Using novel cell patterning approaches, highly organised, regular grid networks of astrocytes on chip, to single-cell fidelity are constructed, permitting a stellate-like in vitro network model to be realised. By stimulating the central cell with a single UV nanosecond laser pulse, the initiation/propagation pathways of stellate-like networks are re-explored. The authors investigate the mechanisms of intercellular Ca2+ communication and discover that stellate-like networks of adult human astrocytes in vitro actually exploit extracellular ATP release as their dominant propagation pathway to cells in the network locally; being observed even down to the nearest neighbour and next nearest neighbouring cells—contrary to the reported gap junction. This discovery has significant ramifications to many neurological conditions such as epilepsy, stroke and aggressive astrocytomas where gap junctions can be targeted. In cases where such gap junction targeting has failed, this new finding suggests that these conditions should be re-visited and the ATP transmission pathway targeted instead.The ‘Astrocyte Network’ and the understanding of its communication has been posed as a new grand challenge to be investigated by contemporary science. However, communication studies in astrocyte networks have investigated traditional petri-dish  By applying pharmacological agents to block the common initiation and propagation pathways in astrocytes, we then proceed to determine the exact initiation and propagation pathways that are repeatedly engaged in astrocyte network Ca2+ communication in the inner and outer regions of the network.Over the past 10 years, scientific research has revealed that the astrocyte –4. Such rk level . Such exrk level , 15, strrk level , focal crk level  and diffrk level . Our grork level , which hrk level , expressrk level , validatrk level  and provrk level , 23, witrk level . Our groits kind . In addios et al . In thisos et al  with ouros et al  organiseCurrent research has only reported the initiation and propagation pathways for clusters or sub-clusters of unorganized monolayers of astrocytes of various species under electrical, mechanical and laser stimulation in networks as follows.2+ initiation was demonstrated to be induced either by extracellular Ca2+ or intracellular Ca2+ stores [et al. demonstrated that Ca2+ waves of primary rat astrocytes induced either by mechanical stimulation was dependent on the presence of external Ca2+ [et al. targeted femtosecond laser pulses to the cell membranes of purified rat astrocytes and hypothesised that the pore allowed for a small influx of extracellular Ca2+ from the extracellular fluid resulting in a localized increase in intracellular Ca2+ [et al. observed that ATP elicited Ca2+ elevations in NT2 derived astrocytes were inhibited by the intracellular store depletory thapsigargin, suggesting that intracellular stores were the main Ca2+ stores for signalling in astrocytes [et al. developed a nanosecond laser stimulation technique for elevating Ca2+ increases in NT2 derived astrocytes and observed that stimulation was still possible in absence of extracellular Ca2+ and thus, hypothesized that localized ablation of the ER was the primary factor in stimulating Ca2+ response by nanosecond lasers [et al. observed that both omitting extracellular Ca2+ and inhibiting Ca2+ stores did not affect Ca2+ increases in primary rat astrocytes by mechanical stimulation [et al. demonstrated that mechanical stimulation may induce Ca2+ release from thapsigargin-insensitive Ca2+ stores.The initiation pathway: The mechanism for intracellular Ca+ stores –29. Venalar Ca2+ . Howevertrocytes . Furtherd lasers . Most remulation . Fujii e2+ propagation via transmission of Ca2+ and inositol 1,4,5-trisphosphate (IP3) ions through gap junctions has become intensively studied [2+ waves can propagate between physically separated cells, indicating that purinergic signalling may also be involved in Ca2+ propagation, presumably by secreted extracellular adenosine triphosphate (ATP) [2+ from Ca2+ stores. Guthrie et al. demonstrated that purinergic antagonists blocked propagation of electrically evoked Ca2+ waves, indicating that extracellular ATP was required for normal Ca2+ wave propagation in primary rat astrocytes [et al. demonstrated that Ca2+ waves of cortical murine astrocytes by mechanical stimulation could propagate to neighbouring astrocytes not only within the same confluent domain but also to neighbouring non-connected astrocyte areas [et al. investigated how to use femtosecond lasers to elevate Ca2+ increases in rat astrocytes and demonstrated that femtosecond laser-induced Ca2+ waves were predominantly mediated by the extracellular messenger ATP in partial collaboration with gap junctions [et al. demonstrated that persistent Ca2+ increases propagated rapidly via gap junctions in the proximal region and transient Ca2+ increases propagated slowly via extracellular diffusion of ATP in the distal region in primary rat astrocytes [et al. investigated the functional properties of NT2 derived neurons and astrocytes and demonstrated that mechanically-induced Ca2+ waves of NT2 derived astrocytes were gap junction and purinergic signalling dependent [2+ responses in the presence of both gap junction blocker and purinergic receptor antagonist.The propagation pathways: Since astrocytes main physical connection is via intercellular gap junctions , 32, the studied –35. Othete (ATP) –38. ATP te areas . Zhao etunctions . Most retrocytes . In addiependent . This wain vitro and to simplify the complexity of communication in vitro cultures, it is important to control where the cells contact with one another. The ability to culture astrocytes in a controlled network configuration with prescribed cell separation distances and connectivity would give insight into the response of astrocyte networks, in a scale that is more physiologically relevant than previously possible. Previous in vitro semi-organized network platforms demonstrated by Jordan et al. patterned the astrocytes in a trench grid network [2 substrates [2+ releases in hNT astrocytes, to investigate the underlying initiation and propagation mechanisms of intracellular Ca2+ waves in organized regular grid astrocytic networks. Specifically, we focus on (1) intracellular Ca2+ storage, (2) influx of extracellular Ca2+, (3) gap junction communication, and (4) ATP pathway contributions in astrocytic networks.Previous studies in astrocytes have been limited to clusters or sub-clusters of unorganized astrocyte monolayers , 38–40.  network . Most rebstrates , 41. Theet al., where hNT astrocytes were patterned on the biomaterial, parylene-C, to high resolution at the single-cell level on nodes in a regular grid array  and 98% sulphuric acid (H2SO4) for 10–15 min and rinsed in Milli-Q water. After the piranha acid wash, chips were immediately placed in a 24-well plate and sterilized in 2% Penicillin-Streptomycin-Glutamine (PSG) solution for 30–60 min at room temperature. Finally, the chips were incubated in fetal bovine serum (FBS) for 3 h at 37°C and 5% CO2 to activate the patterns.Photolithography was used to construct the parylene-C grid networks on parylene-C/SiOes as in , 42. A p2 chips.hNT astrocytes were differentiated from the NTera2/D1 (NT2/hNT) cell line over 9 weeks according to the protocols , 22, 29 2 chips at a density of 50 cells/mm2 [2 for 2 days to allow the astrocytes to migrate onto the patterns  connected to an Olympus BX53 upright microscope and controlled by Andor iQ software. The microscope was set up to operate in reflection mode with 10× water immersion objective. The laser system consisted of a pulsed nitrogen laser producing single 200 μJ, 3 ns duration pulses as determined optimal to induce Catrocytes . The wavtrocytes  by a dye2. For intracellular Ca2+ measurements, cells were stained with a live 1 μM Fluo-4 AM dye and Hoechst 33342 (Blue) for 40–60 min at room temperature. Cells were rinsed in FluoroBrite with 1% FBS twice to improve signal-to-noise ratio of fluorophores and preserve cell health.hNT astrocytes were stained with a live 1 μM Cell Tracker Green Dye (CMFDA) and Hoechst 33342 (Blue) at two drops per mL in 5% FBS/DMEM: F12 for 30 min at 37°C and 5% COVarious pharmacological blocking agents were administered to the hNT astrocytes before the experiments in order to identify the initiation and propagation pathways mechanisms of hNT astrocytes. The initiation and propagation pathways were blocked as follows:ER Ca2+store: Thapsigargin (Tg) has been shown to deplete the Ca2+ store inside the endoplasmic reticulum (ER) of hNT astrocytes manufactured by Hill et al. [l et al. . Thus, al et al.  the astrExtracellular Ca2+: Ca2+free DMEM and Ca2+free HBSS were demonstrated to deplete extracellular Ca2+ in hNT astrocytes [2+free DMEM/HBSS was used to replace the regular medium after the Fluo-4 AM loading. This treatment was used to remove the extracellular Ca2+ sources immediately before the laser stimulation experiments.trocytes . Thus, CGap junction: Carbenoxolone (CBX) has been demonstrated to block the gap junction pathways in hNT astrocytes [trocytes . Thus, tExtracellular messenger ATP: Suramin has been reported to be a non-selective antagonist of purinergic receptors and used in primary rat astrocytes [in vitro hNT astrocytes.trocytes , 30. We 0).Fluo-4 fluorescence images were captured by an Andor Clara-E interline CCD camera that was controlled by the Andor iQ software package. The Fluo-4 fluorescence images were imported into the image processing software ImageJ ©. The region of interest (ROI) of each cell was created manually by the ImageJ’s ROI management toolbox and the fluorescence of each ROI during every frame was measured automatically. The fluorescence data was then processed in MATLAB ©  to determine the change in fluorescence over the baseline fluorescence prior to stimulation  where 90 μm was the maximum field of view using the laser ablation system which permitted a (5 × 5) grid network. 2+ propagation from the central cell through to adjacent cells (the nearest neighbours NI) and the outer cells (the next nearest neighbours NII). Additionally, inter-node distances greater than 90 μm were associated with network magnifications of less than 5 × 5, which would not be sufficient to examine the Ca2+ propagation in the networks of at least the nearest neighbours and next nearest neighbours. Thus, networks with a 65-μm node size, 5-μm interconnecting line width, and a node-distances of 75 or 90 μm  , we showor 90 μm  were useet al. [2+ propagation from the central cell to the outer neighbouring cells in 5×5 network for both 75 and 90μm inter-node distances [The neighbouring hNT astrocytes in the organised networks were classified into two regions according to their distance from the stimulated central cell as follows: cells in the inner neighbourhood (N I)–namely the nearest neighbours directly adjacent to the surrounding the central cell, and cells in the outer neighbourhood (N II)—namely the next nearest neighbours directly adjacent to the inner neighbourhood, as shown in et al.  deterministances , shown F2+ signals propagated from the stimulated astrocyte to the adjacent cells  in the patterned network and then to the outer cells  in the patterned network. 