Daily milk production and reproductive performance of cows vaccinated with a live double-deleted Bovine Viral Diarrhoea Virus (BVDV) vaccine were compared to those of non-vaccinated cows, cohabitating in endemic BVDV herds. All animals in the treatment group were vaccinated on study day 0 irrespective of lactation or gestation status, while control animals did not receive any treatment. 1463 animals were enrolled in the study from four different farms in three different countries . Endemic presence of BVDV in study herds was demonstrated by the detection of BVDV in the bulk tank milk, and seroconversion was evaluated at the beginning of the study. For individual animals, the day of calving was taken to be the start of lactation for the calculation of days in milk (DIM). The standard lactation period of 305 days was divided into three periods: early lactation , mid lactation . For each farm and each lactation period, a mixed model statistical analysis was performed with daily milk production as response, and group, day as well as the interaction between those two factors as fixed factors. Chi-square test was used to compare abortion rate and prolonged inter-oestrous interval rate between treatment and control groups. A significant increase in milk production in the vaccinated group was observed in farms 1 (1.023 L/day) and 3 (0.611 L/day) during EL (p<0.001) and in farm 2 (1.799 L/day) during ML (P<0.001). In addition, at farm 2, vaccinated cows produced more milk than non-vaccinated cows starting from 80 DIM. No differences were found between groups in abortion rates or prolonged inter-oestrous interval rates. Data demonstrate that cows in herds endemically infected with BVDV and vaccinated with live double-deleted BVDV vaccine produce more milk; the difference in milk production occurs during early lactation. Pestivirus within the family Flaviviridae , a general health observation was performed and animals with an abnormal finding underwent clinical examination, including lameness evaluation. Unhealthy animals, including those with lameness grade 4 or 5 (severe lameness), were excluded from participation in the study; however, these animals remained present in the herd . At SD0 Ultimately, to be eligible for inclusion, a proof of BVDV infection within the herd during the six months prior to the start of the study was required. Animals were housed and fed according to the usual standards for each farm. The duration of the study was 365 days, starting from the day of vaccination [Study Day 0 (SD0)], and the study was performed in compliance with good clinical practice (GCP) guidelines, which ensure the maintenance of acceptable levels of animal welfare. The commercial product was administered as part of standard disease prevention measures, in accordance with the summary of product characteristics guidelines, by registered veterinary practitioners. For these reasons, and the absence of invasive procedures, no further approval from animal research ethics committee was required.Herds were considered to be endemically infected with BVDV if the virus was detected by polymerase chain reaction (PCR) in the bulk tank milk within the six months preceding SD0. The samples were collected in tubes containing a preservative agent (bronopol) and shipped at ambient temperature to the laboratory within 24 hours. The presence of BVDV in milk and serum was detected by PCR using a commercial kit . According to the manufacturer, this pan-pestivirus assay is capable of detecting other pestiviruses such as border disease virus. This kit employed a one-step real time RT-PCR protocol. One-step real time reverse transcriptase-PCR allows reverse transcription followed by PCR amplification and fluorescent binding of genotype-specific probes in a single well. Samples were considered positive for a target if the cycle threshold (Ct) value was ≤ 37. Samples with Ct values > 37 were considered inconclusive and those with undetermined Ct values were considered negative.At SD0, a screening blood sample was harvested from lactating cows (n = 10 per treatment group) on each farm in sterile dry sample tubes for detection of BVDV antibodies. Antibodies to BVDV in serum were detected by using an indirect enzyme linked immunoassay . Samples were taken to be positive for antibodies to BVDV when the sample optical density (OD) divided by the OD of the positive control minus the OD of the negative control was greater than or equal to 0.30. For animals with a negative result in the ELISA test, a BVDV PCR was performed to detect possible presence of the virus. During the study, the presence of BVDV was monitored by monthly BVDV PCR analyses of the bulk tank milk. Bulk tank milk sampling procedure and analyses were identical to those described for samples collected during the six months prior to SD0.®, Boehringer Ingelheim Vetmedica GmbH, Germany) by intra-muscular route in the neck region, using sterile single use materials. The G2 animals did not receive any treatment, thus there was no injection procedure. A placebo was not administered due to the requirement that Bovela be used within the terms of its marketing authorisation and no unjustified medical procedures be performed on any study animals, as discussed earlier.At SD0, all healthy animals were restrained for vaccination either in head lockers or in reproduction rails. Animals were allocated to treatment group  according to order of appearance in the retention area; first animal was vaccinated and then every other animal either non-vaccinated or vaccinated accordingly. The G1 animals were vaccinated with one dose of double gene-deleted monovalent BVDV vaccine  and study monitors were not blinded to allocation to treatment groups as the study parameters measured were objective.BovelaPost-mortem examination of animals that died during the study period, including abortions and stillbirths were performed according to the accredited regional veterinary laboratories standard protocol. In all cases, the minimum requirement was that fetal samples and blood samples of the dam were analysed for the presence of BVDV antigens and antibodies. Other diseases such as Neospora, Q fever, Salmonellosis, Leptospirosis and Brucellosis were analysed according to the decision of the investigators and farmers.The investigator was responsible for all aspects of the conduct of the study at the individual study site during the entire duration of the study (365 days). The farmers were encouraged to contact the investigator for health issues of the study animals at any time. At the farmer’s judgement, the animals were examined and treated by either the farmer or the investigator. All incidences of disease were reported to the investigator at least once a month and further investigations conducted when deemed necessary. The treatment data from the official farm register was used, either in a paper version (farm 2) or in an electronic version (farms 1. 3 and 4). Data on daily milk production, reproductive performance and animal movements  were collected for 365 days, starting on SD0. The daily milk yields per cow were measured by inline milk meters. For each farm, the equipment was tested and calibrated before the start of the study by a local accredited organisation. Reproductive performance was measured by the abortion rate (number of abortions during the study / number of lactating cows at SD0*100) and embryonic mortality rate. Since embryonic mortality is associated with increased oestrous cycle length, embryonic mortality rate was estimated from the number of cycles of more than 24 days, referred to as prolonged inter-oestrous intervals . The intFor individual animals, the day of calving was taken to be the start of the lactation (DIM 1). The standard lactation period of 305 days was divided into three separate periods: early lactation , mid lactation . For each farm and each lactation period, a mixed model was used to analyse daily milk production. Group and day, as well as the interaction between those two factors, were considered fixed factors in the model. Repeated measures were made on each cow across days, and days were modelled as a categorical variable. To capture the correlations between observations from the same subject, the autoregressive (AR(1)) variance and co-variance structure was used. The compound symmetry (CS) was used if the models suffered from a lack of convergence. The lactation rank was considered as a covariable in the model. If the interaction between group and day was significant, G1 and G2 cows were compared for each day. If the interaction between group and day was not significant, the interaction was removed from the model and G1 and G2 cows were compared with all days confounded. Normality of residuals was checked by visualization of the Pearson residuals distribution. For each farm  a Chi-square test was performed to compare abortion rate and the prolonged inter-oestrous interval rate between G1 and G2. If the theoretical numbers observed were inferior to 5, a Fisher Exact test was used. Statistical analyses were performed with a type I error set at α = 5%, with two-tailed tests, using SAS BASE 9.4 SAS/STAT 13.1 .BVDV was detected in the bulk tank milk by PCR from all farms on at least one occasion during the six months prior to vaccination. All blood samples (100%) at SD0 (n = 20 per farm) were positive for antibodies against BVDV in farms 2, 3 and 4. On farm 1, 75% (15/20) of the serum samples were positive for antibodies against BVDV. Seroconversion can either be induced by natural infection with BVDV or by vaccination against BVDV. Cows located at farm 3 had been vaccinated against BVDV prior to inclusion in the study; thus for this herd, the seroconversion does not provide additional information regarding the nature of exposure to BVDV. BVDV was not detected by PCR in any of the serum samples that were without detectable antibody. In farms 2, 3 and 4, BVDV continued to be identified in the bulk tank milk in the samples collected each month . On farmA total of 1463 animals were enrolled from the 4 farms in the study, and consisted of 734 G1 animals and 729 G2 animals  distribuDuring the total study period of 365 days, milk production data were eligible for analyses from 1197 cows, and these cows represented 1559 lactations. Data from 28 lactations  could not be used due to short lactation length. For these cows, either vaccination occurred less than 30 days before dry off or the animal calved close to the end of the study, such that less than 30 days of milk production data was collected. Distribution of the lactations according to treatment group, lactation rank and lactation stage are presented in Leptospira hardjo, Neospora caninum and Bovine Herpesvirus-1 were the pathogens most commonly suspected as being the causes of abortion based on the laboratory reports. In no cases were BVDV antigen detected in an abortion sample.Abortion rates and prolonged inter-oestrous interval rates at farms 1 to 4 varied between 2.0 and 6.4% and 45.7 and 73.0%, respectively. No differences were found between groups. The aim of this study was to measure the effect of vaccination with a novel commercially-available double gene-deleted monovalent BVDV vaccine (Bovela®) on milk production in commercial dairy herds with evidence of exposure of the milking herd to BVDV. Since milk production is importantly influenced by farm-related factors such as nutrition, housing and management, we sought collaboration with farms in which large numbers (>100 lactating dairy cows) of vaccinated animals and control animals could cohabitate under identical farming conditions . NumerouIn order to investigate the efficacy of Bovela® in preventing milk production losses in commercial herds with endemic BVDV, the first necessity was to establish the presence or absence of BVDV infection throughout the duration of the study. Lactating cow serological screening reported that 75–100% of samples were positive for antibodies