Experiments were conducted in an experimental setup with Gaussian white noise, frequency, and amplitude control to stimulate myocytes. Cell shortening, maximal shortening velocity, time to peak shortening, and time to half relaxation variables were recorded to cell shortening. Ca2+ transient amplitude and raise rate variables were registered to measure Ca2+ transients. Our results for cell shortening, Ca2+ transient amplitude, and raise rate suggest that cell response improve when myocytes are noise stimulated. Also, cell shortening, maximal shortening velocity, Ca2+ transient amplitude, and raise improves in control cells. Altogether, these findings suggest novel characteristics in how cells improve their response in a noisy environment.Noise is present in cell biology. The capability of cells to respond to noisy environment have become essential. This study aimed to investigate whether noise can enhance the contractile response and Ca Intracellular Ca2+ is an important secondary messenger for ECC. Anticancer therapies could induce alterations in Ca2+ handling3.Excitation–contraction coupling (ECC) is a physiological process that transforms action potential into contractile force. The process involves a series of chemical reactions that converts the calcium ions inside the cardiomyocyte into mechanical movement4. Despite the highly effective antineoplastic DOX properties, one side effect that limits its clinical utility is the dose-dependent cardiotoxicity caused by the drug, which can lead to irreversible cardiomyopathy and subsequent heart failure5. Thus, the doxorubicin-induced cardiomyopathy (DIC) murine model is characterized by systolic dysfunction and Ca2+ dysregulation, such as a decrease in the intracellular Ca2+ transient amplitude and decay rate and increased diastolic Ca2+ levels; as well as reduced sarcoplasmic reticulum Ca2+ ATPase (SERCA), and ryanodine receptors (RYR2) expression6. In addition, multiple mechanisms are associated with DIC, including increased reactive oxygen species production (ROS), disruption of Ca2+, and mitochondrial bioenergetics7.Doxorubicin (DOX) is an anthracycline drug used as a chemotherapeutic to treat numerous cancers, including leukemia, lymphomas, and solid tumors8. Physiological systems are permeated by noise and variability at all observational levels, ranging from molecular processes to complex interactions and dynamics of tissues, organs, and organisms9. Noise is often regarded as an unwanted system component, even though it sometimes enhances system performance in medicine and biology10. Stochastic resonance describes the positive impact of noise in nonlinear systems11. For instance, stochastic noise might provide new treatment alternatives to enhance vestibular function in patients with vestibular pathology12. External noise enhances sensory perception in primary neurons13. In the cardiovascular system at the whole organ scale, synchronization between heart contraction and external electrical stimuli is improved by noise14. A positive effect of noise in promoting skeletal muscle regeneration in neuropathological disease condition was studied three years ago15. Low-intensity noise favors calcium dynamics in H9c2 cells16.One of the major challenges in modern biology is to understand how a cell operates in a highly noisy environment2+ handling? Our main goal is to understand whether cell shortening, and Ca2+ dynamics responses are improved when myocytes are noise stimulated. To this end, we analyzed shortening and Ca2+ dynamics in cardiomyocytes in a short-time mouse model of DOX-induced cardiomyopathy.In summary, there has been substantial progress in understanding the effects of noise on physiological systems. However, understanding how cells of a heart with cardiomyopathy respond to noisy stimuli has received little attention. The present work is devoted to partially filling this gap in knowledge. We address a key question: How does noise in the cellular environment affect the response of cardiomyocytes with impaired contractile response and Cathe National Centre for the replacement Refinement & Reduction of Animal Research, ARRIVE guidelines. A total of 50 mice were divide in four groups: DOX produces myocyte atrophy , DOX effect in cell shortening and calcium fluxes , mitochondrial function , effect of noisy stimulus on cellular shortening and calcium fluxes . In general, 12 mice were studied in each group, except in some non-invasive experiments where projections of sample size from available studies allowed us to use a smaller number of animals. Non-responding mice were dismissed.All animal experimental procedures were conformed to the National Institutes of Health Guide for the care and use of laboratory animals (1996) and were approved by institutional Ethics Review Committee for