2+ magnitude of both stimulated cell and neighbouring cells, and cell response time interval for both 75 μm and 90 μm node distances networks, respectively. Ca2+ magnitude refers to the maximum value of the fluorescence change (ΔF/F0) in Ca2+ traces.In addition, we observed that when the astrocyte in the centre of the network was stimulated by a single laser pulse of 12 μJ, the intracellular Ca2+ responses of nearest neighbours (in the first neighbourhood) was reduced by a factor of 2.13 from the central stimulated cell and the Ca2+ responses of next nearest neighbours (in the second neighbourhood) was reduced by a factor of 3.41 from the central stimulated cell. It was found that there was no significant difference in the Ca2+ magnitude between 75 μm and 90 μm node distances networks in either the N I or N II neighbourhoods. Furthermore, we observed that the mean response time interval of cells in N I was 17.3±16.25 s and the mean response time interval of cells in N II was 36.4±28.2 s. No significant difference of response time was observed between 75 μm and 90 μm node distances networks in either N I or N II.It was found in in vitro culture of hNT astrocytes as compared to organised networks of hNT astrocytes. We first stained the Cx43 gap junction protein to visually observe the amount of gap junctions that exist in standard non-patterned 2+ intensity of the laser-stimulated cell increased dramatically in response to the stimulation. Further, intracellular propagation of Ca2+ to the outer neighbourhoods of the network was also observed [2+ concentration decreased back to the baseline level. It was observed that the propagation of the intercellular Ca2+ is not equal in every direction, which was different from the mathematical models demonstrated by Lallotuette et al. [2+ responses of the stimulated cell, the astrocytes in the neighbourhood N I and N II of the organised grid network in control group. Preliminary observations of simple bulk Ca2+ flow in a grid network of hNT astrocytes stimulated by a UV laser pulse was reported by Li et al. [In order to analyse the results of pharmacological blocking agents, cells that were not treated with any pharmacological agents were used first as the control group. The central astrocyte in the network was stimulated using a single 12 μJ laser pulse. The Caobserved . Finallye et al. . Fig 5 si et al.  at confe2+ initiation mechanism of the central stimulated cell for the hNT astrocyte. The mechanisms underlying the Ca2+ initiation was pharmacologically examined by treating cells with 1 μM Tg or Ca2+-free DMEM/HBSS. Tg inhibits Ca2+ storage in the ER, while the use of Ca2+-free DMEM/HBSS was used to inhibit Ca2+ influx from the medium.We then proceeded to investigate the CaIntercellular Ca2+of the ER blocked using Tg. In 2+ in the stimulated cell as no fluorescence was observed in the central laser stimulated cell. Therefore, no Ca2+ responses were observed in the NI and N II neighbourhoods.Extracellular Ca2+from media blocked using Ca2+-free DMEM/HBSS. In 2+ responses with the presence of Ca2+ free media. We observed that the stimulation was still possible in the absence of extracellular Ca2+, which were consistent with the results reported by Raos et al. [2+ responsive peak in the stimulated cell was observed immediately after the laser stimulation. Further, the Ca2+ peak magnitude of astrocytes in neighbourhoods NI and NII to be much lower than that observed in the stimulated cell.s et al. . A Ca2+ 2+ propagation occurs from the central stimulated cell to neighbouring cells. The mechanisms underlying the Ca2+ propagation during Ca2+ waves were pharmacologically examined by treating cells with 100 μM CBX and 100 μM suramin, to block the gap junction communication and the extracellular ATP pathway, respectively.Next, we investigated the mechanism by which CaGap junction communication blocked using CBX. In 2+ propagation still occurred in the absence of the gap junction pathway.Extracellular ATP pathway blocked using suramin.2+ responses of the stimulated cell and cells in neighbourhoods N I and N II when the extracellular ATP pathway was blocked by suramin. We observed the Ca2+ responses of astrocytes in neighbourhoods N I and N II to be severely inhibited due to the absence of the ATP pathway, with only one cell provided a heavily attenuated fluorescence in neighbourhood N I. No fluorescence was observed in N II.2+ initiation, the percentages of responsive stimulated central cells in the different pharmacological groups (N = 10 experiments) were first calculated, as shown in 2+ responses induced by the laser activation of the central astrocyte were abolished in the group treated with Tg (p<0.0001). It was found that in 8 out of 10 experiments (80%) of Ca2+-free group, the stimulated central cells responded to the stimulation. It was found that the Ca2+-free group did not have a significant difference compared to the control group (p = 0.556). We found that there was a 100% stimulation response in astrocytes treated with CBX, which implied that initiation did not require gap junctions. Further, there was a 90% stimulation response with the treatment of suramin. From this, it was determined that the CBX (p = 0.3306) and suramin (p = 0.99) groups were not significantly different to the control group.To investigate the mechanism of Ca2+ magnitudes of stimulated central cells in each pharmacological group were then calculated. 2+ magnitudes of stimulated central cells in different pharmacological groups. The peak Ca2+ magnitude of stimulated central cells in the control group was (ΔF/F0 = 18±1.836 × 10−3). The peak Ca2+ magnitude of the stimulated central cell in Tg group (0.3833 × 10−3) was significantly lower than the control group (p = 0.0002). It was observed that the Ca2+ magnitude of stimulated central cells in groups with the treatment of Ca2+-free DMEM/HBSS was 14.13±0.9514 × 10−3 (p = 0.091). In addition, the Ca2+ magnitude of the stimulated central cells in the groups that were treated with CBX and suramin were 16.88±1.574 × 10−3 and 15.72±2.067 × 10−3, respectively. These results were also not significantly different to the control group .The peak Ca2+ propagation, the percentages of responsive cells of N I and N II were separated and compared in different pharmacological groups as shown in 2+ responses. As we described above, there were no responsive cells in the N I and N II in the Tg group, given that there was no Ca2+ response in the stimulated cell. The percentages of responsiveness in the N I and N II in the Ca2+-free group were 54.17±15.1% (p = 0.5884) and 34.13±8.3% (p = 0.6017). However, these values were not significantly different to the control. It was observed that 50±7.9% of N I (p = 0.1449) and 30±13.1% of N II (p = 0.0326) were responsive in CBX group, respectively. It was found that only 27±9.4% cells in N I (p = 0.0003) and 7.3±7.3% cells in N II (p = 0.0002) responded in suramin group. This response was significantly lower than that recorded in the control group. In addition, we also observed that the percentage of responsive N II cells was significantly lower than that of N I cells.To investigate the mechanism of Ca2+ released from the Ca2+ store of the ER, blocked by Tg, is responsible for Ca2+ initiation in hNT astrocytes under the nanosecond laser stimulation, instead of the influx of extracellular Ca2+, and the ATP pathway, blocked by suramin, is responsible for Ca2+ propagation instead of the gap junctions in organised astrocyte networks.In conclusion, these results indicated that the Ca2+ concentration that spread from cell to cell provide a mechanism for astrocytes to coordinate many cellular activities. In this study, we investigated the Ca2+ initiation mechanisms and Ca2+ propagation mechanism from stimulated cell to neighbouring cells by UV nanosecond laser stimulation, in organised hNT astrocyte grid networks for the first time.Increases in Ca2+ initiation in hNT astrocytes, we examined the effect of removing the Ca2+ in the extracellular media (by using a Ca2+-free media) in order to halt Ca2+ influx intercellularly, which Raos et al. [in vitro cultures under UV nanosecond laser stimulation. And we employed Thapsigargin (Tg) to deplete the intercellular ER Ca2+ store, which Hill et.al [in vitro cultures under ATP stimulation. These results demonstrated that Ca2+ increases in hNT astrocytes stimulated by a UV nanosecond laser pulse were mainly dependent on intracellular ER Ca2+ storage instead of the extracellular Ca2+. This result was found to be similar to Hill et al.’s work [in vitro cultures albeit under ATP stimulation.In order to determine which pathway was responsible for Cas et al.  used forll et.al  used on .’s work  on hNT a2+ responses was by Zhao et al. [et al. hypothesised that the pore allowed for the influx of extracellular Ca2+ into the cytoplasm resulting in a localised increase in intracellular Ca2+, which is then propagated throughout the stimulated cell by calcium induced calcium release (CICR). However, absorption dynamics are different between femtosecond and nanosecond laser ablation. Nanosecond laser pulses are less confined in the z-axis directions. This implies that a single nanosecond laser pulse could cause both the stimulation of the cell membrane and stimulation of the ER. Most recently, Raos et al. [2+ and hypothesised that localised stimulation of the ER could be a primary factor in producing a Ca2+ response. Therefore, in this study, we provided further evidence to support the hypothesis of Raos et al. [2+ increases stimulated by nanosecond UV laser, resulted in the mobilisation of Ca2+ from the intracellular Ca2+ stores (ER) causing in a localised increase in Ca2+ in the cytoplasm.The closest laser stimulation regime to evoke Cao et al.  who targs et al.  demonstrs et al.  that the2+ wave propagation was based on inositol trisphosphate (IP3) diffusion through gap junction pores, which triggers Ca2+ release from IP3-sensitive Ca2+ stores in neighbouring cells. However, recent studies have demonstrated that ATP was an important extracellular messenger in propagating Ca2+ waves in many cell types. Previous researchers have investigated the role of ATP in astrocyte Ca2+ signalling. Takano et al. and Lee et al. [2+ waves in purified cortical primary astrocytes from mice could propagate to neighbouring unconnected astrocytes, which suggested that the signalling pathway was extracellular diffusion of the signalling molecule. After adding phosphate−6-azophenyl-2‘,4‘-disulfonate (PPADS), a purinergic receptor antagonist, Takano et al. [2+ wave propagation did not propagate to neighbouring non-connected astrocyte areas, which suggested that intercellular Ca2+ wave propagation was mediated by ATP. Zhao et al. [2+ waves of primary rat astrocytes were significantly blocked by ATP receptor antagonists, but were not blocked by gap junction blockers. In this work, we demonstrated that the propagation of Ca2+ wave was significantly impeded by the ATP receptor antagonist, suramin. It was found that chemical blocking of gap junctions did not significantly affect the Ca2+ wave propagations. These results indicated that ATP, rather than gap junction channels, were responsible for mediating the transmission of laser-induced intracellular Ca2+ signalling in hNT astrocytes which occurred at the neighbours and next nearest neighbour regions in organised networks.For many years, the most intensively investigated mechanism for intercellular Cae et al. , 39 obseo et al.  have als2+ propagation. Hill et al. [2+ propagation region of mechanically stimulated NT2 astrocytes in non-patterned well-plates