against BVDV, indicating that the herds contained a PI animal . In addiThe study was designed to evaluate the efficacy of Bovela® in protecting dairy animals from the negative impact of BVDV on milk production, during the 12 months following vaccination. With the objective to mimic field conditions, all animals  of the treatment group (G1), were vaccinated on the same day (SD0), regardless of their age or reproduction status. No drop in milk production in the period immediately after vaccination was observed when compared to the control animals. An important finding when considering that the control group did not receive any placebo i.e. did not undergo an injection procedure.Cows vaccinated with Bovela® produced more milk than the non-vaccinated cows on farms 1, 2 and 3 during the first 100 days of lactation, significantly more during early lactation on farms 1 and 3, and from 80 days until 200 days on farm 2. No improvement in milk production after vaccination with Bovela® was found in farm 4 . Farm 4 Although the reduction of milk production due to both epidemic and endemic BVDV has been reported before, the observation that vaccinated animals produce more milk than cohabitating non-vaccinated herd mates in endemic BVDV herds is novel , 19. FurDue to the study design, early lactation does not coincide with the first period after vaccination. On SD0, animals at different stages of the lactation cycle entered the study at the same time; lactating animals, dry cows, pregnant heifers and heifers that would experience future pregnancies. Accordingly, milk production data were not analysed relative to the start of the study but relative to the DIM of individual cows according to the three stages of lactation: EL, ML and LL. Therefore, resulting changes to the epidemiology of BVDV infection due to vaccination of 50% of the herd are not likely to have been responsible for the observed difference in milk production.Early lactation is a critical phase of the lactation cycle. Modern dairy cows experience an energy deficit during early lactation and this period is associated with impaired immune function. Recently, Contreras et al. suggested that inadequate adipose tissue remodelling that accompanies transition and early lactation compromises immune function . Potentithe additional stress on the immune system caused by BVDV added to the already impaired immune function during early lactation competes with bodily resources otherwise used for milk productionsecondary infections encountered during that period which have a negative impact on feed intake and production.Data from this study demonstrates that, even under the condition of endemic BVDV infection, in three out of four commercial herds with cohabitating Bovela®-vaccinated and non-vaccinated animals, the vaccinated cows overproduced the non-vaccinated cows by 0,61 to 1,8 l/day during early lactation. These findings, for periods of respectively 94 to 101 days , give a compounded amount of gain in milk produced of 57,43 L to 181,69 L. It is difficult to precisely quantify the financial gain associated with this increased production due to the variation of milk price in different countries, but if we take into consideration average milk price in the European Economic Area (EEA) during the first quarter 2020, the financial gain would range from 20 to 63 € per cow per lactation . Since tThe findings of this study are additional considerations for the cost-benefit analyses of BVDV control programs. In addition, the observations provide an additional motivation for farmers to adhere to BVDV control programs. Especially when this finding is combined with the observation that in none of the herds were differences found in important reproductive parameters and incidence of disease, i.e. farm data did not demonstrate the problems that usually signify BVDV infection. Thus, BVDV infection can negatively impact milk yield without affecting the reproductive performance of a herd; milk production losses may be hidden.In summary, cows in herds endemically infected with BVDV and vaccinated with Bovela® produce more milk than the non-vaccinated cows in the same herds. The difference in milk production occurs during early lactation.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Geometric crystal structure analysis using three-dimensional Voronoi tessellation provides intuitive insights into the ionic transport behavior of metal-ion electrode materials or solid electrolytes by mapping the void space in a framework onto a network. The existing tools typically consider only the local voids by mapping them with Voronoi polyhedra vertices and then define the mobile ions pathways using the Voronoi edges connecting these vertices. We show that in some structures mobile ions are located on Voronoi polyhedra faces and thus cannot be located by a standard approach. To address this deficiency, we extend the method to include Voronoi faces in the constructed network. This method has been implemented in the CAVD python package. Its effectiveness is demonstrated by 99% recovery rate for the lattice sites of mobile ions in 6,955 Li-, Na-, Mg- and Al-containing ionic compounds extracted from the Inorganic Crystal Structure Database. In addition, various quantitative descriptors of the network can be used to identify and rank the materials and further used in materials databases for machine learning. One of the prerequisites for both inorganic solid electrolytes and electrodes is high ionic conductivity, which requires a connected mobile ions pathways. Currently, widely used computational approaches to predict or simulate ionic conduction pathways include both ab initio and empirical methods. The methods based on the first-principles calculations include ab initio molecular dynamics9 and the first-principles nudged elastic band method (FP-NEB)10. They provide high accuracy but they are limited by high computational cost and often involve complicated manual pre- and post-processing of the input data and results. At the opposite side of the spectrum, the low-cost empirical methods, such as geometric analysis15 and bond valence method (BV)21, have gained popularity in high-throughput screening29 and obtaining preliminary insights quickly for further accurate calculations. In this context, we have developed a high-throughput screening platform30 that integrates material database with hierarchical ion-transport calculations realized by implementing empirical algorithms to assist in FP-NEB calculation. To meet the requirements of our platform for automated unsupervised workflow, we also independently developed the bond valence site energy (BVSE) calculation program (has been reported in our paper31) and the geometry-based ion-transport analysis library CAVD (presented in this paper).All-solid-state batteries are the promising candidates for electric vehicles32. The atom subspace can be characterized by the composition, symmetry, topology, and geometry, which enables efficient classification of structures. On the other hand, the analysis of the void subspace of crystal structures allows to identify materials favorable for transport of ions or molecules, e.g. in solid electrolytes and metal organic frameworks, respectively33.In the geometric model considering atoms as spheres of characteristic radii, the crystal space can be divided into two complementary subspaces: the subspace occupied by atoms and that of interatomic voids34, ToposPro35 and Zeo++22, have already shown the ability to analyze the void space36. PLATON provides a number of techniques to detect and handle voids in crystal structures by grid method. Based on the Voronoi theory, both ToposPro and Zeo++ characterize the void space as a 3D Voronoi network37 with Voronoi polyhedra vertices and edges indicating the centers of the local cavities and pathways between them, respectively. Although these tools have been successfully used for void space analysis, some improvements are still desirable. For example, since accuracy of the grid method depends on the grid density, the computational cost of the method very rapidly increases with the grid resolution. The Voronoi approach implemented in ToposPro and Zeo++ only identifies the largest cavities in the local environment, however the experimentally determined positions of the mobile atoms which are not close enough to those Voronoi vertices cannot be easily automatically associated with them, which in turn complicates automatic search of transport pathways. In addition, the standard Voronoi decomposition 38 adopted by ToposPro does not consider the atomic radii that affects the simulation results for polydisperse particle systems39. The radical Voronoi decomposition40 and the ability to consider radii are implemented in Zeo++, but its built-in radius table and radius assignment scheme do not take into account dependence for the ionic radii on different coordination environments.Current structure analysis tools, including PLATON42 and the table of the effective ionic radii of Shannon43. Moreover, we consider the Voronoi polyhedra faces as the possible sites for mobile ions. In our tool, the void space is characterized as the 3D network consisting of interstices, channels between interstices, and bottlenecks of the channels, which are identified and quantified from the position and geometry of Voronoi polyhedra vertices, edges, and faces, respectively. The geometric and topological parameters of the network are then translated into ion transport descriptors. These descriptors form the foundation of machine-learning algorithms to predict and optimize materials properties.Inspired by Zeo++ and ToposPro, we developed a high-throughput ion-transport analysis tool CAVD based on the radical Voronoi decomposition. To address the challenge in the calculation of ionic radius calculation, we calculate the ionic radius by combining the rigorous definition of coordination number proposed by O’Keeffe44 and Zeo++ (based on a modification of Voro++45 software library) for symmetry analysis and periodic radical Voronoi decomposition, respectively. The code can be accessed in the repository (https://gitee.com/shuhebing/cavd) based on the FAIR principles for the benefit of the broad research community.All calculations discussed in this work were performed with the developed python package CAVD, which calls the libraries Spglib47. The resulting Voronoi diagram is composed of Voronoi cells, characterized by vertices, edges and faces. The Voronoi faces are the perpendicular planes between two neighboring framework atoms, and the vertices and edges of the Voronoi cell are by definition at the largest possible distance from the framework atoms around them. The Voronoi network, built of Voronoi vertices and edges, maps the void space in the framework. Based on these features, the procedures of our method are summarized as following  p and pi  is denoted by d, the standard Voronoi cell49 corresponding to pi satisfiesThe standard Voronoi decomposition proposed by Georgy VoronoiZ. Each Voronoi cell is a convex polyhedron, which is obtained by constructing the perpendicular bisector (for monodisperse system) planes between two neighboring generators. Replacing the generators with ions, the Voronoi cell can be used to describe the local environment in an ionic crystal structure. The Voronoi vertices can be used to describe the sites of local largest voids, and the Voronoi edges refer to the least restrictive paths from one local largest void to the next because they are farthest away from the ions around themselves.The Voronoi diagram V in Fig. 52, as following:However, the standard Voronoi cell constructed by bisecting the interatomic distances is not adequate for structures with atoms of different size  The radical Voronoi cell is a convex polyhedron.(2) The overall shape of radical Voronoi cell is similar to Voronoi S cell.ri = rj.