In addition, OS also markedly promoted the expression of both Krox20 and myelin basic protein (MBP) in SC culture medium containing dBcAMP/NRG1, which induced differentiation. We found that the effects of OS are dependent on the intracellular Ca2+ level. OS induces elevated intracellular Ca2+ levels through the T-type voltage-gated calcium channel (VGCC) and mobilization of Ca2+ from both inositol 1,4,5-trisphosphate (IP3)-sensitive stores and caffeine/ryanodine-sensitive stores. Furthermore, we confirmed that OS significantly increased expression levels of both Krox20 and MBP in SC-motor neuron (MN) coculture, which was notably prevented by pharmacological intervention with Ca2+. Taken together, our results demonstrate that OS of SCs increases the intracellular Ca2+ level and can regulate proliferation, differentiation, and myelination, suggesting that OS of SCs may offer a new approach to the treatment of neurodegenerative disorders.Schwann cells (SCs) constitute a crucial element of the peripheral nervous system, by structurally supporting the formation of myelin and conveying vital trophic factors to the nervous system. However, the functions of SCs in developmental and regenerative stages remain unclear. Here, we investigated how optogenetic stimulation (OS) of SCs regulates their development. In SC monoculture, OS substantially enhanced SC proliferation and the number of BrdU Dedifferentiated SCs can undergo redifferentiation through upregulation of myelin-associated genes and remyelinate the regenerating axons for complete functional recovery, but regeneration of the PNS with an injured area more than 3 cm in size remains relatively unexplored8. The potential efficacy of SCs is dependent on patient age, regeneration time, and injury extent9. Intensive treatment strategies have been shown to promote axon regeneration, but the molecular mechanisms underlying the regenerative response and long-distance axon regeneration are not fully understood. More advanced practical methods for determining the roles of SCs in promoting axon regeneration are essential.Axon regeneration and remyelination after injury are limited in the central nervous system (CNS) of adult mammals, but recovery capacity is considerably greater in the peripheral nervous system (PNS). Schwann cells (SCs), a glial cell type of the PNS, are crucial for regeneration in the PNS. After peripheral nerve injury, SCs immediately transform into a dedifferentiated state and activate proliferation 13. This revolutionary technique can control the electrical processes of specific types of cells, including cell signaling, modulation, etc.; in particular, it affords unprecedented control of neural activity with high spatiotemporal precision in many types of nervous system investigations. To date, numerous optogenetic studies have demonstrated modulation of the neural system at the molecular and cellular levels through neuronal excitation16, inhibition21, and biochemical control26 of intracellular membranes, the endoplasmic reticulum, nuclear complexes, and mitochondria. More recently, a few studies on optogenetic applications have focused on glial cells, such as astrocytes, in the CNS31. Regarding optogenetically manipulated astrocytes, their activation can be separated from that of nearby neurons, which may shed light on the role of glial cells in highly complex brain functions including modulation of synaptic transmission31, regulation of response selectivity of visual cortex neurons30, control of the sleep-wake cycle in the posterior hypothalamus28, and activation of ATP release27. In contrast to astrocytes, the possibility of optogenetic SCs has not been reported. When optogenetic-mediated dorsal root ganglia cells are stimulated, SCs have been shown to respond to nerve stimuli, and the proliferation and migration of SCs were regulated by optogenetic neuronal stimulation32. Accumulating evidence has shown that SCs play essential roles in neuronal survival34, axon growth36, and myelination38, but it has proven difficult to investigate their role in neuron-glia complexes. Although the actions of SCs are crucial to neurons, fundamental questions regarding their significance to the PNS remain unresolved.Optogenetics has gained attention as a promising biological technology, in which genetically modified cells within complex neural tissues are regulated using light of a specific wavelength after introducing light-sensitive microbial opsins, such as channelrhodopsin-2 (ChR-2), halorhodopsin (NpHR), and archaerhodopsin2+ level. We discuss below how OS affects the differentiation and myelination of SCs in a coculture model of SC and motor neuron (MN) cells.Here, we investigated an optogenetic approach that serves as a useful extension of the tools currently available to study SC function in the PNS. We focused on how optogenetic stimulation (OS) of SCs affects SC development, such as proliferation, differentiation, and myelination, and demonstrated that OS of SCs not only induces SC proliferation but also promotes the differentiation and myelination of SCs in SC monoculture. Moreover, we provided evidence that OS activity is correlated with a change in the intracellular Ca+-S100ß+-SCs increased by about 2.6-, 2.7-, 2.4-, 2.4-, and 2.3-fold at 1, 3, 6, 12, and 24 h, respectively  were transfected with 5 µg of calcium-translocating channelrhodopsin (CatCh) DNA. Before examining SC proliferation, we verified the efficiency of CatCh transfection in the SC monoculture with a YFP-expressing plasmid, which was strongly expressed in the majority of SCs at DIV 7, with 92.4 ± 2.6% of SCs being transfected , which is a critical component of myelin sheaths. In a traditional SC culture environment, SCs without axonal contact progressively downregulate the expression of myelin-associated genes. Consistent with previous reports, we confirmed that SCs rapidly decrease their expression of myelin-associated proteins, and that the number of MBPV 7 Fig. . OS of SV 7 Fig. .Figure 240. To further examine the effect of OS of SCs in SC culture medium treated with dBcAMP/NRG1, we added a high concentration of dBcAMP/NRG1 to the SC culture medium at DIV 3. SCs gradually increased their MBP expression in SC culture medium including dBcAMP/NRG1 at DIV 7 and, surprisingly, MBP expression was substantially enhanced by OS of SCs  and neuregulin1  in an axon-free culture environmentSCs Fig. . When cuion Fig. . Furtherion Fig. . Howeverion Fig. . Taken t2+ influx into the SC plasma membrane and Ca2+ mobilization in the internal Ca2+ stores. When constructing the DNA plasmid used for transfection, CatCh exhibited an approximately 70-fold increase in light sensitivity compared to that of wild-type ChR2 following enhanced Ca2+ permeability41. To examine whether OS induces a change in intracellular Ca2+ signals, SCs were treated with various pharmacological Ca2+-related interventions. Before examining the intracellular Ca2+ level of SCs, we removed extracellular Ca2+ and the intracellular Ca2+ chelator with BAPTA-1 AM by washing with a standard buffer and regulated extracellular Ca2+ during analysis . We observed a very small difference between the intensity of the resulting peak and the baseline intensity in the Ctrl, Onlystim, and Transf. groups. Indeed, there was no significant difference among the Ctrl, Onlystim, and Transf. groups  was 3.4-fold higher than that of Transf. 44, transfected SCs were treated with a T-type VGCC blocker  or an L-type VGCC blocker , which are conventional VGCC blockers used with SCs44. The ΔF/F value decreased significantly when mibefradil was added for OS of SCs, and the ratio of ΔF/F was reduced by approximately half compared to that of OS without mibefradil -sensitive stores and caffeine/ryanodine-sensitive stores, are reportedly associated with internal Ca2+ movement in SCs46. To examine how Ca2+ released within the cell membrane through OS is involved in Ca2+ mobilization from these Ca2+ stores, we first tested the Ca2+ signal from IP3-sensitive stores. We used a specific inhibitor of phosphoinositide-specific phospholipase C beta (PLC-β) activation, U73122, to block IP3 formation and calcium mobilization from internal IP3 stores. After treatment with U73122 (500 nM) during OS of SCs, the peak intensity was significantly inhibited and the ΔF/F value was 0.53-fold attenuated compared to that during OS of untreated SCs  to deplete their IP3-sensitive stores through inhibition of sarco/endoplasmic reticulum Ca2+-ATPase. In OS of SCs treated with Thaps., the ΔF/F value was significantly reduced, whereas treatment with a mixture of Thaps. and caffeine  during OS of SCs led to partial recovery of the ΔF/F value . To acquire samples from DIV 7, the SC-MN cocultures were incubated for 8 h after the third LED stimulation and then fixed. Consistent with previous findings, we did not detect MBP expression in the transfected SC-MN coculture without LED stimulation (Transf.) at DIV 7. In contrast, with OS of SCs, MBP expression was initiated when observed at DIV 7, and MBP proteins were growing around the axon  promoter to activate only glial cells based on the method of Kleinlogel et al.41 and has higher Ca2+ permeability with increasing light sensitivity. Due to the powerful effects of these ChR2 variants on Ca2+ contribution, we favor the hypothesis that the potential effect of OS is mainly correlated with Ca2+, although other complex signaling mechanisms related to the proliferation and myelination of SCs might be involved. In particular, it is well known that increased intracellular Ca2+ in SCs affects their proliferation, differentiation, and myelination51. During the SC proliferation process, the elevation of intracellular Ca2+ via a calmodulin -dependent mechanism regulates SC proliferation during the development and regeneration stages. At this point, Ca2+ may act as a second messenger for the transduction of mitogenic signals on cultured SCs50. In the SC myelination process, neuregulin-1 (NRG1), an initiator of myelination, stimulates an increase in cytoplasmic Ca2+ and simultaneously binds to erbB2 on SCs. The binding of NRG1 to erbB2 activates PLC-γ, resulting in an elevated intracellular Ca2+ level and the activation of calcineurin, which results in the formation of the downstream transcription factor nuclear factor of activated T cells (NFAT) protein complex, NFATc4. The role of the calcineurin/NFAT pathway in Krox20 induction requires an increase in the Ca2+ level, which activates the expression of myelin-related genes52. Furthermore, the blockade of SC function in differentiation and myelination observed in mice lacking calcineurin B1, which inhibits the de-phosphorylation of NFAT proteins, prevents their nuclear entry into NFAT transcription complexes via a Ca2+-dependent mechanism51. In particular, PLC-dependent Ca2+ influx stimulated by NRG1 leads to differentiation in SC precursors51. When SCs are cocultured with sensory neurons, the