Enhanced Ca2+ release through the mutant channels induces excessive heat development upon exposure to volatile anesthetics. However, the mechanism by which Ca2+ release is accelerated at an elevated temperature is yet to be identified. Fluorescence Ca2+ imaging with rapid heating by an infrared laser beam provides direct evidence that heat induces Ca2+ release through the RyR1 channel. And the mutant channels are more heat sensitive than the wild-type channels, thereby causing an increase in the cytosolic Ca2+ concentration in mutant cells. It is likely that the heat-induced Ca2+ release participates as an enhancer in the cellular mechanism of MH.Malignant hyperthermia (MH) is a life-threatening disorder caused largely by mutations in ryanodine receptor type 1 (RyR1) Ca 2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum–targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels’ heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca Malignant hyperthermia (MH) is a life-threatening disorder, triggered by volatile anesthetics or depolarizing muscle relaxants , 2. In M2+-release channels, dihydropyridine receptors , and Src-homologous-3 and cysteine-rich, domain-containing protein 3 in skeletal muscles (32+ is released from the sarcoplasmic reticulum (SR) through RyR1 and causes reversible sarcomere contraction during excitation–contraction coupling. In skeletal muscles expressing these mutants, however, anesthesia-enhanced Ca2+ release from the SR elevates the intracellular Ca2+ concentration ([Ca2+]i) and causes uncontrolled hypermetabolism and hyperthermia  in the progression of MH. However, the intrinsic feature of this positive feedback loop remains elusive because it is still unclear how elevated body temperature affects Ca2+ release.MH is caused by mutations in ryanodine receptor type 1 (RyR1) Camuscles 3. Most hurthermia –8 and in mutants . These s2+ in the intracellular space as well as in the endoplasmic reticulum (ER). Our quantitative analysis demonstrated that the heat-induced Ca2+ release (HICR) mechanism contributes an additional positive feedback loop between Ca2+ and thermogenesis. The RyR1 mutants related to MH displayed greater heat sensitivity than did wild-type (WT) RyR1, and the sensitivity varied among mutants. Likewise, skeletal muscles from MH model mice were found to be more heat sensitive than those from WT mice. These findings led us to propose the following cascade regarding the progression of MH: 1) MH is triggered by Ca2+ release from the SR via the RyR1 mutants upon exposure to volatile anesthetics; 2) a small magnitude of heat stress causes Ca2+ release via the heat-hypersensitive RyR1 mutants; 3) the released Ca2+ causes hypermetabolism and hyperthermia, accelerating Ca2+ release; and then 4) the thermogenic cascade possibly results in lethal hyperthermia if not treated properly.In the present study, by applying optically controlled local heat pulses –14 to hu2+ release in HEK293 cells expressing either WT or RyR1 mutants. We selected three mutants across the rank order of the activity of RyR1 mutants in the N-terminal region, with Q156K, R164C, and Y523S having the lowest, intermediate, and highest ranking, respectively  (152+ leakage (2+] and hence lower [Ca2+] in the SR or ER)  15  , 19.2+ imaging was performed simultaneously (T) was adjustable down to 1 °C or lower by reducing the laser power or by increasing the distance from the point at which laser irradiation was focused . The present experimental system, therefore, allowed us to apply the heat pulse to cells with various ΔTs in the same field of view (138 µm × 138 µm).Heat stimulation was applied using a focused 1,455-nm near-infrared (IR) laser beam –14, 20, aneously B. Changeaneously –13. The aneously C. The am2+]i changes were observed in control cells with no induced RyR1 expression  or in cells expressing WT RyR1 when heat pulses of ΔT = 10 ± 2 °C were applied at the base temperature T0 = 24 °C (Movie S1). In contrast, there were rapid (<∼500 ms) and large [Ca2+]i increases  upon heating, and the Ca2+ bursts were sustained until recooling in most R164C cells in the field of view (Movie S2). In Q156K and Y523S cells, [Ca2+]i decreased during heating, and Ca2+ bursts were observed after the onset of recooling . The decrease in [Ca2+]i during heating in those cells  is likely attributable to heat-activated Ca2+ uptake via activation of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), as suggested by us . Therefore, the differential magnitudes of the heat-induced decrease in [Ca2+]i across the mutant lines are not likely coupled with a difference in the SERCA expression levels. The maximum changes in the Ca2+ bursts of fluorescence intensity (ΔFmax)/F0 during the 20 s after the onset of heating were significantly larger in WT, Q156K, R164C, and Y523S cells than in −Dox cells . Significant Ca2+ bursts were observed in −Dox and WT cells with mean ΔFmax/F0 values comparable to that of Q156K  at T0 = 36 °C. R164C was chosen as the RyR1 mutant of the middle-rank order. To compare the mutant response with its WT, WT RyR1 cells were examined. −Dox was used to examine the contribution of endogenous Ca2+ channels. The Ca2+ burst was preserved in all WT, R164C, and −Dox cells in Ca2+-free medium, whereas the burst was suppressed when Ca2+ was depleted from the ER by 2 µM thapsigargin (an inhibitor of SERCA). These results indicate that the Ca2+ source for the heat-induced Ca2+ burst is the ER, not the extracellular space.The RyR1 mutant−dependent variation in heat-induced Ca2+ channels that function physiologically on the ER membrane are ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Here, Ca2+ bursts in WT RyR1 and R164C cells were blocked by 100 µM ryanodine (an inhibitor of RyRs) , indicating that these RyR1 channels operate in Ca2+ bursts. The suppression of Ca2+ bursts by ryanodine was much less effective in −Dox cells than in WT RyR1 and R164C cells , providing confirmatory evidence that the contribution of the endogenous RyRs , demonstrating a major contribution of IP3Rs to Ca2+ bursts in −Dox cells. The relatively large Ca2+ bursts in −Dox cells at T0 = 36 °C compared with those at T0 = 24 °C  was reduced by 2-APB in WT RyR1 cells, Ca2+ bursts were observed . Furthermore, Ca2+ bursts were not significantly suppressed by 2-APB in R164C cells  via expression of the ER-targeted fluorescent Ca2+ probe G-CEPIA1er . The quick recovery suggests that the decrease during the heat pulse was due to thermal quenching of G-CEPIA1er fluorescence. After heating, the signal decreased and reached the minimum at ∼5 s after the cessation of the heat pulse . Furthermore, the second decrease appeared to coincide with the Ca2+ burst in the cytosol  and that of the Ca2+ burst (ΔFmax/F0 of fluo-4) (2+ burst arises from Ca2+ release from the ER through RyR1.To further strengthen the idea that the ER is the source of CaCEPIA1er  in WT Ry cytosol E. TherefWT cells B. These  fluo-4) C, strengT, as well as by evaluating the fraction of cells exhibiting Ca2+ bursts . At T0 = 36 °C, the Ca2+ burst was more frequently observed when ΔT was larger in WT cells and in all mutant cells. However, the heat sensitivities varied between them. For example, ∼40% of R164C cells showed Ca2+ bursts in response to a heat pulse of ΔT = 1 °C, but only ∼20% of WT and other mutant cells showed such Ca2+ bursts . This variation in heat sensitivity was quantitatively compared by using the threshold values of ΔT (ΔTth) at which 50% of cells responded to heating . We further examined R164L, which had a rank order of activity similar to that of R164C . Resting [Ca2+]i was higher in R2509C cells than in WT cells , which is consistent with the increased resting leak of Ca2+ and the consequent enhancement of store-operated Ca2+ entry . But the [Ca2+]i increases were observed in R2509C muscles during heating (Movie S7). An increase in the amplitude of heating to ΔT = 9 ± 1 °C elevated [Ca2+]i by a slight magnitude in WT muscles  but caused significantly larger Ca2+ bursts in R2509C muscles . The heat-induced Ca2+ bursts were likewise observed in HEK293 cells expressing rabbit RyR1-mutant R2508C, which corresponds to mouse R2509C . At the physiologically relevant temperature of 36.5 °C, heat-induced [Ca2+]i increases were likewise observed in R2509C muscles upon ΔT = 4.0 ± 0.5 °C  thermal damage to biomolecules and cells caused by long periods of heat exposure.MH is a life-threatening disorder caused by mutations in RyR1, manifested as a sudden and irrepressible body temperature elevation. In the present study, we directly demonstrated that Ca release  through elease 15, 38–44. ease 1538, 45. Thi2+ indicators used in the present study. It has been reported that in the range of 20 to 37 °C, a rise in temperature lowers the dissociation constant (Kd) of fluo-4 for Ca2+ , as well as the fluorescence intensity, due to thermal quenching  (2+]i in HEK293 cells expressing RyR1 mutants is within the range of 40 to 90 nM at room temperature (23 to 25 °C) . This is because an increase in the fluorescence intensity of fluo-4 was observed after the cessation of heating in these cells . However, ΔFmax/F0 of fluo-4 could be reached during heating in cells expressing R164C or R164L. Then, the response probability may have been either overestimated or underestimated in these cells. The elevation of the fluorescence intensity of fluo-4 was insignificant because the signal was not sufficiently increased in cells expressing WT, Q156K, and Y523S during heating . In other words, the ΔTth values of these cells could have been smaller than those obtained in the present study and, hence, substantially more heat hypersensitive. In this case, the rank order of ΔTth  and that of the Ca2+ burst (ΔFmax/F0 of fluo-4) . These findings clearly demonstrate that heat-induced Ca2+ bursts are not the temperature-dependent properties of fluorescent Ca2+ indicators.Here, we discuss the possible effects of the local heat pulse on the Cactively) . Becauseo 25 °C) , it is l heating E. If a s of ΔTth C will rent study B. During fluo-4) C. In ske2+ release and thermogenesis accelerates the progression of MH. It has been reported that metabolic activity is higher in R163C mice than in WT mice , the resultant increase in [Ca2+]i exacerbates the SERCA-based thermogenesis. The present study documented that an increase in ambient temperature of <2 °C causes Ca2+ release through the channels (2+ release (HICR). HICR was observed in WT cells and all RyR1 mutant cells as Ca2+ bursts during heating with a relatively strong pulse . With a weaker pulse, HICR was somewhat unclear during heating; rather, it appeared as sustained Ca2+ bursts after recooling. We previously reported that prolonged Ca2+ bursts occur upon recooling in HeLa . Both SERCA and RyR1 are localized in the ER membrane; it is therefore possible that HICR plays a critical role in the progression of MH. We previously examined the increase in rectal temperature induced by isoflurane in R2509C mice and stated that the elevation of the rectal temperature appeared as a two-phase reaction: it rose slowly at first and then surged at ∼39 °C . In future studies, therefore, rapid