(3) The radical Voronoi cell will degenerate into standard Voronoi cell when The radical Voronoi cell constructed by radical Voronoi decomposition has the following three main features:R in Fig. 58 and our derivation (S1 of the Supplementary Information) demonstrates that it has little effect on ionic transport analysis.The radical Voronoi decomposition is easy to implement and provides more adequate description of the void space than standard Voronoi decomposition for a structure with atoms of unequal radii. Although the vertices of the radical Voronoi cell may no longer precisely coincide with the centers of local maximally large interstices 60, the potential mobile ion diffusion path can be further evaluated by determining the parts of this network through which the mobile ions could theoretically migrate. A set of local geometrical descriptors, rSD, rChan23 and G363 has been introduced for the evaluation of ionic transport in previous work. rSD is the radius of a spherical domain whose volume equals to the secondary Voronoi cell around the vertices of the primary Voronoi cell. rChan describes the channel size, which is the radius of a circle passing through the bottleneck area constituted by three framework atoms. G3 is the second moment of inertia of the Voronoi cell, which is necessary to filter out unreasonable rSD values caused by the severe distortion of the secondary Voronoi cell. However, for these local descriptors, Voronoi decomposition needs to be performed twice, and the value of rChan is affected by the distortion of the bottleneck region. Moreover, two different criteria are required to determine the accessibility of the interstices and channels. To meet the goal of making the calculations automatic and robust, we introduce the distance from the interstice/channel to the surface of the nearest framework atom as the weights for the evaluation (see S2 of the Supplementary Information for details). After that, the accessible subspace of the void space can be easily determined by comparing the interstitial/bottleneck size in the network with the statistical values for mobile ions in different coordination environments (see subsection “Ionic transport descriptors”).For the analysis of ionic transport, it is necessary to characterize the void space formed by the framework atoms and then determine the subspace which mobile ions can accessWVN =  is comprising a set V of interstices together with a set E of channel segments, which are 2-element subsets of V. Only with all the sites of mobile ions matched with the interstices that have been included in the WVN, can we get all migration pathways between them. However, in some structures, we cannot find the interstices contained in the network that correspond to the lattice sites of target ions. For example, the lattice positions of Li1 and Li2 in α-Li3N do not coincide with any Voronoi vertices. Li1 locates at the center of the Voronoi face, and Li2 occupies the bottleneck site of the channel connection two adjacent interstices . In this work, instead we focus on more general geometrical/topological descriptors that are frequently used in ionic transport.A descriptor, an effective input data representation for material characteristics, is critical to study structure–property relationshipsTl) and upper threshold (Tu) of ion migration can be easily determined. In order to cover as many samples as possible and avoid excessively long distances with high coordination numbers, the thresholds are obtained from about 90% of the data , the interstitial network dimension (IND), the radius of largest ion that can freely pass through the void space (RT), the critical radii for main crystallographic directions a (RTa), b (RTb) and c (RTc).Based on the TC: The transport channel is formed by connected interstices and channel segments whose size is within . The TC characterizes the continuous subspace of the void space accessible by mobile ions14 and is useful for identifying the approximate minimum energy path of ion migration69 Each interstice that belongs to the transport channel should have the pathways to its periodic images in adjacent cells. The algorithm22 , and push all interstices that directly connected to the interstice into a stack. When the stack is not empty, remove the topmost interstice and performs following analysis:Repeat step (3) until all interstices have been recorded.It should be noted that there may be more than one transport channel in a structure, and all of them form a transport network.RT: The size of restricting interstice or bottleneck in the interstitial network. RT, characterizes the radius of largest ion that can freely pass through the void space. This descriptor is analogous to the radius of largest free sphere (RLFS), a currently widely used description of the pore geometry72 in crystalline porous materials. The RLFS refers to the maximum size of a spherical probe that can travel through the void space in a structure by at least one periodic lattice translation, and can be calculated for three crystallographic directions. Taking the same approach, we define RT as the size of restricting interstice or bottleneck in the interstitial network to characterize the ion transport. The RT for three crystallographic directions a (RTa), b (RTb) and c (RTc) are also obtained, and RT = max{RTa, RTb, RTc}.IND: The interstitial network dimension. The IND provides the information about the dimensionality of the network that mobile ions can diffuse through in the void space14. The interstitial network may be one- (1D), two- (2D) or three-dimensional (3D) or otherwise, zero dimensional (0D) if no infinite channels are found. The value of IND is determined from RTa, RTb and RTc with Tl. For example, if RTa larger than  the Tl, the interstitial network will be called connected in the a direction, and if only one crystallographic direction connected, the system will be called 1D.74 for Li, Na, Mg and Al containing compositions were extracted from the Inorganic Crystal Structure Database 75. For these compounds, the repairable format errors (such as inconsistent bracket and excessive blank lines) were corrected at first. The data cleaning procedure , reduced the set to 17,206 entries. After removing entries with duplicated and undetermined atom sites, 6,955 entries having Li , Na , Mg  and Al  were selected with fully occupied metal sites whose positions are accurately determined. The detailed process of data cleaning is described in S4 of the Supplementary Information.To support our work, 31,499 Crystallographic Information Files (CIFs)IN) using our approach as described in section “Methods”, and standard Voronoi network (VN) (for comparison) for the framework and accumulate the possible mobile ions sites  in a set VIN and VVN, respectively. The experimental sites are stored in set M. The recovery rate for every compounds can be determined by the algorithm68  based on the distance between m and the nearest element (v) in VIN (or VVN): if d < = 0.5 Å, pm = 1; otherwise, pm = 0. (The threshold 0.5 Å is adopted from the Yang’s work68).Check the recovery state M is n, the recovery rate P of the compound satisfiesIf size of set Since one of the main purposes of our tool is to identify pathways for further FP-NEB calculations, the prerequisite for the CAVD analysis is that all experimental lattice sites of mobile ions are matched to the interstitial network. As a first step of the methodology validation, we assess the ability of the calculations to recover the positions of the mobile ions for all 6,955 compounds using only framework structure information. For each compound, we remove mobile ions, then calculate the interstitial network (P = 1), partial recovery (0 < P < 1), and a miss (P = 0). The recovery rates of all 6,955 compounds for IN and VN are presented in Table d are just slightly larger than the threshold 0.5 Å. If the threshold is increased, those compounds are also completely recovered (S5 of the Supplementary Information). All of these discussions clearly demonstrate that our method successfully identifies mobile ion sites and the channels between them.We classify the recovery rate of every compound into three types: complete recovery . The calculated transport channel for each CIF is provided by *.vesta files and *.net files (archived in Channels.zip). The remaining numeric descriptors are recorded in Descriptors.xlsx, and a short description of all data is given in S6 of the Supplementary Information.2O3, are consistent with previous reports (shown in Table 4Zr2(SiO4)3 and Li7La3Zr2O12 with 3D transport channel are presented in this section, LiFePO4 with 1D channel and Na2O(Al2O3)11 with 2D channel are shown in the S7 of the Supplementary Information.The results, including 17 well known ionic conductors, including Li-nitride, NASICON, Garnet, LIPHOS, perovskite, and β-Al4Zr2(SiO4)380. It is a good ion conductor due to the network of wide channels in the three-dimensional rhombohedral R-3c framework of corner sharing ZrO6 octahedra and SiO4 tetrahedra 82. Mobile Na ions are distributed over the two types of interstices: Na1  and Na2 (in an 8–10 oxygen environment at each bend of the conduction channels) and the transport involves the Na1-Na2-Na1 migration.The first material is a NASICON (Na superionic conductor) type compound Na4Zr2(SiO4)3, all Na ions are removed from the structure and the following steps are carried out to locate the transport channels:Calculate the interstitial network based on the framework structure;Calculate the connected transport channels by applying the geometric transport analysis algorithm to the interstitial network;Simplify the transport channels by identifying crystallographically equivalent interstices;Identify the experimental sites, and the pathways between them form simplified channels.Before applying our method to Na+ network consisting of 13 symmetrically distinct interstices and 24 channel segments between the interstices  has two phases: high-temperature cubic phase (cubic-LLZO) and low-temperature tetragonal phase . In the tetragonal-LLZO, Li ions occupy three different sites: the tetrahedral sites (Li1), the octahedral sites (Li2), and the tetrahedral sites (Li3). The 3D transport network in tetragonal-LLZO consists of face-shared tetrahedra and octahedra. After analysis by our method, we reproduce the same 3D transport channel consisting of 24 symmetrically distinct interstices 3 and Li7La3Zr2O12 also confirmed the validity of our method. In addition, 6,955 descriptor data that can be further studied, e.g. material machine learning, structure classification, and structure–property relationships study, are shared. CAVD can be used not only to identify the position of mobile ion sites and conduction channels and quantify the accessible void space, but also to provide insights for BVSE or FP-NEB calculations for the newly established high-throughput screening platform for battery materials 30.We developed the Crystal structure Analysis by Voronoi Decomposition (CAVD) tool to characterize the void space of crystal structures as interstitial network by the radical Voronoi decomposition. The obtained interstitial network can be used to calculate crystal structure characteristics relevant for ionic transport properties. We validated our code by predicting the lattice sites of mobile ions in 6,955 compounds extracted from the ICSD with the success rate > 98%. The detailed study of the migration channels in NaSupplementary Information


