COVID-19 cases  either delayed or lacked sufficient access to reliable testing for various reasons Fig. . One criA central takeaway from the COVID-19 experience in these 6 countries is the strong association between initial action and disease severity. Countries that fared relatively well during the pandemic acted swiftly—from day 1—with testing, contact tracing, travel restrictions and lockdowns, and by circulating factual news, unlike those countries that delayed a rigorous response and rapidly fell behind in diagnosing and treating COVID-19 patients. The situation in the United States continues to unfold, and the outbreak has been characterized by conflicting and often unsubstantiated statements from government officials to the American public.8 Second, understaffing and reduced ICU occupancies over the years,9 in a bid to reduce hospital-acquired infections,10 have undermined the ability to provide critical care during mass-casualty incidents. Although these changes reflect the progress of healthcare and society, they have seemingly “backfired” during the COVID-19 pandemic. However, this is not the case because advanced health care  in the United States would have easily handled COVID-19 if officials had instead chosen to face COVID-19 with tenacity at its inception like its Asian counterparts, which it actually had much time to do (Fig. Many have questioned how the healthcare system of a prosperous country could be overwhelmed so drastically in this era of sophisticated medical treatment and care, most notably with acute shortages of hospital beds, respirators, and ventilators. The answer lies in 2 concluding observations that bear on the capacity of the modern healthcare system in developed countries. First, the number of hospital beds has fallen as the length of stay is reduced in response to pressures from lower insurance costs. Furthermore, increasing numbers of patients are receiving medical and surgical care in outpatient facilities to increase hospital efficiencies.Hopefully, the world will learn from the COVID-19 pandemic and will be in a better position to face the next inevitable pandemic. Perhaps diligent initial responses to outbreaks are indispensable for preventing future outbreaks from exploding into pandemics.


Both hepatic methionine concentration and methionine adenosyltransferase activity were increased, but S-adenosylmethionine level did not change. Hepatic cysteine was increased significantly from 4 h after CCl4 treatment. Glutathione (GSH) concentration in liver was elevated in 4~8 h and then returned to normal in accordance with the changes in glutamate cysteine ligase activity. Cysteine dioxygenase activity and hypotaurine concentration were also elevated from 4 h after the treatment. However, plasma GSH concentration was increased progressively, reaching a level at least several fold greater than normal in 24 h. γ-Glutamyltransferase activity in kidney or liver was not altered by CCl4, suggesting that the increase in plasma GSH could not be attributed to a failure of GSH cycling. The results indicate that acute liver injury induced by CCl4 is accompanied with extensive alterations in the metabolomics of sulfur-containing amino acids and related substances. The major metabolites and products of the transsul-furation pathway, including methionine, cysteine, hypotaurine, and GSH, are all increased in liver and plasma. The physiological significance of the change in the metabolomics of sulfur-containing substances and its role in the induction of liver injury need to be explored in future studies.Impairment of hepatic metabolism of sulfur-containing amino acids has been known to be linked with induction of liver injury. We determined the early changes in the transsulfuration reactions in liver of rats challenged with a toxic dose of CCl


International asthma guidelines recommend the monitoring of peak expiratory flow (PEF) as part of asthma self-management in children and adolescents who poorly perceive airflow obstruction, those with a history of severe exacerbations, or those who have difficulty controlling asthma. Measured with a peak flow meter, PEF represents a person’s maximum speed of expiration and helps individuals to follow their disease evolution and, ultimately, to prevent asthma exacerbations. However, patient adherence to regular peak flow meter use is poor, particularly in pediatric populations. To address this, we developed an interactive tablet-based game with a portable game controller that can transduce a signal from the user’s breath to generate a PEF value.The purpose of this study was to evaluate the concordance between PEF values obtained with the game controller and various measures derived from conventional pulmonary function tests  and to synthesize the participants’ feedback.1), and forced expiratory flow at 25%-75% of pulmonary volume (FEF25-75), using Spearman correlation. A Bland-Altman plot was generated for comparison of PEF measured by the game controller against PEF measured by