was reduced both by a gap junction blocker, CBX, and a purinergic receptor antagonist, PPADS. Thus, Hill et al. [2+ propagation was at least partly dependent on gap junction communication and mediated by the ATP. Fujii et al. [2+ waves of mechanically stimulated rat primary astrocytes on glass coverslips propagated proximally via gap junctions and distally via extracellular diffusion of ATP. Although the Ca2+ propagation inhibited by gap junction blockers was not measured to be highly significant, several studies have demonstrated that blocking gap junction might slightly inhibit the ATP release. For example, Cotrina et al. [et al. [2+ communication. Thus, we suggested that in hNT astrocytes, the Ca2+ wave is mainly mediated by the extracellular ATP and partially collaborated with gap junctions.However, various studies have reported the contribution of gap junction to astrocytic Cal et al.  observedl et al.  demonstri et al.  also dema et al.  demonstr [et al.  reported2+ responses of neighbouring cells were significantly lower than that of the stimulated cells. This could be attributed to different mechanisms of Ca2+ production in the stimulated astrocytes and neighbouring astrocytes. In this work, we demonstrated that Ca2+ increases in laser-stimulated cells were dependent on intracellular Ca2+ storage, which can then be subsequently amplified by the CICR. The Ca2+ responses of neighbouring cells are mainly dependent on ATP binding to the membrane-bound receptors, resulting in the generation of IP3 which triggers the release of intracellular Ca2+ from Ca2+ stores. In addition, there can be ATP drift due to the fluid dynamics of the media, which affected the ATP diffusion and how much ATP is available for uptake.We observed that the magnitudes of the CaWe suggest that the organised chip model approach is a more representative model of astrocytes in the brain albeit in 2D and regular, over the conventional petri dishes culture models where unpatterned hNT astrocytes are typically observed to be a ‘fried egg’ shape and touch all of the adjacent astrocytes at their circumference as shown in 2+ waves in organised 5×5 hNT astrocyte stellate-like networks under UV nanosecond laser stimulation, at the single-cell level, for the first time. First, we demonstrated that Ca2+ increases in organised laser stimulated cells were mainly dependent on Ca2+ released in Ca2+ stores (ER). Furthermore, we demonstrated that the Ca2+ propagation from the stimulated cell to neighbouring cells is primarily mediated by extracellular ATP rather than the contributions of gap junctions. This work provides valuable insight, for the first time, on the mechanisms of Ca2+ wave initiation and propagation in organised adult human stellate-like astrocytic networks following precision activation, using UV nanosecond laser stimulation, demonstrating that extracellular ATP transmission is responsible for Ca2+ release at the local network scale rather than gap junctions; being observed even down to the nearest neighbour and next nearest neighbouring cells—contrary to the reported gap junction.In this article, we carried out an investigation to identify the underlying initiation and propagation mechanisms of intercellular CaThis discovery has significant ramifications to many neurological conditions such as epilepsy, stroke and aggressive astrocytomas where gap junctions can be targeted. In cases where such gap junction targeting has failed, this new finding suggests that these conditions should be re-visited and the ATP transmission pathway targeted instead.


Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.[Ca Howevervesicles , the datgin (TG) , was shoIn each scallop-striated muscle cell, SR is a single tubular system in the zone just beneath the cell surface and surrounds only one myofibril at the cell center . When th2+-ATPase molecules of rabbit SR act as a calcium-sensitive membrane-endoskeleton in addition to their primary role as a calcium pump  concentration (0.002 µM) can be found in Ref.  employed was plotted at even intervals from ~0.002 to ~59 μM, along the horizontal axis  was 1.6% . At ≥1.4 μM Ca2+, the percentage was 0% . On the other hand, elongated vesicles containing monomer arrays appeared up to 1.4 μM Ca2+  at ≤1.4 μM Ca2+. In contrast, the percentage was only 0.08%  at ≥18 μM Ca2+  was 4.2%  at ≤1.4 μM Ca2+ and decreased to 0%  at ≥1.4 μM Ca2+.There were 0–17 tightly elongated vesicles with a crystalline array in each view over the calcium concentrations range 0.002–59 µM in the presence of 5 mM ATP, and their average appearance rate (percentage) was at most 6.8%; the crystalline arrays were mainly of the two-rail and/or monomer type, as in vesicles . Namely,axis see a. The amectively a. At ≤0. μM Ca2+ b; The pe μM Ca2+ b. These 2+ in the presence of ATP  jump-up and –down experiments suggest that the ATPase molecules, at least in part, rapidly and reversibly form their crystalline array, depending on calcium concentration.Considering the above-mentioned damage to the vesicles, the observed crystalline vesicles seem to be survivors, which overcame the difficulty in the crystal-reversibility test. Nevertheless, the small number of images obtained from the results of the  = 10–20 µM, the concentration at which both the ATPase activity and muscle contraction are almost maximum, the elongated SR loses its endoskeleton backbone and contracts  in the rabbit SR were reported earlier; a model of monomer-dimer transition of the ATPase molecules ,21,22 an profile  of the Ae SR see  that is channels  and to fchannels ,27,28 anchannels , and thechannels . The preacts see . (iii) T2+] ≤ 0.6 µM  associated with half-maximum Ca2+-ATPase activities  jump-up experiment, SR preparation (0.3 mg protein/mL) was incubated in buffer solution (250 µL) containing ~0.002 µM Ca2+ and 5 mM ATP at 12 °C for 1 min, and the incubation mixture was added with 1/10 volume of either water or 28.5 or 20.0 mM CaCl2; the calcium additions changed the calcium concentration to ~9.8 or ~1.1 µM, respectively. In the [Ca2+] jump-down experiment, SR preparation (0.3 mg protein/mL) was incubated in buffer solution (250 µL) containing ~16.0 µM Ca2+, 5 mM ATP at 12 C for 1 min, and the incubation mixture was added with 1/10 volume of either water or 30.0 mM EGTA; the EGTA addition changed the calcium concentration to ~0.003 µM. The water additions in the jump-up and jump-down experiments were always carried out as controls. After 1 min of water addition, CaCl2, or EGTA, an aliquot of the incubation mixture was applied to an electron microscopy grid.In the [CaElectron microscopic study of the negatively stained SR was performed, also as mentioned in the previous study . The spe2+-ATPase crystalline array and crystal patch assembly in scallop SR and demonstrated that this is accompanied by the elongation and contraction of the isolated SR vesicles under the physiological conditions of the scallop muscle cell. It has provided some fundamental insights into the active behavior of the scallop and rabbit SR. The data suggest that the tubular scallop SR autonomously elongates and contracts with the help of the calcium-sensitive network of its ATPase molecules, which is called the membrane-endoskeletal motor. The elongation and contraction seem to develop stretching and contractive forces, respectively, in the tubular SR. These forces might regulate the gating of RyRs. The above-mentioned stretching or contractive force, developed in the single solid tubular network of the SR, might be uniformly transmitted to all mechanosensitive RyRs within the cell to engage in the opening, resulting in the unified control of the contraction at the sarcomeres.The present study has shown that there is a reversible conversion between Ca


Introduction: Bioproduction of plant-derived triterpenoids in recombinant microbes is receiving great attention to make these biologically active compounds industrially accessible as nutraceuticals, pharmaceutics, and cosmetic ingredients. So far, there is no direct method for detecting triterpenoids under physiological conditions on a cellular level, information yet highly relevant to rationalizing microbial engineering.Methods: Here, we show in a proof-of-concept study, that triterpenoids can be detected and monitored in living yeast cells by combining coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques. We applied CARS and SHG microscopy measurements, and for comparison classical Nile Red staining, on immobilized and growing triterpenoid-producing, and non-producing reference Saccharomyces cerevisiae strains.Results and Discussion: We found that the SHG signal in triterpenoid-producing strains is significantly higher than in a non-producing reference strain, correlating with lipophile content as determined by Nile red staining. In growing cultures, both CARS and SHG signals showed changes over time, enabling new insights into the dynamics of triterpenoid production and storage inside cells. Saccharomyces cerevisiae was shown to be a desirable host for producing triterpenoids due to its endogenous mevalonate and sterol pathways providing the common triterpenoid precursors squalene and 2,3-oxidosqualene  phenoxazine-5-one] is a lipophilic dye that is fluorescent in the presence of a hydrophobic environment, while the fluorescence is quenched in the presence of an aqueous medium . As a hiS. cerevisiae (Coherent Anti-Stokes Raman Scattering (CARS) and Second Harmonic Generation (SHG) are nonlinear spectroscopy variants that deliver sub-micrometer resolution in a multimodal imaging setup. CARS is a nonlinear, non-destructive and nonperturbative  variant revisiae . The apprevisiae . SHG is revisiae  optical revisiae . It has revisiae  or proterevisiae  and mighrevisiae . Furtherrevisiae , detectirevisiae , and visrevisiae .To our knowledge, no studies exist in which the combination of CARS and SHG is applied for the analysis of intracellular heterologous compounds synthesized in yeast. Therefore, we aimed to test the applicability of the combination of these non-invasive techniques to analyze triterpenoid and lipid accumulation in recombinant yeast cells on a qualitative level. As a model triterpenoid, we used the lupane-type triterpenoid betulinic acid  and its Saccharomyces cerevisiae CEN. PK 113-17A  and two strains, S. cerevisiae BA6 and S. cerevisiae BA6∆, originating from S. cerevisiae CEN. PK 102-5B  and engineered for betulinic acid production were used in this study. Strain S. cerevisiae BA6 has been described in HMG1  is a pah1 deletion mutant of BA6, generated through replacement of PAH1 with a hygromycin resistance cassette (YMR165C∆:hphMX). in HMG1 , a singl4)H2PO4 0.25 g/L, NH4Cl 2.80 g/L, sodium glutamate 10 g/L, MgCl2·6H2O 0.25 g/L, CaCl2·2H2O 4.60 g/L, KH2PO4 2 g/L, MgSO4·7H2O 0.55 g/L, myoinositol 100 mg/L, ZnSO4·7H2O 6.25 mg/L, FeSO4·7H2O 3.50 mg/L, CuSO4·5H2O 0.40 mg/L, MnCl2·4H2O 0.10 mg/L, MnCl2·2H2O 1.00 mg/L, Na2MoO4·2H2O 0.50 mg/L, nicotinic acid 11.00 mg/L, pyridoxin-HCl 26.00 mg/L, thiamin-HCl 11.00 mg/L, biotin 2.55 mg/L, calcium pantothenate 51.00 mg/L, Na2EDTA 2.92 g/L, CoCl2.6H2O 0.300 mg/L, H3BO3 1.00 mg/L, KI 0.10 mg/L, and p-aminobenzoic acid 0.20 mg/L. For the reference strain, the WM8+ medium was complemented with 400 mg/L leucin and 400 mg/L uracil. Static cultivations for real-time microscopy measurements were performed with WM8+ medium complemented with 16 g/L ethanol as sole carbon source, which previous studies suggested