release of intracellular Ca2+ in myelinating SCs into Ca2+ stores appears to depend upon the progress of myelination, which occurs via ryanodine receptors45. In addition, exogenous ATP treatment or electrical stimulation not only induces higher intracellular Ca2+ levels in SCs through the P2y-purinergic pathway upstream of the SC plasma membrane, but also releases Ca2+ into IP3- and ryanodine-dependent Ca2+ stores; the mitochondrial Ca2+ uniporter can promote the myelination process in the presence or absence of axons54. Chronic suppression of Ca2+-activated Ca2+ release attenuates the development of myelinating SCs54. The detailed mechanisms of Ca2+ signaling remain unknown, but Ca2+ is clearly a pivotal regulator of the proliferation and myelination of SCs. Consistent with previous results, we herein demonstrated the contribution of intracellular Ca2+ to OS-induced response of SCs, as well as the physiological consequences of SC activation in terms of regulating the proliferation, differentiation, and myelination of SCs.Cell proliferation and differentiation are orchestrated by various proteins related to CaUsing optogenetics, we focused on boosting SC signaling to reveal their contribution to proliferation, differentiation, and myelination processes in the PNS. Functional regulation of SCs is important, as neurons should be supported by SCs for complete formation of the nervous system. Importantly, injured neurons can scarcely survive without SCs; therefore, modulation of the strong effects of SCs on regeneration and remyelination may be necessary to overcome the limitations of current SC therapy. This study offered an exceptional opportunity to elucidate SC functions in proliferation, differentiation, and myelination using optogenetics. Optogenetic SC manipulation may constitute a technical innovation for elucidating the vital role that SCs play in complex higher PNS functions in developmental and neurodegenerative disorders.All acquisition procedures of biological samples were approved by the Institutional Animal Care and Use Committee of the Yonsei University Health System (IACUC of YUHS), and all experiments were conducted in accordance with the relevant guidelines and regulations set by the committee.55. In brief, sciatic nerves from postnatal day 4 mice were harvested in Ca2+- and Mg2+-free phosphate-buffered saline  and incubated with 2.5% trypsin (Gibco) and 1 mg/ml collagenase A (Roche) at 37 °C for 30 min. After trypsinization, the pellets were washed with high-glucose Dulbecco’s modified Eagle’s medium  containing 10% horse serum  and then suspended in SC culture medium: high-glucose DMEM with 10% HS, 4 mM L-glutamine , 1% penicillin/streptomycin , 0.5 μM forskolin (Sigma), and 2 ng/ml human heregulin beta-1 (Sigma). After suspension, SCs were cultured on coverslips coated with 10 μg/ml PLL (Sigma). To remove fibroblasts from the SC culture, complement-mediated cytolysis was performed at DIV 2 or 3 as previously described33. Briefly, cells were treated with HMEM (DMEM containing 20 mM hydroxyethyl-piperazineethane-sulfonic acid (HEPES) buffer (T&I), 10% HS, 4 mM L-gln, and 1% p/s) for 5 min at room temperature before washing with 20 mM HEPES in Ca2+- and Mg2+-free Hank’s balanced salt solution . After HMEM solution was removed, 4 ng/ml anti-mouse CD90 (Serotec) in HMEM was added and incubated at 37 °C for 30 min, and 200 µl of rabbit human leucocyte-associated antigens A, B, and C (HLA-ABC)-complemented sera (Millipore) were added and incubated at 37 °C for 1–2 h. Cytolysis was terminated by washing cells with 20 mM HEPES buffer in HBSS.SC culturing was performed as previously described55. MNs were harvested in Hank’s balanced salt solution  containing 1% Toll-like receptor (TLR) trypsin (Worthington) for 15 min at 37 °C, followed by treatment with 1% TLR trypsin inhibitor (Sigma). To collect purified MNs, dissociated spinal cord pieces were incubated in an immunopanning culture dish pretreated with p75NTR antibody (Abcam) at room temperature for 1 h. The immunopanning culture dish was washed three times with neurobasal medium (Gibco) containing 1 × Glutamax Ι (Gibco) to remove nerve fragments and p75NTR-negative cells. MNs bound to the bottom of panning dish were treated with depolarization solution  and gently harvested in coculture medium.MNs were isolated from the spinal cord of CD-1 mice (embryonic day 12–13 fetuses) as previously described55. To form the SC feeder layer, SCs cultured on growth factor-reduced Matrigel for 7 days and SCs were transfected with CatCh at DIV 4 in coculture with MNs. MNs were then seeded on top of an SC feeder layer and the SC-MN coculture was grown in coculture medium composed of neurobasal medium, 2% HS, 0.5 mM L-gln, 0.5 μM forskolin, 1% P/S, 1 × B27 supplement (Gibco), 1 mg/ml bovine pituitary extract (Gibco), 10 μg/ml brain-derived neurotrophic factor , 50 μg/ml ascorbic acid (Sigma), and 1 mM β-mercaptoethanol (Sigma). For the coculture experiment, the SC and MN seeding densities were 2 × 104 and 1.5 × 104 cells/well, respectively, and the SC-MN cocultures were plated onto a 12-well plate.The coculture of SCs and MNs was performed as previously described2+ permeability, was used for the transfection of SCs. The GFAP promoter sequence was amplified by PCR from the pAAV-GFAP-EGFP vector (Addgene #50473) using MluI and EcoRI adaptor primers and then cloned into the pAAV-MCS vector (Stratagene). The CatCH-EYFP (L132C) sequence was changed from pAAV-CamKIIa-hChR2(H134R)-EYFP (Addgene #26969) using a mutagenesis kit (Enzynomics), amplified by PCR using EcoRI and BglI adaptor primers and cloned into the pAAV-MCS vector. To transfect SCs, the SCs were treated with a mixture of 5 μg of CatCh and Lipofectamine 2000 reagent (Invitrogen) in Opti-MEM™ medium at DIV 3. After 24 h, SCs were washed three times with PBS and transferred to SC culture medium.pAAV-GFAP-CatCh-EYFP (pCatCh) was constructed and provided by Dr. Eun Mi Hwang of the Center for Functional Connectomes of the Korea Institute of Science and Technology, Seoul, Korea. CatCh, which is ultra-light-sensitive and enhances Ca4 cells) were plated into a 24-well plate. At DIV 7 after CatCh transfection, SC samples were fixed with 4% paraformaldehyde (PFA) and immunostained with anti-green fluorescent protein (GFP)  and anti-S100ß , followed by the secondary antibodies Alexa Fluor® 488 donkey anti-rabbit immunoglobulin G  and Alexa Fluor® 568 goat anti-mouse IgG . The GFP protein used herein shares approximately 97.8% sequence identity with YFP protein. SC transfection was confirmed by calculating the number of GFP protein-stained SCs. Samples were then counterstained with DAPI (Life Technologies). The number of GFP+-S100ß+ SCs was calculated from multiple fields of view in confocal fluorescence images and quantified. The total number of S100ß+ SCs stained with DAPI was first calculated, and the number of GFP+ cells in S100ß+ SCs was confirmed .Prior to examining the effect of optogenetics on SCs, the efficiency of transfected SCs was tested. Coverslips seeded with SCs  on a custom-designed printed circuit board (PCB)  were grown for 3 days in SC culture medium containing 10% HS and then transfected with CatCh in SC culture medium containing 5% HS. After 24 h, the SC culture medium was replaced with fresh SC culture medium containing 1% HS and 10 µM of BrdU, and the culture was then incubated at 37 °C for 24 h. The presence of a non-mitogenic concentration of 1% HS was necessary for SC survival. The following day, transfected SCs were either exposed to LED irradiation or not, and fixed with 4% PFA at 0, 1, 3, 6, 12, and 24 h after LED stimulation. As a control, non-transfected SCs were also exposed to LED irradiation or not, and fixed at the times indicated above. Fixed samples were incubated with 1 N HCl on ice and then with 2N HCl at room temperature for DNA hydrolysis. To detect incorporated BrdU, samples were stained with BrdU  and S100β . The number of BrdU+-S100ß+ SCs was calculated from multiple fields of view under the confocal microscope using ImageJ .To examine the effect of optogenetics on SC proliferation, we used the BrdU assay, which measures the incorporation of BrdU, a thymidine analog of DNA. SCs  to obtain a similar number of cells from different DIV samples on the day when the samples were collected. In addition, the MBP expression level of transfected SCs after OS was compared to those in the presence and absence of a mixture of dBcAMP  and NRG1  that induced SC differentiation and myelination in the SC monoculture without neurons. Transfected SCs at DIV 3 were treated with or without the dBcAMP/NRG1 mixture at DIV 4. The following day, transfected SCs were exposed to the LED irradiator at 20 Hz for 1 h, and LED stimulation was applied for 3 days; a parallel culture was not stimulated. Culture samples were fixed at DIV 1, 3, and 7. To harvest samples at DIV 7, SCs were stimulated and, after 8 h, samples were fixed. SC monocultures were stained with S100ß, MBP, and DAPI at the times described above and the number of MBP-expressing SCs was calculated from five different fields of view under the confocal microscope.To examine the MBP expression level of SCs in the SC monoculture with OS, transfected SCs were stimulated with the LED irradiator or not. SCs seeded onto a coverslip were plated onto a 24-well plate and the plating densities were adjusted , U73122 , U73433 , caffeine , mibefradil , thapsigargin , or dantrolene , and each reagent was maintained in the culture until analysis of calcium imaging and protein expression levels. In Fig. 4 cells/coverslip). After 24 h, SCs were rinsed three times with standard buffer containing 10 mM HEPES, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 139 mM NaCl, and 10 mM glucose, followed by treatment with a mixture of 2 mM Oregon Green 488 BAPTA-1 AM and 10% (w/v) pluronic F-127 (at a 1:1 ratio) in standard buffer for 1 h in the dark at room temperature. SCs were washed three times with standard buffer and then subjected to an OS experiment in the presence or absence of each reagent. A single optic fiber was used instead of the LED irradiator , which was placed near cells under the confocal laser microscope ; the intensity of the optical fiber was adjusted to 5 mW/mm2, the same intensity as that of the LED irradiator. To examine the intracellular Ca2+ level of SCs, the laser power of the confocal microscope was first adjusted to the minimum intensity at which SCs did not respond. Time-lapse images were taken every second with pulse pattern (10 s stimulation/50 s rest) for 5 min at 512 × 512 pixel resolution, and cells were excited at the same intensity using the 488 nm laser line. Under all experimental conditions, the data were excluded after photobleaching was observed during the experiment. Within a given imaging field, only active SCs were chosen for analysis . The intensity of intracellular Ca2+ was measured using