spirometry. A post-game user feedback questionnaire was administered and analyzed.In this cross-sectional multicenter study, 158 children (aged 8-15 years old) with a diagnosis or suspicion of asthma performed spirometry and played the game in one of two hospital university centers. We evaluated the correlation between PEF measured by both the game controller and spirometry, forced expiratory volume at 1 second . The PEF measured by the game controller was reproducible in 96.2% (152/158) of participants according to standardized criteria. The PEF measured by the game controller presented a good correlation with PEF measured by spirometry , with FEV1 , and with FEF25-75 . The PEF measured by the game controller presented an expected mean bias of –36.4 L/min as compared to PEF measured by spirometry. The participants’ feedback was strongly positive, with 78.3% (123/157) reporting they would use the game if they had it at home.The participants had a mean age of 10.9 (SD 2.5) years, 44% (71/158) were female, and 88% (139/158) were White. On average, the pulmonary function of the participants was normal, including FEVThe game controller we developed is an interactive tool appreciated by children with asthma, and the PEF values measured by the game controller are reproducible, with a good correlation to values measured by conventional spirometry. Future studies are necessary to evaluate the clinical impact this novel tool might have on asthma management and its potential use in an out-of-hospital setting. Asthma is a chronic pulmonary disorder that affects more than 339 million people globally . It is tSerious games, defined as games with a primary purpose other than pure entertainment , can be TikiFlow game for portable electronic devices to monitor PEF in children with asthma using conventional spirometry as the comparison standard. We evaluated the concordance between PEF measurements obtained with the game controller and several spirometry measurements, and the reproducibility of PEF measured by the game controller without coaching. Furthermore, we assessed the participants’ feedback both quantitatively and qualitatively to guide the continuous development of the game.In this study, we evaluated the validity of an airflow-based game controller paired with the TikiFlow game by the means of airflow through the game controller  over a 3-week period in November 2019. The respective institutional research ethics board approved this study .We included children aged 8-15 years old with a diagnosis of asthma or a suspicion of asthma presenting to pulmonary function test laboratory for a spirometry, with or without bronchodilator reversibility testing, at their physician’s request. We excluded children with cystic fibrosis, bronchopulmonary dysplasia, and other conditions where the indication for pulmonary function test was unrelated to asthma . We also excluded children who were unable to cooperate for either conventional spirometry or assessment via the game controller according to American Thoracic Society guidelines. We identified eligible participants through the pulmonary function test laboratory database of both pediatric university health centers.TikiFlow with the game controller without coaching within 10 minutes of their conventional spirometry. Specifically, participants were given a brief overview of the game and its purpose and were asked to play on their own without instructions to exert more forceful exhalations or to increase the effort of breathing. Following the game, they also filled out a self-reported general health questionnaire and a satisfaction questionnaire.We asked eligible and consenting participants to play 1), the forced expiratory flow at 25%-75% of pulmonary volume (FEF25-75), and the forced vital capacity values from the participants’ best curve, and the highest PEF value (PEF measured by spirometry). For PEF measured by spirometry, we evaluated the reproducibility of the spirometry in accordance with the American Thoracic Society guidelines, defined as the 2 highest values falling within 40L/min of each other [Conventional spirometry measurements were obtained from a Jaeger MasterScope spirometer , using the Global Lung Initiative 2012 reference values  and intech other . For PEFP<.05. Analyses were performed with R software, version 3.5.0 [We portrayed a descriptive analysis of the participants’ baseline health and demographics based on the results of the self-reported questionnaire. A Bland-Altman plot was used to compare the game controller method to that of conventional spirometry and to evaluate the concordance of both methods to measure PEF. We calculated the spearman correlation between PEF measured by the game controller and by conventional spirometry measures. Statistical significance was set at on 3.5.0 . ParticiWe identified 337 children aged 8-15 years old presenting to both centers for a spirometry, with or without bronchodilator reversibility testing, 187 of whom were eligible based on inclusion criteria. We