heating combined with simultaneous imaging of Ca2+ and temperature should be performed in skeletal muscles of living mice to quantitatively analyze the contribution of HICR in the progression of malignant hyperthermia.The heat hypersensitivity of RyR1 mutants suggests that a positive feedback between Ca WT mice . In a sinditions . It is achannels . We desi in HeLa  and WI-3 in HeLa , 50. It t ∼39 °C . This fit ∼39 °C , 52 in i2+]i activates the production of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) in heterozygous knock-in mice with a Y524S mutation (Y522S in humans) for a longer period of time compared with the present study. Either ROS or RNS, or both may modify RyR1 and other skeletal muscle proteins to promote Ca2+ release via unknown mechanisms. Because of the relatively short time scale of HICR, the slower processes of ROS and RNS production and posttranscriptional modifications may enhance HICR and thereby aggravate MH.We consider that HICR coincides with the positive feedback loop in a relatively long time scale that Durham et al.  and LannTth is smaller in R164C and Y523S cells than in WT cells  (Tth was greater in Y523S cells than in R164C(L) cells  . This analysis revealed strong responses in R164C and Y523S cells, suggesting that the ΔFmax/F0 values in 2+]ER is low . These data support the relevance of the comparison of heat sensitivity between mutants as determined on the basis of response probability to 35 °C . We alsocussion) , 19, ΔTtL) cells C. BecausFmax/F0)  by the rbability .2+]i and [Ca2+]ER in mutant cells were significantly higher and lower, respectively, than those in −Dox and WT cells . Because the SERCA activity depends on [Ca2+] in both the cytosol and ER . The Q156 and R164 in domain A interact with domain B to form interface 1 within the ABC subunit at the N terminus  RyR1 mutant structures are unstable under environmental heat stress (2+ levels in R164C(L) and other N-terminal mutant cells relative to WT cells are lower at 36 °C than at room temperature  was observed in a fashion different from that in muscle cells . Because homozygous mice are lethal  and the neighboring domains at the N-terminal “hotspot” of residues 35 to 614, one of three known hotspots related to MH in RyR1  , and hygromycin  were purchased from Nacalai Tesque Inc. . Fetal bovine serum , penicillin and streptomycin , Lipofectamine 2000 , and fluo-4 AM  were purchased from Thermo Fisher Scientific . Blasticidin (ant-bl-1) was purchased from InvivoGen . Collagen type 1  was purchased from the Research Institute for the Functional Peptides . Dox , 2-APB , and poly (methyl methacrylate)   were purchased from Sigma-Aldrich . Thapsigargin  was purchased from Merck . Ryanodine  was purchased from FUJIFILM Wako Pure Chemical Corporation . Europium (III) thenoyltrifluoroacetonate trihydrate (Eu-TTA)  was purchased from Acros Organics . The stocks of 1 mM fluo-4 AM in dimethyl sulfoxide (DMSO), 2 mM thapsigargin in DMSO, 10 mM ryanodine in distilled water, and 100 mM 2-APB in DMSO were stored at −20 °C until use.Dulbecco's Modified Eagle Medium (DMEM) , HEK293 cells stably transformed with rabbit skeletal muscle RyR1 or its mutants , Y523S, or R2508C; human Q155K, R163C(L), Y522S, or R2508C) were generated, and the expression levels of WT RyR1 and the RyR1 mutants were confirmed to be similar in previous studies 15, 66. Thel-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin) with 100 μg/mL hygromycin and 15 μg/mL blasticidin at 37 °C in 5% CO2. To coat the flasks and dishes with collagen, they were filled with a 0.001% collagen type 1 solution in distilled water for 1 h at 37 °C. The flasks and dishes were washed with the fresh culture medium just before use.The HEK293 cells were cultured in flasks or on dishes coated with collagen  in culture medium  at 37 °C in 5% CO2+ dynamics were studied using the fluorescent Ca2+ probe fluo-4 AM. The cells were incubated in HEPES-buffered saline  containing 1 µM fluo-4 AM for 30 min at room temperature. The solution was replaced with fresh HBS, and the cells were incubated under the microscope for at least 10 min before observation to allow temperature stabilization to 24 ± 1 °C or 36 ± 0.5 °C. In some experiments, the cells were observed in Ca2+-free solution  at least 15 min after incubation. To inhibit the activity of SERCA or IP3R, the cells were incubated in HBS containing 1 µM fluo-4 AM and either 2 µM thapsigargin or 100 µM 2-APB for 30 min at 24 °C, and then observed in HBS containing 2 µM thapsigargin or 100 µM 2-APB. To inhibit RyR1, the fluo-4–loaded cells were observed in HBS containing 100 µM ryanodine.Cytoplasmic Ca2+ dynamics in the ER, HEK293 cells stably expressing G-CEPIA1er (er construct were produced by replacing the complementary DNA of enhanced green fluorescent protein (GFP) in pCL20c-MSCV-AcGFP-WPRE   . HEK293Ter, and Eu-TTA were excited by a solid-state illuminator . The fluorescence and the bright-field images were observed with an inverted microscope  equipped with a dichroic mirror , an emission filter , an objective lens , and an electron-multiplying charge-coupled device camera . The temperature of the solutions on the sample stage was adjusted to 36 ± 0.5 °C using a thermostatically controlled incubator ; otherwise, the temperature of the solutions was 24 ± 1 °C.The microscope with the local heating system was described in detail in our previous reports –14, 20. F/F0 = (Fheating − Ilaser)/(Fbefore − Iback) − 1, where Fheating was the intensity at the end of the heating period , and Ilaser was the background intensity caused by light scattering of the IR laser beam. Iback was the background intensity when the excitation light was off. Photo-bleaching was corrected by fitting a single exponential curve. ΔF/F0 of Eu-TTA was converted to ΔT using the relationship between ΔT and ΔF/F0 (−2.7% °C−1 at 24 °C and −4.1% °C−1 at 36 °C) . The changes in local temperature were measured by thermal quenching of Eu-TTA that had been spin-coated on a glass base dish by a solution containing 5 mg/mL Eu-TTA and 10 mg/mL PMMA in acetone –13. Relat 36 °C) , 13.er, the outlines of cells were manually tracked in the bright-field images. Then the fluorescence intensities of fluo-4 and G-CEPIA1er were measured within the outlined areas. The distance between the area center and a laser spot was defined as the distance between the cell and the heat source. The ΔF of fluo-4 was calculated from F − Fbefore, where F was the fluorescence intensity at an arbitrary time, and Fbefore was the intensity just before heating was initiated . The basal fluorescence intensity of fluo-4 (F0) was calculated from Fbefore − Iback. The peak intensity of fluo-4 (ΔFmax/F0) was calculated from the maximum ΔF/F0 during the 20 s after heating initiation. Light scattering by the IR laser beam (Movie S1) was subtracted to calculate ΔFmax/F0 during heating. No noticeable photo-bleaching of fluo-4 was observed during the measurement /(Fbefore − Iback). To equalize the exposure time of excitation light (485 nm) and that in the heating experiments, Fbefore was set to the fluorescence intensity 10 s after starting the observation. Fmax was the maximum intensity obtained from 10 to 30 s after starting the observation. The cumulative probability of ΔFmax/F0 was fitted by a cumulative distribution function of the Gaussian distribution (x) is an error function. The threshold ΔFth of ΔFmax/F0 was defined as µ + 1.96σ. If ΔFmax/F0 induced by a heat pulse was higher than ΔFth, the [Ca2+]i increase response induced by the heat pulse was considered significant. The threshold of ΔT (ΔTth) was defined as the ΔT that induced a significant [Ca2+]i increase (ΔFmax/F0>ΔFth) in 50% of cells /(Fbefore − Iback), where Fmin was the minimum intensity of G-CEPIA1er obtained from 2.4 s to 10 s after heating initiation. In this calculation of ΔFmin/F0, photo-bleaching was corrected by fitting with a single exponential curve in Excel 2016 (Microsoft).The Δ2, 1 mM MgCl2, 11 mM glucose, 5 mM HEPES, pH 7.4) containing 2 mg/mL bovine serum albumin  for 2 h at 37 °C, followed by gentle trituration in collagenase-free HEPES–Krebs solution. The procedures for experiments with skeletal muscles are described in SI Appendix, Supplemental Materials and Methods.Animal-related procedures were in accordance with the guidelines of The Jikei University School of Medicine. Mice were housed in isolator cages, fed with food and water ad libitum, and kept under controlled lighting conditions (12 h-light/12 h-dark) in specific pathogen-free conditions at The Jikei University School of Medicine. Isolated skeletal muscle cells from mice were prepared based on a previously described procedure with modifications . BrieflyU test was used. These tests were performed using EZR (version 1.51) . The temperature and ΔT are reported as central value ± range where applicable.Multiple groups were compared using the Steel or Dunnett’s test. For comparisons of two independent samples, the Mann–Whitney on 1.51) . StatistSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations corresponded with the rate of temperature decrease. We did not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance but showed that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As intertidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signaling interacts with osmotic signaling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signaling pathways may play an important role in their cellular responses to multiple simultaneous stressors.Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater, and intertidal environments. The latter can experience substantial long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We found that the diatoms  A calcium signaling pathway in marine diatoms is activated by cold temperature and enhances survival during simultaneous hypo-osmotic stress. Diatoms are a group of silicified unicellular algae that represent one of the most important primary producers in modern oceans. They are abundant in diverse marine environments, most notably in polar and temperate upwelling regions, where they play a critical role at the base of the marine food web . Diatom 2 emissions are set to have profound influence on marine ecosystems  cation channel family of temperature-sensitive ion channels  elevation, which leads to changes in gene expression and the establishment of cold tolerance , support for the role of high temperatures in inducing [Ca2+]cyt elevations is mixed, although Ca2+ elevations are observed within the chloroplast , which is proposed to either function as a Ca2+ channel or to activate other Ca2+ channels , hypo-osmotic shock, and the detection of toxic aldehydes (Phaeodactylum tricornutum cells expressing the bioluminescent Ca2+ reporter aequorin did not detect [Ca2+]cyt elevations in response to low (4°C) or high (37°C) temperature . Note that actual temperatures in the perfusion dish differed by ±2°C from these target temperatures due to equilibration of the small volume of warm or cold perfusate with room temperature (RT). Actual dish temperatures were therefore recorded and are displayed for all experiments. We routinely observed a single transient [Ca2+]cyt elevation in cells exposed to a cold shock from 30°C to 12°C   ( n = 63) . In contevations . No cyt elevations observed during cold shock were represented by a >10-fold increase in R-GECO1 fluorescence. Assuming a Kd of 480 nM for R-GECO1 and comparison with published maximum F/F0 values  representing large [Ca2+]cyt elevations were analyzed further.The cyt elevations were also observed when a cold shock (30°C–12°C) was applied to cells held at 22°C, rather than 30°C, indicating that the cold shock response was not a consequence of prior warming of the cells  resulted in [Ca2+]cyt elevations in 100% of cells examined (n = 45), whereas only 7% of cells exhibited a [Ca2+]cyt elevation at a cooling rate of 0.4°C s−1 (n = 45)  . The ampng rates . Examinapulation . These d2+]cyt elevations (−1. A temperature decrease of 8.8 ± 0.4°C induced a [Ca2+]cyt elevation in 38.4% of cells (n = 126), whereas greater decreases in temperature resulted in [Ca2+]cyt elevations in nearly all cells , we next examined the absolute temperature decrease required to initiate signaling. Cells were perfused at 30°C for 1 min and then perfused at a constant flow rate with cold artificial seawater (ASW) (4°C) for different durations to vary the decrease in temperature whilst maintaining similar rates of cooling. A very brief perfusion (4 s) lowered the temperature by 2.4 ± 0.6°C but did not induce cyt elevations in all cells within a field of view , which is proposed to mimic cold-induced membrane rigidification    . Rutheni channel . RR alsoic shock , 2021b. evations . HoweverF0 >1.5) , indicatPhaeodactylum tricornutum contains three EukCatA channels, which represent a novel class of voltage-gated Na+/Ca2+ channel in eukaryotes related to single-domain voltage-gated Na+ channels in bacteria (BacNav) (P. tricornutum strains with knockout mutations in EUKCATA1 (deletions of 38–124 bp in the region of the gene encoding the pore of the ion channel) exhibited an altered response to cold shock. The percentage of responding cells and the mean maximal amplitude of the [Ca2+]cyt elevations did not differ significantly from control PtR1 cells  . As BacN(BacNav) , we examR1 cells , indicatrization , this re2+ signal was required for P. tricornutum cells to survive following a cold shock. We applied cold shocks to cells in the absence of external Ca2+ to inhibit the cold-induced [Ca2+]cyt elevations during cooling and then returned cells into ASW + Ca2+ to monitor their growth at either 4°C or 18°C  administered to cells at 25°C resulted in a single [Caeviously   . The mean maximal amplitude of the [Ca2+]cyt elevations caused by the three different treatments were all significantly different from each other, with the cold shock alone causing the lowest and the combined cold- and hypo-osmotic shock causing the highest [Ca2+]cyt elevations (A stronger hypo-osmotic shock (100% ASW to 50% ASW) resulted in a rapid [Ca applied . In compitiation . Combiniriments) . These cevations .2+]cyt elevations induced by hypo-osmotic shock exhibit significant differences in amplitude and timing in the presence of a simultaneous cold shock. This indicates that the cold shock stimulus is additive and of sufficient magnitude to influence cellular Ca2+ signaling during hypo-osmotic stress. We therefore investigated whether Ca2+ signaling during cold shock may influence the short-term survival of P. tricornutum under hypo-osmotic stress.Taken together, cyt elevations in P. tricornutum do not play an obvious role in acclimation to low temperatures. We found no longer-term growth effects of experimentally blocking the cold shock Ca2+ signal. Cold signaling in P. tricornutum therefore differs from plants and insects  is most likely initiated by activation of mechanosensitive channels due to the increase in cell volume . Mechano2+ was small compared to the much greater reduction in mortality in the presence of external Ca2+. We were unable to identify pharmacological inhibitors to selectively inhibit either osmotic or cold associated Ca2+ signaling and the removal of external Ca2+ completely inhibited both signaling pathways. Dissecting the individual contributions of these signaling pathways to cell survival during simultaneous shocks is therefore not currently easily achieved. Selective inactivation of the underlying molecular mechanisms through genetic approaches will most likely be required to fully understand the nature of the cross-talk between the signaling pathways.We should also consider that low temperature may have a direct effect on reducing mortality during hypo-osmotic stress that is independent of the signaling component, for example by increasing cell wall rigidity. However, the protective effect of cold shock in the absence of CaCellular responses to stressors are commonly examined in isolation in the laboratory in order to simplify the elucidation of the signaling pathways responsible. However, organisms often have to respond to inputs from multiple stimuli simultaneously in their natural environment, leading to cross-talk between signaling pathways. Cross-talk in cell signaling can occur when two distinct stimuli trigger a shared cellular response that confers tolerance to both stressors. This may involve activation of a common receptor or activation of independent receptors that converge on a specific node in the signaling pathway . Cross-tSaccharomyces cerevisiae exhibits a high osmolarity (HOG) response to hyper-osmotic stress that results in increased production of the compatible solute, glycerol. The HOG response is mediated by a mitogen-activated protein kinase pathway that can also be activated by other stimuli including both cold and heat shocks. Heat shock activates the HOG pathway indirectly by stimulating loss of glycerol, leading to hyper-osmotic stress  regulon are also inducible by drought . The colc stress .2+-mediated cellular signaling mechanisms is an important consideration in the response of marine organisms to multiple stressors. While our results are discussed primarily in the context of the intertidal zone where rapid substantial changes in temperature are a regular occurrence, the conserved nature of cold-induced Ca2+ signaling in T. pseudonana suggests that this pathway may be important more widely in diatom ecology. The cold-induced [Ca2+]cyt elevations in T. pseudonana exhibit different characteristics from P. tricornutum that may reflect differences in their physiological response. Planktonic diatoms will undoubtedly encounter substantial fluctuations in temperature and salinity in near-shore and estuarine environments or when they are mixed through the thermocline, although the magnitude and rate of the temperature changes are likely to be lower. Diatoms inhabiting sea ice environments may also experience rapid changes in temperature and salinity, for example, during flushing of hyper-saline brine channels with melt water  encased in acrylic resin, recording in 30-min intervals with a resolution of 0.1°C. The Envlogger was secured to the substrate using Z-Spar A-788 epoxy resin roughly 3 cm below the surface waters of a shallow mid-shore rockpool measuring ∼8-cm deep at Looe Hannfore, Cornwall, UK  from July 1, 2019 to July 7, 2019.P. tricornutum strain used in this study was CCAP 1055/1 . A P. tricornutum strain transformed with the R-GECO1 Ca2+ biosensor (PtR1) was generated as described previously  were generated by CRISPR–Cas9-mediated gene editing, with two single-guide RNAs ∼50-bp apart targeted to the pore region of PtEUKCATA1 resulting in deletions of 38–124 bp (T. pseudonana strain expressing the R-GECO1 biosensor (TpR1) was generated as described in 2SiO3.5H2O and the exclusion of vitamins (P. tricornutum only). For imaging experiments, cells were acclimated to an ASW medium for minimum 10 days prior to analysis. ASW contained 450-mM NaCl, 30-mM MgCl2, 16-mM MgSO4, 8-mM KCl, 10-mM CaCl2, 2-mM NaHCO3, 97-µM H3BO3, f/2 supplements, and 20-mM HEPES (pH 8.0). Cultures were grown at 18°C with a 16:8 light/dark cycle under illumination of 50 µmol m−2 s−1.The wild-type eviously . Three e8–124 bp . The T. Cell culture measuring 500 µL of was added to a 35-mm microscope dish with glass coverslip base  coated with 0.01% poly-L-lysine  to promote cell adhesion to the glass surface. Cells were allowed to settle for 5–20 min at RT under light. R-GECO1 was imaged using a Leica DMi8 inverted microscope  with a 63 × 1.4NA oil immersion objective, using a Lumencor SpectraX LED light source (4% intensity) with a 541–551-nm excitation filter and 565–605-nm emission filter. Images were captured with a Photometrics Prime 95B sCMOS camera  with a 300-ms exposure. Images were captured at 3.33 frames per second using Leica application suite X-software v.3.3.0.−1. To achieve rapid changes in temperature in the dish, the perfusion was switched between solutions of different temperature to achieve target temperatures of approximately 10°C, 22°C, or 30°C respectively. Actual dish temperature was recorded using a Firesting micro optical temperature sensor . For the majority of experiments, cells were perfused with warmer media (dish temperature 30°C) for 1 min prior to application of the cooling shock (these conditions reflect those observed in the rockpool observations). The perfusion flow rate was altered to achieve different temperature change rates. As cooling rate was not linear, the maximum cooling rate was defined as the largest decrease temperature within a one second period.The dish was perfused with ASW without f/2 nutrients at a standard flow rate of 16 mL min2+ was removed by perfusion with ASW without CaCl2 containing 200-μM EGTA. RR was added to cells at a final concentration of 10 µM, 5 min prior to cold shock treatment. Menthol was prepared as a 1-M stock solution in DMSO and used at concentration of 1 mM, resulting in a final DMSO concentration of 0.1% v/v.External CaF-values. The change in the fluorescence intensity of R-GECO1 was then calculated by normalizing each frame by the initial value (F/F0). [Ca2+]cyt elevations were defined as any increase in F/F0 above a threshold value (1.5). The duration of a [Ca2+]cyt elevation was defined as the peak width at half maximal amplitude. To visualize the spatial distribution of a [Ca2+]cyt elevation, each frame was divided by a corresponding background image generated by applying a rolling median (30 frames) to the image-series (Image J). The resultant time series images were pseudo-colored to indicate changes in fluorescence.Images were processed using LasX software (Leica). The mean fluorescence intensity within a region of interest (ROI) over time was measured for each cell by drawing an ROI encompassing the whole cell. Background fluorescence was subtracted from all cellular Graphs and statistical analyses were performed using Sigmaplot v14.0 . Error bars represent standard error of the mean. Unless indicated otherwise, imaging experiments were repeated three times with independent cultures on different days to ensure reproducibility of the response.t test, and when not passed with Mann–Whitney’s rank sum test. Statistical analysis of datasets with more than two groups were