Zen software, and the intracellular Ca2+ level was determined by calculating the ratio between the change in fluorescence signal intensity (∆F) and baseline fluorescence (F).To measure the calcium responses of SCs to OS, we used Oregon Green 488 BAPTA-1 AM following the manufacturer’s instructions. Transfected SCs were treated with each reagent at DIV 4 , Tuj1 , or S100ß  in 1% BSA (Millipore) at 4 °C overnight, and the secondary antibodies used were goat anti-rat IgG H&L , goat anti-chicken IgY H&L , and goat anti-mouse IgG H&L . Nuclei were stained with DAPI (Life Technologies) for 10 min. All images were acquired using an inverted confocal laser-scanning microscope  equipped with solid-state lasers . Five regions in each condition were chosen at random and analyzed for the number of MBP-positive cells.All culture samples were fixed with 4% PFA for 20 min at room temperature, followed by treatment with 0.2% Triton X-100 for 15 min. Samples were soaked with 1% bovine serum albumin  at 4 °C overnight. In Figs Expression levels of MBP and Krox20 were quantitatively analyzed using western blot analysis. Samples were lysed in RIPA buffer (T&I) containing 1% SDS and protease inhibitor . The protein concentration of cell lysates was measured using the Bradford assay (Sigma). The samples were prepared in an SDS sample buffer and heated for 5 min at 95 °C; next, 10 μg of protein from each sample was loaded in SDS loading buffer and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skim milk for 1 h, followed by incubation with anti-mouse Krox20  and anti-rat MBP  antibodies at 4 °C overnight. The membranes were washed three times in Tween-20 and incubated with goat anti-mouse IgG or anti-rat IgG conjugated to horseradish peroxidase  for 2 h. Bands were visualized using an ECL system. The intensity of the blots was quantified with ImageJ software.To determine cell viability, the LIVE/DEAD cell staining kit (Abcam) was used according to the manufacturer’s instructions. In brief, SC-MN co-cultures were washed once with PBS, followed by labeling with a mixture of solution A  and solution B  in PBS at DIV 10. Samples were incubated for 30 min at 37 °C and then rinsed three times with PBS. Samples were examined under a confocal microscope . The numbers of live and dead cells were counted in images acquired from confocal microscopy .p < 0.05, 0.01 and 0.001, and calculated p-values are specified in the figure legends.All statistical analyses were performed with Prism software (GraphPad Software) and the normality of the data was examined using the Shapiro-Wilk test. All statistical data are shown as means ± standard error of the mean (SEM) and comparisons between more than two groups were performed using repeated measurement analysis of variance (ANOVA), while an unpaired two-tailed t-test with Welch’s correction was used to compare the values between two groups. The levels of statistical significance were set to Suppl. Figure 1, 2, 3Calcium imaging in CatCh-transfected SCs by OS and VGCC.Calcium imaging in CatCh-transfected SCs by calcium mobilization from internal calcium stores.


In response to circulating hormonal stimuli, hepatocytes exhibit intracellular Ca2+ responses that propagate through liver lobules in a wave-like fashion. Although intracellular processes that control cell autonomous Ca2+ spiking behavior have been studied extensively, the intra- and inter-cellular signaling factors that regulate lobular scale spatial patterns and wave-like propagation of Ca2+ remain to be determined. To address this need, we acquired images of cytosolic Ca2+ transients in 1300 hepatocytes situated across several mouse liver lobules over a period of 1600 s. We analyzed this time series data using correlation network analysis, causal network analysis, and computational modeling, to characterize the spatial distribution of heterogeneity in intracellular Ca2+ signaling components as well as intercellular interactions that control lobular scale Ca2+ waves. Our causal network analysis revealed that hepatocytes are causally linked to multiple other co-localized hepatocytes, but these influences are not necessarily aligned uni-directionally along the sinusoids. Our computational model-based analysis showed that spatial gradients of intracellular Ca2+ signaling components as well as intercellular molecular exchange are required for lobular scale propagation of Ca2+ waves. Additionally, our analysis suggested that causal influences of hepatocytes on Ca2+ responses of multiple neighbors lead to robustness of Ca2+ wave propagation through liver lobules.Dynamics as well as localization of Ca Once in the cytosol, Ca2+ can be sequestered by mitochondria, released into the extracellular region, or pumped back into the ER, thus reducing the cytosolic Ca2+ levels, yielding a Ca2+ spike. Intracellular Ca2+ spiking has been reported to arise primarily due to fast activation and slow inhibition of IP3R by cytosolic Ca2+ operating in conjunction with active pumping of cytosolic Ca2+ into the ER by SERCA pumps  region of the lobule and propagate toward the periportal (PP) region , 0.1% BSA, pH 7.4) equilibrated with 100% O2 at 30°C. A Ca2+-sensitive indicator, fluo-8 AM (5 μM) was loaded into the hepatocytes in vivo by recirculating the perfusion buffer supplemented with fluo-8 AM plus 0.02% Pluronic F-127 and 2% BSA for 40–50 min. Confocal images were acquired with an EC Plan-Neofluar 10x/0.30 M27 objective using a Zeiss LSM510MP confocal microscope. Fluo-8 images  were captured every 4 s. Periportal and pericentral zones were identified by differential dye loading and perfusion of fluorescein-conjugated BSA.All animal procedures used in this study were handled in accordance with mandated standards of humane care and were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. Confocal imaging of intact perfused livers was performed as previously described  in R platform for statistical analysis  in R (version 3.2.3). This function removes frequencies corresponding to the highest nth percentile from the power spectrum of a given time series. The signal in the lowest 27.5 percentile of the frequency range in every time series was retained for subsequent analysis. The amplitudes of cytosolic Ca2+-dependent fluorescence signal intensity in the resultant time series were then rescaled to between 0 and 1.High frequency components in each baseline-corrected Caigraph (version 1.0.1) package in R (version 3.2.3).Undirected correlation networks were constructed using pairwise Spearman rank correlation coefficient estimates of baseline corrected, low pass filtered, and rescaled time series data. Edges corresponding to correlation values < 0.75 or those that were between hepatocytes lying at a distance > 100 μm were discarded. The resultant networks were analyzed for isolated clusters, their sizes and node degrees. Analysis of the correlation network was performed using the 2+ responses of hepatocytes was estimated based on Shannon's conditional entropy, as follows:Transfer entropy (TE) is a measure of the directed influence between two random processes. TE from a process X to another process Y is defined as the amount of reduction in uncertainty of future values of Y by knowing the past values of X, given past values of Y. In the present study, pair-wise TE between Caxt and yt represent the cytosolic Ca2+ levels in hepatocytes X and Y, respectively, at time t; 2+ in respective hepatocytes X and Y; yt+1 given yt+1, and Y are used to predict the current value of Y compared to past values of Y alone. Information transfer is considered as occurring from X to Y if TEX→Y> 0  or low (= 0) cytosolic Ca2+ at each time point using the 85th percentile of the cell-intrinsic intensity distribution as considered the threshold. As an additional filter to minimize the effect of noise, all high cytosolic Ca2+ values in the time series were changed to zero unless the value at an immediately preceding or following time point was also high, i.e., cytosolic Ca2+ intensity was sustained above the 85th percentile within the cell for at least 8 s.Directed causal networks between hepatocytes based on TE were constructed using quantized CaIn the absence of a good value of TE to infer cell-to-cell influence, hepatocyte-specific significance testing was employed to identify influence edges and construct TE-based causal networks. For every hepatocyte, pairwise TE values from all other hepatocytes were estimated to obtain an empirical distribution. If the TE value to the given hepatocyte from another adjacent hepatocyte was greater than the 95th percentile of its empirical TE distribution, a positive causal influence was considered from the neighbor to the hepatocyte of interest. The similarity in TE networks identified by our method based on binarized data and a continuous TE estimation method implemented in JIDT Lizier,  can be f2+ signal propagation in a cord of hepatocytes detailed in Verma et al. -based model of Caa et al. . Here, wa et al. . In the r) including non-specific binding was modeled as shown in Equation (4), where H represents the hormone level—a model parameter. The total number of receptors for each hepatocyte was assumed to be constant. Intracellular IP3 concentration (IP3) balance was modeled using saturation kinetics for synthesis influenced by hormone binding to the receptor and cytosolic Ca2+ and mass action kinetics for degradation (Equation 5). IP3 exchange between adjacent hepatocytes was modeled as a mass transfer term assuming fast kinetics, with Gij being the mass transfer coefficient. Increase in cytosolic Ca2+ (CaI) in the model was regulated IP3R (g) activation, cytosolic IP3 levels, store Ca2+ content (CaT), and a constant leakage from the ER (L), whereas decrease in cytosolic Ca2+ caused by SERCA pump activity was modeled as a Hill function (Equation 6). IP3R activation (g) in the model was regulated by cytosolic Ca2+, whereas a constitutive rate of IP3R was considered (Equation 7). The model assumed constant total intracellular Ca2+ for all hepatocytes. Additional details of model development can be found in Verma et al. , a Gij was sampled ϵ . Otherwise Gij = 0. pth = 0.2 or 0.5 correspond to cases where 20% or 50% Gij values are likely to be 0 respectively.To identify the effects of non-uniformity of gap junction conductivity between adjacent hepatocytes in our simulations, GSimulation results presented in the current work were reproduced independently using the parameter values and hepatocyte adjacency information provided as 2+ content of each hepatocyte is considered to be a constant (= 500 μM). In this alternative model, Equation (8) is excluded and Equation (6) was changed as follows:We also considered an alternative modeling scheme, in which the store Ca2+ dynamics in 1300 hepatocytes across different liver lobules over a period of 1600 s . At low vasopressin stimulus levels (0.1–0.5 nM), hepatocytes in intact mouse