included 158 participants, as 9 were unable to perform spirometry and 20 refused to take part in the study, mostly due to time constraints see .Participants had a mean age of 10.9 (SD 2.5) years , and mos1, and 86.2% (SD 25.5%) for FEF25-75. However, varying degrees of airway obstruction are represented in our participants as demonstrated by the range of percent predicted values and z scores  performed a reproducible spirometry. On average, the baseline lung function of participants was normal, with a mean percent predicted value of 103.7% (SD 11.7%) for forced vital capacity, 99.6% (SD 13.2%) for FEVz scores .r=0.83, P<.001), with FEV1 , and with FEF25-75   of PEF values measured by the game controller were reproducible. Spearman correlations indicated that PEF measured by the game controller presented a good correlation with PEF measured by spirometry ( P<.001) . The Bla P<.001) . We iden1 of ≥12% post-bronchodilator [Of the 84 participants who underwent a spirometry pre- and post-bronchodilator administration, 7 were considered to have reversible airway obstruction as defined by a change in FEVodilator . Using todilator . Based oOverall, users highly appreciated the game . No partOf the 21 participants that gave negative narrative feedback, we noted the older users (aged 12-15 years) sometimes felt that the game content was intended for a younger population (n=6).This study shows that the PEF measurements obtained through our game controller present a good correlation with conventional spirometry measurements and have a high reproducibility. Additionally, the participants reported overall strongly positive feedback on the game controller and the game, with 78.3% (123/157) reporting that they would play the game if they had it at home. These findings suggest that our game and controller could provide an objective measure of airway obstruction and that its use in an out-of-hospital setting could be explored.PEF self-monitoring has been shown to reduce asthma exacerbations through the development of asthma awareness and control –12. HoweSpecifically, peak flow meters do not seem to consistently report PEF values that correlate with conventional spirometry, and a different measurement bias is associated with each peak flow meter ,31. Our TikiFlow appealed to the vast majority of children (see One suggested approach to increase adherence to medical treatments and to engage and empower the population in management of diseases is the use of serious games. Serious games have found their utility in different disciplines of medicine, whether to distract patients from a painful procedure, to engage adolescents in psychotherapies, or to educate patients on complex medical topics. Importantly, studies report a high adherence of serious games whether at home or in clinical settings -19. Onlydren see . AdditioAnother approach to increase adherence specifically to PEF monitoring in asthma is the automatic generation of diaries as opposed to the traditional handwritten personal diaries of PEF measures. This approach decreases the effort required by the user and reduces the risk of false data generation. It also offers the possibility to depict measures graphically over time to identify trends. A recent study in the United Kingdom  suggestsTikiFlow, is the use of a game controller with which the child uses their breath to control the game, bringing the focus to the play, rather than education alone. The acquisition of good asthma self-care habits early in childhood and adolescence may translate into long-lasting benefits in adulthood [Serious games may be particularly beneficial among children with chronic conditions, where they were found to improve knowledge level and self-management . The roldulthood .One notable strength of our game is its accessibility. Asthma is a global disease and the development of innovative tools to help patients and their families in their asthma management must be accessible to individuals in low- and middle-income countries. Smartphones and tablets represent ideal mediums to this end, as 66% of the world’s population has access to such a device . Our gamThere are several limitations to this study. First, our study does not include children aged 5-8 years old despite the international asthma guidelines recommending PEF monitoring in children aged ≥5 years old . Our decOur study evaluated the use of a novel airflow-based game controller coupled with a smart device serious game in children with asthma. Our game controller produced reproducible PEF measures that correlated well with several conventional spirometry measures and was highly appreciated by participants. Thus, our game controller, coupled with a serious game, may have the potential to increase adherence to PEF self-monitoring in children. Further studies are necessary to evaluate users’ adherence over time, the validity of the game controller in different situations, and its impact on clinical outcomes, specifically its role in the prevention of asthma exacerbations.