Animal Experimentation of Cinvestav-IPN . Male C57B1/6j mice (30–40 g weight) were obtained from the experimental Animal Care center from CINVESTAV-IPN Mexico. Mice were acclimated for one week before experiments in metabolic cages , and maintained in a temperature-controlled room (22° ± 2 °C) on a 12 h light–dark cycle (50%) fed a balanced diet and had water ad libitum. Experimental protocols were designed according to the Directrices from 17. Mice received a total of 25 mg/kg of DOX  suspended in saline solution, administrated periodically in five intra-peritoneal injections of 5 mg/kg weekly/once a week. The control group received saline solution injections (NaCl solution 0.9%). Experiments were performed one week after the last intra-peritoneal injection.Cardiac dysfunction model was established based on previous literature18. Signals were digitized and stored on a computer and analyzed offline.Mice were anesthetized by mask inhalation of isoflurane vaporized at concentrations of up to 3%, while taped to a temperature-controlled laminated plastic board. Electrodes were placed in such a way that all four limb leads could be used for electrocardiographic monitoring. Aortic and mitral blood flow velocities were measured from the apical view using a 2-mm diameter 10-MHz pulsed Doppler probe and a real-time Doppler spectrum analyzer probe . The probe was placed just below the sternum using minimal pressure angled toward the ventricular inflow and outflow tracks. In real-time, the spectral Doppler signal was displayed along with the ECG on a computer monitorhttp://imagej.nih.gov/ij/, NIH, Bethesda, MD, USA); a % blue/% red ratio was made. Two blinded researchers analyzed the complete slides. H&E micrographs from the papillary muscles were used to quantify the cardiomyocyte area at 10×.Hearts were excised and weighted from each experimental group and fixed in 4% (wt/vol) paraformaldehyde in PBS at room temperature for 12 h. Afterward, tissues were embedded in paraffin and stained with hematoxylin/eosin (H&E) and Masson’s trichrome. Images were obtained in an Imager.M2 Zeiss microscope with an AxioCam HRm and analyzed with the AxioVision software. The micrographs from the whole of Masson’s trichrome slides were taken at 2.5×; then, images were decomposed in more than seven fields at 5×. The fibrotic index was assessed by quantification of blue and red pixels, using ImageJ  to evaluate brain natriuretic peptide (BNP) mRNA expression. Purity of all samples was confirmed by measuring their 260/280 nm absorbance ratio using a Take3 multivolume plate in a Synergy HT microplate reader (BioTek Instruments). cDNA was reverse-transcribed from 1 μg of total RNA using the SensiFAST cDNA Synthesis Kit  and used for qPCR using the SensiFAST SYBR Lo-ROX Kit  in a QuantStudio 3 RT PCR System (Thermo Fisher Scientific). Data were analyzed by the 2−ΔΔCt method to estimate each gene’s mRNA expression. The primers specific for each gene were: 5′ CTCCAGAACAATCCACGAT 3′/5′ CTTGAACTATGTGCCATCTTG′ (BNP) and 5′ CGTGATTAGTGATGATGAACC 3′/5′ GAGCAAGTCTTTCAGTCCT 3′ (HPRT).19. Briefly, the thorax was open to expose the heart after complete anesthesia. After flushing with EDTA buffer into the right ventricle, the heart was sequentially digested with EDTA buffer , perfusion buffer , and collagenase buffer  into the left ventricle (LV). Constituent chambers  were then separated and gently pulled into 1-mm pieces using forceps. Cellular dissociation was completed by gentle trituration, and collagenase activity was inhibited by addition of a 5 mL stop buffer (5% of SBF in perfusion buffer). Next, the cell suspension was passed through a 100-μm filter, and extracellular Ca2+ concentration was raised to 1 mM over four sequential rounds of gravity settling, using three intermediate Ca2+ reintroduction buffers . Finally, cells were collected and resuspended in Tyrode`s solution  for further studies.Mouse cardiomyocytes were isolated using the simplified, Langendorff-free method20. Briefly, cells were incubated with calcein-AM  in Tyrode’s solution for 30 min at room temperature. Afterward, cells were rinsed with fluorophore-free Tyrode’s solution, and a stack of 2D images was taken every 1 µm in the z-axis covering the whole cell depth. The measurements were acquired using a Leica TCS SP5 confocal microscope equipped with a D-apochromatic 40×, 1.2 NA, oil objective , using a 488 nm excitation wavelength and a 500–600 nm emission window. Only rod-shaped cells with visible striation without spontaneous contractions were assessed. Analysis was carried out using ImageJ software (NIH). First, the pixel intensity frequency histogram corresponding to the calcein-AM signal was obtained. The sum of the pixel was multiplied by the pixel size and z-step between focal planes and expressed as