livers did not exhibit sustained cytosolic Ca2+ spikes  of 0.75 and maximum inter-hepatocyte distance cutoff (dth) of 100 μm to assign network edges between two hepatocytes. The resulting network consisted of 669 hepatocytes with 565 edges between them. The node degree distribution for all hepatocytes in the network suggested that a large number of hepatocytes were synchronized with one or two other hepatocytes, for the chosen Rth and dth values  for further analysis. For each large cluster, we estimated node degrees for individual hepatocytes as well as the average node degree for all hepatocytes within the cluster. Node degrees of individual hepatocytes residing in large clusters ranged between 1 and 7 Figure . Eighty 2+ response, which were generally situated close to the central veins. The typical spatial dimension of these synchronized clusters was less than the lobular dimensions . It must be noted that other smaller clusters, which are not shown in the figure, may be present in the intermittent space between the larger clusters.Mapping of the large clusters onto their physical locations Figure  suggeste2+ response showed that, despite an apparent global concurrence of Ca2+ peak intensities, synchronized hepatocytes formed localized clusters spanning small regions within liver lobules. Ca2+ responses of up to 7 hepatocytes were synchronized within these clusters. However, only a small fraction of hepatocytes was included in clusters with sizes 8 or greater  based approach to identify causal connectivity between neighboring hepatocytes (see Methods). We considered molecular exchange between hepatocytes as the basis of causal influence between spatially co-localized hepatocytes and therefore allowed causal edges to exist only between closest neighbors. In our analysis, a unidirectional alignment of causal edges would suggest an organized, wave-like information flow along hepatocytes. We analyzed the resulting causal network for average node degree, total node degree, in and out node degrees, and direction of causal edges, to identify how many neighbors typically influence a given hepatocyte and the directional orientation of cell-cell interactions.We constructed causal networks between hepatocytes to identify whether adjacent neighbor-driven intercellular interactions can account for lobular scale CaThe causal network comprised of 1,162 hepatocytes with 1,491 edges between them. The number of hepatocytes included in the causal network far exceeded that in the correlation network (669 hepatocytes with 565 edges) suggesting that causality analysis describes intercellular interactions between neighboring hepatocytes better than a correlation-based analysis. We analyzed total node degree and cluster sizes for all nodes in the graph. The total node degree distribution of the all hepatocytes in the network peaked at a value of 2, pointing to causal connections between a given hepatocyte and multiple neighbors Figure . Upon deFigure 2+ response can yield propagating Ca2+ waves consistent with experimental observations. We started with a previously-published dynamic model of hepatocyte Ca2+ response  and Phospholipase C β-1 (Plcb1). Zonation profiles for Avpr1a and Plcb1 in the data from , and IP3 synthesis rate (model parameter kIP3), respectively, in the present dynamic model. For the subsequent analysis using integrated causal network and dynamic modeling, we considered a large cluster of hepatocytes identified using the causal network analysis. Experimentally determined Ca2+ patterns in this cluster of hepatocytes are shown in Figure 2+ wave propagation through the cluster were: (1) Ca2+ waves propagated through the cluster from the pericentral region toward the periportal region consistent with the prior expectation, and (2) Ca2+ waves started from multiple hepatocytes located closer to the approximate location of the central vein residing closest to the cluster. We evaluated the dynamic model of this large cluster to identify the spatial patterns of intracellular signaling components as well as gap junction connectivity patterns that are consistent with experimentally observed Ca2+ wave propagation. In the dynamic model, Ca2+ response coupling is caused by gap junction mediated IP3 exchange, a phenomenon that has been reported previously : (1) no hepatocyte exchanges IP3 with its neighbors, and (2) each hepatocyte exchanges IP3 with all its neighbors. Gap junction conductivity parameter between any pair of hepatocytes, modeled as a mass transfer term assuming fast IP3 diffusion kinetics, was set to either 0 (no IP3 exchange) or 0.9.We simulated the dynamic model to identify the effect of gap junctions on coupling of Car and kIP3 were sampled from uniform distributions over nominal parameter ranges listed in Verma et al. . In this model formulation, store Ca2+ could be interpreted as a driving force for influx of Ca2+ within the cytosol instead of being trafficked between the cytosol and the Ca2+ store. Ca2+ wave propagation characteristics in this case were similar to the case in which store Ca2+ was dynamic (see Figure Since the store Ca2+ spikes and compared the cluster sizes and localization for correlation and causal networks (Figures We used a step-wise increasing vasopressin stimulus over the course of the live tissue imaging duration Figure . To iden Figures –S10. Our Figures . In cont Figures .2+ signal propagation in a two-dimensional optical slice of a perfused and intact mouse liver at the lobular scale. We generated a large-scale data set on cytosolic Ca2+ responses of 1300 hepatocytes to hormonal stimuli over a period of 1600 s. We analyzed the synchronization of Ca2+ response of a large population of hepatocytes in intact liver using correlation analysis and TE-based causal network analysis to identify directional flow of causal influence across hepatocytes in a lobule. We employed a computational model-based analysis to identify spatial patterns of intracellular Ca2+ signaling components and gap junction conductivity that can yield lobular scale Ca2+ waves consistent with experimental observations.In this work, we analyzed Ca2+ responses in a population of cells under a global stimulus. For instance, correlation networks constructed using Ca2+ dynamics in Islets of Langerhans exhibit stimulus-dependent synchronization characteristics when stimulated by glucose (StoŽer et al., 2+ waves observed in our experiment. In contrast, causal network analysis of the experimental data elucidated prominent features of lobular scale Ca2+ wave propagation such as existence of “islands” of causally connected hepatocytes within liver lobules and lack of directional alignment of causal edges between hepatocytes from the pericentral region to the periportal region. Although causal network analysis yielded misaligned causal connections between hepatocytes residing in a cluster, it pointed toward zonation and intercellular communication as cell-level dynamics that yield lobular scale organization of Ca2+ response. Spatially organized heterogeneity leads to location-based differences in Ca2+ signaling capacity of hepatocytes. Intercellular communication results in entrainment of Ca2+ responses of adjacent hepatocytes which extends throughout liver lobules via local interactions to yield Ca2+ waves.Identification of functional networks within a population of colocalized cells has gained prominence in recent decades (Bullmore and Sporns, 2+ dynamics used in our study is limited in its scope. Our dynamic model-based analysis, parametrized using lobular scale spatial patterns of liver gene expression, represents a specific case of a more generalized concept of functional gradients that control Ca2+ wave propagation in liver lobules. Functional zonation results from zonal differences in micro-RNA expression (Sekine et al., 2+ spiking dynamics have previously been observed in rat hepatocytes due to activation of adrenergic (Rooney et al., 2+ signaling events (Török et al., 2+ signaling cascade with zonal information, a stochastic modeling framework, and integration of experimental data can potentially capture the complexity observed in lobular scale Ca2+ dynamics in the liver, such as lack of directionality of causal linkages between hepatocytes.The computational model of Ca2+ as well as IP3 could be exchanged between neighboring hepatocytes through gap junctions and lead to Ca2+ efflux from intracellular stores, the effective diffusivity of IP3 is higher than Ca2+ because Ca2+ is heavily buffered within hepatocytes (Allbritton et al., 3 is strongly involved in coordinating Ca2+ responses at the lobular scale. In addition, a loss of wave-like propagation of Ca2+ signals has been shown upon disruption of cell-cell contacts using Ca2+ free buffer (Gaspers and Thomas, 2+ mobilization could arise from paracrine ATP release and subsequent purinergic receptor activation. The relative contribution of Ca2+ response synchronization via gap junctions or paracrine ATP would depend on the tissue and zone-specific expression of Connexin subtypes and purinergic receptors. Disrupting cell-cell contacts between hepatocytes in perfused livers results in asynchronous Ca2+ spikes in hepatocytes under a vasopressin stimulus and the Ca2+ signals do not spread to neighbors (Gaspers and Thomas, 2+ signal propagation observed experimentally. That said, explicit consideration of other potential paracrine factors such as ATP will expand the scope and applicability to time scales of cell-cell interaction beyond the relatively fast timescale considered in this study.Although CaWe note that a variety of fluorophores are available for reporting intracellular calcium levels, including genetically encoded calcium reporters. For example, Fluo-8 AM and Rhod4 have been utilized for cytosolic calcium reporting in hepatocytes with small differences in Kd values and photostability (Lock et al., 2+ signal propagation within liver lobules is the three-dimensional arrangement of hepatocytes. Although the proximity of hepatocytes to either a portal triad or a central vein within a two-dimensional slice can be ascertained, information regarding the cellular adjacency and spatial localization along a third dimension is lacking in our experimental data. The lack of directional alignment of causal edges along a pericentral to periportal orientation could arise due to the presence of micro-environmental cues from other pericentral or periportal regions above or below the optical slice corresponding to the imaged area. Alternatively, multidirectional alignment of causal edges may be due to cell-autonomous Ca2+ responses of hepatocytes within a small region which appear independently of the global stimulus and do not propagate beyond a few cells. High spatial and temporal resolution imaging of three-dimensional tissue structure sufficient to study spatial organization of Ca2+ signaling in liver lobules remains a challenging problem. However, imaging techniques are constantly evolving to produce accurate three-dimensional reconstructions of tissues with high spatial resolution (Arganda-Carreras et al., 2+ signal propagation in liver lobules.An important consideration in analyzing and modeling CaAV, JH, and RV designed the study. AA conducted the experiments. AA, AN, JH, and RV analyzed experimental data and interpreted the results. AV performed causal network analysis. AV developed computational model and performed simulations. AV, BO, and RV analyzed and interpreted the network analysis and simulation results. AV and RV drafted the manuscript. AA, BO, JH, and RV edited the manuscript. JH supervised the experimental aspects. RV supervised the computational aspects.