performed using an analysis of variance (ANOVA) followed by a Holm–Sidak post hoc test when the normality test was positive. When the normality test was negative, Kruskal–Wallis’ one-way ANOVA on Ranks was used instead. All statistical tests were performed with Sigmaplot v14.0.3.192 (Systat software Inc).Normal distribution of respective datasets was tested using Shapiro–Wilk’s normality test. When passed, statistical analysis of datasets with two groups were done with Student’s 6 cells mL−1). The culture was divided into 10-mL aliquots and cells were pelleted by centrifugation (1250 g at 18°C). Cells were washed in 40-mL ASW ± Ca2+ and pelleted again by centrifugation. Cells were then resuspended in their respective treatments (20 mL of ASW ± Ca2+ at 18°C or 4°C to administer a rapid cold shock). The temperature of the ASW was monitored after mixing and found to have remained at 4°C. After 10 min, 2 mL of each culture was used to inoculate culture vessels containing 18 mL of standard ASW F/2 media containing 10-mM Ca2+ (approximately starting density of 6.8 × 104 cells mL−1) and cultures were grown at 18°C or 4°C for 5 days or 30 days, respectively. The cells were therefore only in media without Ca2+ for 10 min, which acts to inhibit Ca2+ signaling during rapid cooling but avoids prolonged exposure to very low external Ca2+.For the growth curves, cells were grown to mid exponential phase (2.73 × 106 cells mL−1) were pelleted by centrifugation (1250 g at 18°C). Cells were washed twice with 10-mL ASW ± Ca2+ and 250-μL aliquots were taken. To apply the hypo-osmotic and cold shock treatments, 750 µL of ASW (±Ca2+) or deionized water at two different temperatures (20°C or 4°C) were added to each tube. Addition of water results in a severe hypo-osmotic osmotic shock  simultaneously with the temperature shock. The cells were then incubated at their respective temperatures for 3 min prior to addition of 5-μM Sytox Green . All treatments were then incubated at 20°C 15 min in darkness. Cell viability was measured with a LUNA-FL Dual Fluorescence Cell Counter  to count live (displaying red chlorophyll fluorescence) versus dead cells (Sytox Green stain) with following settings: Excitation intensity green = 11, red = 7, count threshold for both = 3.To examine the effect of temperature on cell viability during hypo-osmotic shock, 10 mL of a late log phase culture  into the pipette tip by suction. Pipettes were then back-filled with the filling solution . The reference electrode was filled with 3-M KCl, and data were recorded using an AxoClamp 2B amplifier, with pClamp v10.6 software . Each K+ microelectrode was calibrated using a two-point calibration with standard KCl solutions. The mean slope of the calibrated electrodes was 53.0 ± 1.3 mV per decade (±se).Keviously . Clark GP. tricornutum cells from exponential culture containing 106–107 cells mL−1 were centrifuged at 1250 g for 10 min and re-suspended in 1 mL of ASW. The cells were then allowed to settle on a poly-L-lysine coated microscope dish. Cells were perfused with ASW or ASW-Ca2+ (0-μM Ca2+ + 100-µM EGTA) and cold shocks were applied as described for the microscopy observations. Control experiments were performed in the absence of P. tricornutum cells to ensure that the change in temperature did not alter the performance of the K+ microelectrodes.For the measurements, 10 mL of The following materials are available in the online version of this article. [Ca2+]cyt elevations in response to repeated cold shocks. Cold shocks from different starting temperatures. Cell volume during cold shock. Proposed Ca2+ signaling pathways in response to osmotic and cold stress.The work was supported by a European Research Council Advanced Grant to CB (ERC‐ADG‐670390) and a Natural Environment Research Council award to GLW (NE/V013343/1).Conflict of interest statement. None declared.kiac324_Supplementary_DataClick here for additional data file.


Polysorbate 80 for injection (TW80) is a common excipient used for injection whose macromolecular impurities, including those that cause anaphylactoid reactions, are frequently ignored. The main aim of this study was to prove that the macromolecular impurities in the excipient are an important cause of anaphylactoid reactions. Component A (containing macromolecules > 100 kDa), Component B (containing macromolecules from 10 to 100 kDa), and Component C (containing substances < 10 kDa) were prepaired from the original TW80 using ultrafilters. The original TW80 contained numerous substances with molecular weights > 10kD. The original TW80 and Components A and B caused strong anaphylactoid reactions in both guinea pigs and rabbits by intravenous administration. Moreover, the original TW80 and Components A and B even caused strong passive cutaneous anaphylactoid (PCA) reactions and pulmonary capillary permeability. The PCA reaction and increased permeability were partly prevented by cromolyn sodium. Additionally, the original TW80 and Components A and B caused vasodilation and severe hemolysis in vitro. The anaphylactoid reactions were associated with histamine release but not with mast cell degranulation. Nevertheless, Component C almost caused no anaphylactoid reactions or hemolysis and was weaker in the few reactions that ocurred. Taken together, these results suggest that macromolecular substances are one of the main risk factors responsible for anaphylactoid reactions and hemolysis caused by TW80.The online version contains supplementary material available at 10.1186/s40360-022-00591-5. Polysorbate 80, also named Tween 80, is a commonly used solubilizer and considered as a safe excipient . The excWith a higher quality standard, polysorbate 80 for injection (TW80) is a popular excipient used in the modern injection industry . Indeed,Anaphylactoid reaction is also known as non-allergic reaction, the symptoms of which are very similar to those of anaphylactic  reaction. It is certain that anaphylactic reaction is mediated by immunoglobin E (IgE) and the symptoms will not appear at the first exposure. Different from anaphylactic reaction, the mechanisms of anaphylactoid reaction may associate the activation of innate immune system, including complements , Toll-liTW80 is a viscous liquid, which is almost colorless or light yellow. Theoretically, 1 mol of polysorbate 80 can be synthesized from 1 mol of sorbitol, 20 mol of ethylene oxide, and 1 mol of oleic acid . The excIn this study, we attempted to isolate the macromolecules of TW80 and verify the risk of anaphylactoid reaction.Polysorbate 80 for injection (TW80)  was manufactured by Nanjing Well Pharmaceutical Co., Ltd.  and was approved by the National Medical Products Administration of China (NMPA). C48/80 was manufactured by Sigma-Aldrich. Normal saline for injection (NS) and water for injection were manufactured by Chenxin Pharmaceutical Co., Ltd. . Enzyme-linked immunosorbent assay (ELISA) kits for Guinea pig IgE (Lot. MM-034001), C-reactive protein (CRP) (Lot. MM-3531202), high mobility group protein (HMGB1) (Lot. MM-8750102), and complement 5a (C5a) (Lot. MM-8750302) were manufactured by Jiangsu Enzyme Industry Co., Ltd. . ELISA kits for rabbit soluble complement terminal complex Sc5b-9 (Sc5b-9) (Lot. MM-8253002), complement factor B (CFb) (Lot. MM-8258102), mannose binding lectin (MBL) (Lot. MM-8258801), bradykinin (BK) (Lot. MM-8258201), β-hexosaminidase (β-HEX) (Lot. MM-8257201), lysozyme (LZM) (Lot. MM-8258601), and histamine (HIS) (Lot. MM-023801) were manufactured by Jiangsu Enzyme industry Co., Ltd. . Cromolyn sodium (Lot. D16J12G137501) and Evans Blue (Lot. L04D11J133367) were manufactured by Shanghai Yuanye Biotechnology Co., Ltd. . Histamine phosphate of the analytic impurity product was manufactured by Beijing Bo’aotuo Technology Co., Ltd . Hematoxylin–eosin (HE) staining kits were manufactured by Boster Biological Technology Co., Ltd. .Non-pregnant guinea pigs weighing 250–350 g, female Sprague–Dawley (SD) rats weighing 180–220 g, rabbits weighing 2.5–3.5 kg, and a male cat weighing 3 kg were provided by Kunming Medical University. The animals were housed at 22 °C temperature, at 45–55% humidity-controlled conditions, and under natural light. At the end of the animal experiments, the animals were intraperitoneally anesthetized with urethane (1.0 g/kg). Under deep anesthesia, the necks of the animals were dislocated for euthanasia. The bodies of the animals were collected in yellow plastic bags and kept in a refrigerator at − 20˚C until they were taken away by a green company for cremation.The KA6600A multiple microplate reader was manufactured by Beijing Kai’ao Technology Development Co., Ltd. . The FDU-1100 lyophilizer was manufactured by EYELA . Ultrafilters of 100 kDa (Lot: 1806047VS) and 10 kDa (1808013VS) were manufactured by Sartorius AG . The biological signal acquisition system (MD3000) was manufactured by Anhui Zhenghua Biological Instrument Equipment Co., Ltd ; ultrapure water was obtained using a Milli-Q water purification system . The other instruments and reagents used in this study were made in China unless otherwise stated.https://arriveguidelines.org).The animal experiments were approved by the Animal Care and Use Committee of Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Kunming, China (2018FF001-002), and animal experiments were conducted under the Guidelines for the Ethical Review of Laboratory Animal Welfare of China . Animal were treated in accordance with ARRIVE guidelines , while the retained part was Component B (from 10 to100 kDa).The abovementioned three components were dried by a lyophilizer. Briefly, the solution was transferred to a plastic bottle of 250 ml, and frozen at –40 oC overnight. The frozen solution was put into the lyophilizer chamber and lyophilized for > 48 h. Subsequently, the bottle was weighed every 3 h, and when the total weight of the bottle decreased no further, the lyophilization was ended. The net weight of the oily substances was calculated by subtracting the bottle weight from the total weight.The original TW80 and its components were dissolved in NS (100 mg/ml). Every six guinea pigs, including three male and three female animals (non-pregnant) were randomly arranged in a group to detect anaphylactic or anaphylactoid reactions. Animals administered NS in the same volume served as a negative control. The detection of the anaphylactic reaction was performed according to the Chinese Pharmacopoeia (CP) of the 2015 edition (Book 4) . BrieflyFor the anaphylactoid reaction test, each guinea pig was intravenously administered with original TW80 and its components . Animals administered with NS of the same volume served as the negative control. The symptoms appeared 30 min after administration, following which, blood was immediately drawn without anticoagulation to obtain serum samples.Scores were recorded based on the symptoms Table . If an aThe coagulated blood was centrifuged at 3,000 g and 4 °C for 5 min to obtain serum. The levels of IgE, HIS, CRP, HMGB1 and C5a in the serum were determined using ELISA kits according to the protocols provided by the manufacturer.Systemic anaphylactoid reaction in rabbits evoked by macromolecules.The original