cell volume.Cellular volume was evaluated following a previous report2+ dynamics were evaluated simultaneously; images were obtained with a laser scanning confocal microscope in the line scan mode. The scanning line was longer than the cell length. Consequently, myocyte length shortening is reflected as an increase in empty space beyond the two longitudinal cell edges, following a previous report22. Briefly, a rectangular region comprising both cellular edges was selected, and a threshold was set to distinguish the intra-cellular from the extracellular space, converting the image into binary  is the time elapsed between the onset and the maximal shortening points, and time to half relaxation (TTHR) is the time elapsed between the maximal shortening time and the moment by which the cell length has recovered to 50% of the maximal shortening.The cell border of the resulting binary image was compared to the border on the original record to ensure a close fit. 2+) for 45 min at 25 °C. Afterward, cells were washed with fluorophore-free Tyrode`s solution and mounted in a stimulation chamber. The solution temperature in the stimulation chamber was maintained at 23 ± 2 °C. Confocal microscopy recorded line scan images taking a longer region than the cell longitudinal axis . Fluo-4 was excited at 488 nm, and the emission window was 500–600 nm. For cell shortening and Ca2+ transient, the cells were field stimulated by four electric pulses at each frequency 0.5, 1, and 3 Hz to achieve a quasi-steady state in Ca2+ transients as previously described23. The train of four pulses of 20 V/cm amplitude and 5 ms duration was generated using a function generator programmed in LabView that controlled an amplifier circuit through a data acquisition board (DAQ)  where the output signal of the circuit is connected to the stimulation electrodes localized in the field stimulation chamber RC-21BRFS (Warner Instruments). In each experiment, a specific value of frequency and noise is examined, the chamber is stimulated whit a pulse train, and the parameters corresponding to the contractile response and Ca2+ transients obtained from the third and four stimuli are averaged. The data are presented as the average of 40 different myocytes studied per animal. To analyze the noisy stimuli effect, three different Gaussian white noise levels with amplitude values  developed in LabVIEW, where the noise was added to the signal from the first to the fourth pulse  . Intracellular Ca2+ dynamics were evaluated by characterizing transient amplitude and raise rate. Ca2+transient amplitude is defined as the maximal F/F0 value, where F0 is the average fluorescence intensity before Ca2+ transient rise; raise rate is defined as F/F0/time to peak, where time to peak is the time between the onset and the peak value; time to 50% of decay (T50%) is the time elapsed between the peak and the moment the peak amplitude has decayed by 50%. All the confocal measurements were acquired using a Leica TCS SP5 confocal microscope equipped with a D-apochromatic 63×, 1.2 NA, oil objective .After isolating, cardiomyocytes were incubated in 10 µM Fluo-4 AM  in Tyrode’s solution  were taken using 543 nm excitation and 555–700 nm emission window.Freshly isolated cardiomyocyte cells were incubated with 300 nM TMRE (tetramethylrhodamine ethyl ester perchlorate)  for 30 min at 25 °C in Tyrode’s solution supplemented with 1 mM Ca2PO4, 3 mM MgCl2, 0.5 mM EGTA, 20 mM HEPES pH 7.4, 0.1% BSA). After signal stabilization, cells were permeabilized with 10 µM digitonin. Succinate was employed as a respiratory substrate. State 3 (phosphorylating state), state 4, and uncoupled state were induced by adding 10 mM ADP, 2.5 µM oligomycin, and 1 µM FCCP, respectively. Non-mitochondrial respiration was obtained by the addition of 1 µM antimycin A. Respiratory control ratios (RCR) were obtained by dividing the mean value of respiration after ADP addition by the mean value after oligomycin addition. At the end of the experiment, cytochrome c oxidase (Cox) activity was determined by the sequential addition of 1 mM Ascorbate and 200 µM N, N′, N′-tetramethyl-p-phenylendiamine (TMPD) into the oxygraphy chamber. Cox activity was determined by subtracting the rate of oxygen consumption in the presence of TMPD/Ascorbate from the oxygen consumption induced by the autoxidation of TMPD. This last parameter was obtained by measuring the TMPD/Ascorbate-induced respiration inhibited by adding 50 µM potassium cyanide. Respiratory data were normalized to citrate synthase activity24. A previous titration protocol obtained optimal concentrations of detergent and respiratory modulators.Mitochondrial respiratory activity in permeabilized cells was determined by High-resolution respirometry . Two thousand cardiomyocytes were suspended in 2 mL of