Colpodella sp. (ATCC 50594) is a free-living biflagellate predator closely related to pathogenic Apicomplexa such as Plasmodium, Cryptosporidium and Toxoplasma gondii. Colpodella sp. (ATCC 50594) obtain nutrients by preying on Parabodo caudatus using myzocytosis. The organization of the myzocytic apparatus and the mechanism of nutrient uptake into the posterior food vacuole of Colpodella species is unknown. In this study, we investigated myzocytosis using light and transmission electron microscopy. We investigated the uptake of 40 nm and 100 nm fluorescent nanoparticles and E. coli BioParticles by Colpodella sp. (ATCC 50594) in a diprotist culture. Transmission electron microscopy was used to investigate the morphology of the tubular tether formed during myzocytosis. E. coli BioParticles were taken up by P. caudatus but not by Colpodella sp. (ATCC 50594). Both protists took up the 100 nm and 40 nm beads, which were observed distributed in the cytoplasm of free unattached Colpodella sp. (ATCC 50594) trophozoites, and also in feeding Colpodella sp. (ATCC 50594) trophozoites and in the pre-cysts. Fragments of the nucleus and kinetoplast of P. caudatus and the nanoparticles were identified in the tubular tether being aspirated into the posterior food vacuole of Colpodella sp. (ATCC 50594). Unattached Colpodella sp. (ATCC 50594) endocytose nutrients from the culture medium independently from myzocytosis. The mechanisms of myzocytosis and endocytosis among Colpodella species may provide important insights into nutrient uptake among the pathogenic apicomplexans. Colpodella species are free-living alveolate predators closely related phylogenetically to the pathogenic Apicomplexa [Colpodella species, along with Voromonas pontica and the dinoflagellates Perkinsus and Psammosa species, possess two heterodynamic flagella and apical complex organelles, and are known as myzozoans [Colpodella species possess an open conoid, known as a pseudoconoid [Plasmodium species and among the gregarines [Colpodella species feed using a process known as myzocytosis, a characteristic of the myzozoans, where they attach to their flagellate and algae prey and aspirate the cytoplasmic contents into a posterior food vacuole. At the conclusion of feeding, most Colpodella species encyst. Variations exist in different Colpodella species regarding cyst formation. Resting cysts are found in the life cycle of Colpodella pseudoedax, in addition to division by longitudinal fission, C. unguis divides by oblique-transversal fission and C. edax does not encyst [C. tetrahymenae and C. gonderi prey on ciliates [Colpodella trophozoites acquire nutrients using endocytosis in addition to myzocytosis. Roles for endocytosis in nutrient acquisition and membrane homeostasis have been suggested. However, the presence of the inner membrane complex (IMC) found in all apicomplexans may hinder membrane invagination and endocytosis [T. gondii, the micropore has been reported to be involved in endocytosis and nutrient acquisition [Colpodella infections have been described with red blood cells infected [Plasmodium-infected red blood cells [Toxoplasma gondii and endocytosis at the cytostome in P. falciparum may contribute to artemisinin resistance [P. falciparum and Toxoplasma gondii [Selenidium pendula Giard, 1884 feeds by myzocytosis; however, unlike Colpodella sp. (ATCC 50594), the food vacuoles along with rhoptries are located in the anterior end of the trophozoite [Colpodella sp. (ATCC 50594) trophozoites using carboxylated-modified fluorescent polystyrene nanoparticles of 40 nm and 100 nm and fluorescent E. coli BioParticles. We performed transmission electron microscopy to investigate the ultrastructure of the initial myzocytic contact between predator and prey and the tubular tether formation. The size of nutrients aspirated during myzocytosis has not been studied previously in Colpodella species.complexa ,2. Amongyzozoans . The apiyzozoans ,5,6. Thedoconoid . Contentegarines ,8. Colpot encyst ,10. Ectociliates ,11 and rciliates . It is uocytosis . The micuisition . Two huminfected  and in ainfected . The modod cells , a mucrood cells ,17 and msistance . The uptsistance . The typa gondii . Howeverphozoite , very liDiprotist culture conditions. Colpodella sp. (ATCC 50594) with Parabodo caudatus as prey was obtained from the American type culture collection (ATCC) . Predator and prey were cultured together at 24 °C in tissue culture flasks containing Hay medium  bacterized with Enterobacter aerogenes as described [escribed ,21. CultStaining and light microscopy. Cells in tissue culture flasks were fixed directly by adding an equal volume of 10% formalin for a final concentration of 5% in the flask (cells “fixed in flight”) as described previously [eviously ,21. Formeviously  followedFluorescent Nanoparticle Uptake. Colpodella sp. (ATCC 50594) was cultured at 24 °C with the prey protist Parabodo caudatus in Enterobacter aerogenes bacterized Hay medium. At 27–28 h post subculture, FluoSpheres and E. coli BioParticles  were added to the cultures and used to evaluate nutrient uptake during myzocytosis, as well as the size of nutrients taken up by predator and prey. To avoid aggregation, FluoSpheres were initially diluted (1:10) and sonicated in a water bath. Dilutions less than 1:10 resulted in a high concentration of beads contributing to a high background and dilutions higher that 1:10 resulted in a dilute bead concentration in culture. For two 20 mL cultures, 20 µL of FluoSpheres and 180 µL of hay medium was added to microcentrifuge tubes and sonicated in a water bath for 10 min. After subculture, FluoSpheres of 4 µm fluorescent red sulfate microspheres (Ex/Em: 580/605 nm), 2 µm yellow-green fluorescent sulfate microspheres (Ex/Em: 505/515 nm), 100 nm red fluorescent Carboxylate-Modified Microspheres (Ex/Em: 580/605 nm), 40 nm, yellow-green fluorescent Carboxylate-Modified Microspheres (Ex/Em: 505/515 nm) were further diluted 1:1000 in Hay medium followed by sonication to disperse the particles. Concentrations of 50 or 100 µL beads from the initial dilution were added to 10 mL of culture and incubated for 1 h at 24 °C to determine the optimum dilution for uptake experiments. A concentration of 50 µL resulted in a dilute bead suspension so 100 µL was used for uptake experiments. pHrodo Red E. coli BioParticles (1 mg/mL) (Ex/Em: 560/585 nm) were diluted by adding 2 mL of hay medium to each 2 mg vial (following manufacturer’s protocols) and sonicated in a water bath for 10 min. An amount of 500 µL of cells were added to 10 mL cultures and incubated for 1h. Cultures were fixed with 5% formalin for 10 min at RT, then centrifuged at 3500 rpms for 10 min in a refrigerated centrifuge. After discarding the supernatant, the pellet was washed by resuspension in 1X PBS, followed by centrifugation. The wash supernatant was discarded and the pellet was resuspended in 1X PBS, for preparation of smears on glass slides. Smears were air-dried and mounted in mounting medium by adding 100 µL of Fluoromount-G containing DAPI . Smears of formalin-fixed cells were also incubated with CellMask Green actin tracking stain (ThermoFisher Scientific) for cells incubated with 100 nm fluorescent nanoparticles and E.coli BioParticles, and CellMask Deep Red actin tracking stain (ThermoFisher Scientific) for cells incubated with 40 nm nanoparticles. Smears were mounted in mounting medium by adding 100 µL of Fluoromount-G containing DAPI . Slides were sealed with two coats of clear nail polish and slides were examined by confocal and differential interference contrast microscopy. Slides were examined at the imaging core . Confocal, fluorescent and differential interference contrast (DIC) images were collected using a Leica SP8 True Scanning Confocal (TCS) DM18inverted microscope . Stained and confocal images were adjusted to 300 dpi using the CYMK color mode, RGB color mode, auto color and auto contrast on Adobe Photoshop (CC). Electron microscopy. An aliquot of Colpodella sp. (ATCC 50594) in culture medium was added to an equal volume of 8% paraformaldehyde in 0.1 M cacodylate buffer and spun down for 10 min at 3500 rpm. The cell pellet was fixed with 2.5% glutarladehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer. The fixation continued in the same fixative solution for a total of 2 h at room temperature. The pellets were thoroughly rinsed in 0.1 M cacodylate buffer, and then postfixed for 2 h in an unbuffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferrocyanide. After rinsing with distilled water, the specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. After another rinse in distilled water, they were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in a EMbed 812 embedding media . Thin sections (70 nm) were cut on a RMC MT6000-XL ultramicrotome. These were mounted on T-300 mesh nickel grids  and then sequentially stained with acidified methanolic uranyl acetate and stable lead staining solution. These were coated on a Denton DV-401 carbon coater , and observed in a FEI Tecnai Spirit (T12) transmission electron microscope with a Gatan US4000 4kx4k CCD.Myzocytosis and tubular tethers are shown in predator–prey attachments with Sam-Yellowe’s trichrome staining in Colpodella sp. (ATCC 50594) trophozoites are shown in both panels. We reported in previous studies the presence of DAPI-stained contents in the food vacuole of Colpodella sp. (ATCC 50594) trophozoites [Colpodella sp. (ATCC 50594) trophozoites were identified with DAPI-stained contents in the cytoplasm as shown in Colpodella sp. (ATCC 50594) trophozoites in active culture were observed to attach to and detach from prey after making contact unlike the irreversible attachments of zoites during host cell invasion in pathogenic Apicomplexans such as Plasmodium species. We performed nutrient uptake experiments using fluorescent particles of 4 µm, 2 µm, 100 nm and 40 nm and E. coli BioParticles in order to determine the size of nutrients taken up in culture by