TW80 and its components were dissolved in NS (100.0 g/L). Every six rabbits, including three male and three female animals (non-pregnant) were randomly arranged in a group to detect anaphylactoid reactions. Animals administered NS of the same volume served as the negative control. Briefly, the solution  was intraperitoneally administered. The symptoms appeared 30 min after the evocation was observed, following which, blood was drawn to obtain serum for the assay.The scores were recorded according to Table Guinea pigs were anesthetized with urethane (1.0 g/kg) by intraperitoneal injection. Next, their abdomens were opened and their anticoagulated blood using heparin was drawn via their abdominal aortae. The blood was spun at 1,000 g and 4 °C for 5 min to remove erythrocytes, and the volume was recovered by adding NS. The suspensions from different guinea pigs were mixed, and a 5 ml suspension containing whole white leukocytes was transferred to the wells of a six-well plate. When the original TW80 or its components of 16 μg  were added to the suspension and mixed, the plate was kept at 37 °C for 30 min. C48/80 (20 μg/ml) served as the positive control. A 1 ml suspension was sampled and centrifuged at 1,000 g and 4 °C for 5 min to obtain the supernatant. The levels of β-HEX, LZM and HIS in the supernatant were determined using ELISA kits.2) was extracted using 4 ml acetone-NS solution (acetone: NS = 7:3) for 24 h. The extract was centrifuged at 3,000 g for 5 min, and the intensity of Evans Blue in the supernatant was determined by a multiplate reader at 610 nm.Every four guinea pigs, including two male and two female (non-pregnant) weighing approximately 300 g, were randomly arranged in a group. The animals were pretreated with cromolyn sodium (50 mg/kg) or NS of the same volume by intraperitoneal injection for 5 days. On the day before the administration, the back fur was removed with a pair of scissors. On the second day, they were intravenously injected with 1 ml Evans Blue solution . Fifteen minutes later, the original TW80 and its components  were subcutaneously injected at one point. The administration of NS of the same volume served as the negative control, and the administration of C48/80  served as the positive control. Thirty minutes later, the animals were anesthetized with urethane (1.0 g/kg), their back skin was cut, and the blue-staining areas were photographed. The Evans Blue in the skin  were perfused into the system and maintained for 10 min. Subsequently, Evans Blue solution  was perfused into the system and maintained for 10 min. Finally, the system was perfused with NS for 5 min, and the lungs were removed from their chests and photographed. The presence of blue staining in the lungs suggested vascular permeability.Female rats were intraperitoneally treated with or without 10 mg cromolyn sodium. Thirty minutes later, the rats were anesthetized with urethane (1 g/kg). Next, their abdomens and chests were opened, and their superior and inferior venae cava were ligated with artery clamps. The NS was perfused into the right ventricle under a pressure of 1,500 mmHThe cat was anesthetized with urethane (1.0 g/kg) and fixed on an operating table, with the local ambient temperature maintained at 30 oC. The skin of the cat’s neck was cut to expose its trachea. After the trachea was linked to a tube to maintain steady breathing, the right common carotid artery was separated and its distal side was ligated. The end of the arterial catheter filled with NS was connected to a blood pressure sensor, and the other end was inserted into the common carotid artery. Blood pressure signals were recorded when the operation was completed. NS, histamine phosphate , or different components of TW80 in the same dose  were successively injected through the cat’s femoral vein. Note that the next injection was only allowed when the blood pressure had recovered.To measure the ability of the macromolecules to affect hemolysis, 5 ml of blood was drawn from a rabbit and decanted into a 100-ml glass beaker. The blood was stirred with a glass stick to remove fibrinogen and fibrous protein and obtain blood without fibrinogen. The blood was mixed with 45 ml NS at 10 °C, and centrifuged at 1,000 g and 10 °C for 15 min to rinse the blood. The blood was rinsed with NS thrice, and clear blood was obtained. Next, the clear blood from the original 5-ml blood sample was mixed with 120 ml NS to obtain the blood suspension that was used for the hemolysis assay. The blood suspension (2.5 ml and NS of 2.2 ml) was transferred to a tube, and a solution of the original TW80 and different concentrations of its components (0.3 ml) was added and gently mixed. Meanwhile, the 2.5-ml blood suspension was mixed with 2.5 ml of water for complete hemolysis. The tubes were kept at 37 °C for 3 h, and centrifuged at 1,000 g for 15 min. The absorbance of the supernatant at 610 nm was read by the microplate reader. The hemolysis rate was calculated using the following formula  or median + inter-quartile range (IQR). If the data exhibited a normal distribution, a one-way analysis of variance (ANOVA) was performed to compare the means between groups. If there was a significance between groups, a post-hoc test  or Tamhane’s test  was used to compare the means between the two groups. If the data did not exhibit the normal distribution, a non-parametric Mann–Whitney test was used to compare the median values between the two groups. Statistical significance was set at The original TW80 and Component A and B caused significant anaphylactic reactions, while Component C almost caused no reactions Fig. A.The levels of IgE, HIS, CRP, HMGB1 and C5a in the serum were measured Fig. . Some faThe original TW80 and Component A and B caused significant anaphylactoid reactions. However, Component C almost caused no reactions Fig. B. In conConsidering that the original TW80 and its macromolecular components (A and B) caused both anaphylactic and anaphylactoid reaction in guinea pigs but with different patterns of serum factor, anaphylactoid reactions were then tested in the larger animals, rabbits. Unsurprisingly, the original TW80 and Component A and B caused significant anaphylactic reactions while Component C caused almost no reactions Fig. C. ComponNext, to understand the possible mechanism underlying the systemic anaphylactoid reactions, the factors in their serum were detected. Compared to the negative control (NS), the original TW80 and Component A tended to increase the factors involved in Fig. To investigate whether leukocytes are involved in anaphylactic reactions, leukocytes were isolated from guinea pig blood and the released factors were measured. The results showed that Component A promoted histamine release only , while Component C showed no tendency to increase the factors mentioned in Fig. The PCA reaction is believed to be a sensitive method for detecting anaphylactoid reactions. Since anaphylactoid reactions increase vascular permeability, the leached Evans Blue from vessels can be used to assess the intensity of the reaction. According to the results in Fig. Considering that PCA reactions are an acute injury to the skin, regeneration rather than necrosis occurred in the area near the injection point. Local hydropic degeneration could be seen in the areas of skin with severe PCA reactions Fig. , the degTo exclude the role of blood cells mediating vascular permeability, pulmonary perfusion experiment was conducted in female rats. The results showed that the lungs treated with normal saline for injection were only slightly stained by Evans Blue Fig. F, While The original TW80 and Component A and B caused strong vasodilation in the cat while Component C almost had no effects Fig. .Fig. 9VaThe original TW80 and Component A and B caused strong hemolysis while Component C caused no hemolysis at a concentration of ≤ 6 mg/ml Fig. . ComponeThis study proves that TW80 contain macromolecular impurities. Though TW80 is a mixture of polymers, it is believed that the agent only contains small molecules with an theoretical molecular weight of 1,310 Da. However, more than 85% the substances were polymers with molecular weights > 10 kDa . AccordiThe original TW80 and its macromolecular components (Components A and B) caused serious anaphylactic and anaphylactoid reactions in guinea pigs. However, there was little difference between the intensity of the anaphylactoid reaction and that of the anaphylactic reaction. Considering that serum IgE both in “anaphylactic” and anaphylactoid guinea pigs was only slightly elevated by the original TW80 and its macromolecular components, the reactions in the anaphylactic experiment are more likely to be anaphylactoid reactions although the possibility of anaphylactic reaction cannot be excluded. The anaphylactoid reactions were further proved in rabbits, and Component A caused the most severe reactions. The finding that Component C partially causes anaphylactoid reactions in both guinea pigs and rabbits indicates that macromolecules are the dangerous factor triggering anaphylactoid reactions.The anaphylactoid reactions must be associated with the release of HIS and the activation of the complement system because Component A tended to elevate the level of SC5b-9, CFB, and MBL. Moreover, the macromolecular substances of TW80 activate the bradykinin system., as shown by the elevation of BK caused by Component A in anaphylactoid reactions in rabbits. The anaphylactoid reaction is likely to be largely unassociated with the activation of mast cells because Component A barely elevated the level of β-HEX and LZM, both in vivo and in vitro, which are most probably released from mast cells .Compared to anaphylactoid reactions ignited by intravenous administration, the PCA reaction is a more sensitive test with visual results . The PCAThe macromolecular substances from TW80 enhanced the vascular permeability in the pulmonary perfusion experiments. Considering that blood cells, including leukocytes, were removed by perfusate, and given that large molecules are unlikely to cross the vascular wall into the interstitial space to activate mast cells where they reside, it can be deduced that the increase in vascular permeability is likely to have resulted from a direct effect on epithelia.Though cromolyn sodium is a mast cell stabilizer with anti-inflammatory activity, it also has other pharmacological effects, including reducing vessel permeability . Thus, iAdditionally, the original TW80 and its macromolecular components caused hemolysis at low concentrations, while Component C did not, even at 6 mg/ml, an impossible concentration in serum by intravenous administration. Thus, the macromolecular substances also demonstrate hemolytic toxicity.Taken together, these results suggest that macromolecular substances > 10 kDa are one of the main risk factors responsible for anaphylactoid reactions caused by TW80, which can be removed by ultrafiltration. The direct increase in vascular permeability participates in the anaphylactoid reactions ignited by TW80, although a role for mast cell degranulation cannot be excluded as one of the main mediators of the reaction.Additional file 1.