as a preferred substrate for triterpenoid production  solution and a 40 h culture in mineral salt medium and stored at −80°C. 500 µL of a single cryogenic stock was used to inoculate a 50 ml pre-culture. All cultivations were performed in WM8+ , a modifoduction .600) of 0.1. The optical density of the cultures was determined using a spectrophotometer  with 0.5% phosphate-buffered saline (PBS) used as blank. A predetermined correlation factor of 0.29 g cell dry weight (CDW) per unit OD600 for strain BA6∆ and 0.2 for BA6 and the reference was used to derive the CDW concentration. The cultivations took place in a rotary shaker (New Brunswick Innova 44R) at 30°C, 150 rpm, and 25 mm shaking diameter. For microscopy measurements, samples were taken during exponential growth on glucose and after the diauxic shift during growth on ethanol.Batch cultivations in 500 ml shake flasks started with a filling volume of 20 ml. A pre-culture was used to inoculate the main cultures to a start optical density measured at 600 nm , 80 μl 1 M HCl, and 800 μl chloroform:methanol  and vigorously agitating in the Mini-Beadbeater 16  for 4 min. Samples were centrifuged for 10 min at 13,000 rpm  and 5°C. 200 μl of the lower organic phase were transferred into a glass vial  containing an insert  for High-Performance Liquid Chromatography (HPLC) analysis. Product quantification was conducted with a reverse HPLC system . Samples were kept a 4°C in an autosampler . Five µL of the sample were injected into a C18 column (Merck Spherisorb ODS-2 (5 μm), 250 × 4 mm, Merck KGaA, Germany) with a guard column , while the column temperature was kept at 40°C in a thermostatic column compartment . Analytes were eluted using gradient chromatography at 1.2 mL/min flow rate using a binary pump . Solvent A was acetonitrile (LC-MS grade). Solvent B was 0.2% (v/v) formic acid of high purity (≥98%). The linear gradient method was as follows: 20% B from 0 to 1 min, 20%–0% B from 1 to 13 min, 0% B held until 23 min, then back to 20% B from 23 to 26 min. Solvents were degassed using an online degasser. Analytes of interest were monitored using a charged aerosol detector  driven by a nitrogen generation system . Technical duplicates were measured, i. e., two times 800 μl were taken from the same cultivation, processed, and analyzed. For quantification of triterpenoids, serial dilutions (df = 2) of standards for lupeol , betulin , betulinic acid , and betulin aldehyde  in chloroform were measured.600 of 1.0 was applied on the agarose layer and protected with a cover glass. Desiccation of samples was prevented by covering the edges of the coverslip with clear lacquer and analyzing samples immediately.Yeast cells were immobilized on agarose beds for microscopic analyses. One ml of warm 1% agarose gel was applied onto a microscope slide and covered with a second microscopy slide to force the formation of a planar surface. After solidification, the upper slide was carefully removed. Ten µL of a culture diluted to an OD600 adjusted to 1, was centrifuged at 12,000 g for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 mL 0.5% PBS. Next, 60 μl of Nile red (80 μg/mL dissolved in acetone) was added to the cell suspension. The samples were incubated for 30 min at RT in the dark. After incubation, samples were centrifuged again at 12,000 g for 5 min, and the supernatant was discarded. Pellets were carefully resuspended in 1 mL 0.5% PBS, and 100 μl of the sample were transferred to a 96-well microplate. Fluorescence was measured at different excitation (Ex) and emission (Em) wavelengths using the Multi-Mode Microplate Reader Synergy; Ex/Em 550/638 nm for phospholipids, 515/583 nm for triglycerides, as recommended by the manufacturer. Data were analyzed with the software Gen5. All reads took place at RT (ca. 23°C) with the following parameters: read height 8 mm, read speed normal, delay 100 msec, measurements/data point 10, gain 100.Nile Red staining was performed based on a protocol described in via a pump/probe beam at 816 nm and 817 nm and a Stokes beam at 1,064 nm while the SHG images were based on the pump/probe beam. CARS spectra were generated by tuning the pump/probe beam in a spectral window from 807 nm to 819 nm  or 787 nm–826 nm . The real-time growth experiments were performed using a Leica HC PL Fluotar 10x/0.30 DRY objective. All other measurements were performed using a Leica Fluotar VISIR 25x/0.95 WATER objective. The generated signals were detected by non-descanned Leica photomultipliers in EPI and forward direction. A more detailed description of the systems is given in CARS and SHG measurements were performed with a modified Leica TCS SP8 CARS  equipped with a picoEMERALD™ laser system . The CARS images were generated Pure lupeol, betulin, betulin aldehyde, betulinic acid, and ergosterol  were analyzed in crystalline conditions and squalene in liquid form. Saturated solutions of ergosterol and the main triterpenoid products, betulin and betulinic acid in ethanol were prepared to analyze the CARS and SHG spectra in their dissolved state. Assuming additivity of the signal intensities, the mean intensities of dissolved substances were corrected by subtracting the mean CARS signal intensity of pure ethanol. The difference in mean intensities is the estimated CARS signal caused by these substances. While betulin and betulinic acid generated only a weak CARS signal, ergosterol produced a stronger CARS signal in solution.All images were re-scaled to equal contrast levels for proper quantification of the colocalization of CARS and SHG. The lower threshold was set equal to the value of the background signal of the respective channel. The upper threshold was chosen so that no oversaturation occurred. For CARS images, the lower contrast value was set to 20 and the upper value to 60; for SHG images, the lower contrast value to 7, and the upper to 30; and for transmission images, the lower value was 25 and the upper 100. An image gradient was calculated for re-scaled transmission images, and a threshold was set that defined which gradient values were considered for evaluation. From this gradient image, objects smaller than 100 pixels were removed since they are too small for cells and, most likely, debris. Subsequently, all holes in determined structures were filled. From the generated masks, the number of cells per object was determined manually and by comparison with the original transmission image. For each object within an image, the colocalization evaluation was performed separately, i.e., for each object the number of all pixels above a colocalization intensity threshold of 25% of the maximum intensity was determined for both channels (CARS and SHG). From these, the colocalizing pixels were determined within the object. The ratio of colocalization of CARS and SHG was calculated with Eqs We also determined the number of SHG pixels not colocalizing with CARS. The term was normalized for each object using Eq. S. cerevisiae CEN. PK strains, BA6 and BA6∆, engineered to produce the lupane-type triterpenoids betulin, betulin aldehyde, and betulinic acid , which catalyzes the committed step for the synthesis of major phospholipids . Samples were taken at an early stage (I) and a late stage (II) of a batch cultivation with distinct changes in specific triterpenoid content . ImmobilBoth triterpenoid-producing strains showed a similar CARS signal at stage I, slightly stronger than the reference. C-H-containing compounds cause a strong CARS signal at 816 nm  and reprBA6∆ cells showed cell-to-cell heterogeneity in SHG, especially during stage I. Similarly, previous studies reported variability in endoplasmic reticulum morphology and lipid droplet abundance between single BA6∆ cells , which mImmobilized cells of the reference and engineered strains sampled during stage II showed comparable CARS signals. Spherical structures were detected by CARS in all three strains , which wThe overlay of the CARS and SHG signals allows speculation of analyte distribution. While most regions show a colocalization (indicated by the yellow color), there are also regions with only or predominantly SHG or CARS activity, and this separation increased in stage II. Notably, areas of presumed lipid droplet localizations showed no SHG signal, suggesting that triterpenoids are either not present in these organelles or are not in a crystal-like form required for exerting SH activity. Contrary, regions with a predominant SH signal suggest the agglomeration of solid triterpenoid particles in a low-lipid environment.To further evaluate the localization of triterpenoids at the subcellular level, we performed a colocalization analysis, where we first determined the number of objects in each image, consisting of one or more cells. Then, the number of pixels above a threshold of 25% maximum intensity was determined for every object for CARS and SHG channels. Using these values, we determined ratios of colocalization as described in detail in the methods section. The ratio of pixels with CARS signal, which colocalize with SHG , was in The large variance in some data sets indicated by the whiskers and outliers may have been caused by biological heterogeneity between individual cells and analytical challenges when cell agglomerates are formed. Despite the significant variance, and important for the scope of this study, these results indicate a correlation of SHG signal with triterpenoid accumulation and the subcellular distribution.Overall and important for the scope of this study, these results indicate a correlation of SHG signal with triterpenoid accumulation and the subcellular distribution.z-direction. This way, the sensitivity was increased and importantly, consistent values were achieved even with vertical movements of the living cells. This improved analytical method allowed better capture of the SHG signal of the reference cells, which increased over time. The cultivations were performed with ethanol as sole carbon source at initial concentrations of 16 g/L. It is known that elevated ethanol concentrations induce ergosterol production in yeast  to analyze lipid compounds and triterpenoids in yeast cells. We could show that the combination of the non-invasive techniques CARS and SHG allow detection of lipids and triterpenoids in living yeast cells. This highlights the great potential of these methods for future analysis of natural product synthesis with special and temporal resolution. Other than conventional methods like Nile red, the application of CARS and SHG allows the detection of both triterpenoids and lipids in individual cells. The application of CARS and SHG signal measurement shows potential for further development and an advanced quantification. For instance, detection of the individual triterpenoid components at different wavelengths would be an option to further improve the existing method towards more specific and quantitative analysis. Using the current state-of-the art techniques, we were able to monitor triterpenoid accumulation in cells of a population over the entire cultivation time. To our knowledge, this is the first time that the combination of both CARS and SHG techniques are applied for the analysis of intracellular product accumulation in living yeast cells. This proof-of-principle study shows the potential of application of CARS and SHG microscopy techniques in applied microbiology allowing new insights into the dynamics of triterpenoid production and storage by recombinant yeasts.