Colpodella sp. (ATCC 50594) and its prey. The 4 and 2 µm particles were not taken up by Colpodella sp. (ATCC 50594). Fluorescent nanoparticles of 100 nm (Colpodella sp. (ATCC 50594) trophozoites, distributed within the cytoplasm. Colpodella sp. (ATCC 50594) trophozoites did not take up E. coli BioParticles. Parabodo caudatus trophozoites did not take up 4 µm or 2 µm particles but took up 100 nm, 40 nm and E. coli BioParticles from the culture. E. coli BioParticles were observed in the cytoplasm of P. caudatus (Different sizes of the prey targeted by hozoites . In actif 100 nm A–C and 4f 100 nm A–H were P. caudatus were observed in the cytoplasm of an unattached Colpodella sp. (ATCC 50594) trophozoite below the nucleus (n) (P. caudatus (red arrow), and in the tubular tether, along with 100 nm nanoparticles (red) also distributed in the cytoplasm of predator and prey (Colpodella sp. (ATCC 50594) trophozoites and in the forming posterior food vacuole (The nucleus and kinetoplast of leus (n) H. In Figand prey A, DAPI-s vacuole B,C,F. Ce vacuole D,E,G–J.Colpodella sp. (ATCC 50594) the food vacuole contained the nucleus and kinetoplast of P. caudatus (Colpodella sp. (ATCC 50594) trophozoite (Colpodella sp. (ATCC 50594) trophozoites using a myzocytic aperture located posterior to the pseudoconoid followed by uptake of the prey’s membrane, dissolution of the membrane and aspiration of the prey’s cytoplasmic contents [In the pre-cyst of caudatus A and thecaudatus B,G. The phozoite D–F,H. Wecontents . The proColpodella sp. (ATCC 50594) shown in the TEM micrograph is pointed away from the contact site (brown arrow), while the attachment site (black arrow) is in direct contact with the prey’s membrane. From this contact site, aspiration of the cytoplasmic contents of the prey occurs following dissolution of the prey’s plasma membrane. The prey’s (red arrows) membrane is pulled in further into the predator’s (yellow arrows) cytoplasm, as shown in Colpodella sp. (ATC C 50594) are seen surrounding the prey’s plasma membrane  trophizoites have been observed feeding on mature P. caudatus trophozoites, and mature Colpodella sp. (ATCC 50594) trophozoites in culture feed on juvenile P. caudatus trophozoites. In culture, Colpodella sp. (ATCC 50594) trophozoites can be identified without prey attachments and trophozoites have been observed to detach from their prey after initial attachment and persist in culture. In this study, we investigated the hypotheses that unattached trophozoite stages would take up nutrients through means other than myzocytosis and that aspirated contents from the prey would be degraded before uptake and incorporation into the posterior food vacuole. We also wanted to identify the point of contact between predator and prey at the initial stages of myzocytosis. Fluorescent particles of 4 µm, 2 µm 100 nm and 40 nm were used to investigate the size of aspirated particles taken up during myzocytosis. Free, unattached, active young trophozoites of Colpodella sp. (ATCC 50594) and mature trophozoites were observed in culture. The size of aspirated nutrients has not been reported for Colpodella species, and in our previous light microscopy and ultrastructural studies, particulate nutrients were not observed in the pre-cysts and cysts of Colpodella sp. (ATCC 50594) [C 50594) ,24,25.Colpodella sp. (ATCC 50594) can feed on different stages of P. caudatus trophozoites, forming tubular tethers that are used for aspirating cytoplasmic contents. In unattached Colpodella sp. (ATCC 50594) trophozoites, DAPI-stained contents were identified in the cytoplasm, suggesting that nutrient uptake can take place independently of myzocytosis. Results of nutrient uptake experiments showing uptake of 40 nm and 100 nm beads into unattached trophozoites of Colpodella sp. (ATCC 50594) confirmed uptake by endocytosis. Although not considered bacteriotrophic, bacteria from culture were observed in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites. However, fluorescent E. coli BioParticles were not taken up. Aspirated cytoplasmic contents of P. caudatus were observed in the tubular tether between predator and prey during myzocytosis. Intact kinetoplast and nuclei of P. caudatus were observed in the posterior food vacuole of Colpodella sp. (ATCC 50594) in the trophozoite and pre-cyst stages of the life cycle  and the prey’s membrane in [Selenidium orientale where the cytostome is used and can re-open for intermittent feeding [Colpodella sp. (ATCC 50594) is within the opening of the pseudoconoid or a separate structure such as the cytostome or micropore. Myzocytosis has been described as a type of endocytosis [Cryptosporidium species may be the mode of feeding used by the common ancestor of the Apicomplexans [Colpodella species and V. pontica [Cryptosporidium species serving as the link between predatory feeding, extracellular parasitism and intracellular parasitism following host cell invasion [Among the Apicomplexa, trophozoites of blastogregarines and archigregarines attach to their host cells using the mucron, through which, nutrients are obtained by myzocytosis ,17. The st cells . Cryptos species . The epi species ,17 similbrane in  and the brane in ,25. More feeding . It is uocytosis  and as a pontica  represeninvasion .Colpodella sp. (ATCC 50594) trophozoites perform endocytosis, with the uptake of 100 nm and 40 nm beads (Colpodella sp. (ATCC 50594) and P. caudatus, since the larger particles were not observed in the cytoplasm of the protists. The rhoptries and micronemes in Colpodella species are emptied during myzocyosis and may participate in the digestion of the prey’s membrane and the uptake and digestion of aspirated nutrients. The unexpected observation in this study of intact nuclei and kinetoplast of P. caudatus in the cytoplasm of free unattached trophozoites and in food vacuoles of Colpodella sp. (ATCC 50594) in pre-cyst stages demonstrates that undigested particulate, as well as digested soluble nutrients, can be taken up by Colpodella sp. (ATCC 50594) trophozoites in diprotist culture, contrary to our hypothesis. A major challenge to performing nutrient uptake studies in diprotist culture with nanoparticles is the presence in the culture of more P. caudatus than Colpodella sp. (ATCC 50594) and the presence of large amounts of bacteria. We are currently investigating synchronization protocols to enrich for each protist and eliminate bacteria. This will facilitate quantitating particle uptake at each stage of the life cycle for each protist in future experiments, as well as performing endocytosis inhibition studies. The synchronization of life cycle stages will also aid the identification of proteins involved in endocytosis. The cytoskeleton participates in tubular tether formation, as we showed previously that cytochalasin D treatment distorts the tubular tether [Colpodella species to the pathogenic Apicomplexa [Plasmodium falciparum [Plasmodium species may provide clarity for understanding nutrient acquisition and the role of the food vacuole during intracellular infection.The IMC is described as a barrier to endocytosis in Apicomplexa, with the micropore serving the role of an endocytotic apparatus, and the cytostome consists of proteins that are associated with endocytosis . The obsnm beads , suggestr tether . CellMascomplexa  and the lciparum ,30, the Colpodella sp. (ATCC 50594) use myzocytosis and endocytosis for nutrient acquisition during growth in diprotist culture. Unattached Colpodella sp. (ATCC 50594) trophozoites take up nutrients in culture independently of myzocytosis and may attach and feed intermittently before detaching to seek other prey. The presence of a “myzocytotic aperture”, most likely a structure similar to a cytostome, may permit intermittent feeding. During myzocytosis, Colpodella sp. (ATCC 50594) trophozoites do not pierce the plasma membrane of its prey to obtain nutrients. Following attachment, the plasma membrane of the prey is taken up into the cytoplasm of Colpodella sp. (ATCC 50594), followed by dissolution of the membrane, to form a channel through which the cytoplasmic contents of the prey are aspirated into the posterior food vacuole. Nutrients as large as intact nuclei and kinetoplasts were identified in the posterior food vacuole of Colpodella sp. (ATCC 50594), suggesting that the organelles can be squeezed through the tubular tether with the cytoplasmic aspirate and deposited in the posterior food vacuole. Fluorescent nanoparticles of 40 nm and 100 nm were taken up by Colpodella sp. (ATCC 50594) and P. caudatus. Nanoparticles taken up by P. caudatus were aspirated by Colpodella sp. (ATCC 50594) during myzocytosis. Fluorescent E. coli BioParticles were only taken up by P. caudatus. In future studies, endocytosis inhibition will be performed to gain insights regarding the cytoskeletal components involved in nutrient uptake and proteins involved in endocytosis will be identified. The use of synchronized life cycle stages of predator and prey in future nutrient uptake studies will permit the quantitation of beads taken up by both protists. Myzocytosis for predation may be the ancestral mode of nutrient acquisition among the Apicomplexa.Our data show for the first time that