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite major scientific discoveries and novel therapies over the past four decades, the treatment outcomes of acute myeloid leukemia (AML), especially in the adult patient population remain dismal. In the past few years, an increasing number of targets such as CD33, CD123, CLL-1, CD47, CD70, and TIM3, have been developed for immunotherapy of AML. Among them, CLL-1 has attracted the researchers’ attention due to its high expression in AML while being absent in normal hematopoietic stem cell. Accumulating evidence have demonstrated CLL-1 is an ideal target for AML. In this paper, we will review the expression of CLL-1 on normal cells and AML, the value of CLL-1 in diagnosis and follow-up, and targeting CLL-1 therapy-based antibody and chimeric antigen receptor T cell therapy as well as providing an overview of CLL-1 as a target for AML. Acute myeloid leukemia (AML) is the most common and fatal hematological malignancy in adult patients where the majority have a poor prognosis. Despite major strides in the field of anti-cancer treatments and breakthroughs in immunotherapies, over the past four decades, no significant change in the conventional chemotherapy for AML including induction and consolidation treatment underscores the urgency to develop new methods to improve the prognosis in this deadly disease , 2. OverC-type lectin-like receptors play a pivotal role in the fight against infection and maintain homeostasis and self-tolerance by recognizing damage-associated—and pathogen-associated—molecular patterns leading to regulation of innate and adaptive immunity , 12. In CLL-1 belongs to group V of C-type lectin-like receptor family. The human gene encoding CLL-1 maps to 12p13 and is within NK gene complex in which NKG2R, low-density lipoprotein receptor-1 (LOX-1), and β glucan receptor (BGR) are included, CLL-1 is highly homologous to LOX-1 and BGR -induced reactive oxygen species (ROS) specifically and highly increased on both bone marrow cells and purified neutrophils of Clec12a−/− mice. Furthermore, significantly increased neutrophil infiltration occurred in Clec12a−/− mice rather than wild-type mice when MSU crystal or dead cells were injected into peritoneum or total body irradiation of X-ray with a dose to kill double positive thymocytes was given, accompanying with elevated level of CXCL1,CXCL10, and TNF-α /CLL-1 was associated with the control of myeloid cell activation during inflammation, it was a negative regulator of granulocyte and monocyte function , detailsid cells ; while iid cells . Additioutophagy . The incutophagy . Furtherutophagy . More re+CD38−, but not on T, B, NK cells and erythrocytes and their precursors [+CD38+CD123+CD45RA+, Marie Bill et al. reported 39.1% common myeloid progenitors (CMPs), 81% GMPs, and 11.9% megakaryocyte-erythroid progenitors (MEPs) express CLL1 whereas the normal stem cell with lin−CD34+CD38− does not express CLL1. In fact, the earliest CLL1+ cell in normal hematopoiesis is CMPs and CLL1+ subsets facilitate colony growth of myelomonocytic lineage [+CD34+CD38− cells isolated from AML patients can produce leukemia in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, but CLL-1 is negative on the CD34+CD38− cells derived from normal bone marrow, regenerating bone marrow after chemotherapy from other diseases and mobilized peripheral blood from non-AML patients [+CD38− cells. Therefore, compared with CD123, CLL-1 is a better marker for LSC. With regard to cell line, HL-60, THP-1, and U937 have a high expression of CLL-1 which facilitate to be selected as a target cell for research [In mouse model, Pyz et al. revealed the ligand of mouse MICL (mMICL) was widely expressed on bone marrow, thymus, heart, spleen, and kidney, indicating a role in immune hemostasis . Howeverecursors . CLL1 isecursors , 13, 29. lineage . In cont lineage , 31. Morpatients . CD123 h+ status [In an analysis of 55 retrospective and 36 prospective samples, Larsen et al. revealed that the expression of hMICL/CLL-1 was restricted to myeloid cells as compared to other stem cell antigens, which indicated CLL-1 can be used as a marker of AML diagnosis . Additio+ status . High le+ status , 39.+ expression and low SSC on flow which is similar to the “blast gate” [Of note, one needs to exclude basophils and part of DCs when CLL-1 is used as a marker to evaluate the MRD, because these cells test positive for CLL-1 and have low CD45st gate” . Harringst gate” . Therefo+ HL60 cell line, which may indicate such an antibody does not have anti-leukemic effect, the possible reason may be the absence of induction of antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Van et al. proposed to combine a toxic moiety to anti-CLL-1 antibody to induce killing effect [Bakker et al. reported CLL-1 can efficiently internalize after ligand binding, indicating CLL-1 as target antigen for antibody-based therapy . Howeverg effect , where tg effect –43. In cg effect . The cong effect . This prg effect . Utilizig effect . MCLA-11g effect . Related+ and CD8+ T cell immune response against cancer, simultaneously and that the antibody binding did not influence phenotype and function of DCs [Compared with other c-type lectin receptors, DACL-2/CLL-1 is mainly expressed on myeloid DC, it can be used as Ag capture receptor due to its internalization after ligand binding and it can also interact with TLR or CD40 to regulate the immune response. Therefore, a strategy of targeting DACL-2/CLL-1 on DCs is also a feasible way for antibody-mediated delivery . Hutten n of DCs . Lahoundn of DCs . The reaUp to now, there is only one clinical trial with MCLA-117 which has recruited relapsed, refractory, and newly diagnosed AML in old patients (≥ 65 years) with high-risk cytogenetics or intolerance of induction therapy since 2016. It is a phase 1, multinational and first in a human study with a planned completion time of December 2018, where 50 patients are scheduled to be recruited with the primary goal to determine the maximum tolerated dose and then assesses the safety and efficacy based on recommended dose. The patients receive treatment weekly for 1 cycle, 28 days is 1 cycle, no dose, and any results are released until now (NCT03038230).+/CLL-1+ AML blasts have a higher risk to be resistant to chemotherapy than their negative counterpart. They generate second CLEC12A CAR-T with 41BB to evaluate the anti-leukemia activity, where the CAR-T cells were highly and specifically effective to CLEC12A cell lines. Although monotherapy with CLEC12A elicited modest anti-leukemia activity, a significant prolonged survival was achieved when it was sequenced after chemotherapy, indicating a preferable option for consolidation to eliminate MRD and LSC [Besides the selective expression on AML blasts and LSC, CLL-1 is also rarely expressed on non-hematological tissues , 13, mak and LSC . Similar and LSC . Related6/kg, 3 × 106/kg, and 9 × 106/kg for escalation in phase I trial. Inspiringly, a 44-year-old male patient with refractory AML  converted to MRD− disease when a dose of 7 × 105/kg CLL-1-CD33 CAR-T cells was firstly used after T cell-depleting conditioning therapy with fludarabine 30 mg/m2 and cyclophosphamide 500 mg/m2 for 3 consecutive days. Before receiving the CAR-T cell therapy, the patient had refractory disease to 4 cycles of chemotherapy including DA, FLAG, and 2 cycles of priming therapy plus decitabine. The patient tolerated the therapy well and experienced pancytopenia, and only grade 1 cytokine release syndrome (CRS). A matched sibling allogeneic stem cell transplantation was successfully followed, and the patient is alive and disease-free at the time of last follow-up [6/kg CAR-T cells were used on day 1 and day 2, respectively and a dramatic elimination of AML cells in bone marrow within 1 week, as evidenced by 98% on day 12 and MRD on day 19, accompanying 36% and 60% CAR-T cells in PBMC and bone marrow, respectively, was demonstrated. The patient also experienced pancytopenia and grade 1 CRS, as well as grade 3 neurotoxicity. The patient went on to receive a non-myeloablative hematopoietic cell transplantation where successful hematopoiesis recovery could be seen 2 weeks after HSCT. Unfortunately, the patients succumbed to severe infection [Bakker et al. reported 67% CD33-AML express CLL1, making CLL-1 a compliment as therapeutic target . On EHA nfection . The teaThe unique expression pattern makes CLL-1 a preferred target for AML. Although most studies regarding CLL-1 are mainly on anti-CLL-1 antibodies, the first successful utilization in human has inspired more interests in targeting CLL-1 for AML, it can be expected an increasing number of researches on CLL-1 CAR-T will emerge, especially the combination with other markers such as CD123 or CD33 or with novel agents such as FLT3 or BCL-2 inhibitor . The eff