TP53-mutant acute myeloid leukemia (AML) respond poorly to currently available treatments, including venetoclax-based drug combinations and pose a major therapeutic challenge. Analyses of RNA sequencing and reverse phase protein array datasets revealed significantly lower BAX RNA and protein levels in TP53-mutant compared to TP53–wild-type (WT) AML, a finding confirmed in isogenic CRISPR-generated TP53-knockout and -mutant AML. The response to either BCL-2 (venetoclax) or MCL-1 (AMG176) inhibition was BAX-dependent and much reduced in TP53-mutant compared to TP53-WT cells, while the combination of two BH3 mimetics effectively activated BAX, circumventing survival mechanisms in cells treated with either BH3 mimetic, and synergistically induced cell death in TP53-mutant AML and stem/progenitor cells. The BH3 mimetic–driven stress response and cell death patterns after dual inhibition were largely independent of TP53 status and affected by apoptosis induction. Co-targeting, but not individual targeting of BCL-2 and MCL-1 in mice xenografted with TP53-WT and TP53-R248W Molm13 cells suppressed both TP53-WT and TP53-mutant cell growth and significantly prolonged survival. Our results demonstrate that co-targeting BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance to individual BH3 mimetics in TP53-mutant cells, thus shifting cell fate from survival to death in TP53-deficient and -mutant AML. This concept warrants clinical evaluation. TP53-mutant acute myeloid leukemia (AML) respond poorly to current therapies. Consequently, TP53 mutations are frequently enriched/selected in refractory/relapsed AML [Patients with psed AML –4, and apsed AML .TP53 mutations have vastly inferior responses to VEN-based therapies [TP53 mutations confer resistance to HMA/VEN in vitro and in vivo [TP53-mutant AML [The combination of the BCL-2 inhibitor venetoclax (VEN) with a hypomethylating agent (HMA) or low-dose Ara-C elicits complete remission/complete remission with incomplete hematologic recovery rates of >65%, is well tolerated by elderly AML patients , 10, andherapies –16, supp in vivo  and CRIS in vivo . Furthertant AML .BAX mutations can confer VEN resistance to AML cells [TP53-WT AML by promoting pro-apoptotic BCL-2 proteins and targeting MCL-1 [TP53-mutant AML.Mitochondria-mediated apoptosis is controlled by pro- and anti-apoptotic BCL-2 proteins. We and others have reported that MCL-1, a major resistance factor for BCL-2 inhibition, is frequently induced in VEN-resistant AML cells –22 and tML cells . We repong MCL-1 . We, theTP53-mutant cells to BH3 mimetics. We determined RNA and protein levels of BCL-2 family members in large cohorts of TP53-mutant AML and investigated if shifting the balance of BCL-2 proteins to pro-death by co-targeting BCL-2 and MCL-1 could overcome TP53-mutant AML resistance to individual BH3 mimetics. We here report that decreased expression of BAX in TP53-defective AML cells contributes to their resistance to BH3 mimetics. Co-targeting of BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance of TP53-mutant AML and AML stem/progenitor cells to individual BH3 mimetics in vitro and in xenografts comprising both TP53-WT and -mutant Molm13 cells in vivo.Alterations in the balance of pro- and anti-apoptotic BCL-2 proteins could restrict the response of http://bioinformatics.istge.it/clima/) [TP53-R248W Molm13 [TP53-knockout (KO) and -mutant  Molm13 cells [TP53 mutations were obtained from patients . U937, KG1, and THP1 cells were purchased from ATCC . Cell lines were validated by STR DNA fingerprinting using the AmpF_STR Identifier kit according to the manufacturer’s instructions (Applied Biosystems). The STR profiles were compared to known ATCC fingerprints, and to the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (/clima/) . The STRW Molm13  and TP5313 cells  were gen13 cells  and p53-13 cells  were genTo generate AML cells with acquired resistance to both BCL-2 and MCL-1 inhibitors, we treated MV4-11 cells with acquired resistance to VEN previously generated  with incTP53-mutant (n = 63) and -WT (n = 668) AML patient samples collected at diagnosis using a dataset from Dr. Torsten Haferlach, MLL Munich Leukemia Laboratory, and the Torsten Haferlach Leukemia Diagnostic Foundation, Munich, Germany. These sample sizes are sufficient to provide 99% power for a 0.5 log fold change, with the false discovery rate being controlled under 5%.We performed differential gene expression analysis between TP53 gene status, who had not been exposed to chemotherapy. The log2-normalized expression levels of BCL-2 family proteins in this cohort were analyzed and compared between the TP53-WT (n = 101) and -mutant (n = 21) samples. These sample sizes are sufficient to provide 90% power for a 0.5 log fold change, with the false discovery rate being controlled under 5%.Proteomic profiling of >200 proteins in samples obtained from newly diagnosed AML patients were performed by reverse phase protein arrays (RPPA) following the custom-built AML array –38. Amon+), and leukemia stem/progenitor cells (CD34+) and expressed as specific apoptosis [Viable cell numbers and apoptosis were determined by flow cytometry as previously described . For pripoptosis , 39:\docWestern blot was carried out and analyzed as described previously  using thC (#612310)  release was determined concomitantly to assess loss in mitochondrial membrane potential.Activation of BAX and BAK was determined following the described assay . Antibodhttps://www.R-project.org) as described previously [TP53-WT, -deficient, and -mutant (R175H and R248Q) leukemia cells treated with DMSO, VEN, AMG176, or both based on the expression levels of the features assessed using flow cytometry. UMAP embedding based on 4 principal components was used to separate different experimental conditions.Cell fates were determined using a multi-metric cell death assay , modifieeviously . The Seueviously  was used6 luciferase/GFP-labeled TP53-WT plus 0.03 × 106 BFP-labeled TP53-R248W cells) were injected into the tail veins of 6- to 8-week-old male NSG mice . Once engraftment was confirmed by luciferase imaging with the IVIS-200 noninvasive bioluminescence in vivo imaging system , mice (5 or 6/group) were treated with vehicle , VEN , AMG176 , or VEN and AMG176 combination. Both agents were provided by Amgen and given via oral gavage. Disease progression and treatment responses were monitored with luciferase imaging and flow cytometric measurement of circulating GFP- and BFP-positive cells on day-3 of each week. Mouse survival was followed.Mouse experiments were conducted in accordance with MD Anderson’s Institutional Animal Care and Use Committee-approved protocols. Molm13 cells . The combination index (CI) [50, ED75, and ED90. A CI <1 indicates synergistic; CI = 1, additive; and CI >1, antagonistic effects. The Kaplan–Meier method was used to estimate mouse survival, and survival data were analyzed using the log-rank test.Cell line experiments were performed in independent triplicates; results were expressed as means ± SEMs. The Student dex (CI) , determiTP53-mutant AML, we first assessed the expression of BCL-2 proteins in AML. RNA-seq analysis compared the expression of BCL-2 family members in TP53-mutant  samples at diagnosis. The volcano plot, generated using the ggplot2 package in R, has a P cutoff of 0.5 Log2 fold change (FC) and 0.05 of adjusted P value cutoff , BAD, and BBC3 (PUMA) were observed (adjust P < 0.05) . As expected, p53 protein levels were significantly higher in TP53-mutant (n = 21) than in TP53-WT AML . TP53 VAFs were not available and could vary greatly among these samples. Even so, we found that only the BAX levels were significantly lower in TP53-mutant than in TP53-WT AML (P = 0.006) among BCL-2 family proteins  Fig. . We nextTP53-deficiency is indeed responsible for BAX reduction, we determined the expression of BCL-2 proteins in isogenic Molm13 cells with TP53-WT, TP53-KO, or various TP53 hotspot mutations [TP53-defective cells show variably altered expression of various BCL-2 proteins. Among them, BAX is markedly decreased in TP53-KO/mutant cells . Interestingly, in addition to increased MCL-1, we recently reported that AML cells with acquired resistance to VEN or to AMG176 had decreased BAX [TP53-deficient AML cells.To validate that utations . Comparells Fig. , and it ased BAX . These rBAX-KD diminished the sensitivity of AML cells to BH3 mimetics, but that this sensitivity was restored by the combined inhibition of BCL-2 and MCL-1 [BAX-KD cells and found that these were largely unchanged  , which were essentially ineffective as single agents in TP53-deficient cells, were able to effectively induce apoptosis and reduce absolute cell numbers, reflected in the exceptionally low CI values, regardless of TP53 mutational status.We previously reported that AML cells with acquired resistance to VEN or AMG176 expressed increased MCL-1 and BCL-2A1 and decreased BAX levels. Although VEN or AMG176 individually were less effective in these cells, the combination of the agents was highly synergistic . We repond MCL-1 . To demo57) Fig.  and effe57) Fig. . AlthougTP53-R248W Molm13 cells and TP53-KD OCI-AML3 cells  Molm13 cells treated with VEN, AMG176, or the combination. As expected, AMG176 increased MCL-1 levels in all cells examined  and stress responses  utilizing dimension reduction and unsupervised clustering to gain a broad overview of the treatment-specific proteomic landscape and assess whether VEN, AMG176, or co-treatment elicited overlapping or distinct proteomic shifts across leukemia cells with different TP53 statuses. TP53-WT, -KO, and -mutant (R175H and R248Q) Molm13 cells were treated with VEN, AMG176, or both. We generated a concatenated data matrix that included data points from 16 different conditions  and subjected the data matrix to UMAP dimension reduction and FlowSOM clustering to partition the proteomic data into distinct subpopulations   Table  and reduy) Table  comparedTP53-WT and BFP-labeled TP53-R248W Molm13 cell mixtures (10:1) in NSG mice - and BFP (TP53-R248W)-labeled cells by flow cytometry. VEN or AMG176 alone had minimal effects on either TP53-WT or TP53-R248W Molm13 cells, and their combination significantly reduced both cell types   Fig. .Fig. 6ThTP53-mutant AML to individual BH3 mimetics targeting BCL-2 or MCL-1. The combined inhibition of both effectively activates BAX and synergistically decreases cell viability primarily by apoptosis induction in AML cells largely independent of TP53 mutational status. Hence, the diminished activity of BH3 mimetics in TP53-defective cells can be circumvented in a p53-independent manner by co-targeting BCL-2 and MCL-1.We demonstrate that the significant reduction of BAX is critical for the decreased sensitivity of TP53 or p53-regulated apoptosis regulators, such as BAX, confers VEN resistance [BAX mutations have been identified as a mechanism of BH3 mimetic resistance in AML [BAX mutations have also been detected in the myeloid compartment of CLL patients with clonal hematopoiesis who were treated with VEN [TP53-mutant patient samples and isogenic AML cells compared to TP53-WT AML cells. We also found that genetic silencing of BAX rendered AML cells resistant to BH3 mimetics [TP53-deficiency or BAX-KD alone sufficiently inhibits BAX and BAK activation. Collectively, these findings show that the reduction or loss of BAX are critical for BH3 mimetic resistance and contributes to the vastly reduced activity of BH3 mimetics in TP53-mutant AML.CRISPR genome editing analyses have demonstrated that the loss of WT sistance . BAX mute in AML . BAX mutwith VEN , which fmimetics  and thatTP53-mutant clones are commonly selected and frequently become dominant, conferring further resistance to other therapies [TP53-mutants [TP53 mutations have been identified in AML [TP53-deleted or mutant-AML to individual BH3 mimetics and synergistically induces TP53-independent profound apoptosis in AML cells and AML stem/progenitor cells in vitro and in vivo. Hence, our approach has potentially broad clinical implications, beyond approaches targeting selective TP53 mutations.Following exposure to chemotherapeutic agents, herapies , 48. Alt-mutants , 50, thed in AML . We hereTP53-deficient/-mutant than in TP53-WT AML. Achieving efficacies in TP53-deficient/-mutant AML that were similar to that in WT AML required higher doses of both agents. Nevertheless, xenograft-bearing mice with TP53-mutant AML cells under combination treatment survived almost twice as long than those treated with either agent alone using the same dose and treatment schedule as we previously described, in which a VEN/HMA resistant TP53-WT PDX model showed a three-fold survival extension [Despite effectiveness, the combined inhibition of BCL-2 and MCL-1 was less robust in xtension . AMG176 TP53-mutant AML cells to VEN and warrant further exploration.Besides direct inhibition, indirect targeting MCL-1 through mechanisms such as inhibition of MAPK or CDK9 could also sensitize AML cells to VEN. Given the critical role of BAX in mitochondria-regulated apoptosis, pharmacologically or genetically increasing BAX levels may improve the lethality of BH3 mimetics and benefit patients whose disease is resistant to BH3 mimetics. In fact, with an improved understanding of the structure-function relationship of BAX, researchers are developing strategies to modulate BAX activity or expression with various approaches, including small molecules and peptides . All appSupplemental Data


Venetoclax (VEN), the first selective Bcl-2 inhibitor, has shown efficacy and safety both as monotherapy and in combination with other agents in the treatment of newly diagnosed acute myeloid leukemia (AML), while its role in relapsed or refractory (R/R) disease is not well defined. Here, we reviewed the latest advances of VEN-based therapy for R/R AML from the 2022 American Society of Hematology (ASH) Annual Meeting, including some novel and encouraging regimes, such as VCA, VAH, and HAM regimes, etc. Further research is still needed to fully understand the optimal use of these agents in R/R AML treatment. To the editor,Relapsed or refractory (R/R) acute myeloid leukemia (AML) is associated with a poor prognosis and limited treatment options . The BclThe combination of VEN and HMA in R/R AML were investigated by several studies from different countries, which showed overall response rates (ORR) of 44% , 52% [4]ASTX727 is an oral agent that combines decitabine with the cytidine deaminase inhibitor cedazuridine. The combination of VEN with ASTX727  achievedChidamide is a subtype-selective benzamide inhibitor of histone deacetylase. In one phase II study , the triP = 0.003), OS  and event-free survival (EFS)  than VEN + HMA group. Subsequently, the data were updated [Homoharringtonine (HHT), a natural alkaloid derived from the cephalotaxus, is a well-defined anti-AML agent. The triplet of VEN, HMA, and HHT (VAH)  had sign updated  with a mIn this series, the novel VCA and VAH regimes appear to have higher response rate, and the efficacy is worth further exploration.Cladribine is an adenosine deaminase resistant analog of adenosine. After one cycle of VEN, cladribine plus low-dose Ara-C (CAV)  for R/R One phase I/II trial  evaluateDesikan et al.  updated In this series, CAV and HAM regimens seem to have deeper remissions. The efficacy and durability of responses need to be further assessed.Pivekimab sunirine (PVEK) can target CD123, which is expressed on the majority of AML blasts and leukemic stem cells. In a phase Ib/II study , the comPegcrisantastase (PegC) is a long-acting crisantastase that exhibits strong anti-leukemia activity through complete consumption of glutamine. An open-label phase Ib clinical trial  evaluateIn conclusion, the 2022 ASH annual meeting presented the new development of VEN-based therapy for R/R AML Tables  and 2, i


Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca The incidence of all causes of cardiovascular disease (CVD) is much lower in premenopausal females in comparison to males of the same age . However2+ from the sarcoplasmic reticulum (SR) mediated by cardiac ryanodine receptors type 2 (RyR2)  entering matrix via MCU. An increase in mitochondrial matrix [Ca2+] activates several dehydrogenases stimulating NADH and FAD2 production that fuel electron transport chain (ETC). Interestingly, studies using mouse models with loss-of-MCU function demonstrated no change in basal cardiac function but showed significant loss of responsiveness to adrenergic stimulation  is significantly lower in female VCMs than males  . An2+] . It was 2+] . A group2+] . Of part2+] , 53. How2+ homeostasis in VCMs under conditions mimicking stress. Our results demonstrate a higher degree of ETC organization into supercomplexes and reduced mito-[Ca2+] uptake in female VCMs vs males. This leads to reduced mito-ROS production and less pro-arrhythmic SCWs due to reduced RyR2 oxidation under β-adrenergic stimulation. Our data suggest that sexual dimorphism in mitochondrial Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.In the present study we investigated the role of sex and COX7RP-dependent changes in mitochondrial supercomplex assembly, mito-ROS production, and intracellular Ca2+ homeostasis, mito-ROS and mito-Ca2+ 22-month-old F344 rats of both sexes were obtained from NIH aged rat colony.All procedures involving animals were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the Institutional Animal Care and Use Committees of The Ohio State University, University of Rhode Island and Rhode Island Hospital. Two-month-old male and female Sprague–Dawley rats (control) were purchased from Charles River Laboratories  and heart tissue samples and isolated ventricular myocytes were studied at 3–4 month of age. In addition seven 6 month old Sprague–Dawley rats were bilaterally ovariectomized (OVX) and additional five rats received a sham surgery (Sham) at Harlan Laboratories Inc. Indianapolis, IN, USA. These rats were used 3 months after surgery to obtain heart tissue samples. To study potential sex differences in intracellular CaHuman left ventricular tissues were obtained from human organ donors as previously described , 39. The2+ sensor mtRCamp1h was constructed by fusing cytochrome C oxidase subunit IV at the N-terminus of RCamp1h coding region as described previously  = Kd × (F − Fmin)/(Fmax − F), where Kd of mtRCamp1h = 1.3 µmol/L for Ca2+, fluorescence was converted to mito-[Ca2+] for each VCM. Analysis parameters included baseline mtRCamp1h mito-[Ca2+] (μmol/L) in resting VCMs; peak mito-[Ca2+] and the time to peak amplitude (s) in VCMs undergoing periodic 1 Hz field stimulation [2+ decay  was derived from single exponential fit of VCM mito-[Ca2+] decrease upon cessation of pacing.Myocytes were infected with mtRCamp1h adenovirus ± COX7RP or COX7RP-shRNA adenovirus and cultured for 36–44 h . mtRCampmulation . The ratThe ratiometric MLS-HyPer7 probe was excited using 405 nm and 488 nm laser lines. Fluorescence emission was collected at 520–540 nm wavelengths, measured in the x–y mode at 400 Hz sampling rate. Minimum fluorescence was obtained by application of dithiothreitol , and maximum fluorescence was obtained by application of 2,2′-dithiodipyridine . Data are presented as a percentage of ΔF/ΔFmax where ΔF = F − Fmin and ΔFmax = Fmax − Fmin .Tissue samples or cultured rat VCMs were lysed in lysis buffer from Cell Signaling , supplemented with phosphatase  and protease inhibitor cocktails  as described previously . SamplesH9c2 cells were plated in 6-well plates and infected with COX7RP-FLAG and shRNA COX7RP adeno-viruses. At 48 h after infection, cells were lysed in lysis buffer from Cell Signaling (Cat # 9803S), supplemented with phosphatase  and protease inhibitor cocktails  as described previously . SamplesFor immunoprecipitation of RyR2, cultured rat VCMs infected with Adv-COX7RP and Adv-shRNA COX7RP for 48 h were lysed using cell lysis buffer from Cell Signaling , supplemented with phosphatase  and protease inhibitor cocktails . An overnight immunoprecipitation of RyR2 was performed at 4 °C using the Catch and Release v2.0 Kit  in accordance with the manufacturer’s instructions using anti‐RyR2 antibody  and a negative control antibody comprising normal rabbit IgG . Samples were analyzed by immunoblotting.To determine oxidation status of RyR2, the Oxidized Protein Western Blot Kit  was used, whereby carbonyl groups of immunoprecipitated RyR2 were derivatized to 2,4 dinitrophenylhydrazone (DNP) by reaction with 2,4 dinitrophenylhydrazine . For conBlots were developed with ECL (Bio-Rad Laboratories) and quantified using Image J  and Origin 8 software.g for 10 min. The pellet consisting of nuclei and cell debris was discarded and the supernatant was then centrifuged at 20,000g for 15 min. The pellet was considered as mitochondrial enriched fraction. Samples were then solubilized using the mild detergent digitonin (1%) from NativePAGE™ Sample Prep Kit  in accordance with the manufacturer's instructions. To demonstrate changes in association of supercomplexes in VCMs infected with adenoviruses, cells were lysed in digitonin (1%) for 30 min on ice, and centrifuged at 20,000g for 30 min. Processed samples were resolved on NativePAGE™ 4–16% Bis‐Tris protein gels, 1.0 mm, 15‐well  in Native-Page cathode and anode buffers electrophoresed at 150 V for 1.5. followed by 250 V for 2 h. Samples were transferred onto nitrocellulose membranes before being probed with antibodies, as described above. The primary antibodies used were as follows: Complex I—anti-NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX IV; all from Abcam. Hsp60 was used as loading control. Anti-Hsp60 antibodies were from Cell Signaling. The secondary antibodies used were goat anti‐mouse and goat anti‐rabbit ones . Blots were developed with ECL (Bio-Rad Laboratories) and quantified using Image J  and Origin 8 software.For the assessment of native protein complexes using blue native polyacrylamide gel electrophoresis (BN‐PAGE) , we used2. Adv-shRNA COX7RP was used for infection on day one and Adv-COX7RP on day 2. On day 3, cells were washed twice with Seahorse assay medium  and incubated in a 37 °C incubator without CO2 for 1 h. Oxygen consumption rate (OCR) was measured according to manufacturer’s guidelines of the XF Cell Mito Stress Test kit (Agilent). Oligomycin A , FCCP (1.5 μmol/L), and a combination of rotenone and antimycin A (1 μmol/L each) were injected on cells as indicated. After the assay, cells were stained with Hoechst dye and nuclei counted on a Cytation 5 cell imaging plate reader (Agilent) to normalize results. Data are expressed as pmol O2/min/100 cells. Plate location was randomized between groups in each assay. Wells with no response to oligomycin, FCCP, or rotenone/antimycin were removed from analysis based on the assumption of premature injection/leak of assay drugs into the well from upper plate reservoir, which confounds interpretation. There were no differences in the proportion of these wells between groups.To assess mitochondrial function, oxygen consumption rates (OCR) of H9c2 cells infected with Adv-COX7RP-FLAG and Adv-shRNA COX7RP were