Ca2+ activity in astrocytes significantly increased during locomotion from a low quiescence level. Ca2+ signals first appeared in the distal processes and then propagated to astrocytic somata, where it became significantly larger and exhibited oscillatory behaviour. Thus, astrocytic soma operates as both integrator and amplifier of Ca2+ signal. In neurons, Ca2+ activity was pronounced in quiescent periods and further increased during locomotion. Neuronal Ca2+ concentration ([Ca2+]i) rose almost immediately following the onset of locomotion, whereas astrocytic Ca2+ signals lagged by several seconds. Such a long lag suggests that astrocytic [Ca2+]i elevations are unlikely to be triggered by the activity of synapses among local neurons. Ca2+ responses to pairs of consecutive episodes of locomotion did not significantly differ in neurons, while were significantly diminished in response to the second locomotion in astrocytes. Such astrocytic refractoriness may arise from distinct mechanisms underlying Ca2+ signal generation. In neurons, the bulk of Ca2+ enters through the Ca2+ channels in the plasma membrane allowing for steady-level Ca2+ elevations in repetitive runs. Astrocytic Ca2+ responses originate from the intracellular stores, the depletion of which affects subsequent Ca2+ signals. Functionally, neuronal Ca2+ response reflects sensory input processed by neurons. Astrocytic Ca2+ dynamics is likely to provide metabolic and homeostatic support within the brain active milieu.Locomotion triggers a coordinated response of both neurons and astrocytes in the brain. Here we performed calcium (Ca  This system of brain elements and their interactions is called the brain active milieu.12 cellular morphology, and structure of extracellular space.2+ imaging is the most popular: Multiple genetically encoded sensors were developed to monitor [Ca2+]i in specific cell types.Optical methods allow monitoring of various types of cellular activity in the brain that include ionic signaling,23 These methods can be combined with a virtual reality environment to design various behavioral paradigms.25 However, whether virtual reality faithfully represents the real world as an experimental animal perceives it remains an open question. In addition, a head-restricted animal cannot turn and always runs in a single direction on treadmills. The air-floating ball allows an animal to turn. Yet, the animal’s posture differs from that of an animal running on a flat surface: It resembles climbing an object, with the tail and the hindlimbs positioned below the head and forelimbs.Two-photon microscopy provides cellular and subcellular resolution for imaging with genetically encoded sensors in vivo.2+]i dynamics in neurons and microglia in mice.27 However, the other elements of the brain active milieu have not been systematically studied in this experimental paradigm. Here, we compared the Ca2+ activity of astrocytes and neurons of mice navigating an airlifted platform.Using a flat airlifted platform rectifies the limitations of the above systems for 2-photon imaging in awake animals. This technique was used recently to monitor . The sample number (2+ sensor GCaMP6f under either astrocytic (AAV5-gfaABC1D-cyto-GCaMP6f) or neuronal (AAV2/9-CamKII-GCaMP6f) promotors  and had low ΔF/F . Consistent with previous reports, the locomotion was accompanied by a rise in astrocytic [Ca2+]i.34 We observed an increase in both the active area  and ΔF/F . Sometimes, the animal ran several times with short intervals of quiescence. Astrocytic [Ca2+]i increased during each running episode, but this increase became smaller in subsequent episodes.First, the imaging was done on astrocytes expressing GCaMP6f in cortical layer 1 . The ani2+ imaging was combined with animal tracking , and the mean ΔF/F per period of quiescence was 3.1 [2.7–4.2]% (n = 37). Locomotion was accompanied by a rise in neuronal [Ca2+]i .During quiescent periods, the mean active area was 6.0 [4.4–9.4]% of the frame  approach implemented in CaImAn, open-source software for Ca2+ imaging analysis.37 With CNMF followed by clustering, we identified neuronal structures activated synchronously within individual recordings  and locomotion (L). The Q/L ratio of ΔF/F was significantly higher in neurons than in astrocytes  in neurons and astrocytes. The cross-correlations yielded reasonably high peak coefficients in both cell types . In astrocytes, the peak had a lag of several seconds  corresponding to 2 locomotion episodes , n = 14; P = .002, mixed-effects model) suggesting a decline in astrocytic response. Similar results were obtained for PRR of active area  as the ratio between the peaks of second and first Caepisodes . The PRR2+ entry through voltage-gated Ca2+ channels. We observed that overall neuronal Ca2+ activity in the somatosensory cortex increased during animal locomotion. However, neurons remained active in quiescent periods between episodes of locomotion. In some neuronal units, activity during quiescence was higher than that during locomotion.Cortical neurons receive sensory input from thalamic nuclei. During locomotion, neuronal firing triggers Ca2+]i elevations in astrocytic leaflets can be mediated by Ca2+ entry through the plasma membrane (through ionotropic receptors or reversed Na+/Ca2+ exchanger), whereas large Ca2+ transients in soma and branches are mediated by Ca2+ release from endogenous stores—endoplasmic reticulum and mitochondria.2+ events. These events can be either spontaneous or triggered by local synaptic activity. During locomotion, astrocytic Ca2+ activity substantially increased. Relative to Ca2+ activity during the quiescent period, this increase was significantly larger in astrocytes than in neurons. Moreover, astrocytic [Ca2+]i elevation was delayed for several seconds after the beginning of the locomotion episode and neuronal [Ca2+]i elevation. These findings suggest that the activity of the local neuronal network may not trigger the astrocytic Ca2+ signal. Most likely, astrocytic response originates from modulatory subcortical projections, such as noradrenergic input from the locus coeruleus targeting adrenergic receptors abundantly expressed in mature astrocytes.2+ response also raises a question of its physiological relevance. This delay suggests that astrocytes do not respond to sensory signals in real time, hence are involved in the real-time processing of sensory information. On the other hand, astrocytic Ca2+ activity may be involved in modulation of synaptic plasticity,44 regulation of vascular tone,45 and metabolic activation of astrocytes46 that occur with a delay. For example, synaptic plasticity and memory consolidation may happen after sensory information is processed, filtered, and compared to previous memories in the brain. The same applies to vasodilatation and metabolic activation of astrocytes, which can represent a systemic response to the increased energy demand of the brain following robust neuronal activity during locomotion.47Local i elevation in astrocytes.53 In moderate quantities, ROS play a signaling role and promote synaptic plasticity in the brain.54 In high quantities, they cause oxidative stress and cell damage. Therefore, the astrocytic refractory period may preserve ROS’s beneficial effects while preventing cell damage.Another example of dissociation between Ca2+]i did not increase simultaneously in the entire astrocytic morphological domain during locomotion. [Ca2+]i elevation started in the distal processes and then propagated into the soma. This finding is consistent with a higher probability for Ca2+ activity generation in distal astrocytic processes.55 This phenomenon may be explained by the specific localization of synapses near distal astrocytic processes. However, a recent report demonstrated a relatively even distribution of synapses near distal processes and astrocytic soma.56 An alternative explanation is that distal astrocytic processes have a higher surface-to-volume ratio (SVR).36 High SVR increases the amplitudes of [Ca2+]i elevations induced by Ca2+ entry through the plasma membrane and consequently increases the probability of Ca2+-dependent Ca2+ release from endogenous stores.At the subcellular level, i regulate distinct molecular pathways.58 Thus, Ca2+ amplification may target specific cellular functions. For example, somatic [Ca2+]i elevation can boost ATP production by promoting the activity of the tricarboxylic acid cycle enzymes, the proteins of the electron transport chain, and the ATP synthase.60 Such enhanced metabolic response of astrocyte can be the physiological outcome of Ca2+ integration, homologues to, and yet distinct from, synaptic integration and action potential generation in neurons.Our observations also suggest that astrocytic soma operates as an integrator of Ca2+ integration in astrocytic soma triggers [Ca2+]i oscillations. [Ca2+]i oscillations in astrocytes were routinely recorded in primary cultures and in slices in response to pharmacological stimulation.2+]i dynamics also predict oscillatory activity due to positive and negative feedbacks.66 Although astrocytes, in principle, should possess mechanisms for [Ca2+]i oscillations, oscillatory behavior was not reported in vivo. A possible reason is that Ca2+ activity in astrocytes in vivo is defined by inputs from many elements of the brain active milieu , which drive the sequence of astrocytic Ca2+ events. However, astrocyte oscillatory mechanisms can re-emerge during somatic [Ca2+]i elevations. These oscillations originate specifically in the soma and fade away when propagating toward the astrocytic periphery. [Ca2+]i oscillations require [Ca2+]i to exceed a certain threshold. Large [Ca2+]i elevations and well-expressed Ca2+ stores in the soma provide optimal conditions for [Ca2+]i oscillations.65 Possibly, the information is encoded in the frequency of [Ca2+]i oscillations that depends on the strength of the external signal and intrinsic properties of an individual astrocyte. The oscillatory Ca2+ signal is decoded by the enzymes containing Ca2+ binding motifs with a distinct affinity that can regulate their phosphorylation and activate specific cellular programs.67In addition, Ca2+ dynamics in astrocytes does not follow neuronal Ca2+ activity that accompanies locomotion, indicating that astrocytic [Ca2+]i is controlled by pathways distinct from sensory inputs that activate neuronal networks. While neuronal [Ca2+]i faithfully follows episodes of animal locomotion, astrocytic Ca2+ signals develop on a different timescale, compatible with distinct roles astrocytes play in sustaining nervous tissue and supporting neuronal information processing and storage.We demonstrate that Cazqad019_Supplemental_MoviesClick here for additional data file.