Ca2+ also acts as a key intracellular messenger regulating transcription underlying hypertrophic growth. Although Ca2+ release via RyRs is by far the greatest contributor to the generation of Ca2+ transients in the cardiomyocyte, Ca2+ is also released from the SR via inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs). This InsP3-induced Ca2+ release modifies Ca2+ transients during ECC, participates in directing Ca2+ to the mitochondria, and stimulates the transcription of genes underlying hypertrophic growth. Central to these specific actions of InsP3Rs is their localization to responsible signalling microdomains, the dyad, the SR-mitochondrial interface and the nucleus. In this review, the various roles of InsP3R in cardiac (patho)physiology and the mechanisms by which InsP3 signalling selectively influences the different cardiomyocyte cell processes in which it is involved will be presented.The contraction of cardiac muscle underlying the pumping action of the heart is mediated by the process of excitation-contraction coupling (ECC). While triggered by CaThis article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. In chandling ,19. Thesart beat –22 and Ihe adult . Moreoveelopment ,25. Furtdulthood . By contectively ,27. Supphythmias ,29. Despmyocytes ,30–32. M. 32+ release from the SR, a role for InsP3Rs in ECC can be explained i in their vicinity. Besides functional interactions with RyR, IICR has recently been reported to regulate atrial Ca2+ transients through a mechanism involving activation of the Ca2+ stimulated adenylyl cyclases, AC1 and AC8  at diastole and slower kinetics of nuclear Ca2+ transients i i ,96. In cm the SR ,42 or vi2+ entry . During lic Ca2+ ,54,56.2+ release through InsP3R2 can induce hypertrophic gene expression additionally via activation of CaMKIIδB in the nucleus [2+ signals arising from InsP3R1 also engages CaMKII. CaMKII regulates transcriptional processes via phosphorylation of transcription factors  and epigenetic regulators and histone deacetylases  [δB induces association with 14-3-3 proteins, resulting in nuclear export of the complex, while phosphorylation by cytosolic CaMKIIδC blocks HDAC nuclear import [2+ source leading to activation of cytosolic CaMKIIδC in the cardiomyocytes is not fully known. However, a recent study by Qi et al. demonstrated that autophosphorylation of CaMKIIδC was prevented by knockdown of ITPR1 [Ca nucleus . In medi7 and 9) . Of part7 and 9) . To brinr import ,100. To of ITPR1 .3-mediated Ca2+ signalling has also been shown to be required for the hypertrophic response to IGF-1 stimulation [3- and nuclear Ca2+-dependent manner [While cues for physiological and pathological hypertrophy are considered to engage distinct pathways leading to different outcomes, InsPmulation . The dowt manner . Whether. 62+ signalling from InsP3Rs mediates increased cellular glucose uptake in response to insulin stimulation through GLUT4 translocation and fusion with the sarcolemma [ECC has a high energetic cost consuming the largest part of ATP produced in cardiomyocytes . The ATPrcolemma .2+ via the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane [2+-regulated manner [2+ in turn stimulates Ca2+-dependent dehydrogenases involved in oxidative phosphorylation [2+ levels are coupled with mitochondrial metabolism. For example, under conditions of increased cytosolic Ca2+ fluxes , mitochondrial Ca2+ accumulation is enhanced, thereby boosting ATP production to provide for the increased demands of ATP consuming pumps [2+ within mitochondria leads to activation of programmed cell death pathways and increased oxidative stress [2+ overload brings about this effect through activating the mitochondrial chaperone cyclophilin D (CypD) that induces opening of permeability transition pore (mPTP) [2+ uptake also contributes to shaping cytosolic Ca2+ dynamics [Mitochondria accumulate Camembrane  and the d manner . Increasrylation ,111 (figng pumps . Under pe stress . Ca2+ ove (mPTP) . Mitochodynamics ,116.2+ uptake by mitochondria occurs at membrane contact sites known as mitochondrial-associated membranes (MAMs) [2+ flux involving InsP3Rs at MAMs is supported and regulated by association with the VDAC1 and the mitochondrial stress 70 protein  in a macromolecular complex [3R1 is invoked and together with VDAC1 are the channels involved in Ca2+ transfer between SR and mitochondria respectively, while GRP75 links both channels through binding to their cytosolically facing regions. The importance of this pathway in the induction of mitochondrial permeability during stress is underlined by the increased interaction between CypD and the InsP3R-GRP75-VDAC1 complex under conditions of greater mitochondrial Ca2+ content [3Rs in endoplasmic reticulum (ER)-mitochondrial signalling and cell death induction, augmented InsP3-mediated Ca2+ fluxes to the mitochondria also have a pro-apoptotic effect in ischaemia-reperfusion (IR) injury [3R-mitochondria transfer is brought about by glycogen synthase kinase 3β (GSK3β) mediated phosphorylation of InsP3R1 [2+ homeostasis and metabolism, mitochondrial Epac1 complex interacts with and promotes InsP3R-GRP75-VDAC1 complex formation under IR conditions, leading to mitochondrial Ca2+ overload and opening of the mPTP [3Rs by the protein kinase Akt has also been shown to suppress ER-mitochondrial Ca2+ transfer and cell death in the heart. Following IR injury, the abundance of Nox4 at MAMs is increased where it, through generation of reactive oxygen species activates Akt, which in turn phosphorylates and inhibits InsP3Rs [2+ transfer via InsP3Rs to the mitochondria in cardiomyocytes, as they do in other cell types, remains to be fully explored [et al. reported that Ca2+ released from the SR via InsP3Rs, activated downstream of ET-1 stimulation, is taken up by mitochondria via mitochondrial type 1 RyR (mRyR1) resulting in increased ATP generation in both quiescent and electrically stimulated cells (Cas (MAMs) . In line complex ,118 (fig content . In line) injury ,120. Durthe mPTP . First dthe mPTP –124, pho InsP3Rs   [2+ uptake into mitochondria and consequently suppress mitochondrial respiration and apoptosis. While both MFN2 and FUNDC1 regulate InsP3R-mitochondria crosstalk by maintaining the integrity of MAMs, FUNDC1 was also shown to directly interact with InsP3R2 and regulate its stability [Further influencing local Ca2 (MFN2)  or FUN142 (MFN2) . Ablatiotability . Remarkatability .. 73Rs are also functionally expressed in sino-atrial node (SAN) and Purkinje cells. In these cell types, InsP3R activation contributes to rhythm regulation and AP propagation, which given the influence of these cells on contraction of the myocardium has significant consequences for cardiac function. In SAN and Purkinje cells, InsP3Rs are relatively more abundant than in atrial and ventricular cardiomyocytes. As shown for atrial and ventricular cardiomyocytes, InsP3R activation in both of these cell types is amplified by Ca2+ release via RyRs leading to more substantial consequences for cellular Ca2+ signalling [2+ release events underlie a Ca2+ clock involved in pacemaker activity [2+ clock interacts with a membrane clock centred on the funny current (If) conveyed by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels [2+ release via InsP3R2 increases the frequency of the spontaneous SR Ca2+ release events and Ca2+ waves, which by stimulating NCX activity, accelerates membrane depolarization resulting in the threshold for AP generation being more rapidly reached [3R2 and under conditions of InsP3R inhibition [2+ wave frequency (the Ca2+ clock) by IICR was independent of NCX, thereby demonstrating the importance of IICR in heart rhythm regulation [2+-sensitive adenylate cyclases (AC1 and 8) [f), or via PKA and its modulation of the Ca2+ handling machinery [3Rs to sub-sarcolemmal regions of the SR in the vicinity of sarcolemmal HCN channels makes this mechanism possible [et al. and Kapoor et al., baseline pacemaking activity was not influenced by loss of IICR in the murine atrial preparations used by Capel et al. The reason for this discrepancy is not clear but could be owing to preparation (cells versus tissue), or the species studied. In Purkinje cells in which the InsP3R1 is predominant [3R activation is proposed to contribute to the generation of pathological arrhythmias [2+ release via InsP3Rs leads to the generation of Ca2+ waves emanating from the nuclear and sub-sarcolemmal regions of the cell where InsP3R expression is enriched [2+ waves may then give rise to triggered activity [In addition to their role in contractile atrial and ventricular cardiomyocytes, InsPgnalling ,130. In activity . By stimchannels ,132. Ca2 reached ,133. Thigulation . Throughgulation ,129,133.1 and 8) . The cAMachinery . The locdominant , InsP3R hythmias . This mehythmias . In thesactivity .. 82+ controls in the cardiomyocyte raises a significant problem for its ability to concurrently and specifically regulate them. Key to the overlapping and non-overlapping functions of InsP3 signalling in the heart is the localization of InsP3Rs to cellular Ca2+ microdomains coupled with the involved effectors — the dyad with RyRs, the nucleus with associated transcription factors and their regulators, and at MAMs with mitochondria and the Ca2+ uptake machinery. The capacity for cardiomyocytes to function in the absence of InsP3Rs despite their involvement in multiple cardiomyocyte functions is not clear. Compensation for InsP3R2 by upregulation of other InsP3R isoforms is one possibility. This potential redundancy is however overcome by use of genetically encoded inhibitors of InsP3 signalling such as of the InsP3 5-phosphatase or of a high affinity version of the ligand binding domain of InsP3R1 (InsP3 sponge) [3 sponge and the InsP3 5-phosphatase have been applied to analysis of hypertrophic signalling, and in the case of the InsP3 sponge, proven to be effective in in vivo studies [3 signalling, including through cardiac development has not however been determined. To allow acute analysis of the role of InsP3Rs in cardiomyocyte physiology without the cell culture required for adenoviral-mediated expression of InsP3 probes or interfering RNAs or for transgenesis, improved drugs that target the InsP3R are required. For inhibition of InsP3Rs, a toolbox including Heparin [Xestospongia exigua and 2-aminoethoxydiphenyl borate (2-APB) is relied upon [3R [3 3-kinase [3R, equally suppresses SERCA pump activity leading to Ca2+ store depletion [2+ mobilizing activity of InsP3 or of an InsP3 generating agonist in experiments in which it is applied. Specifically, IICR may be lost owing to store depletion rather than InsP3R inhibition. Xestospongin B was reported to elicit a more selective effect on InsP3R without the off target effects on SERCA [2+ release via RyR [3Rs was detected, thereby further supporting an indirect mechanism of action for these agents on IICR. Of the InsP3R inhibitors used in cardiomyocytes, 2-APB is most reliable and widely used. Like the aforementioned inhibitors, off target effects of 2-APB on store-operated Ca2+ entry, mitochondria and SERCA pumps have however been reported. Careful titration of 2-APB in cardiomyocytes, showed that when applied at a low concentration of ∼2 µM, selective inhibition of InsP3Rs is achieved with no effects on Ca2+ transients or SR store loading detected [3Rs but owing to its potent activation of RyRs has limited use in cardiomyocytes [3R inhibition, InsP3R may also be activated pharmacologically. To these ends, cell permeant forms of InsP3 or caged derivatives are employed in intact cells and InsP3 salts and caged derivatives introduced via patch pipettes [3R activation [3Rs, it has multiple other targets including induction of Zn release from cellular stores [3R probes such as those involving modified versions of InsP3 [3Rs. Development of strategies to selectively modulate InsP3Rs in distinct cellular microdomains and to relocalize InsP3Rs to different cellular microdomains to selectively influence discrete functions may also prove of use — for example to prevent arrhythmogenic Ca2+ signals. Moreover, application of these approaches in large preclinical models of cardiac disease as well as in human cardiomyocytes is necessary to fill our gap in knowledge regarding the role of InsP3R signalling in human pathology.The diversity of functions that Ca sponge) , which h studies ,39. Whet Heparin , Xestosp Heparin , derivedied upon ,141. Whiupon [3R , it is n3-kinase . Xestospepletion . This efon SERCA . Xestosp via RyR . In a mo via RyR , no inhidetected ,33,144. myocytes . As a copipettes ,37. The tivation . While tr stores . The issof InsP3  or of thof InsP3 . These n