The coupling of appropriate sequencing strategies and imputation methods is critical for assembling large whole-genome sequence datasets from livestock populations for research and breeding. In this paper, we describe and validate the coupling of a sequencing strategy with the imputation method hybrid peeling in real animal breeding settings.We used data from four pig populations of different size  that were widely genotyped at densities between 15,000 and 75,000 markers genome-wide. Around 2% of the individuals in each population were sequenced . We imputed whole-genome sequence data with hybrid peeling. We evaluated the imputation accuracy by removing the sequence data of the 284 individuals with high coverage, using a leave-one-out design. We simulated data that mimicked the sequencing strategy used in the real populations to quantify the factors that affected the individual-wise and variant-wise imputation accuracies using regression trees.Imputation accuracy was high for the majority of individuals in all four populations . Imputation accuracy was lower for individuals in the earliest generations of each population than for the rest, due to the lack of marker array data for themselves and their ancestors. The main factors that determined the individual-wise imputation accuracy were the genotyping status, the availability of marker array data for immediate ancestors, and the degree of connectedness to the rest of the population, but sequencing coverage of the relatives had no effect. The main factors that determined variant-wise imputation accuracy were the minor allele frequency and the number of individuals with sequencing coverage at each variant site. Results were validated with the empirical observations.We demonstrate that the coupling of an appropriate sequencing strategy and hybrid peeling is a powerful strategy for generating whole-genome sequence data with high accuracy in large pedigreed populations where only a small fraction of individuals (2%) had been sequenced, mostly at low coverage. This is a critical step for the successful implementation of whole-genome sequence data for genomic prediction and fine-mapping of causal variants. Sequence data has the potential to empower the identification of causal variants that underlie quantitative traits or diseases –4, enhanSuch a strategy is likely to perform well in livestock breeding populations, in which individuals have a high degree of relatedness, allowing low-coverage sequence data to be pooled across individuals that share a haplotype and imputed to individuals who share that haplotype. Due to the implementation of genomic selection in livestock breeding populations, many individuals in breeding nucleus populations have already been genotyped with marker arrays. This genotype data can be used to identify the individuals that share haplotype segments and to select individuals for sequencing that will be more informative from an imputation perspective given a limited budget , 14.We have recently proposed ‘hybrid peeling’ , a fast These properties make hybrid peeling a very appealing imputation method for the cost-effective generation of whole-genome sequence data for large pedigreed populations that have already been extensively genotyped using marker arrays and in which a small proportion of the individuals have been sequenced with variable coverage. In the situations described, the sequence data will be sparsely distributed across the pedigree and there may be great variability in the amount of data to which each individual is exposed. Understanding which factors affect individual-wise and variant-wise imputation accuracy and how their effects are mediated is important for determining how this sequencing strategy, together with hybrid peeling, performs in real settings that are common in animal breeding and for enabling accuracy-aware quality control of the imputed data before downstream analyses. Such knowledge could be used in the future to design cost-effective routine whole-genome sequencing strategies.The objectives of this study were to: (i) demonstrate whether whole-genome sequence data could be imputed with high accuracy in a variety of pig pedigrees when small subsets of individuals are sequenced, mostly at low coverage; (ii) quantify the factors that determine the individual-wise and variant-wise imputation accuracy; and (iii) quantify the impact of data misassignment and pedigree errors on imputation accuracy. Our results showed that high overall imputation accuracies can be achieved for whole-genome sequence data in large pedigreed populations using hybrid peeling provided that the individuals are connected to a sufficient number of informative relatives with marker array or sequence data. Our results have implications for the practical implementation of sequencing and imputation strategies.We structured the study in three tests. In Test 1, we evaluated the imputation accuracy of hybrid peeling in four populations of different sizes. In Test 2, we used regression trees and simulated data based on three real pedigrees to quantify which factors determined the individual-wise and variant-wise imputation accuracy of hybrid peeling. Then, we used the observations in the real data to validate the findings and to predict individuals with low imputation accuracy. In Test 3, we evaluated the potential impact that data misassignment and pedigree errors could have on the imputation accuracy. In the following sections, we first describe how the data was generated and then how the different tests were performed.We performed whole-genome sequencing of 4427 individuals from four commercial pig breeding lines  using a total coverage of approximately 18,514×. To account for a range of population sizes, the number of individuals in each population was 18,349 (20 k), 34,425 (35 k), 68,777 (70 k), or 107,815 (110 k). Approximately 2% (1.7–2.5%) of the individuals in each population were sequenced, mostly at low coverage. The number of individuals sequenced and the coverage at which they were sequenced are summarized for each population in Table We selected the individuals and the coverage at which they were sequenced using a three-step strategy: (1) first, we selected sires and dams that contributed most genotyped progeny in the pedigree (referred to as ‘top sires and dams’) to be respectively sequenced at 2× and 1×; (2) then, we used AlphaSeqOpt part 1  to identWe sorted the pedigrees of each population so that parents appeared before their progeny. Thus, relative position in the pedigree was used as a proxy for the generation to which an individual belonged.Tissue samples were collected from ear punches or tail clippings. Genomic DNA was extracted using Qiagen DNeasy 96 Blood & Tissue kits . Paired-end library preparation was conducted using the TruSeq DNA PCR-free protocol . Libraries for sequencing at low coverage (1× to 5×) were produced with an average insert size of 350 base pairs and sequenced on a HiSeq 4000 instrument . Libraries for sequencing at high coverage (15× or 30×) were produced with an average insert size of 550 base pairs and sequenced on a HiSeq X instrument . All libraries were sequenced at Edinburgh Genomics . Most pigs were also genotyped either at low density  using the GGP-Porcine LD BeadChip  or at high density  using the GGP-Porcine HD BeadChip .Sscrofa11.1 . Single nucleotide polymorphisms (SNPs) were identified with the variant caller GATK HaplotypeCaller  using t03025.6; . DuplicaK 3.8.0; , 22) usihttps://github.com/pysam-developers/pysam), which is a wrapper around htslib and the samtools package  an an15] anThe coupling of an appropriate sequencing strategy , 14, 27 In this paper, we focused on individual-wise imputation accuracy as an indicator of the value of this data for applications such as genomic prediction. Previous studies on imputation accuracy of whole-genome sequence data focused on variant-wise imputation accuracy rather than individual-wise , 37, 43.We demonstrate the high accuracy of hybrid peeling for imputing whole-genome sequence data of hundreds of thousands of individuals from real livestock populations in which only a small fraction of the individuals (2%) had been sequenced, mostly at low coverage. Using data from pig populations, we show that imputation accuracy was very high for individuals that were genotyped with marker arrays with densities that ranged between 15,000 and 75,000 markers genome-wide. The coupling of an appropriate sequencing strategy and hybrid peeling is a powerful method for generating whole-genome sequence data in large pedigreed populations, as long as the individuals are connected to enough informative relatives with marker array or sequence data, and regardless of population size. The characterization of the factors that affect the individual-wise and variant-wise imputation accuracy of hybrid peeling can inform genotyping and sequencing strategies as well as provide accuracy-aware quality control guidelines for the imputed data before downstream analyses. The success of this sequencing strategy demonstrates the possibility of obtaining low-cost whole-genome sequence data on large pedigreed livestock populations, which is a critical step for the successful implementation of whole-genome sequence data for genomic prediction and fine-mapping of causal variants.


Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies. The autophagy-related (Atg) proteins are essential for this pathway, which starts with the nucleation and expansion of the phagophore. Around 20 Atg proteins are considered the core Atg machinery, and they are subdivided into six clusters based on their functions. Atg9 is the only transmembrane protein among the core Atg machinery and belongs to the Atg9-containing vesicle cluster. The Atg9-containing vesicles play a seed role in both the nucleation of the phagophore and the assembly of the Atg machinery. Moreover, the expansion of the phagophore requires the scramblase activity of Atg9 to rearrange the lipids transferred from the endoplasmic reticulum by Atg2. In yeast, Atg9 is present in tubulovesicular compartments, also called the Atg9 reservoirs, which are derived from the Golgi apparatus and are recruited to the phagophore assembly site (PAS) upon the induction of the formation of an autophagosome  and Dnm1 (dynamin-related 1), are involved in autophagy [ATG1 (TAKA) assay. This analysis revealed that Vps1 is involved in Atg9 transport to the PAS and, by using specific point mutants, we also showed that the GTPase and oligomerization activities of Vps1, which are essential for its membrane remodeling, are required for this function in autophagy. Mutations in human dynamins are the cause of some neurological disorders and other pathologies. We found that insertion of several of the corresponding mutations in Vps1 blocked Atg9 trafficking and autophagy, suggesting that a defect in autophagy may be a contributing etiological factor in these diseases.Dynamins are GTPases that can remodel membranes and mediate fission, and they play an important role in trafficking in both the endomembrane system and organelle dynamics. In our study we explored whether the two yeast I422G, blocks autophagy without affecting Atg9 trafficking. This observation implies that Vps1 is probably involved in one or more additional steps of autophagy as well.In summary, we show that the dynamin-like GTPase Vps1 associates with Atg9 at the Atg9 reservoirs to mediate Atg9 transport to the PAS [


ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a ‘programmed’ process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP. Living cells require energy that is provided by the principal intracellular energy carrier, adenosine-triphosphate (ATP). Free energy from ATP that is released upon hydrolysis is utilized in various vital processes including the generation and maintenance of the plasma membrane potential , remodelApoptosis is a form of programmed cell death, with important roles in development, tissue homeostasis, and immunity. It is characterized by distinctive morphological changes, such as membrane blebbing, nuclear condensation, and externalization of phosphatidylserine . AlthougIt has been reported that cells die from necrosis rather than apoptosis when intracellular ATP is depleted prior to otherwise apoptotic stimuli . It has In this work, we established a method for imaging both ATP concentration and caspase-3 activity in a single apoptotic cell with newly developed genetically encoded Förster resonance energy transfer (FRET)-based biosensors for ATP and caspase-3 activity. We found that the intracellular ATP level starts to decrease following the activation of caspase-3 and that the caspase-triggered opening of the plasma membrane channel pannexin-1 (PANX1) is the major cause of the decrease in intracellular ATP.oF1-ATP synthase ε subunit and yellow fluorescent protein . Unfortunately, we found that the original ATeam (AT1.03) was cleaved into its constituent pair of separate fluorescent proteins in apoptotic cells, most probably by activated caspases  cells expressing AT1.03CR. Overall, the intracellular ATP levels remained almost constant for several hours after stimulation. The cytosolic ATP levels in these cells started to decrease after a variable time interval , in which the caspase recognition sequence close to the C-terminus of PANX1 is mutated . When PAΨm, which is required for mitochondrial ATP regeneration, is almost lost upon activation of caspase-3  in mitochondria for the regeneration of ATP, while cells in embryonic tissues and tumors use glycolysis . In the aspase-3 . Even inaspase-3 . This reIf efflux of adenine nucleotides through PANX1 channel causes the decrease in the cytosolic ATP concentrations of apoptotic cells, extracellular adenine nucleotides would suppress the decrease by counteracting the efflux of its cytosolic counterpart. We monitored cytosolic ATP dynamics in apoptotic cells in the presence of AMP, ADP, or ATP in the culture medium, and found that extracellular AMP suppressed the decrease in intracellular ATP levels of apoptotic cells, while extracellular ADP and ATP exhibited no or negligible effects . Thus, i2+ dynamics of apoptotic cells. Mg2+ is an essential divalent cation in cells, required for various cellular processes, including the activity of endonucleases and the compaction of chromosomes during cell division  on plasma membrane, one of the hallmarks of apoptosis . Cycloheximide (CHX) was from Calboichem. Tetramethylrhodamine ethyl ester (TMRE) and alexa647-annexin-V were purchased from Molecular Probes. Recombinant human Trail and anti-His tag antibody were purchased from R and D. Trail was dissolved in DPBS(-) supplemented with 0.1% BSA at a concentration of 20 µg/mL, and then stored at −80 °C. Anti-His antibody was dissolved in DPBS(-) at a concentration of 1 mg/mL, and then stored at −80 °C. 2-Deoxyglucose were from Wako Pure Chemicals . Other chemicals were purchased from Nacalai Tesque  unless otherwise noted.The HeLa cell line was a kind gift from Prof. Shin Yonehara. The cell line was authenticated by short-tandem repeat profiling and was checked for mycoplasma infection. The cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . The SW480 cell line was obtained from American Type Culture Collection. The cells were cultured in Leibovitz’s L-15 medium  supplemented with 10% FBS. Apoptosis of HeLa cells was initiated either by adding anti-FAS antibody (125 ng/mL) and CHX (10 µM) or by adding staurosporine (1 µM). Apoptosis of SW480 cells was initiated by adding Trail (50 ng/mL) and anti-His tag antibody (1 µg/mL). Knockout of PANX1 gene was carried out using pre-designed PANX1-KO CRISPR-Cas9 plasmids (Santa Cruz Biotechnology). Briefly, cells were transfected with CRISPR-Cas9 plasmids using PEI-Max (Polysciences Inc) as described previously . After 2The expression vector for the caspase-resistant AT1.03 (pcDNA-AT1.03CR) was constructed by introducing caspase-resistant mutations (D242N/D339G) into pcDNA-AT1.03  using PCCells were transfected with the AT1.03CR plasmid and the O-DEVD-FR plasmid using PEI-Max as described previously . For exoHeLa cells were transfected with the pcDNA-SCAT3.1 plasmid  using Li2+ imaging was performed using a synthetic Mg2+ indicator MGH (2 were transfected with the plasmid pcDNA-3.1-(+)-Halo-Tag using PEI-Max (Polysciences). After 48 hr, the cells were washed twice with HBSS and incubated with 5 µM MGH(AM) for 30 min, then 50 nM Halo-TMR for 100 min. After washing the cells twice with HBSS, the medium was replaced by phenol red-free DMEM containing 10% FBS. After 4 hr, the cells were washed with HBSS. Imaging of the cells was started just after replacing the medium by phenol red-free DMEM containing 10% FBS, 50 ng/mL anti-FAS and 10 µM cycloheximide. Cells were visualized with a Ti-E inverted microscope using a Plan Apo 40×, 0.95 numerical aperture, dry objective lens (Nikon). Cells were maintained on a microscope at 37°C with a continuous supply of a 95% air and 5% carbon dioxide mixture by using a stage-top incubator (Tokai Hit). For imaging of MGH, an FF01-497/16 excitation filter, an FF516-Di01 dichroic mirror and an FF01-535/22 emission filter were used. For imaging of TMR, an FF01-562/40 excitation filter, an FF593-Di02 dichroic mirror, and an FF01-641/75 emission filter were used. All filters were purchased from Semrock.Mgator MGH . HeLa ce2 (1 mM), propidium iodide (1 µg/mL), Annexin-V-Alexa647 (10 µL), anti-FAS (250 ng/mL) and CHX (10 µM). Cells were visualized with a Ti-E inverted microscope  using a Plan Apo 20×, 0.75 numerical aperture, dry objective lens (Nikon). All filters used for fluorescence imaging were purchased from Semrock : for imaging of propidium iodide, an FF01-504/12 excitation filter, an FF593-Di02 dichroic mirror, and an FF01-562/40 emission filter; for imaging of Annexin- V-Alexa647, an FF02-628/40 excitation filter, an FF660-Di02 dichroic mirror, and an FF01-692/40 emission filter. Fluorescence emissions from cells were imaged using a Zyla4.2 scientific CMOS camera (Andor Technologies). Image analysis was performed using MetaMorph software (Molecular Devices). Fluorescence intensity of Alexa647 within a whole-cell area of each cell that was shrunk but did not exhibit propidium iodide fluorescence was quantified.HeLa cells were cultured on a 35 mm glass-bottom culture dish. At 6 hr before imaging, the medium was replaced by 2 mL of phenol red-free DMEM supplemented with FBS (10%), CaCl6) were cultured in 60 mm dish in DMEM (1 g/L glucose) supplemented with 10% FBS. After 24 hr, the medium was replaced by HBSS. Subsequently, apoptosis was induced either by replacing the medium with 4 mL of phenol red-free DMEM (1 g/L glucose) supplemented with 10% FBS, 250 ng/mL anti-FAS antibody and 10 µM cycloheximide, or by irradiating the cells with 20 mJ UV-C, followed by replacing the medium with 4 mL of phenol red-free DMEM (1 g/L glucose) supplemented with 10% FBS. Small aliquots of culture medium from the cell cultures were sampled at defined intervals. After centrifugation at 3000 x g for 3 min at 4 °C, the supernatant from each sample was stored at −30°C until the glucose and lactate quantification assay. Glucose and lactate concentrations of the aliquots were determined using Glucose CII-test Wako  and Lactate Assay Kit-WST , respectively.HeLa cells , which cross-reacts with both CFP and YFP, and a monoclonal anti-actin antibody (Millipore), respectively. Protein expression levels of PANX1 were examined by western blotting using a rabbit monoclonal antibody against human PANX1 . Actin was also detected as a control by western blotting using a mouse monoclonal anti-β-actin antibody (Santa Cruz). Horseradish peroxidase (HRP)-labeled anti-mouse or anti-rabbit IgG antibody  was used as a secondary antibody. Chemi-Lumi One L reagent  was used as a HRP substrate. A LAS4000 imager  was used to detect the luminescence. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:By designing an assay that allows continuous real-time dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells, the authors have gained detailed knowledge of the kinetics of the decline in cellular ATP relative to caspase activation. This allowed the authors to nail down that adenine nucleotide via caspase activation of pannexin-1 channels early in apoptosis not only recruits phagocytes to the apoptotic cells, but also makes a major contribution to the eventual arrest of energy-dependent functions in the dying cells.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by four peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: George Dubyak (Reviewer #3).Thank you for submitting your work entitled "Pannexin-1 mediates programmed intracellular ATP decrease during apoptosis" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Your manuscript "Pannexin-1 mediates programmed intracellular ATP decrease during apoptosis" has been examined by four expert reviewers whose detailed comments are enclosed. As you can see, the reviewers were very appreciative of the means that you have established for monitoring the dynamics of changes in the cytosolic ATP concentration on a single-cell level during apoptosis. It is certainly a valuable addition to the analytical toolbox applied in the study of cell death. The reviewers were reserved, however, about the fact that too much of the paper (including its title) concerns the finding that Pannexin-1 mediates the decrease of ATP in apoptosis – a finding that has been already documented before. They also raised many questions and requests about the evidence for the new biological insights that the paper aimed to present.eLife, I am unfortunately unable to recommend further handling of current submission. However, we do hope that you will submit the paper anew to eLife after addressing the comments of the reviewers.Since the breath of the requests made by the reviewers is too wide to allow addressing them within the two month period allowed by A simpler and quicker alternative would be to resubmit the paper, not as Research Article, but in the Tools and Resources category (after adding more detailed description of the ATP monitoring technique). This category is appropriate for a new technique that will be generally useful to a field, and although new biological insight is not required in this category you should be able also to include your major observations in the "Tools and Resources" format.Reviewer #1:Overall this is an interesting study that adds incremental insight to the original story from Chekeni et al., 2010, follow up study by Sandilos et al., 2012, and other more recent work. The use of imaging to assay ATP levels creates a potential for revealing spatial information but this potentially novel aspect is unfortunately not fully realized. Most importantly, clear interpretations of some of the key pieces of imaging data are hampered by the lack of controls, including controls demonstrating that the loss of signal is not a loss of cell area/cell collapse. Further, rather than relying primarily on the use of the blocker probenecid for a large portion of the study, the systematic use of a more complete genetic deletion model like Crispr Cas9, which should be readily feasible in the Hela model, would also make the interpretation and model more convincing. There are also some kinks in the logic/model that could be smoothened out (clarified) by more detailed explanations. In summary, there is potential here, but the manuscript seems premature.Detailed comments:Abstract: I'm not sure this sentence in the Abstract makes sense (nor is it substantiated by the data) – "Thus, the pannexin-1-mediated "programmed intracellular ATP decrease" of the apoptotic cell might benefit surrounding healthy cells by preserving environmental glucose."Results first paragraph – Why is cell-specific information important? This isn't very well explained.The first sentence should specify whether "between cells" means between cell types or between cells of a given cell type to emphasize the point being made.Variable time interval range should be described in the text.