In AML with NPM1 mutation causing cytoplasmic dislocation of NPM1, treatments with Menin inhibitor (MI) and standard AML chemotherapy yield complete remissions. However, the causal and mechanistic linkage of mtNPM1 to the efficacy of these agents has not been definitively established. Utilizing CRISPR-Cas9 editing to knockout (KO) or knock-in a copy of mtNPM1 in AML cells, present studies demonstrate that KO of mtNPM1 from AML cells abrogates sensitivity to MI, selinexor (exportin-1 inhibitor), and cytarabine. Conversely, the knock-in of a copy of mtNPM1 markedly sensitized AML cells to treatment with MI or cytarabine. Following AML therapy, most elderly patients with AML with mtNPM1 and co-mutations in FLT3 suffer AML relapse with poor outcomes, creating a need for novel effective therapies. Utilizing the RNA-Seq signature of CRISPR-edited AML cells with mtNPM1 KO, we interrogated the LINCS1000-CMap data set and found several pan-HDAC inhibitors and a WEE1 tyrosine kinase inhibitor among the top expression mimickers (EMs). Additionally, treatment with adavosertib (WEE1 inhibitor) or panobinostat (pan-HDAC inhibitor) exhibited synergistic in vitro lethal activity with MI against AML cells with mtNPM1. Treatment with adavosertib or panobinostat also reduced AML burden and improved survival in AML xenograft models sensitive or resistant to MI. Cytoplasmic dislocation and loss of nucleolar localization of the chaperone protein NPM1 occurs in 30% of AML, most commonly following a heterozygous, frame shift, C-terminal mutation in exon 12 of NPM1 (creating NPM1c) –7. PatiemtA/wt). Findings presented here demonstrate that, compared to control OCI-AML3, CRISPR-edited OCI-AML3 cells with KO of mtNPM1 exhibited profound depletion of NPM1c and suppression of MI-induced loss of viability. Additionally, NPM1c depletion significantly reduced sensitivity of OCI-AML3 cells to apoptosis induced by KPT-330, cytarabine, and daunorubicin. In contrast, OCI-AML2 NPM1mtA/wt cells exhibited increased levels of HOXA9, MEIS1, and c-Myc, and notably these cells were sensitized in vitro and in vivo to MI-induced anti-AML efficacy. Utilizing the RNA-Seq signature of OCI-AML3 cells with CRISPR-edited depletion of NPM1c, we interrogated the LINCS1000-CMap data set of gene expression signatures and determined that the top expression mimickers (EMs) [In present studies, we first determined the mechanistic linkage of the presence/absence of mtNPM1 to the active epigenome/transcriptome, protein expression, as well as to cell growth, differentiation, and viability of AML cells. Additionally, we interrogated the causal linkage of the presence/absence of mtNPM1 on therapy responsiveness to standard anti-AML agents, including cytarabine and daunorubicin, as well as to novel agents, including MI, exportin-1 inhibitor KPT-330) , arsenicrs (EMs) , 30. The , arsenirs (EMs) , 32. AddSNDX-5613, SNDX-50469, ziftomenib (KO-539), ATRA, SY-1425, Cytarabine, Daunorubicin, Selinexor (KPT-330), Entinostat, Panobinostat, and Adavosertib were obtained from MedChem Express . Cycloheximide was obtained from Santa Cruz Biotechnology, Inc. . All compounds were prepared as 10 mM stocks in 100% DMSO and frozen at −80 °C in 5–10 µL aliquots to allow for single use, thus avoiding multiple freeze-thaw cycles that could result in compound decomposition and loss of activity. For in vivo studies, SNDX-5613 was obtained from Syndax Pharmaceuticals under an MTA and reconstituted per the manufacturer’s instructions.The cell lines utilized in these studies were authenticated in the Characterized Cell Line Core Facility at M.D. Anderson Cancer Center, Houston TX utilizing STR profiling.Patient-derived AML cells samples were obtained with informed consent as part of a clinical protocol approved by the Institutional Review Board of The University of Texas, M.D. Anderson Cancer Center. Mononuclear cells were purified by Ficoll Hypaque  density centrifugation following the manufacturer’s protocol. Mononuclear cells were washed once with sterile 1× PBS then suspended in complete RPMI media containing 20% FBS. Cells were counted to determine the number of cells isolated prior to immuno-magnetic selection. CD34 + AML blast progenitor cells were purified by immuno-magnetic beads conjugated with anti-CD34 antibody following the manufacturer’s protocol  prior to utilization in the cell viability assays, RNA expression, and immunoblot analyses.Following designated treatments (72–96 hours), cultured cell lines or patient-derived (PD) AML blast cells, were washed with 1× PBS, stained with TO-PRO-3 iodide , and analyzed by flow cytometry on a BD Accuri CFlow-6 flow cytometer .t test in GraphPad V9. For the in vivo mouse models, a two-tailed, unpaired t test was utilized for comparing total bioluminescent flux. For survival analysis, a Kaplan–Meier plot and a Mantel–Cox log-rank test were utilized for comparisons of different cohorts. P values of <0.05 were assigned significance.Significant differences between values obtained in AML cells treated with different experimental conditions compared to untreated control cells were determined using the Student’s Utilizing OCI-AML3 cells that harbor heterozygous mutation in NPM1 and DNMT3A, along with homozygous NRAS mutation , we firs1Ep < 0.05), log2 fold-decline in the H3K27Ac peak-density at the enhancers/promoters as well as a log2 fold-decline in the H3K4Me3 peak-density at promoters of genes in OCI-AML3 cells. Rank ordering of super-enhancers (SEs) according to their H3K27Ac peak occupancy (ROSE plot) revealed a decline in H3K27Ac mark and associated SE activities of important AML-relevant oncogenes, including HOXA9, MEIS1, MYC, MYB, BCL2, MCL1, GFI1 and SPI1   in these cells. As shown in Fig. 5) Figs. . These fwt/wt), thus creating the isogenic OCI-AML2 NPM1mtA/wt cells with heterozygous mtNPM1  mice. Following engraftment, mice were treated via oral gavage with SNDX-5613 versus vehicle control. After 5-weeks of treatment, SNDX-5613-treated mice demonstrated significantly less AML burden   models expressing mtNPM1 and FLT3-ITD in NSG mice. AML cells in these models are transduced with Luciferase/GFP for bioluminescence imaging. Following tail-vein infusion and engraftment, cohorts of mice were treated with vehicle control or with panobinostat or adavosertib alone. The doses of each drug employed here were previously determined to be safe , 32. Com05) Fig. . Co-trea05) Fig. . These iThe presence of NPM1 mutation in AML stem/progenitor cells (LSCs) has been documented to be associated with but not shown to be causally linked to increased sensitivity to AML therapies, including treatment with menin inhibitor, the exportin-1 inhibitor selinexor, RARA or RXR agonists, or with the chemotherapeutic agents cytarabine and daunorubicin. To address this, we created via CRISPR-Cas9 isogeneic AML cell models with depletion (OCI-AML3) or knock-in of mtNPM1 (OCI-AML2). Utilizing these cellular models, we demonstrate that the presence of mtNPM1 is causally linked to the sensitivity of OCI-AML3 cells to loss of viability induced by menin inhibitor, selinexor, or cytarabine.cA and FLT3-ITD, double mutant NPM1cA and NRAS-G12D model developed AML with a longer latency and exhibited better prognosis [Compared to a previous report , our finrognosis . This marognosis .Results of preclinical studies and early clinical trials have demonstrated that treatment with MIs, e.g., SNDX-50469, revumenib, and ziftomenib, effectively induce cell differentiation and loss of viability in the preclinical models and induce complete remissions in patients with AML with mtNPM1 with or without FLT3 mutation but not in AML without mtNPM1. Our findings go further and show that KO of mtNPM1 desensitizes AML cells to treatment with MI, selinexor, and cytarabine. Similar to findings presented here that CRISPR-mediated KO of mtNPM1 repressed HOXA genes and MEIS1, in a recent report the targeted degradation of NPM1c or treatment with selinexor of AML cells, was shown to disrupt the binding of the residual and nuclear NPM1c to exportin-1, which led to repression of a small subset of normal MLL1-regulated genes, including HOXA genes and MEIS1, without affecting MLL1 occupancy at the target loci . AdditioGiven that in most elderly AML patients with mtNPM1, especially with FLT3 co-mutations, MI treatment is either ineffective, or after yielding initial clinical CR, AML relapse and poor clinical outcome ensues. Therefore, we adopted an approach to find agents that would be effective against AML with mtNPM1 regardless of sensitivity/resistance to MI treatment. Utilizing the RNA-Seq signature following NPM1c depletion in OCI-AML3 cells, our findings from LINCS1000-CMap analysis yielded several EMs, including HDAC inhibitors, e.g., THM-I-94, trichostatin-A, apicidin, dacinostat, vorinostat, and panobinostat, as well as adavosertib. Although we elected to fully interrogate the activity of panobinostat, the other FDA-approved pan-HDAC inhibitors, including vorinostat or belinostat, with biologic activity like panobinostat, would be as attractive for further evaluating as monotherapy against AML expressing NPM1c. Nonetheless, findings presented validate that monotherapy with panobinostat or adavosertib exerts in vitro lethal activity and in vivo efficacy against AML expressing NPM1c. Previous reports have documented that, among its diverse mechanisms of activity, as a pan-HDAC inhibitor panobinostat transcriptionally perturbs large numbers of gene-expressions beyond what would be regulated by menin-MLL1 in AML cells, including repression of pro-growth and pro-survival gene-expressions . This woRecent reports have highlighted the promising preclinical and clinical anti-AML efficacy of other novel agents and their combinations in AML expressing NPM1c . These iSupplemental Figure LegendsSupplemental Materials and Methods