Acinetobacter baumannii (MDRAB) clinical isolates in macrophages, after phagocytosis, to determine their virulence characteristics. After ATCC 19606 and 5 clinical isolates of MDRAB were phagocytosed by mouse and human macrophages, the bacterial count of MDRAB strains, R4 and R5, increased in the mouse macrophages, 24 hours after phagocytosis. Bacterial count of the strains, R1 and R2, was almost equal 4 and 24 hours after phagocytosis. Intracellular reactive oxygen species was detected in the macrophages after phagocytosis of these bacteria. Further, the strains R1, R2, R4, and R5 showed higher catalase activity than ATCC 19606. Additionally, strains R1, R4, and R5 grew more efficiently than ATCC 19606 in the presence of H2O2, whereas growth of strains R2 and R3 was marginally more than that of ATCC 19606 in the presence of H2O2. The MDRAB clinical isolates altered the expression of TNF-α, IL-1β, IL-6, and MIP-2 mRNA induced in J774A.1 cells, 24 hours after phagocytosis. These results provide insights into the renewed virulence characteristics of MDRAB clinical isolates. Finally, tigecycline killed MDRAB phagocytosed by the macrophages more effectively than colistin, although colistin and tigecycline are both considered effective antibiotics for the treatment of MDRAB.We investigated the intracellular survival of multidrug-resistant  Acinetobacter baumannii is an important opportunistic pathogen, associated with nosocomial infections such as bacteraemia, pneumonia, meningitis, urinary tract infections, and wound infections2. The recent increase in outbreaks of multidrug resistant A. baumannii (MDRAB) worldwide is a cause for concern5. Additionally, A. baumannii is included among the 6 nosocomial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) that acquire multidrug resistance and virulence7. Therefore, A. baumannii, especially MDRAB, has further gained importance as a human pathogen in the hospital environment.A. baumannii is regarded as a low-virulence pathogen8, recent studies have clarified that A. baumannii shows several forms of pathogenicity such as biofilm formation, adherence, and invasion of lung epithelial cells11, host cell death14, and iron acquisition15. The pathogenicity of A. baumannii depends on various virulence factors, especially, the outer membrane proteins ‘Omps’ being vital in this respect16. Additionally, we have reported that clinical isolates of MDRAB show different levels of omp expression and exhibit different cell adherence capacities across strains17. Moreover, the clinical isolates show different degrees of biofilm formation in the presence of sub-minimum inhibitory concentrations of antibiotics18. These results suggest that A. baumannii is emerging as a highly pathogenic bacterium and that the characteristics of A. baumannii vary in different environmental stress conditions, such as multiple antimicrobial agents and host immune responses.Although 19. These cells ingest and eliminate microorganisms by the phagocytic process, which involves the formation of phagosome and subsequent maturation of this phagosome into a phagolysosome. A robust antimicrobial environment such as low pH, oxidative conditions, nutrient depletion, and antimicrobial peptides are provided within the phagolysosome20. The production of reactive oxygen species (ROS) within the phagolysosome is especially potent, leading to the destruction of microorganisms21. In fact, malfunctioning of ROS production in patients suffering from severe recurrent infections can lead to death in many cases23. In A. baumannii infections, the production of ROS or NO appears to contribute to bactericidal function of neutrophils and macrophages and plays a crucial role in host defence and survival25. As a defence mechanism, S. aureus expresses the enzymes super oxide dismutases and catalase that protect it against ROS and enable its survival within the phagolysosome20. Likewise, A. baumannii is a catalase-positive bacterium, where in, catalase is encoded by the katE/katG genes. Additionally, the universal stress protein UspA protects it against H2O2 stress27, suggesting that A. baumannii survives within phagolysosomes of macrophages through the degradation of H2O2 by its catalase activities. Although, the uptake of A. baumannii by alveolar macrophages and murine macrophage cell line J774A.1 has been explored25, few studies have focused on the intracellular survival of A. baumannii in macrophages because it is regarded as an extracellular pathogen.Phagocytic cells such as neutrophils and macrophages represent the first line of defence against invading bacterial pathogens in the hostA. baumannii clinical isolates depend on its ability to adhere to human epithelial cells, and on the expression level of omp mRNAs17. These results might imply that since the clinical isolates of A. baumannii may have been exposed to various environmental stress conditions in the hospital, numerus virulence factors in the clinical isolates may have been modulated. Therefore, in this study, we have focused on the intracellular survival of MDRAB clinical isolates in macrophages, and their catalase activity. We have further evaluated the expression levels of ROS and proinflammatory cytokines in macrophages after phagocytosis with the aim of exploring the influence of intracellular bacteria on the functioning of macrophages. Finally, colistin and tigecycline, which are considered effective antibiotics for the treatment of MDRAB, have been evaluated for their ability to kill intracellular MDRAB clinical isolates within macrophages.We have previously reported that the renewed virulence characteristics of A. baumannii in vitro without the presence of antibody or complement opsonisation25. Therefore, we examined the MDRAB clinical isolate counts in J774A.1 and human macrophages at 4 and 24 hours after phagocytosis. As shown in Fig. E. coli, ATCC 19606, and 5 clinical isolates of MDRAB were detected in J774A.1 cells at 4 hours after phagocytosis. The bacterial count of E. coli and MDRAB strain R3, at 24 hours after phagocytosis, was significantly decreased compared with that at 4 hours after phagocytosis, whereas the bacterial counts of strains R4 and R5 at 24 hours after phagocytosis were increased compared with that at 4 hours after phagocytosis. Bacterial counts of the strains R1 and R2 were almost equal at 24 hours as well as 4 hours, after phagocytosis. Additionally, we examined the counts of E. coli, ATCC 19606, and a representative MDRAB strain, R1, in human macrophages at 4 and 24 hours after phagocytosis. As shown in Fig. E. coli and ATCC 19606 at 24 hours after phagocytosis were significantly decreased compared with that at 4 hours after phagocytosis, whereas a slight decrease in the count of strain R1 was observed at 24 hours compared with that at 4 hours, after phagocytosis. We next examined whether A. baumannii was phagocytosed by the macrophages or they invaded into the macrophages. J774A.1 cells were co-cultured with the bacteria in the presence of cytochalasin D (CytD), a potent inhibitor of micropinocytosis/phagocytosis. As shown in Fig. E. coli, ATCC 19606, and 3 representative MDRAB strains R1, R3, and R5 in CytD-treated J774A.1 cells were significantly lower than that in non-treated J774A.1 cells at 4 hours after phagocytosis. These results indicate that MDRAB clinical isolates were phagocytosed by the macrophages, following which, they managed to survive within the cells.Previous studies have shown that mouse macrophages can rapidly and efficiently phagocytose 23. Therefore, we examined whether macrophages produced ROS in response to intracellular MDRAB. As shown in Fig. E. coli, ATCC 19606, and 3 representative MDRAB strains. However, the induction of ROS was of about the same level in E. coli, ATCC 19606, and MDRAB strains R3 and R5, whereas the ROS level in J774A.1 cells co-cultured with MDRAB strain R1 was slightly and significantly lower than that when J774A.1 cells were co-cultured with E. coli  . This was consistent with the observation that the MDRAB strain R2 exhibited high catalase activity, however, could not survive in a medium containing H2O2.ROS plays a crucial role in the rapid killing of ity Fig. . Therefo2O2 Fig. . Howeveriod Fig. . Further2O2 Fig. . Moreove2O2 Fig. , whereas2O2 Fig. . These r10) Fig. . However17. Therefore, in this study, we evaluated the mRNA levels of proinflammatory cytokines in J774A.1 cells at 24 hours after phagocytosis of E. coli, ATCC 19606, and the MDRAB representative strains R1, R3, and R5. The expression of TNF-α, IL-1β, IL-6, and MIP-2 mRNA was induced in J774A.1 cells at 24 hours after phagocytosis of the bacteria , used for the killing of extracellular MDRAB in the phagocytosis assay, did not successfully kill intracellular MDRAB 30 regulates the major H2O2-degrading enzymes, encoded by katE and ahpF1, , in A. baumannii31. In the present study, the expression of katE mRNA in MDRAB clinical isolates was significantly higher than that in ATCC 19606, whereas the expression of ahpF1 mRNA in MDRAB clinical isolates was slightly higher than that in ATCC 19606 (data not shown). Considering these results, novel transcription factors may regulate the expression of the katE gene. Additionally, the MDRAB strain R2 showed high catalase activity but could not survive in media containing H2O2. Further studies are required for in depth analysis of these clinical isolates.The universal stress protein UspA plays a crucial role in protecting A. baumannii has emerged as a major pathogenic species, this bacterium exhibits varying pathogenicity in different environments. Since A. baumannii clinical isolates may have been previously exposed to environmental stress conditions such as multiple antimicrobial agents, bactericidal substances in the serum, and host immune responses such as oxidative stress, they may have altered the expression of several genes in order to adapt to these stress conditions. Wright et al. found insertion sequence (IS) elements throughout the genome of A. baumannii, and these elements contributed to genome variation by interrupting genes or altering gene expression32. In addition, A. baumannii carrying an ISAba1 element upstream of the catalase-peroxidase gene katG, was selected by serial subculture in the presence of sub-inhibitory concentrations of H2O232, suggesting that A. baumannii has the ability of adapting to oxidative stress. In the present study, although the expression of katG mRNA in strains R4 and R5 was significantly higher than that in ATCC 19606, no insertion sequence in the upstream region of the katG gene in MDRAB clinical isolates was found (data not shown). The DNA sequence upstream of the katG gene in MDRAB clinical isolates was the same as that in A. baumannii BJAB0868, BJAB07104, AC29, and AC30 clinical isolates34. As MDRAB clinical isolates exhibit enhanced catalase activity, further studies are required to determine novel transcription factors of katG as well as katE in these clinical isolates.Considering that 28. Tigecycline acted synergistically with colistin, to exert antibacterial effects on A. baumannii in vitro35. However, cohort studies have reported that a combination of colistin and tigecycline against MDRAB, showed disappointing results36 and tigecycline-based therapy may not be the best option for treating MDRAB infection37. We clarified the antimicrobial effect of tigecycline on intracellular MDRAB that had survived in macrophages after phagocytosis in vitro. Our laboratory has previously reported a novel bacterial transport mechanism, where A. baumannii exploits human neutrophils by adhering to the cells and inducing IL-8 release for bacterial portage38. Moreover, this study clarified the intracellular survival of MDRAB clinical isolates in macrophages, and the results imply that A. baumannii spreads systemically by exploiting the macrophages. As colistin is an effective antibiotic for extracellular MDRAB, it may block the systemic spread of A. baumannii by targeting the neutrophils. Likewise, if tigecycline is an effective antibiotic for the killing of intracellular MDRAB, it may block the systemic spread of A. baumannii by targeting the macrophages. Further studies are required to clarify the role of colistin and tigecycline in controlling MDRAB infection.Colistin