measured by the Agilent Seahorse XFe96 extracellular flux analyzer . Briefly, cells were seeded in XFe96 cell culture plates at 10,000 cells/well in standard DMEM + 10% FBS medium and placed in a 37 °C incubator with 5% CO2, 1.70 MgSO4, 24.88 NaHCO3, 6.00 glucose, 1.20 KH2PO4, and 2.00 Na pyruvate) to wash blood and supply oxygen to the heart during working heart preparation. Pulmonary veins were ligated, the lungs removed under a microscope and the heart rapidly moved to working heart apparatus . Hearts were then slowly increased to a constant perfusion pressure of 70 mmHg over 5 min. A cannula was inserted into the left atria and a pressure catheter inserted into the LV through the base of the heart with care to avoid large coronary vessels. A small catheter attached to an in-line optical O2 sensor  and withdrawal syringe pump was placed in the pulmonary artery to continuously monitor O2 tension and temperature of coronary effluent. An additional in-line O2 sensor monitored O2 and temperature of perfusate. Hearts were switched to working mode via opening gravity flow of oxygenated Krebs buffer to the left atria (preload set to 10 mmHg). Langendorff perfusion was reduced and hearts allowed to equilibrate for 5 min before O2 consumption measurements. Coronary flow was assessed via eluent collection over 1 min. O2 consumption was normalized to heart weight and coronary flow and calculated according to Neely et al. , given that changes in matrix [Ca2+] are often associated with changes in mito-ROS production [2+ probe mtRCamp1h we determined that mitochondrial [Ca2+] is significantly lower in field-stimulated female VCMs pretreated with 50 nmol/L ISO for 5 min  and female (red) VCMs derived from mRCamp1h signal. To convert mtRCamp1h fluorescence into [Ca2+], cells were permeabilized with saponin at the end of recording and exposed to 2 mmol/L Ca2+ buffer EGTA to obtain Fmin followed by application 20 μmol/L [Ca2+] to obtain Fmax. Although no significant changes in mito-[Ca2+] were detected at rest, field stimulation of VCMs at 1 Hz revealed that in female VCMs it takes significantly longer to reach maximum mito-[Ca2+], and that this maximum concentration is significantly lower than in male VCMs  was confirmed in H9c2 cells 48 h after transduction with western blot Fig. A. As seeCMs Fig. .Fig. 11C2+ release. Representative line scan images and corresponding Fluo-3 fluorescence time profiles are depicted in Fig. 2+ release in female VCMs promoting generation of pro-arrhythmic SCWs, while COX7RP overexpression led to opposite results in males.Next, we tested the effects of adenoviral-mediated COX7RP-FLAG overexpression in male VCMs and shRNA-mediated KD in females treated with ISO  on RyR2-mediated spontaneous SR Ca2+ release governed by hyperactive RyR2s due to oxidation by mito-ROS. We found that lower mito-ROS production in females is accompanied by lower mito-[Ca2+] and heightened ETC supercomplex assembly. We found the more efficient electron transport in females is likely related to increased expression levels of the estrogen-dependent mitochondrial assembly protein COX7RP.In the present study we investigated the mechanisms underlying reduced mito-ROS production in female VCMs. We have shown that male rat VCMs exhibit higher propensity to pro-arrhythmic diastolic SR Ca2+ release is directly coupled to mitochondrial metabolic output in stress conditions  in VCMs rapidly changes in response to changes in frequency and amplitude of cytosolic Ca2+ transients, directly linking contractile activity and mitochondria ATP production to closely match varying metabolic demand with output. Our experiments in VCMs expressing mito-[Ca2+] biosensor mRCamp1h (Fig. 2+] is drastically lower in field-stimulated ISO-treated VCMs from female rat hearts vs males (Fig. 2+ transient amplitudes (Fig. 2+ uptake is lower in isolated female rat cardiac mitochondria than in males  accumulation to satisfy increased metabolic demand during high workload or stress.Mitochondria are increasingly acknowledged as a key target for sex differences in pathologies . However agonist . Importap1h Fig. A showed les Fig. B, C, des2+] in females vs males. We confirmed that despite lower mitochondrial [Ca2+] in female VCMs, female working hearts have the same myocardial O2 consumption as male hearts when operating at similar work levels (Fig. 2+] uptake in female cardiac mitochondria , underlies lower mito-ROS emission rates, reduced RyR2 oxidation, and lesser propensity for pro-arrhythmic spontaneous SR Ca2+ release under stress conditions. We conclude that sexual dimorphism in ETC organization and mitochondrial Ca2+ homeostasis may contribute to cardioprotection in healthy premenopausal females.To summarize, our data highlight the fundamental differences in bidirectional SR-mitochondria communication in VCMs from heathy adult male and female hearts. Higher COX7RP-dependent ETC supercomplex assembly in female VCMs, combined with lower levels of mito-[Ca


TP53-mutated (TP53m) acute myeloid leukemia (AML) comprises only 5-15% of de novo AML, associated with poor survival outcomes due to its resistance to conventional therapy. Ring chromosomes, an even more rare subset of genetic anomalies, occur in only 2% of cases. We report a unique case of de novo AML with both TP53 and ring chromosome anomalies leading to a catastrophic outcome in a 72-year-old male who initially presented with gastrointestinal bleeding (GIB) and urethral stone status post-cystoscopy with J-stent placement. He had no history of chemotherapy use, radiation, benzene exposure, or any other risk factors except for his age. He was noted to have pancytopenia, for which bone marrow biopsy, flow cytometry, and cytogenetic studies were done. Biopsy reported an interesting next-generation sequenced TP53-mutated AML, which correlates with a low rate of response to standard chemotherapy except for bone marrow transplants. Notably, with a complex aberration of 45 XY with multiple translocations (t), deletions (del), inversions (inv), derivative (der) breakpoints, aneuploidy, and rare ring and maker chromosomes, his case was complicated with rapid-onset and very severe hyperleucostasis, reflecting the prognostic value of this rare cytogenetic configuration. The patient expired within 48 hours of diagnosis, despite the urgent initiation of cytoreductive therapy and the mitigation of tumor lysis syndrome with Rasburicase. To the best of our knowledge, this is one of the first AML-M4 patients with rapid-onset leucostasis and the demise of next-generation sequences (NGS) in a de Novo AML patient with this rare complex combination. Acute myeloid leukemia (AML) is a neoplastic proliferation of clonal myeloid cells that are truncated in their growth and associated with grave outcomes ,2. AML hPatients are usually expected to at least survive months, and palliative team discussion for hospice care would be considered at the time of diagnosis. A systematic review and meta-analysis was carried out to compare treatment outcomes in newly diagnosed, treatment-naïve patients with TP53-mutated (TP53m) AML, and despite improved responses seen with IC and VEN + HMA compared to HMA, survival endpoints remained poor with very limited health benefits cut across all treatment groups, including treatment-naïve patients . This shCytogenetic abnormalities involve either complete, partial loss, or gain of a chromosome; however, they play a significant role in the diagnostics, classification, prognostic value, and choice of therapy in AML . The preA 74-year-old man with a history of Benign prostatic hyperplasia, type 2 diabetes, and nephrolithiasis presented with a three-day history of bright red blood per rectum and an infected wound on the right hand. Initial workup showed pancytopenia  with no evidence of hepatic or splenic disease. He was started on empiric antibiotics for cellulitis. While awaiting endoluminal evaluation, he developed severe right-sided flank pain and an oliguric acute kidney injury (creatinine 6 uptrend from 0.8), concerning obstructive uropathy. An emergent cystoscopy did not show obstruction or signs of infection. Postoperatively, he developed acute hypoxic respiratory failure requiring mechanical ventilation and a medical intensive care unit, septic shock, and anuric renal failure requiring renal replacement therapy. Despite broad-spectrum antibiotics, he had persistent leukocytosis (WBC 33k). Peripheral smears showed metamyelocytes, warranting a bone marrow biopsy and flow cytometry. Results showed acute myeloid leukemia with a TP53 mutation, complex aberrations including multiple translocations (t), deletions (del), inversions (inv), derivatives (der), breakpoints, aneuploidy, and a rare ring chromosome 15 . The aberrations identified are not specific to a particular AML French-American-British classification (FAB) subtype, with clinical and hemopathology correlations suggested. Furthermore, G-G banding shows a rare ring and marker nonspecific aberration for a particular FAB subtype. G-G banding shows a rare ring chromosome Figure .Within 48 hours of diagnosis, he developed rapid-onset leukostasis and hyperleukocytosis WBC 110-120k). Despite initiating urgent cytoreductive therapy with hydroxyurea and prophylactic treatment for tumor lysis syndrome, the patient expired. Our patient had no history of radiation, benzene exposure, or any other risk factor except for age. His biopsy reported an interesting next-generation sequenced TP53-mutated AML with ring chromosome 15. To the best of our knowledge, this is one of the first AML-M4 with next-generation sequences (NGS) in de novo AML patients. Ring chromosome 15 [r (15)] Mosaic is a very rare genetic makeup requiring further research due to the diversity of both its phenotype and genotype, with further complexity in AML. Further analysis of the patient's bone marrow differentials showed 34% myeloblast with varying degrees of normoblast . The number of blasts : 34% by cytomorphology; 50% by CD34 immunohistochemical (IHC) stain. The leukemia-associated phenotype (LAP) blasts show aberrant loss of human leucocyte antigen-DR (HLA-DR). His marrow cellularity was 90%, with marrow fibrosis absent (MF-0). Positive for the TP53 mutation (VAF 90%), per the next-generation sequence analysis of a recent blood sample collected on June 26, 2023. Iron storage increased: WBC 130 K/Ul, absolute neutrophil count (ANC) 24.26 K/Ul, absolute monocyte count 39,16 K/Ul (53.1%), hemoglobin 7.9 G/DL, platelet 34 K/Ul. Our patient's 10-color flow cytometry analysis for acute leukemia and myeloid/lymphoid neoplasm demonstrated hypercellular bone marrow with markedly increased immature and scattered foci of trilineage hematopoiesis. Phenotype of blasts: CD13+|CD33+ (moderate), CD64+|CD34+|CD38+|CD117+, and HLA-DR. Percentage of abnormal cells: 23% myeloblasts, cell size: large, Immature myeloid precursors positive for CD13| CD33|CD64 (dim), and CD117 are increased (23%) (myeloid-associated antibody) ] mosaic is a very rare genetic makeup requiring further research due to the diversity of both its phenotype and genotype, with further complexity in AML.Furthermore, large-scale cytogenetic studies are warranted in bigger population groups of AML patients in order to identify newly acquired chromosomal aberrations that may aid in cloning novel genes involved in the neoplastic process, with the ultimate goal of developing targeted therapeutic therapy. Research is needed to study these rare driver mutations in AML with TP53 mutations and chromosomal aneuploidies, deletions, derivatives, and translocation.