The analysis showed that the inhibition and retardation effects occurred when the Ca2+/OD loading rate exceeded 8.46 M. The critical value was equal to the proportional constant for obtaining carbonate precipitation rate (CPR) from OD. Due to this, the blending design of materials became possible, with no risk of inhibition. In conclusion, the inhibition and retardation of the MICP process are governed by the Ca2+ load and the linear standard line (LSL), which may be attributed to the capacity or tolerance of viable cells, i.e., CPR/OD = 8.46 M or Ca2+/OD = 8.46 M.The estimation of optical density (OD) with viable cells is challenging for engineering purposes. In this study, the OD conversion based on previous study was used. The inhibited and retarded behavior of the microbially induced carbonate precipitation (MICP) process was examined. The experimental results showed that high Ca The 2NH2+ becomes a dominant factor for the CaCO3 precipitation rate. However, this type of reaction is not characterized by the controlled type of biomineralization (Reaction (9)), but the induced type [Accordingly, the concentration of Caced type .Ca2+ + 2+. The interface between the cells and electrolyte solution forms the diffuse double layer [2 and NH3 can be inhibited or retarded by the diffuse double layer.The surface of the cell is negatively charged and surrounded by cations, i.e., Cale layer ,29. It lle layer . For exa2+ overload effects. The experimental elapsed time was 24 h [2+ load.was 24 h . It is aOne of the objectives in this study is to understand the condition of the inhibition and retardation on the MICP process. It is noted that the analyses due to the OD conversion may be a new aspect for the MICP study.It is important to note that in bac is the thawed volume of concentrated bacteria required to prepare the OD* solution, Cf is the concentration factor due to centrifugation and VBC is the volume of the BC solution.In the previous studies, to prepare the assumed OD* of the biocement solution, BCS, the following relationship was used for various states of the bacteria stored in a freezer:2+ on bacteria is experimentally studied to establish the design for biocement technology.Thus, the optimization of the blending of materials is important to avoid the failure of engineering design, although this has not been established yet. In addition, it is also important from the economic and environmental point of view. In this study, the inhibition mechanism by CaS. pasteurii, which is often used by other researchers [3. A 100 L of culture liquid was continuously centrifuged at 4 °C. The centrifugal concentration factors are usually 250 to 300. The centrifuged bacteria are quickly frozen and stored in a freezer at −80 °C until utilized.The bacteria used in this study were isolated from the alluvial soils in Japan in 2008, which were named as NO-A10, and the microbiological aspects have been described in the literature where the nucleotide sequence of NO-A10 showed 93% similarity to earchers . A batch2+ on bacteria and CPR. The maximum concentration of Ca2+ used was 1.0 M and the 1.1 M urea was used. A 20 mM ammonia buffer solution was used for the biocement solution consisting of the reaction solution and bacteria. The concentration of bacteria was represented by the viable OD value, which was converted from the tentative OD*, as described earlier.The reactive agents used were urea, calcium chloride, distilled water and ammonia buffer. These agents are those being used for MICP techniques due to the ureolytic bacteria . The bacThe CPR measured by using the apparent OD, i.e., OD* is compared with the CPR–OD relationship . The con2+. Various combinations are needed for different methods of preparation. The preparation method for each case is briefly described in the corresponding section.Test biocement solutions were prepared by a combination of basic OD* and Ca3. The mass of bacteria was measured with an electronic balance. Note that the concentrated bacteria were diluted by about 1/8000 for preparation of the undiluted solution, in which the weighed bacteria are poured into the mixed solution of other agents or the OD* solution is prepared for mixing with other solution. In this study, the preparation methods are divided into the following three types, i.e., constant Ca2+ and various OD*, various Ca2+ and constant OD*, and constant Ca2+/OD ratio.The OD* value for the undiluted solution was usually given, or estimated by CPR and Rcv, if known. The volume of bacteria required was calculated by Equation (10). As the mass of bacteria is easier to manage than volume, the volume of bacteria was converted to mass using the density of the concentrated bacteria, 1.07 g/cmThe experimental results are analyzed using OD, which is converted from OD*. The OD-conversion is described as follows. The OD* values used are tentative, which neglect the effects of unknown factors on the Rcv value such as drainage of bacteria in the supernatant during centrifuging, the effects on Rcv by adding glycerin to concentrated bacteria and unexpected temperature changes. In addition, the ODi and Cf values are not constant, but vary. In this study, both are fixed as 4.0 and 250, respectively, which are approximate averages of the actual data. The OD conversion enables the solving of the problem previously mentioned because OD is expressed by “Rcv OD*”, which can be converted from the one measured CPR for a given OD* at an elapsed time of 24 h.The standard CPR–OD, Equation (6) is expressed by the following quadratic equation:The quadratic formula is as follows:Taking the maximum CPR for three evaluations, each Rcv was determined by the following:2+ were prepared and the various OD* values were adjusted by calculation of Vbac in Equation (10). Twelve 10 mL aliquots of different BC solutions were prepared to investigate CPR as a function of OD and time.Reactive solutions of 0.5 M Ca3 adsorbed in the test tubes and filtered on the paper filters [The time dependency of CPR was obtained in terms of low OD, as shown in  filters . Thus, t3 is higher with a larger OD value. This trend is common [2+ used, it means that the OD value used was sufficient. As the carbonate crystals induced sometimes contain H2O, the CPR induced becomes a little greater than the Ca2+ used [2+/OD is very high. The effects of the high Ca2+/OD cannot be neglected.s common . If the a2+ used ,32. When2+ loading line. As the bacterial cell becomes the nuclei of carbonate precipitation [3 during the MICP process.The CPR values with OD and time are shown in pitation ,34,35,36pitation  summarizAs the urease enzyme of bacteria is an embedded type, the products of urease activity, i.e., ammonia and carbon dioxides, are discharged outside of the cell, where calcium ions are abundant . It is p2+/OD loading rate. The linear standard line (LSL) provides the maximum CPR per OD. In other words, the LSL is the limited capacity of a bacterial cell. The existence of LSL means limit theory of ureolytic bacteria in MICP. Therefore, the Ca2+/OD ratio influences the CPR. The software curve fitting would not give the correct curves, particularly between an OD of 0 to 0.04. For example, the CPR at an OD of 0.01 and 0.02 may be lower than 2+/OD value increases with the decreasing of the OD value, as indicated at the top of 2+/OD of 8.3 M and 12.5 M because the average capacity of bacteria cells is 8.46 M. The higher the Ca2+/OD is, the more damaged the cells are. The inhibition and retardation in the MICP process may be dependent on the degree of damaged cells.Thus, the OD-CPR relationships abruptly changed between a Ca2+/OD value is the highest at the beginning. On the other hand, if the reaction occurs at the beginning, it continues until Ca2+ is consumed, as the Ca2+ concentration necessarily decreases according to the increased CaCO3.If inhibition or retardation occurs, it occurs at the beginning of the reaction, as the Ca2+/OD ratio decreases, which means less possibility of inhibition and retardation. On the other hand, increasing the Ca2+ concentration may not necessarily increase CPR. It increases the risk of inhibition. Note that retardation is not necessarily a risk. The reaction rate is a function of OD, i.e., representative of viable cells. Strictly speaking, the rate is a function of OD, Ca2+, pH, Ca2+/OD and temperature. The period of 24 h was used to measure CPR, for Rcv and CPR tests, which is defined as 24 h OD-CPR relationship:The increasing bacteria in the BC solution means that the CaThe time to reach a given CPR for different OD is more important than how much the CPR will be over time.2+.Similar test series were performed using an initial Ca ion concentration of 1.0 M. The OD values used were as low as the test series of 0.5 M Ca2+ load, as shown in 2+/OD method can decrease Ca2+ according to the decreased OD. Therefore, the risk of inhibition can be obtained by this method.The results showed a clear inhibition due to 1.0 M Ca2+/OD loading rate is twice that for a 0.5 M loading rate at the same OD values. It may be the reason that inhibition occurred. The details are discussed later.The test results show that the CPRs obtained are 0.1 M for a range of OD from 0.01 to 0.06 and at an elapsed time from 5 h to 213 h. This means that the precipitation stopped at an elapsed time less than 5 h and a concentration of about 0.1 M. The 1.0 M Ca2+ loads were examined in the previous sections. Therefore, when a low Ca2+ load is examined, it is considered that the whole picture will be revealed. Then, the effects of a 0.3 M Ca2+ load was examined using bacteria with Rcv = 0.25. The test solutions were prepared by five levels of OD* concentration. The test solution was prepared and CPRs obtained are presented in The 0.5 M and 1.0 M Ca2+ used. Next, at a low range of OD, the CPR was controlled by bacteria activity, i.e., OD control in contrast to Ca control. The former means that theoretically the products followed stoichiometry. On the other hand, the latter did not follow the chemical equation, but showed a reaction proportional to the viable cell number, which is presented by the linear standard line, LSL.The results obtained are a little deviated as expected because of the different temperature, as shown in On the deviation of data, further study remains, which may be the effect of temperature. As shown in As the temperature affects the precipitation rate CPR, the 24 h CPR for the Rcv test is a little low. For the same reason, the results of 0.3 M CPRs were a little higher than those expected. Particularly, the 72 h CPR test was cured at 28 °C for a long time.2+/OD loading rate were used for Rcv and CPR tests [2+. The urea concentration was 1.1 M. Therefore, the blend was sufficient to complete the reaction. In fact, the OD conversion shows an excellent fit to Equation (5).Test solutions of constant CaPR tests . The tes2+ and increasing CaCO3 which was confirmed experimentally, as shown in Similar OD–CPR relationships to 3 yield to be induced cannot be beyond the LSL. The competitive inhibition is common in enzyme reaction; however, there is no proof of this. The inhibition effects increase with the proceeding of the reaction. Therefore, this case may indicate product inhibition.In 2+/OD is lower than 8.46 M, all CPR measurements must show the full MICP behavior, which must accord with the standard curve, Equation (5), which is obtained by the OD conversion. Then, all results were obtained at an elapsed time of 24 h, the OD CPR relationship is fitted by the unique 24 h standard curve, as shown in Therefore, when the initial Ca2+/OD loading rate and will continue until the CPR reaches the LSL because the Ca2+ load will decrease due to the decrease in Ca2+ by producing CaCO3. Accordingly, the highest loading of Ca2+ is 1.0 M for the example in The different behavior from 2+ and OD values, i.e., 1.0 M/OD. It is possible that if the loading rate is less than 8.46 M, no inhibition may occur. In fact, the loading rate Ca2+/OD was 11.1. However, the result was not inhibition, but a retarded reaction. By the reaction for 24 h, MICP was 40%. It is expected that the reaction will be complete within 48 h because of the lower loading rate. Note that the retarded point remained as a retarded point after the OD-conversion. Accordingly, when the measurements cannot be fitted by Equation (5) after the OD conversion, it means that the result(s) was retarded or inhibited.Whether or not the inhibition occurs depends on the Ca2+/OD loading rate was evaluated by the OD conversion from the OD*–CPR relationships, which was presented in the previous study [2+ load, and another is the inhibited or retarded MICP behavior, which occurred at a 1.0 M Ca2+ load. It was understood that at a 1.0 M Ca2+ load, the greater the Ca2+/OD or Ca2+/OD, the stronger the inhibition