RUNX1::RUNX1T1 fusion. The other two patients showed karyotypically visible three-way translocations t and t, respectively. Each resulted in RUNX1::RUNX1T1 fusion. Our findings demonstrate the importance of recognizing variant forms of t translocations and emphasize the value of applying RUNX1::RUNX1T1 FISH for the detection of cryptic and complex rearrangements when abnormalities involving chromosome band 8q22 are observed in patients with AML.Acute myeloid leukemia (AML) represents a heterogeneous disease entity that is continuously moving to a more genetically defined classification. The classification of AML with recurrent chromosomal translocations, including those involving core binding factor subunits, plays a critical role in diagnosis, prognosis, treatment stratification, and residual disease evaluation. Accurate classification of variant cytogenetic rearrangements in AML contributes to effective clinical management. We report here the identification of four variant t translocations in newly diagnosed AML patients. Two patients showed a t and a t variation, respectively, with a morphologically normal-appearing chromosome 21 in each initial karyotype. Subsequent fluorescence in situ hybridization (FISH) on metaphase cells revealed cryptic three-way translocations t and t. Each resulted in  Acute myeloid leukemia (AML) is a clonal hematopoietic disorder resulting from genetic alterations in normal hematopoietic stem cells. These alterations disrupt normal differentiation and/or cause excessive proliferation of abnormal immature leukemic cells. The classification of AML has shifted from a morphology-based classification to a classification algorithm based primarily on genetic abnormalities. The current World Health Organization (WHO) classification includes a major entity termed AML with recurrent genetic abnormalities, meaning with specific chromosomal or mutational alterations ,2,3.RUNX1 (runt-related transcription factor 1) on 21q22 to RUNX1T1  on 8q22. The fusion resides on the derivative 8q22. It leads to the disruption of the normal function of the core-binding factor in hematopoietic differentiation and maturation and recruits transcriptional repressors to block the expression of genes involved in normal hematopoiesis, thus impairing apoptosis [Translocation t8;21) is a recurrent cytogenetic abnormality and is one of the most common subtypes of AML, occurring in approximately 5% of AML patients. This subtype has predominantly neutrophilic maturation and is associated with a high rate of complete remission and favorable long-term outcomes [poptosis .RUNX1 occurs between exons 5 and 6; the translocation juxtaposes the 59 end of the RUNX1 gene, including the RHD domain, with the 39 end of the RUNX1T1 gene with its four NHR domains. The generated fusion protein consists of 752 amino acids; the first 177 amino acids are derived from RUNX1, whereas 575 amino acids are from RUNX1T1. Structurally, RUNX1::RUNX1T1 therefore has five domains: the RHD from RUNX1 and NHR domains 1 to 4 from RUNX1T1 [At the molecular level, the translocation breakpoint in While the t translocation is a primary chromosomal abnormality in a specific subtype of AML, secondary, non-random cytogenetic abnormalities have also been identified. Frequent secondary abnormalities include the loss of a sex chromosome (-X or -Y) or a deletion in 9q .RUNX1::RUNX1T1 fusion on the derivative 8q22. Identification and accurate classification of these RUNX1::RUNX1T1 fusion-positive variants in the t subtype plays an important role in the diagnosis of this specific subtype of AML and in the optimal clinical management of patients with this subtype.Variant and complex rearrangements in the form of t8;V;21), with V being a variable third chromosome, have been reported in approximately 3–4% of AML patients with t ,8. Simil;V;21, wiRUNX1-RUNX1T1 probe revealed cryptic three-way translocations t and t, which resulted in RUNX1::RUNX1T1 fusion in both patients. The other two patients showed karyotypically visible three-way translocations t and t. Each resulted in RUNX1::RUNX1T1 fusion. We conclude that RUNX1::RUNX1T1 FISH is critical in the detection and classification of variant t.We report here the identification of four variant t translocations in newly diagnosed AML patients. Significantly, two patients showed t and t respectively, with a morphologically normal-appearing chromosome 21 in each initial karyotype. Subsequent fluorescence in situ hybridization (FISH) on metaphase cells using a Bone marrow or peripheral blood samples from the four patients described in this report were received in our cytogenetics laboratory for diagnostic purposes. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the University of Maryland, Baltimore (HP104494).RUNX1T1-RUNX1 dual-color dual-fusion probes  on metaphase and interphase cells from each sample following the standard procedures described in the AGT Cytogenetics Laboratory Manual [Each received sample was set into cultures and cultured in RPMI 1640 medium for 24 to 48 h. The cultured cells were harvested and G-banded following standard cytogenetic procedures as described in the AGT Cytogenetics Laboratory Manual . Metaphay Manual . Two hunRUNX1::RUNX1T1 fusion.NGS-based cDNA sequencing was performed in the molecular lab. RNA was extracted from patients’ bone marrow or blood samples, which was followed by a reverse transcription reaction to generate cDNA. The library was prepared on the Ion Chef System. Massive parallel cDNA sequencing was performed using an Ion S5 OMA Workflow  for the detection of a set of 29 fusion genes, including A peripheral blood sample was received from a 25-year-old man admitted to our Cancer Center in February 2022. His hemoglobin level was 8.5 g/dL, the platelet count was 51 K/uL, and the white blood cell count (WBC) was 10.8 K/uL with 42% blasts. The blasts expressed the myeloid markers CD33, CD117, and cytoplasmic MPO along with the immature marker CD34 by flow cytometry. Additionally, there was aberrant dim and partial expression of CD19, typically a B-cell marker. These findings indicated a diagnosis of AML.RUNX1-RUNX1T1 probe to rule out a variant rearrangement. FISH analysis of metaphase cells revealed a cryptic three-way translocation  translocation, with a sub-clone showing t and -Y. Both copies of chromosome 21 appeared normal a. Becauslocation b, and th4 transloRUNX1::RUNX1T1 RUNX1::RUNX1T1.R3R3 chr21:36231771—chr8:93029591.Molecular testing confirmed the presence of gene fusion A bone marrow sample was received from a 57-year-old man admitted to our Cancer Center in February 2022. He had hypercellular bone marrow with 70% cellularity. His hemoglobin level was 5.2 g/dL, the platelet count was 14 K/uL, and the WBC was 2.8 K/uL with 31% blasts. The blasts expressed the myeloid markers CD33, CD117, and cytoplasmic MPO along with the immature marker CD34 by flow cytometry. Additionally, there was aberrant CD56 expression, typically a natural killer cell marker, along with dim expression of CD19. These findings indicated a diagnosis of AML.RUNX1-RUNX1T1 probe to rule out a variant rearrangement. FISH analysis of metaphase cells revealed a cryptic three-way translocation  translocation, with a sub-clone showing t and -Y. Both copies of chromosome 21 appeared normal a. Becauslocation b, and th translocRUNX1::RUNX1T1 RUNX1::RUNX1T1.R3R3 chr21:36231771—chr8:93029591.Molecular testing confirmed the presence of gene fusion A bone marrow sample was received from a 49-year-old woman admitted to our Cancer Center in May 2022. She had hypercellular bone marrow with 80% cellularity. Her hemoglobin level was 12.6 g/dL, the platelet count was 21 K/uL, and the WBC was 2.8 K/uL with 18% blasts. The blasts expressed the myeloid markers CD33, CD117, and cytoplasmic MPO along with the immature marker CD34 by flow cytometry. Additionally, there was aberrant CD56 expression along with dim, partial expression of CD19.RUNX1-RUNX1T1 fusion in 76% of the 200 examined nuclei.Karyotype analysis detected a three-way translocation: 46,XX,t  Figure . FISH anBecause no sample was submitted for this patient, molecular testing was not possible.A peripheral blood sample was received from a 20-year-old man admitted to our Cancer Center in December 2022. His hemoglobin level was 6.8 g/dL, the platelet count was 10 K/uL, and the WBC was 5.1 K/uL with 25% blasts. The blasts expressed the myeloid markers CD33, CD117, and cytoplasmic MPO along with partial expression of the immature marker CD34 by flow cytometry. Additionally, there was aberrant CD56 expression along with partial expression of CD19.RUNX1-RUNX1T1 fusion in 75% of the 200 examined nuclei.Karyotype analysis detected a three-way translocation: 45,X,-Y,t  Figure . FISH anRUNX1::RUNX1T1 RUNX1::RUNX1T1.R3R3 chr21:36231771—chr8:93029591.Molecular testing confirmed the presence of gene fusion RUNX1T1::RUNX1 fusion, is a recurrent cytogenetic abnormality in AML. Although the RUNX1T1::RUNX1 fusion is most commonly seen in cases with cytogenetically visible t, detection of the fusion transcript in the absence of visible t(8:21) has been reported in a few cases with cryptic rearrangements, including sequence insertions [The t translocation, with resulting sertions  and thresertions ,14,15,16RUNX1T1::RUNX1 fusion is known to be associated with a complete remission rate and a high cure rate after high-dose cytarabine consolidation therapy; thus, allogeneic hematopoietic stem cell transplantation in first remission is not recommended for these patients. Due to a limited number of reported cases and studies, it is not clear whether the clinical outcomes of patients with three-way translocations are different from those of patients with the t translocation. At the molecular level, all t translocations resulted in a RUNX1T1::RUNX1 fusion, which is the same as the consequence of t translocations. All of our four patients achieved remission. While three of the four patients currently remain in remission, Patient 2 passed away after achieving remission due to end-stage renal disease status post failed renal transplantation. In comparison to the typical t translocations, the long-term clinical outcomes of patients with three-way translocations remains to be evaluated.Accurate classification of AML involving core-binding factor (CBF) subunits, especially those with cryptic and complex translocations, is of important significance in clinical management. AML with KIT gene mutations in CBF AML confers a higher relapse risk [KIT mutations in CBF AML has both prognostic and therapeutic significance. The activated KIT can potentially be targeted with novel tyrosine kinase inhibitors. The molecular testing results from our three tested patients with RUNX1::RUNXT1 fusion showed no evidence of KIT mutation in these patients.AML with t that disrupts core-binding factor (CBF) subunit α and inv(16) that disrupts CBF subunit bata are collectively referred to as CBF AML. When compared to other cytogenetic groups, patients with CBF AML have relatively favorable outcomes. Multiple studies have shown that the presence of pse risk ,18. TherRUNX1::RUNX1T1 FISH when abnormalities