respiratory buffer .All data are presented as mean ± SEM. Student’s t-tests or Mann–Whitney U-tests were used to perform simple statistical comparisons. Two-way ANOVA followed by Tukey’s post-hoc was performed to analyze experimental groups with multiple comparisons. Differences were considered significant when 2+ cell transients.As previously mentioned in the cardiovascular systems, the role of noise in cardiac cells still needs to be better understood. We studied how a noisy stimulus in cardiomyocytes from a cardiomyopathy model improves cardiac contractility and CaWe performed experiments at different scales to validate the cardiomyopathy model. First, mice were administered DOX and checked for depression in cardiac function. Second, the atrophy of myocytes from DOX-treated mice was verified. Third, it was verified that the cellular response was diminished. The experiments are described in the following three sections.Cardiac function was assessed by pulsed doppler system to investigate the DOX effects at systemic level. Table Furthermore, mitral inflow represents the flow from the left atrium into the LV during diastole. The mitral flow velocity signal is biphasic; one phase represents an early (E) peak during the rapid filling phase, while the other (A) peak occurs during atrial contraction. Thus, E and A peak velocities decreased in the DOX-treated compared to the control group. A representative Doppler displays of aortic and mitral signals are shown in the Supplementary Fig. These data suggest that mice treated with DOX led to a more pronounced reduction in aortic outflow velocity and a slight reduction in mitral inflow velocity which indicates low cardiac output or poor LV systolic function and low diastolic function, respectively; these effects depend on impaired cardiomyocyte function.After the impaired cardiac function found in DOX-treated group, the characterization of the cardiac structure was performed. Histochemical analysis of collected hearts demonstrated no significant fibrosis in DOX-treated animals Fig. A as dete2+ dynamics were evaluated under three stimulation frequencies  in isolated cardiomyocytes. Cell shortening was characterized with the following parameters: cell shortening, maximal shortening velocity, and TTHR. The DOX-treated group showed compromised cellular shortening to all frequencies tested compared to the control group  was measured. Thus, we observed that DOX-treated cells presented the lower activity of this enzyme, which indicates that this enzyme limits the respiratory activity only at high respiratory rates. Since all respiratory data were normalized to citrate synthase activity, we can rule out that the mitochondrial mass content per se affected the bioenergetic profile observed in our samples.Moreover, these lower respiratory rates influenced the mitochondrial coupling, as observed in the respiratory control ratios in DOX-treated cells. These results indicate that some respiratory components cannot sustain high respiratory activities, but at rest, they present unaltered activities. This was corroborated when the activity of the terminal respiratory complex, cytochrome 2+ handling and contractile response. We perform experiments that electrically stimulate two cell groups: cardiomyocytes of DOX-treated mice as a cardiomyopathy model and cardiomyocytes from the control group. We registered cell shortening and Ca2+ dynamics when cell groups were electro-stimulated. Stimuli were applied at three frequencies with three noise levels added. Each data in DOX-treated animals was normalized by the data in control myocytes without noise to have a relative response to control myocytes.We aimed to investigate whether a noisy stimulus affects the response of cardiomyocytes with impaired CaCell contraction was evaluated by characterizing cell shortening and maximal shortening velocity. We observed that cell shortening increases by 20% at 0.5 Hz and 16% at 1 Hz, with 10% noise-induced, while maximal shortening velocity increases by 17% at 0.5 Hz in control cells. In the absence of noise, cell shortening was 17% reduced, and maximal shortening velocity was 12% reduced in DOX-treated cells. Additionally, we found that 20% of noise-induced reach the control cell basal levels without noise in the stimulus. Maximal shortening velocity did not change compared with the control group Fig. A. TTSP a2+ dynamics coordinate cell contractility, we evaluated Ca2+ dynamics by characterizing transient amplitude and raise rate. As noise level increases (10–20%), both Ca2+ transients and raise rate increase at 0.5 Hz in control myocytes. At 1 Hz, Ca2+ transient amplitudes increase as the noise level increases (10–20%). Raise rate increase with 10% noise level added. At 3 Hz, we observe that Ca2+ transients increase with 10% noise