and/or retardation are. Thus, the inhibition and retarded behavior are not only influenced by Ca2+, but also OD in terms of Ca2+/OD. The details are discussed later. However, constant Ca2+/OD load rates mean the increase in Ca2+ load for increasing OD values.The influence on the MICP behavior due to the increase in the initial Caus study . The res2+/OD values have been used for the Rcv tests because CPR can easily be obtained without inhibition of Ca2+. In those cases, the CPR–OD* relation was well expressed by Equation (6). The Rcv tests can conveniently be used for determining the optimum blend of the biocement solution.In fact, constant low Ca2+ concentration, which is the Ca2+ loading. In addition, the Ca2+/OD loading rate also affects the MICP behavior. The inhibition and retardation for the reaction occur at the beginning because the initial Ca2+ load is the highest. For discussion, the OD* axis of 2+/OD*. Therefore, the average Rcv value was used for the conversion. The red results are the converted values of the peak points of CPR for different Ca2+/OD* values. It is interesting that these results are on the 24 h SC, as shown in It was found that uncommon CPR performances such as inhibition and retardation were influenced by the initial Ca2+ loads used in this study are illustrated in 2+/OD increases, the inhibition zone expands to the left, which accords with The initial Ca2+ seems to be sufficient to cause inhibition. An OD of 0.118, shown by the red broken line, is the limit where the bacteria can induce 1.0 M CPR. The OD value is defined by the linear standard OD–CPR relationship, OD = 1.0 M/8.46 M. Usually, if a half of the full CPR, i.e., 0.5 M is achieved, the reaction can be completed within 48 h. This is because Ca2+ concentration decreases with time and the overload decreases.From 2+ loads is due to the effects of intensity of the initial Ca2+ load and the initial Ca2+/OD loading rate. A load of 1.0 M at low OD showed the clear inhibition, but not for a 0.5 M load. However, the retarded behavior was observed in terms of OD values under 0.5 M Ca2+.The different behavior between the 0.5 M and 1.0 M Ca2+/OD loading rates under the various Ca2+ loadings. In 2+/OD = 8.46, which is possibly the tolerance of the biomass. The figure indicates that the higher the initial Ca2+ concentration, the OD required to resist the loading effect should be higher. In addition, the lower the OD value, the greater the Ca2+/OD loading rate is.2+/OD value. In fact, the bacteria at an OD of 0.04 was subjected to a Ca2+/OD of 12.5 M, which exceeded 8.46 M, as a critical standard value  is the designed Ca2+ (M). Equation (14) has been experimentally confirmed.The example for the application of this study is given by the blending of BCS for liquefaction measures. Simply, the following relationship is available to determine the OD value:a is the proportional constant (kPa) depending on the soil type, and C should be lower than five percent. The relation between C and CPR is presented by the following:3) of soil particles. Note that Equations (15) and (16) are applied for marine sediments [2+ loading is minimized, and economic design is achieved. The flow chart concerning Equations (14) to (16) is shown in The design CPR is determined by the design carbonate content, C (%), in which the design strength UCS is as follows:ediments . Saturat3 calculated from injected Ca2+ and ultimate carbonate content is of importance and should be studied. The inhibition of carbonate precipitation causes the imbalance of masses. As was discussed by Lai et al. [2+ concentration determines the CPR in soils. There are unknown factors which govern MICP in soils, which may include the existence of LSL, the segregation of bacteria, Ca2+ load and inhibition.There is no doubt that the soil strength can be developed by carbonate precipitation. The mass balance between the masses of CaCOi et al. , there i2+ on MICP process. This study dealt with the basic interaction properties and behavior for ureolytic bacteria and Ca2+ in a urea solution. This manuscript consisted of three main themes. Firstly, the standard OD-CPR relationship was presented, which provides the OD conversion method from aged unknown bacteria, based on previous study. Secondly, OD-CPR relationships under four types of Ca2+ loads and constant Ca2+/OD loading rates were experimentally examined. Thirdly, the inhibition and retardation in the MICP process were investigated by the OD conversion method and examined to enable CPR prediction.There has been little research on the inhibition of CaThe main highlights in this study are as follows.2+/OD loading rate and high initial Ca2+ load possibly cause the inhibition of MICP. The initial condition to avoid the inhibition is given by Ca2+ < 8.46 OD or OD > Ca2+/8.46. Thus, the higher the Ca2+, the higher OD is required to avoid inhibition and/or retardation. However, in this study, the initial condition Ca2+ ≦ 1.0 M should be added because no experimental data for Ca2+ is higher than 1.0 M. The optimum blending condition is given by Ca2+/OD = 8.46.High initial CaConsidering OD is a function of the viable number of cells, the condition to avoid inhibition is given by the function of viable cell number. In other words, the condition given is due to the ability of the cell or the limit or capacity of urease activity.The retardation should not be avoided because the slow reaction usually contributes to a higher strength of soils unless a shorter time frame is required. If the CPR at 24 h is about half of the full precipitation, the ultimate precipitation usually reaches full precipitation. The retardation condition is included in avoiding inhibition.The OD conversion is enabled to compare the experimental data obtained using aged bacteria. This can give knowledge about MICP. In addition, there may be the possibility that by coordinate transformation, the OD*–CPR relationship obtained by using any species of bacteria can be converted to the standard OD–CPR relationship proposed in this study. Future work will examine the relationship between the unconfined compressive strength of biocement-treated soils and carbonate content, which can be estimated from CPR, and also the dry density of the soil as an application to civil engineering is the ultimate objective.


PML::RARA fusion. The occurrence and progression of AML are often accompanied by the emergence of gene fusions such as PML::RARA, CBFβ::MYH11, and RUNX1::RUNX1T1, among others. Gene fusions are the main molecular biological abnormalities in acute leukemia, and all fusion genes act as crucial oncogenic factors in leukemia. Herein, we report the first case of LYN::LINC01900 fusion transcript in AML with a promyelocytic phenotype and TP53 mutation. Further studies should address whether new protein products may result from this fusion, as well as the biological function of these new products in disease occurrence and progression.Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem/progenitor cells characterized by the abnormal proliferation of primitive and naive random cells in the bone marrow and peripheral blood. Acute promyelocytic leukemia (APL) is a type (AML-M3) of AML. Most patients with APL have the characteristic chromosomal translocation t, forming  Acute pcurrence . Initialcurrence , 4. Howecurrence , 6. Thuscurrence , 8.CBFB::MYH11, PML::RARA and RUNX1::RUNX1T1  is defined as a non-coding RNA molecule with a length exceeding 200 nucleotides that was once considered as transcriptional ‘junk’ DNA . HoweverLYN::LINC01900 fusion transcript, which was predicted to express no fusion protein in an AML patient with TP53 mutation. Moreover, clinical evidence showed that the bone marrow cells of the patient retained a promyelocytic phenotype; but there was no PML::RARA fusion. This indicated the potential function of this fusion in a new AML subtype. Hence, we hope that this case report on LYN::LINC01900 transcript will provide a new perspective for understanding the occurrence and progression of AML.Herein, we identified the 2A 76-year-old man was admitted to our hospital on October 12, 2022, with dizziness and fatigue for 6 months that had aggravated one month prior. The patient had a history of hypertension for over 20 years and type 2 diabetes mellitus and renal insufficiency for 8 and 2 years, respectively. After admission, a complete routine peripheral blood examination showed a white blood cell count of 1.26*10^9/L, red blood cell count of 1.44*10^12/L, monocyte cell count of 0.05*10^9/L, neutrophil cell count of 0.38*10^9/L, hemoglobin level of 51.0 g/L and platelet count of 32*10^9/L. The lymphocyte count was normal. The fibrinogen and D-dimer levels were 3.30 g/L  and 0.55 mg/L (ref. 0.00–0.55 μg/mL), respectively. The prothrombin and activated partial thromboplastin time were 12.5 s (ref. 10.0–14.0 s) and 27.2 s (ref. 25–31.3 s), respectively.To understand the cytological morphology, we observed the bone marrow smear of the patient and found that bone marrow cells proliferation and granulocyte proliferation were markedly high. The proportion of primitive granulocytes was 28%, and that of promyelocytes was as high as 56.5%. In addition, Wright Giemsa staining showed that erythrocyte and lymphocytic proliferation were inhibited  but showed a higher fluorescence signal , indicating gene amplification or +15,+15,+17,+17, which may originate from aberrant cells  of 81.4%. WT1 p.Thr277Ile was detected, and the WT1 mutation was suspected to be a germline mutation, based on the MAF of 67.10%. We used STAR software for bioinformatics analysis to predict fusion genes, with the reference genome being hg38. The LYN::LINC01900 transcript, which is a fusion of LYN (NM_001111097.3) exon 8 with LINC01900 (NC_000018.10) exon 2, was detected at a relative transcript expression level of 15.92% normalized to the reference gene ABL1 , +8, –9, –9, –11, –11, –12, +13, +14, +15, +15, +16, ?17, der (17)?i(17q), +19, +19, +20, +21, +22, +mar, inc[CP3]/45, X, –Y[5]/46, XY[2]. FISH showed no evidence of 2 from days 1 to 7), subcutaneous injection (s.c.), was recommended according to the 2022 European LeukemiaNet (ELN) recommendations for the diagnosis and management of AML in adults (2 from days 1 to 7), s.c., supplemented with antiemetic treatment at the same time from November 02, 2022. The therapy regimen was adopted after communicating with the patient and their family members. The patient discharged due to economic reasons on November 09, 2022. After discharged, we conducted a follow-up finding that the patient continued the therapy of venetoclax  as we suggested until November 22, 2022. Unfortunately, no further clinical outcomes were monitored because the patient died of COVID-19 infection on December 2022 , PO QD, and azacitidine , SH3, proline-rich hinge region (P), and tyrosine kinase domain , and myeloproliferative neoplasms (MPN) (GATAD2A::LYN transcript in B-cell acute lymphoblastic leukemia (B-ALL). However, no other relevant clinical data have been found in this paper  and complete molecular remission (CMR), respectively  –24. The is paper . Yano etin B-ALL , 27. Alsectively , but there were higher fluorescence signals indicating gene amplification. Studies have reported that APL-like AML is often accompanied by mutations in NPM1 and/or FLT3 mutation (NPM1 mutation