“It should be noted that the timing and the speed of the decreases in ATP did vary between cells, emphasizing the advantages of single-cell ATP analysis of apoptotic cells over the conventional cell population-based methods.” Great, but why is that precise information important?2, however this is noted in the Mg2+ level measurements section). Some minimal details of the imaging/acquisition should be provided in the main body of the text.Figure 1—figure supplement 2 should include a negative control consisting of a cohort of cells treated with a control antibody to control for cell death due to imaging conditions  quite spatially restricted but it is not clear that the space where the signal is absent is still "cell". Without a cell membrane marker like WGA (which can be used in live cells), or a DIC image (or similar) it is impossible to ascertain whether or not this is the case.It seems that Figures 1 and 2 could be collapsed into one figure.It seems that Figures 3 and 4 should be combined. To more convincingly show that PANX1 is the mediator of the caspase mediated ATP release, the authors should completely delete it using Crispr-Cas9 and then re-express the caspase resistant mutant (and compare with re-expressed WT). There should be at least 3 biological replicates and the traces from the individual biological replicates should be distinguishable from one another.Figure S6: how many biological replicates here? Reporting on number of cells and number of biological replicates should be consistent throughout the figures.Figure 5: The logic behind the interpretation of the results of Figure 5 isn't clear, please elaborate on "Strikingly, extracellular AMP and ATP, but not ADP, suppressed the decrease in intracellular ATP levels of apoptotic cells . Thus, it is most likely that the intracellular ATP decrease of apoptotic cells is a result of a reduction in adenosine nucleotide pools inside apoptotic cells, which is caused, at least in part, by release of ATP and AMP from the cells."2+ and glucose consumption this also be assayed with Crispr-Cas9 deletion of Panx1, and the compared with wildtype and mutant caspase-defective PANX1 expression.Figures 6 and 7: Cytosolic free MgThe conclusions drawn from Figure 7 don't make intuitive sense to me. While the cells aren't consuming the ATP they are still in effect losing ATP by releasing it, so less ATP is available for cellular processes, so they should still "want to consume" glucose.Finally, given the nature of their studies the authors should take the following papers into consideration and add genetic models to strengthen their investigation of these aspects:1) Probenecid interferes with renal oxidative metabolism: A potential pitfall in its use as an inhibitor of drug transport https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1572299/2) Probenecid potentiates MPTP/MPP+ toxicity by interference with cellular energy metabolism. https://www.ncbi.nlm.nih.gov/pubmed/23802648Reviewer #2:The major novelty of this paper is the generation of new probes to study intracellular ATP, and using this probe the authors address the involvement of Pannexin-1 in decreasing intracellular ATP (ATPi) during apoptotic cell death via FRET-based single cell imaging. Additionally, albeit in less detail, the authors begin to show that apoptotic cells can use glycolysis during the cell death process but the loss of intracellular ATP impedes this.eLife as it adds another level to the understanding of cell death process.Overall usage of the probe to track single cells and their intracellular ATP and some of the controls are well done and the findings are backed up with experimental evidence. However, as the authors acknowledge and based this work on, it is known that ATP is released via pannexin channels during apoptosis and this occurs via caspase-mediated cleavage of the channel. What is potentially novel is the possible differential involvement of glycolysis and OXPHOS during apoptosis when Panx1 function is blocked – however, the data supporting this claim is weak and will require additional experiments to fully understand the contributions of these metabolic processes. Additionally, the authors do not begin to explore what impact this process has on the neighboring tissue, and what the larger importance of it could be. If the authors can successfully address the major concerns, especially major concern 1, 2, and 3, then this work would potentially be a strong candidate for publication in 1) Although the authors show intracellular ATP decreases during apoptosis, what is the importance of this process? They speculate that there will be more glucose for surrounding cells  because the apoptotic cells cannot use it as they will have lost their ATPi. However, they never actually provide evidence for this and by mentioning it in the Abstract it is misleading. This needs to be addressed much more in detail.2) This paper primarily addresses one form of death – anti-Fas + methotrexate treatment of HeLa cells. The authors make much larger claims on what this would mean for the larger process of apoptosis and the glycolysis vs. OXPHOS may be impactful for the tissue, etc. But these are not justified. At the minimum, the authors should use one or two other cell lines and another form of cell death.3) Lastly, the authors provide some indirect evidence for the function of glycolysis during apoptosis. By inhibiting Panx1 channels, thereby increasing ATPi , the authors show an increase in glucose uptake. However, this does not necessarily indicate increased glycolysis. Can the authors measure lactate release to more directly show that there is increased glycolysis during Panx1 inhibition (or even better via Seahorse Assay)? Interestingly, this may indicate that glycolysis is still occurring during apoptosis even if Panx1 is active although it may be with lower levels of ATPi and therefore to a less extent. This is an important point and needs to be proven.4) The authors attempt to rule-out the involvement of mitochondrial depolarization as a mechanism for decreased ATPi by treatment of CCCP on live cells and showing that ATPi does not decrease. However, this is not a fair comparison because this does not indicate that it cannot be occurring during apoptosis (or in dying cells). Can the authors more clearly rule out this issue? For example, can the authors induce cell death, but inhibit mitochondrial depolarization, and show that ATPi is still decreasing regardless of mitochondria membrane potential? Does this affect the dynamics of ATPi decrease?5) The authors state that "the ATP decrease in apoptotic cells is a caspase-3-dependent process", but they reach this conclusion based on timing events . Can the authors inhibit caspases with zVAD/QVD and show that ATPi levels do not decrease after anti-Fas treatment to directly prove this statement?6) Can the authors show that a cellular ATPi levels still drop in a cell that endogenously/naturally uses OXPHOS and that this is still dependent on Panx1 activation? In this paper they use a system where glucose has been depleted and this can impact results. Also, does Panx1 inhibition still increase glycolysis in these cells? It would be interesting to know whether these cells attempt to switch their metabolism during cell death.Reviewer #3:This study describes a novel on-line optical probe to track the kinetics of intracellular ATP concentration in single HeLa cells during the induction/progression of apoptosis by Fas activation. HeLa are "type II" cells wherein extrinsic apoptotic  activation of the executioner caspase-3 is indirectly mediated by a caspase-8> Bid > mitochondrial permeabilization> caspase-9 cascade. This contrasts with type I cells wherein extrinsic apoptosis involves a direct caspase-8 > caspase-3 cascade. The major findings are that the decrease in intracellular ATP: 1) occurs shortly after collapse of mitochondrial membrane potential and accumulation of active caspase-3; and 2) is mediated by caspase-3 mediated proteolytic activation of plasma membrane pannexin-1 channels as conduits for efflux of ATP and other adenine nucleotides. Importantly, genetic or pharmacological suppression of pannexin-1 function markedly delays the decrease in intracellular ATP even when mitochondrial function or ATP synthesis is inhibited. That proteolytically-gated pannexin-1 channels can be major determinants of intracellular and extracellular adenine nucleotide metabolism/ distribution basically confirms several previous studies with populations of apoptotic cells. However, extending such findings to the single cell level is a significant contribution. One major issue requires attention.Major concern: In the Figure 7 experiments, the authors use 2-deoxyglucose (2DG) as a "glycolysis inhibitor" to conclude that ongoing ATP synthesis via glycolysis is sufficient to sustain the intracellular ATP levels of apoptotic HeLa cell for several hours when pannexin-1-mediated ATP efflux is blocked by probenecid. There's major problem with this interpretation. 2DG acts an intracellular ATP "sink". Hexokinase readily phosphorylates 2DG, but 2DG-phosphate can't be further converted to fructose-6P. Thus, it's difficult to deconvolute the ATP-consuming action of 2DG from subsequent suppression of glycolytic ATP production. This needs to be addressed and alternative experimental support for the stated conclusion is required. Inhibition of glucose uptake by cytochalasins or other GLUT blockers might be an approach.Reviewer #4:Too much of this paper is dedicated to demonstration of the role of PANX1 cleavage in ATP release, and in the resulting depletion of the cellular ATP pool, which are, both, not new.  been reported that over-expression of PNAX1-CR (a dominant negative mutant) significantly suppresses the release of ATP from apoptotic cells").What the paper should have focused at, are the following two things that are new and potentially important:a) The more accurate information of the kinetics of ATP-loss during apoptosis, and its relation to the kinetics of other changes, which one can gain by applying the ATPsensor that the authors devised to monitor the changes on a single-cell level.b) Novel knowledge of the functional significance of the changes in the cellular ATP level in the course of apoptosis.Both subjects are addressed, but in a way that is not thorough enough to yield solid conclusions.With regard to the documentation of the kinetics of ATP loss, the paper claims to present findings that challenge a prior claim that the occurrence of apoptosis requests maintenance, or in fact – elevation, of the ATP cellular levels till a rather late stage in the process . However, the quality of the evidence that the current paper presents in support of its claims is harmed by the following shortcomings:a) The means applied for arrest of PANX1 function have limitations. Knock down is incomplete. Probenecid has unrelated effects. The authors should have knocked outthe PANK1 gene by Crispr/Cas9 instead.b) The cause for the marked intercellular variations in the effect of PANK1 inhibition on the kinetics of ATP decrease is not clear. The authors did not exclude contribution of variation in the extent of arrest of PANK1 function/expression, which makes it particularly important to use cells in which PANK1 was knocked out.c) The apparent lag in the decrease of ATP pursuant to caspase activation/mitochondrial potential loss , which might actually be related to the observations of Zamaraeva et al., and the intercellular variation in the length of this lag, requests further analysis and documentation. Does the length of the lag depend on the mode of cell death induction, on the type of cell, on growth condition? The possible mechanisms for this lag should be discussed.Particularly in view of the dissimilarity to the study of Zamaraeva et al., where various kinds of cells and various means of death induction have been examined, there is need also to apply the tests of the current study in various cells and with various means of death induction. It is of particular importance to repeat the tests in cells induced to die through the type 1 extrinsic apoptotic pathway – a direct Fas (or TNF) >casp8>casp3>Panx1 cascade independent of perturbed mitochondrial function.d) The evidence presented for some of the major conclusions in this paper  is much too