and tigecycline are considered effective antibiotics for the treatment of MDRABA. baumannii infections. Further studies are required to understand the resistance mechanisms employed by MDRAB to evade killing by macrophages.In summary, we demonstrated that MDRAB clinical isolates acquired renewed virulence characteristics after phagocytosis by macrophages. High catalase production by MDRAB may impair the intracellular killing by macrophages and the consequent spread of All methods were carried out in accordance with relevant guidelines and regulations.A. baumannii were isolated from the Teikyo University hospital during an outbreak that occurred around 2010. The bacteria were isolated on CHROMagar™ Acinetobacter and incubated for 24 hours at 37 °C. As shown in Table A. baumannii by DNA sequencing of a partial RNA polymerase β-subunit (rpoB) gene . Additionally, the isolates were confirmed as non-clonal by pulsed-field gel electrophoresis (data not shown). After identification, these isolates were stored in glycerol stocks at −80 °C at the Department of Microbiology & Immunology, Teikyo University School of Medicine. Antimicrobial susceptibility testing was performed using 5 strains of A. baumannii based on the minimum inhibitory concentrations (MICs) of imipenem, amikacin, and ciprofloxacin. Against these 5 strains, the MICs of imipenem, amikacin, and ciprofloxacin were >8, 16, and 2 mg/L, respectively. These strains were thus identified as MDRAB strains. The A. baumannii ATCC19606 strain (AB) and the Escherichia coli ATCC 25922 strain (EC) were used as standard strains. The MIC of colistin against both ATCC 19606 and MDRAB strains, was determined as 2 mg/L. The MIC of tigecycline for ATCC 19606, and strains R1, R2, R4, and R5 was found to be 0.5 mg/L. The MIC of tigecycline for the R3 strain was 1 mg/L  measurements at 595 nm. The bacterial suspensions thus obtained were used for the phagocytosis assay.R1, R2, R3, R4, and R5 strains of 2 in RPMI-1640 medium supplemented with 10% FBS. J774A.1 cells were seeded at a concentration of 2 × 105 cells/well in 24-well plates and cultured overnight. Prior to co-culture with A. baumannii, the J774A.1 cells were washed twice with PBS. The cells were then co-cultured at a multiplicity of infection of 100 bacteria per cell at 37 °C under 5% CO2 for 2 hours. In order to determine the intracellular viable bacteria at 4 and 24 hours, after 2 hours of co-culture, cells were washed 3 times with PBS, following which they were maintained in RPMI-1640 medium, supplemented with 10% FBS and 50 mg/mL colistin to kill the extracellular bacteria. To inhibit the phagocytosis of J774A.1 cells, they were pre-incubated for 1 hour with cytochalasin D (CytD)  (5 μg/mL) before co-culture with bacteria and were kept with CytD during co-culture with bacteria. To analyse the number of intracellular bacteria, J774A.1 cells were washed thoroughly 3 times with PBS after co-culture. The bacteria were harvested after lysing the J774A.1 cells by adding sterile distilled water (1 mL) to each well; bacterial count was confirmed by the growth of serial dilutions of the bacterial suspension on LB agar in terms of CFUs after 24 hours of incubation at 37 °C.J774A.1 cells were purchased from the JCRB cell bank , and maintained at 37 °C under 5% COHuman peripheral blood mononuclear cells (PBMC) were isolated from the peripheral venous blood of healthy volunteers. Briefly, whole blood (20 mL) was mixed with 7 mL of a 6% dextran solution and 15 mL of HBSS and allowed to stand for 30 minutes at 25 °C until stratification occurred. The upper leukocyte-rich plasma layer was transferred to a new tube containing endotoxin-free Ficoll-Paque PLUS gradient  and was centrifuged . The cell layer was harvested and subsequently, the cells were resuspended at 100 million PBMC per mL in Monocyte Attachment Medium . The cells were cultured for 10 days as per the manufacturer’s protocol and used for the phagocytosis assay, as described above. The protocol was approved by the Ethical Review Committee at the Teikyo University School of Medicine (no. 15-192-2). All participants gave a written informed consent prior to their inclusion in the study.A. baumannii cultured in LB broth for 6 hours at 37 °C was extracted using an RNeasy Protect Bacteria mini kit (Qiagen). Harvested RNA samples were quantified using the NanoDrop spectrophotometer . Total RNA was reverse-transcribed to cDNA using PrimeScript™ 1st strand cDNA Synthesis Kit . To analyse the mRNA levels of all genes, cDNA was amplified using the SYBR Green PCR Master Mix (Thermo Fisher Scientific) with consensus primers for detecting rpoB, katE, katG, mouse GAPDH, mouse TNF-α, mouse IL-1β, mouse IL-6, mouse MIP-2, and mouse IL-10. The primer sequences are listed in Table rpoB was used as an internal control for the quantification of katE and katG. Mouse GAPDH was used as an internal control for the quantification of mouse TNF-α, mouse IL-1β, mouse IL-6, mouse MIP-2, and mouse IL-10. Real-time PCR was performed as follows: 40 cycles of denaturation at 95 °C for 15 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 1 minute. The amplified PCR products were quantitatively monitored using a StepOne Real-Time PCR System . Fold changes in the expression level of each gene were calculated by the 2−ΔΔCt method using rpoB or mouse GAPDH gene as an internal control. The relative expression of each gene was evaluated relative to the control sample (ATCC 19606 or uninfected cells), which was assigned a value of 1 arbitrary unit.To analyse the expression of proinflammatory cytokines, total RNA was extracted from J774A.1 cells after 24 hours of co-culture with bacteria, using an RNeasy Plus Mini kit . Total RNA of ® Green Flow Cytometry Assay Kit (Thermo Fisher Scientific). Briefly, J774A.1 cells cultured for 24 hours after phagocytosis, were washed with PBS and incubated in RPMI-1640 medium supplemented with 10% FBS and CellROX® reagent at 37 °C for 30 min, as per the manufacturer’s protocols. The cells were harvested and washed with PBS, and subsequently fixed with BD Cytofix™ Fixation Buffer . The stained cells were analysed by using a FACSCanto II flow cytometer (BD Biosciences) equipped with a FACS Diva software. All flow cytometric data were analysed using FlowJo software (BD Biosciences). ROS levels were measured as the Median Fluorescence Intensity (MFI) for CellROX® Green marker. The relative ROS level was evaluated relative to the control sample (uninfected cells), which was assigned a value of 1 arbitrary unit.ROS production level in J774A.1 cells cultured for 24 hours after phagocytosis was detected by CellROX2O at a concentration of 2.5 × 107 cells/mL, with the concentration being adjusted via OD measurements at 595 nm. Thereafter, the bacterial cell suspension was used for catalase activity assay.Catalase activity of the bacteria was measured using Catalase Colorimetric Activity Kit  by following the manufacturer’s protocols. Briefly, the bacterial strains were cultured on LB agar plates  for 16 hours at 37 °C, after which, they were suspended in H2O2 growth assay was conducted according to a previous study31. Bacterial strains were cultured on LB agar plates  for 16 hours at 37 °C, after which they were suspended in LB broth at a concentration of OD595 = 0.05. The bacterial cell suspensions were subcultured for 1 hour prior to treatment with H2O2  at the concentrations indicated in the figures. All growth assays were carried out in 96-well plates in a 100 μL volume, and growth was measured by OD595.Ht-test or one-way analysis of variance (ANOVA), followed by the Dunnett’s multiple comparison test. Pearson correlation analysis was used to compare the intracellular bacterial count in macrophages and % growth of bacteria in the presence of 51 mM H2O2. In all analyses, a 2-tailed probability of < 5% (i.e. *p < 0.05) was considered statistically significant.Quantitative results were combined from 2 independent experiments (n = 6) and presented as mean ± standard error of the mean (SEM). Comparisons of numerical data were performed using Student’s


A key aspect of this regulation is the compartmentalization of Ca2+ in various cytoplasmic organelles that act as intracellular Ca2+ stores. Whereas Ca2+ release from the large-volume Ca2+ stores, such as the endoplasmic reticulum (ER) and Golgi apparatus, are preferred for signal transduction, Ca2+ release from the small-volume individual vesicular stores that are dispersed throughout the cell, such as lysosomes, may be more useful in local regulation, such as membrane fusion and individualized vesicular movements. Conceivably, these two types of Ca2+ stores may be established, maintained or refilled via distinct mechanisms. ER stores are refilled through sustained Ca2+ influx at ER-plasma membrane (PM) membrane contact sites (MCSs). In this review, we discuss the release and refilling mechanisms of intracellular small vesicular Ca2+ stores, with a special focus on lysosomes. Recent imaging studies of Ca2+ release and organelle MCSs suggest that Ca2+ exchange may occur between two types of stores, such that the small stores acquire Ca2+ from the large stores via ER-vesicle MCSs. Hence vesicular stores like lysosomes may be viewed as secondary Ca2+ stores in the cell.Cells utilize calcium ions (Ca Under resting conditions, [Ca2+]cyt is low (~100 nmol/L), while [Ca2+]s are at least several thousand fold higher in the extracellular environment (~2 mmol/L) and in the lumen of organelles, such as the endoplasmic reticulum  and lysosomes (0.4–0.6 mmol/L), which serve as intracellular Ca2+ stores  or Ca2+ release channels in the ER, such as inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) , pumps and secondary Ca2+ transporters in the PM and membranes of intracellular Ca2+-sequestering organelles  can also undergo regulated Ca2+ release   that is about 5,000 times higher than resting [Ca2+]cyt . SOCE relies on the collaborative actions of stromal interaction molecule (STIM) proteins, which serve as ER luminal Ca2+ sensors, and Orai proteins, which act as store-operated Ca2+ channels in the PM. Upon ER Ca2+ store depletion, STIM1 and STIM2 become activated and oligomerized  SERCA pumps  in the inner membrane and mediated by voltage-dependent anion channels (VDACs) in the outer membrane and mitochondrial Ca2+ uniporters in the inner membrane  in non-ER Ca2+ stores  have indicated that the Ca2+ concentration in the lysosome lumen ([Ca2+]Ly) is about 0.5 mmol/L, which is comparable to [Ca2+]ER . Hence, lysosomes and other acidic stores, such as secretory granules, are referred to as small Ca2+ stores.Lysosomes are acidic membrane-bound organelles responsible for degrading macromolecules from both intracellular and extracellular sources , are Ca2), an endolysosome-specific phosphoinositide, may serve as an endogenous TRPML agonist PTPCN1 and TPCN2, respectively, are localized on endosomal and lysosomal