Compared to the CHT alone group, complex karyotypes were significantly increased in the CHT + RT group . In the PSM cohort, t-AML patients with prior CHT and RT had significantly worse 3-year OS than those with de novo AML . Our results suggest that prior CHT and RT for primary malignancy may be associated with increased relapse and worse OS of allo-HSCT in t-AML.Therapy-related acute myeloid leukemia (t-AML) is a therapeutic challenge as a late complication of chemotherapy (CHT) and/or radiotherapy (RT) for primary malignancy. Allogeneic hematopoietic stem cell transplantation  presents itself as a curative approach. To establish the optimal allo-HSCT strategy for t-AML, we evaluated the relationship between characteristics of primary malignancy and allo-HSCT outcomes. Patients with t-AML or de novo acute myeloid leukemia (AML) who underwent first allo-HSCT in Japan from 2011 to 2018 were identified using a nationwide database. The detailed background of t-AML was obtained by additional questionnaires. Multivariate analysis and propensity score matching (PSM) analysis were performed to detect the prognostic factors associated with t-AML and compare outcomes with de novo AML. We analyzed 285 t-AML and 6761 de novo AML patients. In patients with t-AML, receiving both CHT and RT for primary malignancy was an independent poor-risk factor for relapse and overall survival (OS) (hazard ratio (HR) 1.62; The online version contains supplementary material available at 10.1007/s00277-023-05356-6. Therapy-related acute myeloid leukemia (t-AML) is an important category of myeloid malignancies. The World Health Organization (WHO) classification defines t-AML as acute myeloid leukemia (AML) that develops as a late complication of cytotoxic chemotherapy (CHT) and/or radiation therapy (RT) for malignant or non-malignant disease , 2. WhilCompared to de novo AML, t-AML patients have a dismal prognosis because of their older age, higher proportion of unfavorable cytogenetic abnormalities, and cumulative toxicity of therapy for the primary malignancy , 6, 8, 9As with de novo AML, prognostic factors such as age, performance status, disease status, and type of cytogenetic abnormality influence the outcome of allo-HSCT for patients with t-AML –16. In aMore accurate assessment of the risk of allo-HSCT for patients with t-AML is important to support their decision-making and provide adequate supportive care. Our study objectives were to evaluate the influence of primary malignancy and its therapeutic approach on the outcome of allo-HSCT for t-AML in a nationwide registry data combined with additional research.For this retrospective cohort study, patient, disease, and transplantation characteristics were obtained from the nationwide registration data of the Japanese Society for Transplantation and Cellular Therapy (JSTCT) and the Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT). We included patients ≥ 16 years old with t-AML and de novo AML who underwent their first allo-HSCT from 2011 to 2018. Among t-AML patients, an additional national survey was conducted in each participating institution to obtain clinical information regarding the primary malignancy and its treatment. This study was conducted according to the Declaration of Helsinki and approved by the data management committees of the Transplant Registry Unified Management Program (TRUMP) , 18 and t-AML was defined as patients who received CHT and/or RT for primary malignancy with AML occurring as a late complication , 2. In tThe endpoints were overall survival (OS), disease free survival (DFS), relapse incidence (RI), non-relapse mortality (NRM), and graft-versus-host disease (GVHD). OS was defined as the time from transplantation to death due to any cause, DFS was the time from transplantation to relapse or death due to any cause, RI as the time from transplantation to relapse of AML, and NRM as the time from transplantation to death due to any cause in remission. Acute and chronic GVHD were assessed according to standard criteria , 22.U test for continuous variables. OS and DFS were estimated by the Kaplan–Meier method and was compared using the log-rank test. To identify risk factors associated with OS, univariable and multivariable Cox proportional-hazard regression models were used. Gray’s method was used to estimate the probabilities of RI and NRM, and acute and chronic GVHD. Competing events were death without relapse for RI, relapse for NRM, and death without GVHD for acute and chronic GVHD. The Fine–Gray proportional-hazard model was used to identify risk factors associated with RI and NRM. The variables to be considered were age (divided by median), sex, Eastern Cooperative Oncology Group Performance Status (ECOG-PS) (0–1 vs. 2–4), hematopoietic cell transplantation-specific comorbidity index (HCT-CI) (0 vs. 1-2 vs. 3-), cytogenetic risk (favorable vs. intermediate vs. poor), disease risk at transplantation (low risk: CR1 and CR2 vs. high risk: CR3- and NR), donor source (related bone marrow (Rel-BM) vs. related peripheral blood (Rel-PB) vs. unrelated bone marrow (UR-BM) vs. unrelated peripheral blood (UR-PB) vs. cord blood (CB)), and conditioning (MAC vs. RIC). In t-AML patients, the type of primary malignancy , treatment , type of chemotherapy agent ), and autologous peripheral blood stem cell transplantation (auto-PBSCT) were also considered as variables. If a variable contained more than 5% missing values, missing data were categorized as a separate item.The analyses were conducted as follows: first, an assessment of patient characteristics with respect to t-AML; second, univariate and multivariate analysis of transplant outcomes using a cause-specific hazard function for OS and a cumulative incidence function for RI and NRM; third, a comparison of patient characteristics between t-AML and de novo AML; fourth, univariate and multivariate analysis of outcomes including all t-AML and de novo AML patients; fifth, validation of the above analyses using propensity score matching (PSM) in both t-AML and de novo AML cases. Patient characteristics were compared in t-AML, and between t-AML and de novo AML using the Fisher’s exact test for categorical variables and the Mann–Whitney p-value of < 0.1 in each univariable analysis was also included in each multivariable model, and hazard ratios with 95% confidence intervals (CI) were calculated. Two-sided p-values of < 0.05 were considered statistically significant. The potential interactions between the outcome and selected significant risk factors in the multivariable Cox model were also tested by adding interaction terms to the model. PSM was performed at a one-to-one ratio using a nearest-neighbor matching method with a caliper width of 0.2 of the standard deviation of the logit of the propensity score. The final cohort was evaluated by the Mantel–Haenszel test and histogram to confirm adequate matching. All statistical analyses were performed with EZR version 1.55 , which is a graphical user interface for R . More precisely, it is a modified version of R commander (version 2.7-1) designed to add statistical functions frequently used in biostatistics [The variables included in the final multivariable models were age, sex, ECOG-PS, HCT-CI, cytogenetic risk, disease risk, donor source, and conditioning for OS; cytogenetic risk, disease risk, donor source, and conditioning for RI; and age, sex, ECOG-PS, HCT-CI, donor source, and conditioning for NRM. In addition, the factor associated with t-AML with a atistics .We obtained information on primary malignancy and its treatment from registry data and the additional nationwide survey. Finally, a total of 285 t-AML patients were included. The characteristics of patients with t-AML are presented in Table p = 0.014) (Table p = 0.045) , along with male sex, PS 2-4, and high disease risk (Table The 3-year OS for t-AML patients was 37.5% 95% CI: 31.6–43.5%) and the median duration was 13.8 months (95% CI: 9.5–21.7)  (Table p = 0.019) , whereas cytogenetic risk was not significant  (Table p = 0.90)   patients with more HCT-CI ≥ 3 values (p = 0.002) and less frequent hematological malignancies as the primary malignancy (p < 0.001) than CHT or RT alone group, whereas other variables were not significantly different . However, in the poor cytogenetic risk group, complex karyotype was significantly increased in the CHT + RT group (p = 0.007), whereas − 5/5q, − 7/7q, -17/17p, and monosomal karyotype were not significantly different  , while chronic GVHD at the 3 years was significantly higher in de novo AML than in t-AML   and RI  compared to de novo AML . RI and NRM were also not significantly different  , disease risk at transplantation (low risk vs. high risk), HCT-CI 0 vs. 1–2 vs. 3-), donor source (Rel-BM vs. Rel-PB vs. UR-BM vs. UR-PB vs. CBT), conditioning (MAC vs. RIC), PS (0–1 vs. 2–4), and cytogenetic risk (favorable vs. intermediate vs. poor). In the final PSM cohort, 197 patients were assigned to each de novo AML and t-AML with CHT or RT alone group