was detected. We found a TP53 (NM_000546.5) p.Arg273Cys mutation with a MAF of 81.4%. To the best of our knowledge, this is the first case of APL-like AML with TP53 mutation, which is the most important tumor suppressor factor and is crucial for maintaining cellular genomic integrity. These findings may be used to identify new AML subtypes. Studies have indicated that AML with TP53 mutations is often accompanied by complex karyotypes, which is consistent with the findings in our case  values from RNA-sequencing of the present case appear to be higher than those of the AML patients with TP53 mutations, indicating that the LYN expression in this fusion may increase, which may cause the progression of diseases by regulating expression of other genes related to AML , forming the mutation , 34. Howour case , 36. Intour case . LYN::LId to AML Figure 4Although the new fusion transcript was confirmed using different methods, no significant differences in expression of the fusion partner genes were observed. Moreover, no fusion protein was predicted because there may have been no termination codons. Further studies should address whether new protein products may result from this fusion, as well as the biological function of these new products in disease occurrence and progression.4PML::RARA. Additionally, TP53 p.Arg273Cys was identified in the present case. Taken together, this patient was diagnosed with AML with TP53 mutation and a promyelocyte phenotype. A novel fusion LYN::LINC01900 was identified in this case. However, reciprocal fusion was not observed. Furthermore, the fusion was predicted to not express fusion proteins. Future studies should focus on the subgroups of fusions involving non-coding RNAs and their biological functions in hematopoietic malignancies. In conclusion, the LYN::LINC01900 fusion transcript from the patient is the first such fusion to be reported on a global scale, to our knowledge. Here, we share this case report a novel LYN::LINC01900 transcript with TP53 mutation in APL-like AML to provide a new perspective for understanding the molecular diversity of AML.Here, we report the case of a 76-year-old man with AML and a significantly higher proportion of promyelocytes. Wright Giemsa compound staining and peroxidase staining showed that erythrocyte and lymphocytic proliferation were inhibited, and the results of peroxidase staining of the bone marrow cell smear were positive. Immunophenotyping showed that the granulocyte population was positive for CD117, CD33, and CD38; weakly expressing CD13, CD4, CD64, and CD45; and negative for CD7, CD34, HLA-DR, CD10, CD20, CD19, CD14, CD2, CD15, CD11B, CD56, CD8, and CD3. Although a complex chromosomal karyotype was identified, FISH, qPCR, and RNA-targeted sequencing confirmed no evidence of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.The studies involving humans were approved by Department of Hematology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Jiangyin, Jiangsu, China. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CH: Data curation, Writing – review & editing. QD: Data curation, Writing – review & editing. RZ: Data curation, Writing – original draft, Writing – review & editing. HY: Data curation, Writing – review & editing. MW: Data curation, Writing – review & editing. KG: Data curation, Writing – review & editing. JY: Data curation, Writing – review & editing. WM: Data curation, Writing – review & editing. PC: Data curation, Methodology, Writing – review & editing. MX: Data curation, Methodology, Writing – review & editing.


The goal of academic mentoring relationships is to enable the mentee to identify/achieve professional advancement. Although mentors of clinician educators (CEs) must understand the criteria for successful career advancement, few have received formal CE mentor training.The National Research Mentoring Network convened an expert panel to develop a 90-minute module for training CE mentors. This module included individual development plans, case studies involving challenges for CE faculty, and examples of the broadened scope of scholarly activity. The workshop was delivered to 26 participants across four institutions and evaluated by a retrospective pre/post survey.very low, 4 = average, 7 = very high), participants rated the overall quality of their CE mentoring as slightly below average preworkshop (M = 3.9) and as above average postworkshop . Areas of greatest self-perceived change in skills on a 7-point scale  included setting clear expectations of the mentoring relationship , aligning mentor expectations with those of mentees , and helping mentees set career goals .Using a 7-point scale (1 = This module trains CE mentors using an interactive and collective problem-solving approach. Workshop participants better defined demonstrable markers for CE progression with potential to impact tailored guidance for mentees. Defining and demonstrating progress towards the mentee's academic objectives and explaining how the objectives contribute to the establishment of a unique identity or area of mastery are critical elements of success that mentors help their mentees navigate. For the purposes of this workshop, we define clinician educators (CEs) as faculty who are practicing clinicians and teaching either in the classroom or in the clinical context.1 A review of CE promotion criteria revealed that only 43% of U.S. medical schools had a defined CE promotion pathway and that less than 10% had fully adopted published evaluation/promotion criteria.2 This deficit in clear promotion criteria potentially leaves CEs being evaluated for academic advancement based on incomplete or unclear criteria and may leave mentors struggling to properly advise CEs.As academic medicine has evolved, so have metrics of success for promotion across the differing academic tracks. Senior mentors including division chiefs, department chairs, and promotions committee chairs are frequently from clinician scientist pathways, where mentoring programs, infrastructure, and metrics are better established than those for CEs.5 While the literature has provided frameworks6 and recommendations for mentorship skills,8 there are fewer opportunities for mentors to enhance their skills at mentoring CEs.9Mentorship is critical for career development in the clinician scientist pathway, but CEs in one study identified an education mentor less than 33% of the time, and the absence of a mentor was felt to have a negative career impact.Promotion as a CE is based on excellence in clinical practice, teaching, and service. Institution-specific markers of excellence necessary for advancement/promotion may include excellence in meeting expectations commensurate with academic rank, regional/national reputation based on opinions from senior faculty members, and productivity commensurate with the service portion  of one's effort. Frequently, service to the institution is expected and can be time consuming, but the amount of service work that is valued for advancement varies across institutions.10 While CEs may play an integral role as contributors to team science (discovery), more commonly their advancement is assessed in integration , application , and teaching. Teaching may occur in multiple settings, including classroom, online, and clinical sites, and is traditionally assessed by teaching evaluations. Learners range from students, residents, and fellows to faculty and nonacademic clinicians.Scholarship and dissemination are expected of a successful CE. We define scholarship broadly and more consistently with Boyer's model of scholarship including domains of discovery, integration, application, and teaching.MedEdPORTAL resources, invited talks, and conference workshops. Other forms of expected recognition include membership/leadership in regional/national academic societies or program review, certification/accreditation committees, and so on.CEs are frequently expected to demonstrate dissemination of their efforts as evidenced by published curricula, papers, 3 To address this need, in 2018 the National Research Mentoring Network (NRMN) convened an expert panel composed of this publication's authors to develop and pilot an interactive module for training mentors of CEs. The resulting module  was designed as a freestanding tool to train mentors of CEs using an interactive and collective problem-solving approach. It expands upon existing training for research mentors that includes aligning expectations, culturally aware mentoring, and self-efficacy.8Although mentors of CEs must understand the academic and professional criteria for successful career advancement, few have received formal training in mentoring CEs.clinician educator, clinical educator, and mentoring and restricted articles reviewed to the English language. We reviewed MedEdPORTAL content and noted resources that taught mentoring skills or were directed at specific training levels, for example, medical students or residents.11 Furthermore, those of us who had participated in NRMN workshop experiences cited frequent questions about mentoring CEs as a practice gap.The curriculum development team (the authors) obtained institutional review board approval through NRMN protocols 2019-0956, 2015-0871, and 2016-0458 and Center for the Improvement of Mentored Experiences in Research protocol 2017-1336. The authors were researchers and CEs with extensive CE mentoring experience. To develop workshop content, we searched PubMed using the terms 8 We developed content and integrated the CE Mentor Training Module into the structure of the Entering Mentoring curriculum, as well as developing brief didactic material on identifying institution-specific promotion criteria for CEs with large-group discussion .scussion  and a coscussion . The resWe served as facilitators at our four respective institutions. We delivered the workshop to 26 participants across these institutions as a stand-alone module, except in one case where the workshop complemented previously implemented mentorship educational sessions. We delivered the session once at each site during preexisting faculty development conference time, with four to 12 participants depending on the site. We used two facilitators per workshop. Workshop participants were mentors of early-career stage CEs. In addition to materials provided in the appendices, resources included a whiteboard/flip chart with markers for group discussion, note cards for participant commitments, and ability to display a PowerPoint presentation.After outlining institution-specific promotion criteria and general markers of success in the large group, each participant reviewed examples of individual development plans IDPs; . In pair12 to evaluate the workshop. We performed a matched t test with significance set at p < .01 for data collectively from all sites as the number of participants at each individual site was too small to determine a meaningful change. We calculated Cohen's effect size. Also, we collated the open comments in the evaluation and surveyed for themes.Nineteen of 26 participants completed a retrospective pre/post surveyOf the 19 respondents, 73% were assistant professors, 8% were associate professors, and 15% were at the instructor level; 4% self-identified as Black, 19% as Hispanic/Latinx, and 66% as White. Sixty-nine percent reported they were on the CE track, acknowledging