membranes P2 can also activate whole-lysosome TPC currents  is the most potent Ca2+ release through lysosomal P2X4 has been implicated in lysosomal membrane fusion in a calmodulin-dependent manner , an ATP-gated cation channel first discovered in the PM of various cell types, also resides on the lysosomal membranes of Cos1 cells where it can be activated by luminal ATP and alkalization   is a Ca2+ permeable non-selective cation channel gated by ADP ribose and Ca2+, is expressed on the PMs of neurons, pancreatic cells, and immune cells , encoded by CACNA1, mediate the Ca2+ entry that triggers neurotransmitter release. In a recent study, Tian et al., found that the α1A subunit of VGCCs is also present on lysosomal membranes in both fruit flies and mice, and is required for autophagosome-lysosome fusion  may drive pH-dependent Ca2+ uptake into lysosomes  also cause lysosomal H+ release ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)], in a recent study  application of various pharmacological reagents, which has many advantages, including amenability to prolonged treatment protocols, as have been used predominantly in previous studies.The aforementioned lysosome-targeted GCaMP and ML-SA assay is the first robust and sensitive method developed with the ability to measure lysosomal Ca2+ stores or their refilling  has little to no impact on naïve Caefilling . This red probes .2+ stores by SERCA inhibitors was shown to abolish lysosomal Ca2+ refilling  docking, involving the formation of ER-lysosome MCSs; and 3) fueling, wherein Ca2+ is transported from the ER to lysosomes through functional ER-lysosome MCSs.A possible critical role of the ER in lysosomal Ca2+ exchange. In ER-PM MCSs, STIM and Orai proteins are concentrated  appositions with tethering, but not fusion of membranes between organelles P2 and PIP2 have been observed in lysosomes  , TRPML1 activity was found to be inhibited by cholesterol accumulation in lysosomes, and increasing TRPML1 activity alleviated lysosomal storage in these cells  exhibit dysregulated lysosomal Ca2+ stores , TRPML1 overexpressing transgenic exhibits constitutive acid secretion (Sahoo et al., 2+ release as a missing link between histamine-protein kinase A signaling and tubulovesicle exocytosis (Sahoo et al., Studying Cao et al.  demonstr2+ stores, the underlying mechanisms of such refilling have not been delineated. Experiments showing that inhibition of ER Ca2+ and, more specifically, IP3Rs can abolish lysosomal store refilling support the notion that the ER may be the primary source of lysosomal Ca2+ under physiological conditions. Hence, small secondary stores, including lysosomes, may acquire Ca2+ from large primary stores, which in turn are refilled by extracellular Ca2+. In the near future, we expect to see advancements in the following areas:Identification of more regulators of ER-lysosome MCS formation.2+ roles in the regulation of ER-lysosome MCS formation.Revelation of lysosomal ∆ψ and Ca2+ sensors enabling the study of ER-lysosome MCS dynamics with super-resolution live imaging.Development of organelle-targeted voltage dyes and luminal CaIdentification of low-affinity uptake channels or transporters in the lysosome.2+ stores, together with their Ca2+ release channels and Ca2+ uptake transporters in these vesicles.Discovery of additional small, vesicular and mobile CaAlthough it is supposed that the ER may refill lysosomal Ca


SBDS gene and characterized by pancreatic insufficiency, skeletal abnormalities, and a bone marrow dysfunction. In addition, SDS patients are predisposed to develop myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), typically during adulthood and associated with TP53 mutations. Although most SDS diagnoses are established in childhood, the nature and frequency of serial bone marrow cell investigations during the patients' lifetime remain a debatable topic. The precise molecular mechanisms leading to AML progression in SDS patients have not been fully elucidated because the patient cohorts are small and most disease monitoring is conducted using standard histological and cytogenetic approaches. Here we report a rare case of a patient with SDS who was diagnosed with AML at 5 years of age and survived. Intermittent neutropenia preceded the AML diagnostic but serial bone marrow monitoring according to the standard of care revealed no cytogenetic anomalies nor signs of clonal hematopoiesis. Using next generation sequencing approaches to find cytogenetically cryptic pathogenic mutations, we identified the cancer hotspot mutation c.394C>T/p.Arg132Cys in IDH1 with high variant allelic frequency in bone marrow cells, suggesting clonal expansion of a major leukemic clone karyotypically normal, in the SDS-associated AML. The mutation was somatic and likely occurred at the leukemic transformation stage, as it was not detected in a matched normal tissue nor in bone marrow smear prior to AML diagnosis. Gain-of-function mutations in IDH1, such as c.394C>T/p.Arg132Cys, create a neo-activity of isocitrate dehydrogenase 1 converting α-ketoglutarate into the oncometabolite D-2-hydroxyglutarate, inhibiting α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Overall, our results suggest that along with previously described abnormalities such as TP53 mutations or monosomy7, 7q-, which are all absent in this patient, additional mechanisms including IDH1 mutations drive SDS-related AML and are likely associated with variable outcomes. Sensitive techniques complementary to standard cytogenetics, such as unbiased or targeted panel-based next generation sequencing approaches, warrant testing for monitoring of myelodysplasia, clonal hematopoiesis, and leukemia in the context SDS. Such analyses would also assist treatment decisions and allow to gain insight into the disease biology.Shwachman-Diamond syndrome (SDS) is a rare and systemic disease mostly caused by mutations in the  Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome characterized by exocrine pancreatic insufficiency, bone abnormalities, progressive cytopenia, and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The risk of worsening cytopenia(s), clonal myeloid evolution or AML is estimated at 20% by age 18 , which is a rare cooperating event in pediatric AML and has never been reported in conjunction with SDS. Gain-of-function mutations in Isocitrate Dehydrogenase 1, an enzyme involved in cell metabolism, are known to modify the epigenetic landscape and to promote cancer progression   on cytogenetic analysis  and intermittent neutropenia with an absolute neutrophil count fluctuating between 0.8 ×10Ser41fs) . He was Ser41fs) . These canalysis .He was treated with a first cycle of induction chemotherapy , but minimal residual disease (MRD) remained positive at 0.4%, as assessed by flow cytometry at the end of the cycle. He pursued treatment with a second cycle consisting of high-dose cytarabine and etoposide, after which he achieved MRD negativity and proceeded to hematopoietic stem cell transplant. A first attempt with intra-osseous cord blood transplant following busulfan, fludarabine, and anti-thymocyte globulin (ATG) was unsuccessful with persistent aplasia. A second attempt with an alternate cord blood unit also failed to engraft after conditioning with total body irradiation, cyclophosphamide and fludarabine. Finally, the patient underwent a matched unrelated donor bone marrow transplant following conditioning with cyclophosphamide, fludarabine, and ATG. Hematopoietic engraftment was successful and the patient remains in first complete remission 5.5 years post-transplant (11-year-old).IDH1 (c.394C>T/p.Arg132Cys), reported to be pathogenic and recurrent in adult AML and other cancer types  while a minor clone with a mutation in BRCA2  was also detected . To estimate the timing of IDH mutation appearance, we performed NGS on DNA material obtained from scraping fixed bone-marrow smears, archived 6 months prior to AML diagnosis. IDH1 c.394C>T (p.Arg132Cys) mutation was not detected in any of the 44 sequencing reads obtained spanning that position, indicating that the mutation was not present or below our detection threshold 6 months prior to AML diagnosis  analysis did not reveal chromosomal copy-number abnormalities (data not shown) in leukemic cells, consistent with a normal karyotype. Then, whole exome and transcriptome sequencing were performed using bone marrow cells at diagnosis. A point mutation in er types , was ideer types . Whole el tissue . Somaticdetected . No fusiiagnosis . These rSDS is a ribosomopathy characterized by genomic instability contributing to the occurrence of chromosomal abnormalities and therefore to the risk of malignant transformation to MDS/AML. Characterization of high-risk clonal chromosomal abnormalities remains a challenge, as some patients only develop transient genetic alterations devoid of malignant potential. The most commonly encountered chromosomal anomalies in SDS comprise isochromosome 7 (i(7)(q10)), resulting in a gain of long arm of chromosome 7 (7q), and deletion 20q (del(20)(q)) , not typTP53 mutation confirmed this hypothesis and revealed a cooperating genetic mutation in the Isocitrate Dehydrogenase 1 gene (IDH1), associated with promoting leukemia.SDS being a rare disorder, thorough understanding of SDS-related AML has remained limited due to the small number of patients, especially in pediatrics. Given the heterogeneity of MDS/AML progression in patients with SDS, we postulated that acquired cooperating somatic mutations must contribute to malignant transformation. This should be especially true for pediatric patients in whom the risk of SDS-related MDS/AML is considerably lower than in adults, prompting our investigations for this case. The unique presentation of an SDS-associated AML in a young pediatric patient lacking chromosomal rearrangements and any pathogenic 2 (IDH1 mutations have been found to be rare in pediatric AML (~1–2%)  of children with Maffucci syndrome, a non-hereditary disorder characterized by multiple enchondromas and hemangiomas  –13, but  (~1–2%) . They ha (~1–2%) , 15 and,patients . Somaticangiomas , 18. In elopment .IDH1 mutation identified in SDS-associated AML. Pediatric AML associated with IDH1 mutations present a favorable overall survival  are not available at the time of publication but will be available upon request.The Institutional Review Board of CHU Sainte-Justine approved the study and informed consent was obtained from the parents.SM, J-RL, MB, AR, and SC wrote the manuscript. SM, J-RL, MB, MR, LL, and AR generated the figures and table. SM, J-RL, MB, MR, LL, LB, AR, LJ, and PG contributed to experiments and analyses. YP, MD, PT, HB, and SC were in charge of the patient. JH and SC supervised the biobanking resource. All authors revised and approved the manuscript.J-RL is a founder and has ownership stake in Streamline Genomics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.