induced. Without noise in the stimuli, Ca2+ transient amplitude was 15% reduced, and the raise rate was 15% reduced in DOX-treated myocytes. As noise level increases in the range (10–20%), both Ca2+ transients and raise rate increases at 0.5 Hz. At 1 Hz, Ca2+ transient amplitudes and raise rate increase with a 20% noise level added. Additionally, we found that in the range of 10–20%, noise-induced cell response reaches the control cell basal levels without noise in the stimulus   at 0.5, 1 and 3 Hz. Adding 10 and 20% of noise in the signal at 0.5 and 1 Hz in control cells, facilitates the CaHz) Fig. .2+ handling are improved in control and DOX-treated myocytes. It might be associated with alterations in the sarcolemma Ca2+ channels/transporters such as L-type voltage-dependent Ca2+ , Na+/Ca2+ exchanger (NCX) or other molecules related to the release of Ca2+ transient; since it has been shown that DOX treatment does not affect the activity or the expression of ICa,L, and NCX45. Recently, Sciancalepore and colleagues reported that “noisy” electrical stimulations were already proved to be more effective in inducing firing, Ca2+ transient changes and contractions in cultured mouse myotubes46. Moreover, Bosutti and colleagues demonstrated that noisy stimulus favors the excitability of skeletal muscle cells and suggested that intrinsic variability characterizing the “noisy” might facilitate a cumulative membrane depolarization responsible for the Ca2+ release from the SR during the excitation–contraction coupling, resulting in Ca2+ transients with increased peaks and area15. Our results agree with these authors (Fig. 13. Therefore in our experiments, we propose that the noisy stimuli may induce fluctuations in the membrane potential, increasing the probability of opening of ICa,L and therefore, improving the intracellular Ca2+ dynamics even in myocytes with compromised cellular contractility, Ca2+ handling, and cellular energetics. We drew this conclusion with reservations, a detailed experimental analysis of the contribution of each voltage sensitive molecule such as ICa,L, NCX, and RYR2 of excitable cells stimulated with noisy electrical signal requires further investigation.Our results show that both cellular contractility and Ca2+ handling and contractile response improves cell shortening, Ca2+ transient amplitude, and raise rate. Dox-treated cells reached the basal level of myocyte control with a specific noise level and frequency.We can conclude from these facts that the noisy stimulation of cardiomyocytes with impaired CaSupplementary Information.


Management of DCL in a patient with FA posed a unique challenge because of the need for a more effective approach than RIC without causing excessive toxicity. Since donor-derived myeloblasts would not be as sensitive to chemotherapy as the DDR-deficient blasts in FA-related MDS/AML, RIC alone would likely not induce a remission. However, intensive chemotherapy would be highly toxic in a patient in whom all the nonhematopoietic cells carried the FA defect. Therefore, she was treated with venetoclax/azacitidine, a regimen with minimal nonhematopoietic toxicity and low DNA damage potential known to induce high remission rates in NPM1-mutated AML, before a second RIC HSCT. The patient's guardian consented to the submission of the case report to the journal.Here, we report a case of a child who developed donor-derived FANCA mutation (Table 2A 5-year-old girl was diagnosed with FA after presenting with thrombocytopenia. BM showed hypocellularity (20%-40%) without dysplasia and normal cytogenetics. BM failure testing was negative for paroxysmal nocturnal hemoglobinuria (flow cytometry) with normal telomere lengths and serum adenosine deaminase levels. FA was diagnosed on the basis of abnormal chromosome breakage with diepoxybutane and mitomycin C and a compound heterozygous on Table .22 daily for 4 days), total body irradiation (300 cGy), and antithymocyte globulin (ATG). She experienced engraftment after 13 days with resolution of BM failure. Forty-five months after HSCT, she developed thrombocytopenia (9k/uL). BM showed myelodysplasia with 10% myeloblasts and 100% donor chimerism, consistent with DCL. NPM1, GATA1, and WT1 mutations were detected, indicative of donor-derived MDS/AML (Table 20She underwent a 4/6 matched cord blood HSCT preceded by RIC with low-dose cyclophosphamide (10 mg/kg daily for 4 days), fludarabine  and the planned use of RIC for a second HSCT, she was considered to be at high risk for post-transplant relapse without remission-inducing antileukemia therapy before conditioning. A pretransplant minimal residual disease (MRD)–negative remission is one of the strongest predictors of favorable outcomes for HSCT in AML.26 In AML, NPM1 mutations are strongly predictive of a high