limited, constituting just of data acquired with a single cell.e) It is not clear to which extent does the apoptotic volume-decrease, such as that seen in Figure 1A, affect the interpretation of the data acquired with the ATP sensor. Towhich extent does the decrease of the signal indeed reflect decrease of ATP and not decrease in cell area? A clearer more complete description of the methodology e.g. more details of how the background subtractions were made, should be presentedWith regard to the functional significance of ATP loss in apoptosis, the paper should have more thoroughly addressed the following three issues:maintenance of high ATP levels in the cells. At which exact stage of the apoptotic process does the maintenance of high ATP in the cells turn, from being requested for the apoptotic process, to become inhibitory to some of its manifestations?a) Apoptosis is largely believed to depend on b) Does the observed increase in glucose consumption indeed reflect enhanced glycolysis (just monitoring glucose levels does not suffice to tell that), and is this increase also occurring in other cell types, including ones that normally resort to oxidative phosphorylation, and in cells dying in response to other triggers?c) Which of the various manifestations of apoptosis depend on the decrease of ATP. The authors suggest that, besides its contribution to arrest of glucose consumption, this decrease accounts for the role of PANX1 in restricting blebbing  and that it also contributes to DNA fragmentation/condensation throughincrease of magnesium. However, they provide no evidence for that. One would expect comprehensive assessment of the impact of arrest of ATP decrease (e.g. by incubating the dying cells in growth medium containing ATP or AMP) on a wide range of functional and mechanistic events that are known to be associated with apoptosis. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:Detailed comments:Abstract: I'm not sure this sentence in the Abstract makes sense (nor is it substantiated by the data) – "Thus, the pannexin-1-mediated "programmed intracellular ATP decrease" of the apoptotic cell might benefit surrounding healthy cells by preserving environmental glucose."We have removed this discussion from the Abstract.Results first paragraph – Why is cell-specific information important? This isn't very well explained.The first sentence should specify whether "between cells" means between cell types or between cells of a given cell type to emphasize the point being made.We have changed the sentence as follows: “In general, the progression of apoptosis varies between individual cells, even in the same cell type.”Variable time interval range should be described in the text.We have added the time information in the text as follows: “The cytosolic ATP levels in these cells started to decrease after a variable time interval .”“It should be noted that the timing and the speed of the decreases in ATP did vary between cells, emphasizing the advantages of single-cell ATP analysis of apoptotic cells over the conventional cell population-based methods.” Great, but why is that precise information important?This sentence has been removed because it was considered disruptive to the flow of the story.2, however this is noted in the Mg2+ level measurements section). Some minimal details of the imaging/acquisition should be provided in the main body of the text.Figure 1—figure supplement 2 should include a negative control consisting of a cohort of cells treated with a control antibody to control for cell death due to imaging conditions . The cells did not die without the apoptotic stimulus. During imaging, cells were maintained at 37˚C and in 5% COFigure 1: The data shown in B for a single cell could also be aggregated/averaged and shown for a larger cohort of cells by adjusting the data as in part C. Part C should be added to Figure 1—figure supplement 2 as is the same data presented with an adjusted time scale, not clear how this really adds anything to the story. The images in Part A are much too small to appreciate and spatial information.The old Figure 1 has been removed because we no longer used TMRE as an indicator of caspase-3 activation in the revised experiments.Perhaps a more important point is that the images provided in Figure 1A suggest that cell has collapsed between minute 297 and 300, rather than lost ATP, as it appears the FRET signal and TMRE signal are both now (at minute 300) quite spatially restricted but it is not clear that the space where the signal is absent is still "cell". Without a cell membrane marker like WGA (which can be used in live cells), or a DIC image (or similar) it is impossible to ascertain whether or not this is the case.The fluorescence might have appeared to be localized to a part of the cell because the apoptotic cells shrank. There is no spatial restriction of the fluorescent signals. We used larger fluorescent images of the cells in the revised manuscript to clearly show cell shrinkage.It seems that Figures 1 and 2 could be collapsed into one figure.These figures have been removed in the revised manuscript.It seems that Figures 3 and 4 should be combined. To more convincingly show that PANX1 is the mediator of the caspase mediated ATP release, the authors should completely delete it using Crispr-Cas9 and then re-express the caspase resistant mutant (and compare with re-expressed WT). There should be at least 3 biological replicates and the traces from the individual biological replicates should be distinguishable from one another.We used PANX1-knockout cell lines, instead of using siRNA and inhibitors, in the revised manuscript. Because there are many panels, we decided not to combine figures. Traces from individual replicates were labeled with bars of different shades.Figure S6: how many biological replicates here? Reporting on number of cells and number of biological replicates should be consistent throughout the figures.Number of cells and number of biological replicates were presented throughout the revised manuscript.Figure 5: The logic behind the interpretation of the results of Figure 5 isn't clear, please elaborate on "Strikingly, extracellular AMP and ATP, but not ADP, suppressed the decrease in intracellular ATP levels of apoptotic cells . Thus, it is most likely that the intracellular ATP decrease of apoptotic cells is a result of a reduction in adenosine nucleotide pools inside apoptotic cells, which is caused, at least in part, by release of ATP and AMP from the cells."I agree that the explanation in the previous manuscript was ambiguous. We have added the following sentences in the revised manuscript: “If efflux of adenine nucleotides through PANX1 channel causes the decrease in the cytosolic ATP concentrations of apoptotic cells, extracellular adenine nucleotides would suppress the decrease by counteracting the efflux of its cytosolic counterpart.”2+ and glucose consumption this also be assayed with Crispr-Cas9 deletion of Panx1, and the compared with wildtype and mutant caspase-defective PANX1 expression.Figures 6 and 7: Cytosolic free Mg2+ dynamics using PANX1-knockout cells.We re-measured glucose consumption and free MgThe conclusions drawn from Figure 7 don't make intuitive sense to me. While the cells aren't consuming the ATP they are still in effect losing ATP by releasing it, so less ATP is available for cellular processes, so they should still "want to consume" glucose.Km will retain their activity even at low ATP levels, the decreased intracellular ATP levels will reduce the net ATPase activity of the cell. We added the following sentence in the revised manuscript: “It is also likely that the reduction of ATP during apoptosis leads to the decrease in the activities of other ATPases because ATPase activity depends on the concentration of ATP.”In general, activity of enzymes, including ATPases, highly depends on substrate concentration. Although I agree that some ATPases with very low Finally, given the nature of their studies the authors should take the following papers into consideration and add genetic models to strengthen their investigation of these aspects:1) Probenecid interferes with renal oxidative metabolism: A potential pitfall in its use as an inhibitor of drug transport https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1572299/2) Probenecid potentiates MPTP/MPP+ toxicity by interference with cellular energy metabolism. https://www.ncbi.nlm.nih.gov/pubmed/23802648Thank you for raising the concern. We decided to remove the data that used probenecid.Reviewer #2:[…]1) Although the authors show intracellular ATP decreases during apoptosis, what is the importance of this process? They speculate that there will be more glucose for surrounding cells  because the apoptotic cells cannot use it as they will have lost their ATPi. However, they never actually provide evidence for this and by mentioning it in the Abstract it is misleading. This needs to be addressed much more in detail.Metabolism is one of the major biological activity of living systems. The metabolic activity of cells is an indicator of "the state of being alive". In other words, in order for a cell to "die a complete death", metabolism must stop. However, it has not been well understood how metabolism of dead cells ceases. Our results showed that PANX1 plays a major role in stopping glycolysis, a central part of metabolism, of apoptotic cells, through facilitating ATP loss. This is the biological importance of ATP depletion in apoptosis that we want to emphasize in the revised work. We discuss this statement in the revised manuscript. We agree that the model that PANX1 stops the waste of glucose by apoptotic cells to benefit surrounding cells is a discussion/speculation. We have deleted this point from Abstract.2) This paper primarily addresses one form of death – anti-Fas + methotrexate treatment of HeLa cells. The authors make much larger claims on what this would mean for the larger process of apoptosis and the glycolysis vs. OXPHOS may be impactful for the tissue, etc. But these are not justified. At the minimum, the authors should use one or two other cell lines and another form of cell death.In the revised manuscript, we analyzed staurospoine-induced apoptosis , as well as anti-FAS + cycloheximide-induced apoptosis for HeLa cells. In addition,TRAIL-induced apoptosis was analyzed for SW480 cells .3) Lastly, the authors provide some indirect evidence for the function of glycolysis during apoptosis. By inhibiting Panx1 channels, thereby increasing ATPi , the authors show an increase in glucose uptake. However, this does not necessarily indicate increased glycolysis. Can the authors measure lactate release to more directly show that there is increased glycolysis during Panx1 inhibition (or even better via Seahorse Assay)? Interestingly, this may indicate that glycolysis is still occurring during apoptosis even if Panx1 is active although it may be with lower levels of ATPi and therefore to a less extent. This is an important point and needs to be proven.Thank you for the suggestion. Lactate release was also measured in the revised manuscript. Consistent with glucose consumption data, lactate release did not cease in PANX1-KO cells. To further support that existence of glycolytic activity, we showed that lactate dehydrogenase inhibitor sodium oxamate also reduces intracellular ATP, like hexokinase inhibitor 2-deoxyglucose does. Please note that we did not show that glycolysis is increased, just showed that glycolysis is retained at substantial levels in apoptotic cells.4) The authors attempt to rule-out the involvement of mitochondrial depolarization as a mechanism for decreased ATPi by treatment of CCCP on live cells and showing that ATPi does not decrease. However, this is not a fair comparison because this does not indicate that it cannot be occurring during apoptosis (or in dying cells). Can the authors more clearly rule out this issue? For example, can the authors