that nomenclature for CE tracks varied institutionally. Furthermore, 73% of participants had never participated in research mentoring prior to this workshop.very low, 4 = average, 7 = very high), participants rated the overall quality of their mentoring of CEs as slightly below average preworkshop  and as above average postworkshop , t(24) = 1.3, p < .001. Cohen's effect size (d = 0.74) suggested moderately high impact. These findings supported our hypothesis that mentors lack formal CE-targeted mentor training in their career paths, a gap this curriculum begins to fill.We delivered the workshop across four geographically diverse institutions with consistent results among the 19 participants (73%) who responded to the postworkshop survey. On a 7-point Likert-type scale . Areas of greatest self-perceived change in skills included working with mentees to set clear expectations of the mentoring relationship , t(24) = 7.9, p < .001; aligning mentor/mentee expectations  t(24) = 7.3, p < .001; and helping mentees set career goals  t(24) = 3.9, p < .001. Cohen's effect (d = 0.96) indicated a large effect size. Perceived changes in all areas on the evaluation form were statistically significant at p < .001 except for balancing work and personal life, a topic that was not as explicitly covered in this brief module. All (100%) respondents found the training valuable, 90% would recommend the training to colleagues, and 92% reported planning to make changes to their mentoring.We asked participants about mentoring skills using a 7-point Likert-type scale (1 = I have a better idea of questions to ask of my mentors and a better idea how to encourage mentees to frame their growth. I simply would have loved to have had this available to me earlier in my career.Qualitative data demonstrated common themes, including mentors’ intention to use the IDPs to set concrete goals with mentees and the need to set clear expectations with mentees, supporting the quantitative data. The following was a typical open comment:Another sample comment was “The most helpful thing for me was the document which offered a description of the work that educators do, providing a guide to discuss mentee's goals in each category.” Unique comments included wanting facilitators to review institution-specific promotion criteria aloud and wishing the workshop covered grant writing. In the future, facilitators may choose to state abbreviated versions of their institution's promotion criteria, but our experience has proven that a thorough review of criteria merits an additional faculty development session devoted solely to the topic. Similarly, grant writing is a multifaceted topic that requires other forms of programming.The CE Mentor Training Module is a tool to train mentors of CEs using an interactive and collective problem-solving approach. It expands upon existing training for research mentors that includes aligning expectations, culturally aware mentoring, and self-efficacy. This workshop was created based on literature review and expert input and uses methods such as review of IDPs and case studies to enhance mentoring skills.Typically, mentoring others becomes an increasingly important activity as one advances in academic rank. While the majority of our participants were assistant professors, we did not distinguish between early versus late assistant professor stage. Importantly, at some institutions, due to their promotion criteria/pathways, some faculty remain at rank for extended periods and do mentor more early-career faculty. Regardless of time spent in rank, it was clear from the evaluation data that many mentors felt unprepared for their roles.We designed our training for any faculty member who mentors CEs. However, most of the participants were themselves on the CE or teaching pathway.13 The data support the generalizability of the content to a diverse population of faculty mentors. Furthermore, open comments and evaluation data support the idea that participants were better able to define markers of success for CEs, use an IDP, and access a toolbox of resources for mentoring CE faculty. It was more challenging to determine if mentors were truly better able to help mentees prioritize tasks based on the case discussion. Longitudinal follow-up with participants would be useful to better characterize changes in this domain.While there were multiple areas of growth noted, the greatest self-perceived changes in skills concerned articulating and aligning expectations, changes also observed in the randomized controlled study of the Entering Mentoring–based curriculum adapted for the mentors of clinical and translational researchers.Limitations to our results include the small number of participants. Despite our anticipating that most of the audience would be clinician scientists, most participants were themselves on the CE track. We may need to further tailor the workshop for an audience that is not familiar with the CE track/promotion criteria. Faculty participants in our cohort were early- to mid-career faculty, and results may vary for more-senior faculty. Whereas we observed significant gains in self-efficacy, the outcomes were self-reported, an inherent limitation. We did not investigate if mentor training improved mentee-reported quality of mentoring. Notably, no participants to date have been full professors, possibly limiting the generalizability of our results. Lack of participation may reflect the paucity of CEs who are full professors and our workshop recruitment strategy.We learned several lessons from the multi-institutional implementation of this module. First, communication/promotion about the session often dictates the participants who register for training. CE mentors do come from clinician scientist pathways, so it is important to develop promotional materials that are more inclusive. Second, users should consider whether the module should be delivered stand-alone versus as part of a larger program with companion modules covering topics such as mentoring across differences and other diversity/inclusion considerations. Lastly, a stand-alone module is attractive to mid-career CEs mentoring other earlier-career faculty. The shorter time commitment for this module may have generated even more interest among actual CEs due to their competing time constraints.Future iterations will target faculty participation at the associate and professor levels, particularly those with significant leadership roles, to ensure their understanding of CE mentoring needs. It would be helpful to pilot this workshop at institutions with larger numbers of senior CE faculty. Furthermore, we recommend the module be included in the portfolio of evidence-based mentorship education. We want to develop an extended workshop incorporating a section on empowering mentees to ask for resources needed for their career advancement. Through broader workshop dissemination, we want to investigate the longitudinal impact on participating mentors. Future work will help us understand who self-identifies as mentors of CEs and how to encourage more faculty to effectively mentor this crucial group. Finally, we are developing content for an additional module that addresses intersectional identities and belonging through the CE lens and what challenges faculty are navigating in their personal /professional spaces.CE Training Workshop.pptxFacilitator Guide.docxIndividual Development and Mentoring Plans.docxCase Studies.docxResource Guide.docxWorkshop Evaluation.docxAll appendices are peer reviewed as integral parts of the Original Publication.


A special problem in the immune therapy of AML is few neo-antigens compared with solid cancers because of a relatively low mutation frequency. Studies of CAR-T-, CAR-NK-adaptor CAR-T- and allogeneic NK-cells are progressing as are approaches using synthetic biology. Presently, there are no convincing data of efficacy of immune therapy in AML.Although safe and effective immune therapies have been developed in several cancers, this has not been so in acute myeloid leukaemia (AML). Studies of antibodies to CD33, CD123 and CLL-1 report with unconvincing efficacy and substantial adverse events. Lacking AML-specific target antigens, these approaches using non-specific antigen targets often cause unacceptable bone marrow toxicity and off-target adverse events. Studies of AML incidence in persons with immune deficiency indicate little if any immune surveillance against AML. In contrast, data studies of recipients of haematopoietic cell transplants support an effective allogeneic anti-AML effect associated with graft- Corynebacterium parvum. Some subjects received allogeneic or autologous leukaemia cells. We concluded immune therapy was ineffective in AML.Forty years ago, we reviewed the role of immune therapy in AML . We inteIn the past 10 years, immune therapy has been developed in B-cell and solid cancers •, 3, 4•.Lymphoid cancers and AML are different in several aspects. For example, only about 10 million lymphoid cells are produced daily compared with about 600 billion myeloid cells. Also, granulocytes and platelets survive only a few hours or days compared with lymphoid cells which live years. The consequence is a disruption of myelopoiesis is much more dangerous than a disruption of lymphopoiesis. Second, this is the different targetability of lymphoid compared with myeloid antigens. The target of immune therapy of lymphoid cancers is B-lineage antigens which are not cancer specific. Killing substantial numbers of normal B-cells is compatible with life, whereas this is not so for killing substantial numbers of myeloid cells. Consequently, immune therapy of AML is limited by the absence of AML-specific target antigens and potential damage to normal bone marrow function.Risks of lymphomas, melanoma and kidney cancer are substantially increased in lymphoma risk in persons with immune deficiency or iatrogenic suppression. In contrast, there is only a small increased risk of AML in these settings suggesting immune surveillance does not operate in AML Fig.  5].Fig..Fig.5].Fversus-host disease (GvHD) outcomes  leukemia-specific antigens. Whether this allogeneic graft-versus-leukemia effect (GvL) is distinct from GvHD is controversial . Aft. Aftversoversial .Fig. 2PrSeveral variables correlate with success of immune therapy including antigenicity, immunogenicity, accessibility, sensitivity to killing and collateral damage to normal cells. In AML, we are faced with the lack of a AML-specific target antigen(s). In solid cancers, efficacy checkpoint inhibitor antibodies correlate with mutation frequency Fig.  8]. How. How8]. Several immune therapies have been studied in AML including monoclonal antibodies, drug-, -toxin and radionuclide antibody conjugates, bi-specific antibodies and others Table  11, 12], 1211, 1Studies of cell therapies in AML include NK- and CAR-T- and CAR-NK-cells and cytokine-induced NK-cells (CIK) •, 14. ThCompared with immune therapy of B-cell lymphomas and several solid cancers, there has been little progress in immune therapy of AML outside the context of an allogeneic haematopoietic cell transplant. The predominant challenges are the lack of an AML-specific target antigen(s) and presently unavoidable collateral damage to normal bone marrow function. Whether these challenges can be overcome is unknown.