MRD-negative response rate (88%) with venetoclax/azacitidine therapy.29 Furthermore, the low DNA-damaging potential of venetoclax/azacitidine was favored given the exquisite sensitivity of the nonhematopoietic FA cells to DNA-damaging agents. Thus, the toxicity risks related to the FA DDR defect, chemotherapy mechanisms of action, and specific molecular profile of the MDS/AML drove the choice of venetoclax/azacitidine.Given the high risk of toxicity with DNA-damaging agents in FA and the high sensitivity of myeloblasts with the FA DDR defect, FA-related MDS/AML is typically treated with RIC HSCT without intensive chemotherapy before conditioning.2 dose once daily) for 1 week, and venetoclax was administered for 21 days instead of the typical 28 days to limit myelosuppression.31 The US Food and Drug Administration–approved adult dose of venetoclax with the recommended package insert reduction for concomitant posaconazole prophylaxis was used . She underwent craniotomy and evacuation; HSCT was delayed by 1 month. BM showed complete remission with MRD negativity by flow cytometry and NPM1 polymerase chain reaction (PCR). She subsequently contracted respiratory syncytial virus, necessitating a further 6-week delay of HSCT. Given the delay, an additional 14-day cycle of venetoclax/azacitidine was administered, a duration demonstrating similar efficacy and reduced myelosuppression relative to the conventional 28-day cycle in a study of elderly patients with AML.35 After the second cycle, the BM showed MRD negativity by flow cytometry; peripheral blood showed low level NPM1 PCR positivity (ratio of 0.0056). She then underwent RIC with low-dose cyclophosphamide, fludarabine, busulfan, and ATG followed by an 8/10 cord blood HSCT. BM was MRD-negative (flow cytometry and PCR) 3 months post-HSCT. She remains in remission over 1.5 years after HSCT.Azacitidine was administered .42 Of note, the presence of an NPM1 mutation is strongly predictive of response .41 Several laboratory studies have uncovered potential mechanisms by which mutant NPM1 increases sensitivity to venetoclax. NPM1-mutated AML cells show 20-fold higher sensitivity to venetoclax than NPM1 wild-type AML cells in vitro.43HOX overexpression, a key leukemogenic mechanism in NPM1-mutated cells,44 is strongly associated with increased sensitivity to venetoclax in ex vivo drug sensitivity studies.45 Venetoclax in combination with azacitidine eliminates leukemic stem cells by inhibiting mitochondrial electron transport chain complex II activity.46 Two studies have shown that inhibition of mitochondrial function sensitizes AML cells to venetoclax.48 Studies in mice and cell lines have demonstrated that mutant NPM1 impairs mitochondrial function, a likely mechanism for the exquisite sensitivity of NPM1-mutated cells to venetoclax.48 Venetoclax/azacitidine induced a morphologic response in six of eight children with MDS or AML; three were MRD-negative.49 To our knowledge, the use of venetoclax/azacitidine to achieve a deep remission in MDS/AML in FA or DCL has not been reported.Hematologic malignancies highly express the antiapoptotic protein B-cell leukemia/lymphoma-2 (BCL2).NPM1 mutation is not adequate for the diagnosis of AML when the blast percentage is below 20%. However, several studies suggest rapid progression to AML and a need for intensive AML-type therapy in MDS with NPM1 mutations, raising the question of whether NPM1-mutated MDS should be classified as AML.50 Since MDS with excess blasts and AML are a continuum and NPM1-mutated MDS tends to rapidly progress to AML, we considered our patient to have MDS/AML and used an AML-type approach to achieve a deep remission before HSCT.AML is distinguished from MDS on the basis of a BM blast cutoff of >20%. Several myeloid neoplasms are classified as AML on the basis of the presence of an AML-defining genomic aberration , regardless of the blast percentage. In the current WHO classification, NPM1-mutated DCL in a patient with FA where MRD-negative remission with limited toxicity was achieved using venetoclax/azacitidine before HSCT, and the patient remains disease-free for over a year after HSCT. This case suggests that venetoclax/azacitidine can induce a deep remission in children with NPM1-mutated myeloid DCL who cannot tolerate intense chemotherapy and may represent a useful approach in the setting of other DDR defects or telomere diseases  that are characterized by poor chemotherapy tolerance; these patients often undergo HSCT for BM failure and are thereby at risk for DCL.51 Studies such as the ongoing multicenter trial  of venetoclax/azacitidine are needed to investigate the outcomes and optimal dosing for this regimen in FA.In conclusion, we report a unique scenario of