involving 8q22 are observed in AML karyotypes. We also recommend taking immunophenotype findings into consideration. It is worth noting that all four patients with t translocations showed aberrant CD19 expression by flow cytometric analysis, and three of the four patients  also showed aberrant CD56 expression. CD19 is normally a B-cell lineage marker, and CD56 is a natural killer cell/stem cell marker. Aberrant expression of CD19 on myeloblasts or co-expression of CD19 and CD56, along with myeloid antigens, has been identified as a characteristic immunophenotype associated with t [RUNX1::RUNX1T1 FISH is also recommended for AML patients with aberrant CD19 expression or those with CD19 and CD56 co-expression. When the classic t translocation is not apparent in a karyotype, immunophenotype detection of these markers may be helpful in suggesting that FISH be performed to look for a variant or cryptic RUNX1::RUNX1T1 fusion.The four patients with t translocations were identified among approximately one hundred and twenty AML patients that were treated in our Cancer Center in 2022. Our findings indicate that the presence of t translocation variants may be more frequent than previously appreciated. We recommend  t ,19,20. TRUNX1::RUNX1T1 fusion. The first two rearrangements were cryptic three-way translocations that were not readily identifiable through conventional karyotyping. These findings indicate the challenges associated with accurate classification, which is required to support the stratified treatment of AML. We recommend an integral approach that combines cytogenetic analysis with molecular and immunophenotypic studies to improve the detection of variant cytogenetic rearrangements in AML.In summary, we identified four variant cytogenetic rearrangements, namely t, t, t, and t, in four patients with newly diagnosed AML. Each rearrangement resulted in a


SIGNOR provides freely-accessible static maps of causal interactions that can be tailored, pruned and refined to build dynamic and predictive models. Each signaling relationship is annotated with an effect (up/down-regulation) and with the mechanism  causing the regulation of the target entity. Since its latest release, SIGNOR has undergone a significant upgrade including: (i) a new website that offers an improved user experience and novel advanced search and graph tools; (ii) a significant content growth adding up to a total of approx. 33,000 manually-annotated causal relationships between more than 8900 biological entities; (iii) an increase in the number of manually annotated pathways, currently including pathways deregulated by SARS-CoV-2 infection or involved in neurodevelopment synaptic transmission and metabolism, among others; (iv) additional features such as new model to represent metabolic reactions and a new confidence score assigned to each interaction.The SIGnaling Network Open Resource (SIGNOR 3.0,  Cell physiology is governed by a complex mesh of interactions between different biological entities: proteins, RNA, metabolites etc. Perturbation of these interaction networks often causes diseases . Some ofExperimental evidence on causal interactions is sparse and, while the vast majority of proteins in the human proteome have at least one identified physical partner, for many proteins little or no experimental detail exists about the impact of modulating their activities on other protein activity in the proteome.Over recent years, we and others have started annotating causal information in public resources ,10. SIGNWe report in this update the increase in proteome coverage. In addition, we present a new website that we have developed to improve user experience and to introduce new graph and search tools. Finally, we describe a new approach to assign to each interaction a score that estimates the trust that the community places on its biological relevance.After the publication of SIGNOR 2.0  the SIGNThe degree distribution of the SIGNOR graph, as that of many biological interaction networks, follows a power law with few nodes connected by many interactions while most nodes are connected by only one or few edges Figure . A novelAlthough SIGNOR is a generalist database that aims at capturing causal information without concentrating on specific biological domains, we have also undertaken some focused curation campaigns . The bar chart in Figure While the SIGNOR database structure remains unchanged, we largely redesigned the SIGNOR 3.0 web site, according to users’ comments and suggestions. One of the aspects we focused on, while developing the new version of the web interface, was the design of a homepage that would improve usability and user experience Figure . To thisThe most important updates of the newly redesigned resource are within the scope of functionality. These include 1) the direct access to SIGNOR-related projects Figure , namely,‘all’ radio button is selected; (ii) a network showing all the direct interactions between the query proteins, when the ‘connect’ radio button is checked; (iii) a network connecting the query entities that are separated by two edges or fewer when both the ‘connect’ and the ‘add bridge proteins ’ are checked. Finally, when two entities are entered and the user selects the ‘find path’ radio button the tool searches and displays all the directional graph paths that connect the two query proteins.The ‘search’ tab gives access to the default search tool. Users can enter in a text field one or more entity names or identifiers. If a single entity name is entered, the tool redirects the user to an entity page showing functional details and all the causal interactions of the query entity. If more than one entity is entered in the search field the tool returns (i) a network formed by all the interactions of the query proteins, if the ‘causal interaction database’, where literature-captured relationships are merged to form a large, intricate and connected interactome. However, part of the SIGNOR curation effort is directed at annotating sub-networks (pathways) underlying the cellular response to specific environmental stimuli. In this model the cellular pathways in SIGNOR are embedded in the global interactome, thereby allowing for exploration of pathway cross-talk. The Pathway Browser tab grants access to 114 manually curated pathways that are organized in five categories: Pathway, Metabolic Pathway, Disease, Tumor and Module. Modules are defined as smaller networks that underlie commonly perturbed signal transduction pathways.SIGNOR—unlike pathway-centric resources like KEGG and Reactome ,19—is a Two new tools that are offered by SIGNOR 3.0 can be accessed via the ‘Disease Browser’ and ‘Advanced methods’ tabs.all, connect and add bridge proteins as described in the ‘Searching SIGNOR 3.0’ paragraph : DisGeNET, IntOGen and Cancer Gene Census . Brieflyaragraph  14). At. Atall, SIGNOR 3.0 also offers new ‘Advanced methods’ designed to ask more complex queries and to filter and refine search results. The tool allows one to search for entries where a query protein or a list of query proteins play the roles of regulators or targets Figure . AdditioThe ‘Advanced methods’ page also includes a tool that infers the regulatory crosstalk between one or more proteins and a curated pathway. This tool takes as input a list of proteins and one of the manually curated pathways and searches for paths (of two or fewer steps) connecting the input entities to the selected pathway. If such paths exist a visual representation of the result is given in output .https://signor.uniroma2.it/documentation/).Since the latest release, the procedure to assign a confidence score to each interaction was updated as detailed in Supplementary materials . BrieflySIGNOR is a public repository of signaling information, fully compliant with the FAIR principles, which grant for data Findability, Accessibility, Interoperability and Reusability .https://signor.uniroma2.it/APIs.php). In addition, SIGNOR data is also regularly released via The Network Data Exchange (NDEx), an open-source collection of biological networks, conceived for manipulation, integration and re-use (https://apps.cytoscape.org/apps/signorapp.Version 3.0 presents significant advancements in the value of the SIGNOR resource. To improve user experience and data accessibility, we redesigned the web resource and added new search and analysis tools. In addition, we also expanded the programmatic access to the resource, by implementing new APIs that are described in the corresponding section and that can be accessed from the Website homepage (d re-use . As a fod re-use . SIGNORAA steady curation effort has led to a significant increase of the coverage of the human proteome. To date, 33% of it is integrated in the causal interactome, whereas the number of interactions and pathways captured grew by 45% and 130%, respectively.Complete capture of published causal information remains the main goal of the SIGNOR project. SIGNOR is presently the primary causal interaction database with the highest coverage in the non-commercial domain ,12. DespIt is important to stress that all the interactions annotated by SIGNOR curators are mapped to the human proteome and displayed as a single network that describes an abstract cell where all the proteins are expressed and active. The functional interactome that is relevant for the physiology of a specific cell, on the other hand, depends on the cell molecular context and on the level of gene expression of the different genes in the specific cell type. Recent advances in methods that allow to reveal the transcription profile of single cells now permit tuning the interactome of this naive cell to the molecular profile of the different cell types . ProduciFinally, although proteins are the main players in the causal interactome, gene expression and signaling are also regulated by other molecular entities. In the new millennium non-coding RNAs have progressively taken a main stage in the gene expression scenery ,31. The https://signor.uniroma2.it/downloads.php.SIGNOR 3.0 interaction data are available and freely downloadable at: https://signor.uniroma2.it/APIs.php.Furthermore, programmatic access is available through the APIs section at https://signor.uniroma2.it/.All described database features are accessible via the main SIGNOR 3.0 website: https://apps.cytoscape.org/apps/signorapp.SIGNORApp can be freely downloaded from gkac883_Supplemental_FileClick here for additional data file.


Similar to the cases described by Martelli and colleagues, bone marrow findings were notable for presence of dysplasia most notable in the megakaryocyte lineage (Figure However, based on the review of data we identified two additional AML patients with ns Table . Patient6 Figure . Patiente Figure  and varie Figure .NPM1 exon 5 in‐frame insertions or duplications are an uncommon but recurrent finding in AML. Because AML with mutated NPM1 has been recognized as a distinct entity based on a single gene mutation by the WHO classification of AML [NPM1 mutations is critical for accurate AML diagnosis and patient management [NPM1 exon 5 were re‐stratified from "Intermediate" to "Favorable" risk based on the 2017 European LeukemiaNet guidelines [NPM1 mutations with IHC, or whole NPM1 gene sequencing to identify rare NPM1 mutations outside of exon 12 for better classification of AML patients. Furthermore, similar to exon 12 mutations, exon 5 NPM1 mutations can be followed either by RT‐PCR based assays [NPM1 exon 5 mutations in AML prognosis.Overall, we confirm that nagement . Both ofd assays  or highlNone.The authors declare no conflict of interest.The study is deemed as exempt from IRB approval.Peng Wang and Sandeep Gurbuxani designed research, performed research, analyzed data, and wrote the paper; Michael W. Drazer, Girish Venkataraman, and Daniel A. Arber performed research and wrote the paper. Michael W. Drazer has served as a consultant to Cardinal Health.