induce cell death, but inhibit mitochondrial depolarization, and show that ATPi is still decreasing regardless of mitochondria membrane potential? Does this affect the dynamics of ATPi decrease?We have no idea to perform the suggested experiment. The relevant text and data have been removed in the revised version for clarity. We do not think that the removal of this result affects the major conclusion of this study.5) The authors state that "the ATP decrease in apoptotic cells is a caspase-3-dependent process", but they reach this conclusion based on timing events . Can the authors inhibit caspases with zVAD/QVD and show that ATPi levels do not decrease after anti-Fas treatment to directly prove this statement?We have confirmed that treatment with zVAD inhibit ATPi decrease after anti-FAS stimulation .6) Can the authors show that a cellular ATPi levels still drop in a cell that endogenously/naturally uses OXPHOS and that this is still dependent on Panx1 activation? In this paper they use a system where glucose has been depleted and this can impact results. Also, does Panx1 inhibition still increase glycolysis in these cells? It would be interesting to know whether these cells attempt to switch their metabolism during cell death.As far as we know, cultured mammalian cells that are generally used for apoptosis research predominantly use glycolysis for ATP synthesis rather than OXPHOS. Therefore, it is difficult to conduct experiments to solve this question at present.Reviewer #3:[…]Major concern: In the Figure 7 experiments, the authors use 2-deoxyglucose (2DG) as a "glycolysis inhibitor" to conclude that ongoing ATP synthesis via glycolysis is sufficient to sustain the intracellular ATP levels of apoptotic HeLa cell for several hours when pannexin-1-mediated ATP efflux is blocked by probenecid. There's major problem with this interpretation. 2DG acts an intracellular ATP "sink". Hexokinase readily phosphorylates 2DG, but 2DG-phosphate can't be further converted to fructose-6P. Thus, it's difficult to deconvolute the ATP-consuming action of 2DG from subsequent suppression of glycolytic ATP production. This needs to be addressed and alternative experimental support for the stated conclusion is required. Inhibition of glucose uptake by cytochalasins or other GLUT blockers might be an approach.We agree that 2DG can potentially act as an ATP sink. In the revised manuscript, we also used sodium oxamate, an inhibitor for lactate dehydrogenase. Supply of NAD+, which is required for the reaction of GAPDH, is inhibited by this chemical. Consistent with our model, the addition of sodium oxamate to the apoptotic PANX1-KO cells rapidly reduced the intracellular ATP levels .Reviewer #4:Too much of this paper is dedicated to demonstration of the role of PANX1 cleavage in ATP release, and in the resulting depletion of the cellular ATP pool, which are, both, not new.  been reported that over-expression of PNAX1-CR (a dominant negative mutant) significantly suppresses the release of ATP from apoptotic cells").The reviewer might think that nucleotide release is synonymous with depletion of intracellular ATP concentration, but we do not think that the idea is correct. For example, astrocytes and vascular endothelial cells are known to release ATP frequently, but the ATP release is unlikely to deplete ATP in these cells. Most of previous PANX1-apoptosis works have focused on nucleotide release. Therefore, we disagree with the reviewer's view that there is no scientific novelty in examining in detail whether the mechanism of intracellular ATP depletion in apoptosis is related to the mechanism of nucleotide release.What the paper should have focused at, are the following two things that are new and potentially important:a) The more accurate information of the kinetics of ATP-loss during apoptosis, and its relation to the kinetics of other changes, which one can gain by applying the ATPsensor that the authors devised to monitor the changes on a single-cell level.b) Novel knowledge of the functional significance of the changes in the cellular ATP level in the course of apoptosis.Both subjects are addressed, but in a way that is not thorough enough to yield solid conclusions.Thank you for the positive comments on our work. We believe that the revised work has strengthen these conclusions.With regard to the documentation of the kinetics of ATP loss, the paper claims to present findings that challenge a prior claim that the occurrence of apoptosis requests maintenance, or in fact – elevation, of the ATP cellular levels till a rather late stage in the process . However, the quality of the evidence that the current paper presents in support of its claims is harmed by the following shortcomings:a) The means applied for arrest of PANX1 function have limitations. Knock down is incomplete. Probenecid has unrelated effects. The authors should have knocked outthe PANK1 gene by Crispr/Cas9 instead.b) The cause for the marked intercellular variations in the effect of PANK1 inhibition on the kinetics of ATP decrease is not clear. The authors did not exclude contribution of variation in the extent of arrest of PANK1 function/expression, which makes it particularly important to use cells in which PANK1 was knocked out.c) The apparent lag in the decrease of ATP pursuant to caspase activation/mitochondrial potential loss , which might actually be related to the observations of Zamaraeva et al., and the intercellular variation in the length of this lag, requests further analysis and documentation. Does the length of the lag depend on the mode of cell death induction, on the type of cell, on growth condition? The possible mechanisms for this lag should be discussed.Particularly in view of the dissimilarity to the study of Zamaraeva et al., where various kinds of cells and various means of death induction have been examined, there is need also to apply the tests of the current study in various cells and with various means of death induction. It is of particular importance to repeat the tests in cells induced to die through the type 1 extrinsic apoptotic pathway – a direct Fas (or TNF) >casp8>casp3>Panx1 cascade independent of perturbed mitochondrial function.Because our results indicate that cytosolic ATP is maintained until caspase-3 is activated , we think that our results do not fully challenge the previous claim that the occurrence of apoptosis requests maintenance of ATP. However, we did not observe an apparent increase in cytosolic ATP just after induction of apoptosis, unlike the observation by Zamaraeva and colleagues. The cause of this discrepancy is unknown at present. According to the reviewer’s suggestion, we have performed the same experiments again using PANX1 knockout cells and using different types of apoptotic stimulations. Again, we observed the short lag in ATP reduction and the kinetic variations in cytosolic ATP reduction after caspase-3 activation. The possible causes for the lag and the variations were discussed in the revised manuscript as described below.“Notably, the intracellular ATP levels of PANX1-KO cells were almost unchanged in the first 30-60 min after the activation of caspase-3, followed by a gradual ATP decline . The lag in the cytosolic ATP decrease observed for PANX1-KO cells might be partially relevant to the previous observation by Zamaraeva , which suggested the enhancement of cytosolic ATP level after apoptotic stimulation.”In addition, we performed the experiments using SW480 cells , which is known to die through type 1 cell death pathway when stimulated with TRAIL .d) The evidence presented for some of the major conclusions in this paper  is much too limited, constituting just of data acquired with a single cell.We disagree with this assertion. We think that the power of single-cell analysis has led to these results. In addition, the single-cell ATP imaging technology, which is the basis of this research, has been proven to be practical by number of researchers over the past decade.e) It is not clear to which extent does the apoptotic volume-decrease, such as that seen in Figure 1A, affect the interpretation of the data acquired with the ATP sensor. Towhich extent does the decrease of the signal indeed reflect decrease of ATP and not decrease in cell area? A clearer more complete description of the methodology e.g. more details of how the background subtractions were made, should be presentedSince we monitor the ratio of YFP fluorescence and CFP fluorescence as a signal, any increase or decrease in fluorescence intensity due to cell shrinkage is offset. That is, this method is much more robust against morphological changes in cells than methods such as obtaining a single fluorescence wavelength. We added the following sentence. “It should be noted that any increase or decrease in fluorescence intensity due to cell morphological change was offset because we monitored the ratios of fluorescence intensities of an acceptor and a donor of the FRET biosensors.”Details of background subtractions and ratio calculation were described in Materials and methods.With regard to the functional significance of ATP loss in apoptosis, the paper should have more thoroughly addressed the following three issues:a) Apoptosis is largely believed to depend on maintenance of high ATP levels in the cells. At which exact stage of the apoptotic process does the maintenance of high ATP in the cells turn, from being requested for the apoptotic process, to become inhibitory to some of its manifestations?Previous studies have shown that the pre-reduction of ATP inhibits the activation of caspase-3  and switches cell death form from apoptosis to necrosis . It is also known that dATP/ATP is also important for the formation of apoptosomes . Our results demonstrated that cytosolic ATP is retained at high level until caspase-3 is activated. Thus, it is very likely that maintenance of high ATP is required until the activation of caspases. We added the above points into the revised manuscript (Discussion paragraph one). We found that PANX1-KO did not affect phosphatidylserine exposure, indicating that ATP loss is not critical to this process . Apoptotic PANX1-KO cells did not immediately die a complete death because they did not completely stop glycolytic metabolism . Also, apoptotic PANX1-KO cells continued to waste environmental glucose for more than 32 hours, which potentially causes starvation of the surrounding cells .b) Does the observed increase in glucose consumption indeed reflect enhanced glycolysis (just monitoring glucose levels does not suffice to tell that), and is this increase also occurring in other cell types, including ones that normally resort to oxidative phosphorylation, and in cells dying in response to other triggers?not enhanced, as evident from Figure 6E-G: Although apoptotic PANX1-KO cells retain glycolytic activity, the glucose consumption rate and the lactate release rate are slower than the wild-type cells.Glycolytic activity of apoptotic cell is c) Which of the various manifestations of apoptosis depend on the decrease of ATP. The authors suggest that, besides its contribution to arrest of glucose consumption, this decrease accounts for the role of PANX1 in restricting blebbing  and that it also contributes to DNA fragmentation/condensation throughincrease of magnesium. However, they provide no evidence for that. One would expect comprehensive assessment of the impact of arrest of ATP decrease (e.g. by incubating the dying cells in growth medium containing ATP or AMP) on a wide range of functional and mechanistic events that are known to be associated with apoptosis.We added new videos as supplementary materials, which clearly shows that an apoptotic PANX1-KO cell immediately stop blebbing after the forced decrease in intracellular ATP concentration by 2DG treatment. We think that this video strongly supports the idea that PANX1-enhanced decrease in intracellular ATP restricts excessive blebbing of apoptotic cells.