We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C–28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca  •2+ rhythms are maintained at 22°C–28°C in SCN neuronsTranscriptional and Ca•2+ increaseRhythms are suspended at 15°C, accompanied by a large Ca•2+ rhythmRewarming instantly resets the Ca•2+ rhythm precedes the transcriptional rhythmsStable Ca Biological sciences; Neuroscience; Molecular neuroscience; Cellular neuroscience Period (Per) 1, Per2, Cryptochrome (Cry) 1, Cry2, Bmal1, and Clock, along with their protein products.10 = 2 to 3), while the Q10 of the circadian period is compensated to 0.8 to 1.2. This phenomenon is referred to as “temperature compensation,” an essential intrinsic circadian clock property.In mammals, daily rhythms in physiological and biochemical events, as well as animal behavior are coordinated by the master circadian clock localized in the hypothalamic suprachiasmatic nucleus (SCN) in the brain.Per1/2 and Cry1/2 through E-box cis-element, and resultant PERs and CRYs interfere with transcriptional activation by CLOCK-BMAL1 heterodimers.,Per1/2 are regulated by Ca2+/cAMP response element binding protein (CREB) through CRE. Because transcriptional activities of CLOCK and CREB are regulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), expression levels of Per1/2 are directly regulated by intracellular Ca2+ dynamics.,Bmal1 is rhythmically transcribed through ROR/REV-ERB responding element (RRE), and this transcriptional regulation forms the sub-loop.Per2 and Bmal1 are reported to regulate Ca2+ oscillations,,2+ oscillation in the SCN neurons. Moreover, it has been suggested that Ca2+ oscillation acts as a cytosolic messenger linking TTFL and physiological activities in the SCN neurons.,2+ oscillation sustains even in the SCN neurons of Cry1/2 deficient mice, in which the TTFL is disrupted.2+ signaling for temperature-compensated oscillations of TTFL.2+ signaling is a primary timekeeping mechanism to sustain TTFL oscillations.In the core transcriptional feedback loop, CLOCK and BMAL1 activate the transcription of ,,Various mammalian species use evolutionarily developed controllable hypothermia, torpor, and hibernation, by lowering thermogenesis and basal metabolism to survive during the harsh winter season for food.,,,,,,The entry of a hypothermic state, in both torpor and hibernation, is reportedly time-dependent with a circadian clock-regulated timing,2+ at the single-cell level in SCN slices,2+ rhythms in the SCN.,,,,2+ rhythms in SCN slices. We observed that the clock gene and Ca2+ circadian rhythms are maintained in the cold at 22°C–28°C, but suspended at approximately 15°C. Rewarming immediately resets the circadian Ca2+ rhythms, whereas the Bmal1 and Per2 rhythms gradually reestablish the phase relation relative to the Ca2+ rhythm. Our data demonstrate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in the SCN neurons.Pioneering work established time-lapse fluorescence imaging of intracellular Ca2-permeable filter, enabling us to monitor the SCN slices without temperature difference-related water condensation and evaporation during the long day recordings , and a red-color Ca2+ probe under a neuron-specific promoter  for visualization. We prepared mouse SCN slices and expressed these fluorescence probes by the adeno-associated virus (AAV)  D. To quaus (AAV) E. Based N slices G, togeth2+ and Bmal1 signals in the SCN slices at various temperatures  and assembled on figures the representative traces  and a nearly anti-phasic relation (13.2 ± 0.6 h) (n = 6 slices), the value of which was consistent with the results of a previous study.First, to assess the stability and continuity of the circadian rhythms under our experimental conditions, we measured Ca at 35°C A. We det,2+ and Bmal1   and the number of spots was considerably lower than that obtained using ionomycin and CaCl2. After rewarming, the signal spots quickly returned to undetectable levels. We observed small tissue shrinkage after rewarming, probably due to the elimination of damaged cells after rewarming. Based on these observations, we concluded that a long cold exposure at 15°C for 3–4 days causes mild cell damage in a small population of cells; however, damage can be restored and the cells can be recovered after rewarming.We further evaluated the condition of SCN neurons using a dying cell maker (SYTOX-Green) and performed time-lapse imaging under cold exposure . We adde0, 600%) A. Next, 0, 600%) C–S2F. At2+ and Bmal1 rhythms with an anti-phasic relationship could be detected for 12 days in the SCN neurons    A1. The Cctively) A2.2+ rhythm phase remained stable, whereas the Bmal1 rhythm was transiently delayed . After this transient delay, the Bmal1 rhythm phase advanced and the phase difference returned to the pre-chilling level on Day 7. At 28°C, the Ca2+ rhythm period slightly lengthened . Our statistical analysis showed that the Bmal1 rhythm amplitudes became significantly smaller , although it was not the case for Ca2+ rhythms   B2. These2+ and Bmal1 rhythms were delayed  . After rewarming, the period was returned to that of the pre-chilling levels   C1. Notabg levels C1. The C 0.0006) C2. These2+ and Bmal1 rhythms were detected , whereas that of Bmal1 was widely distributed on Day 1 after rewarming (SD = 7.9 h). Both rhythms reached a stable phase after a longer transient state . At 15°C, the Ca2+ baseline level increased rapidly and reached significantly higher levels on Day 1  and Day 4 . After rewarming to 35°C, the baseline levels returned quickly to lower values. In contrast, the Bmal1 baseline level gradually increased during the 22°C–28°C exposure  .Cold exposure at 22°C–28°C increased the Cae levels A. Notablor 22°C) C and 5D.2+ during cold exposure, we increased the intracellular Ca2+ concentration to the maximum level by supplementing the medium with 10 μM ionomycin and 20 mM CaCl2 at 15°C. This treatment further increased nes-jRGECO1a signal during cold exposure at 15°C  . FluoresBmal1 baseline levels at 28°C, but not at 22°C and 15°C, were significantly higher than Venus intensity . The Ca2+ baseline levels on Day1 and Day4 at 15°C, but not at 22°C–28°C, were significantly higher than mApple intensity   were virally expressed in the SCN slices. As expected, we observed increased Venus and mApple fluorescent signals upon cold exposure at 15°C  . Bmal1 b t test) B and 5D.2+ signal increase and rhythm suspension at 15°C, and rhythm resetting after rewarming, we performed time-lapse imaging using an FRET Ca2+ probe, Twitch2B, a troponin C-based Ca2+ probe.2+ signals under cold exposure.Fluorescent probes often suffer from pH sensitivity. To confirm the findings of CaBmal1 and Per2. The Bmal1 transcription depends on RORs and REVERBs present in the upstream region of Bmal1, whereas Per2 transcription is regulated through the cAMP responding element (CRE) or E-box by CREB or CLOCK.2+-CaMKII (32), Per2 is directly regulated by Ca2+ signaling.Next, we compared the cold responses of two transcriptional rhythms, Per2 promoter  together with nes-jRGECO1a in the SCN slices . Then, the SCN slices were cooled at 15°C for 4 days, followed by rewarming to 35°C for another 4 days . The Ca2+ increase was confirmed to be significantly larger than mApple in mouse SCN neurons (p = 0.0002). Notably, unlike in the case of Bmal1, both the Ca2+ and Per2 rhythms were restarted quickly on Day 1 after rewarming . In particular, the phase differences between Ca2+ and Per2 rhythms were large on Day 1 after rewarming (14.9 ± 0.5 h), but gradually recovered toward the original phase difference after a transient state for 4 days (9.2 ± 0.6 h) (We expressed Venus under N slices A. Simila neurons B. We obsr 4 days C. We obs± 0.6 h) D, right.2+ rhythm restarted on Day 1 and the Bmal1 rhythm gradually converged to a similar phase on Day 4, re-establishing the anti-phase relationship  . Based on these results, we conclude that most fluorescence signals observed in our analysis originated from SCN neurons.Astrocytes in the SCN reportedly exhibit circadian rhythms, synchronizing the SCN network and regulating animal behavior. slices) . The dou2+ rhythm after rewarming. The SCN slices were exposed to 15°C and the duration of cold exposure was systematically varied for 6–48 h. 2+ and Bmal1 rhythm traces and phase plots, respectively. Short cold exposures (6–24 h) delayed the phase but did not reset the rhythms , but they gradually converged into an identical phase on Day 4 (SD = 1.6 h) (2+ rhythms (13.0 ± 1.2 h).We examined the time necessary to reset the Ca rhythms A–S11C. Nst peak phase of the rhythms after rewarming. After 6–24 h of cold exposure at 15°C, the Ca2+ rhythms were not reset , but a 48-h cold exposure fully reset the Ca2+ rhythms (13.9 ± 0.7 h). In contrast, the Bmal1 rhythms were not reset by any of the cold exposures . These results suggest that a cold exposure over 48 h would be required for the phase resetting of the Ca2+ rhythms.Mesocricetus auratus)  with a phase difference (8.6 ± 1.0 h) in hamster SCN at 35°C (n = 3 slices) (2+ level (72.2 ± 16.5%). Interestingly, the magnitude of Ca2+ increase in Syrian hamster SCN neurons was significantly smaller than that in mice (p = 0.0001), implying a species difference in cold response. After rewarming, the circadian rhythms resumed and returned to identical phases  . Syrian  neurons A–S12B. W slices) C–S12E. Ul phases F. These ,,,2+ signaling compensates for the slow transcriptional oscillation speed at low temperatures.2+ rhythm persistence in SCN neurons lacking key clock genes Cry 1/2.2+ rhythms important for TTFL but they themselves are important for self-sustained, temperature-compensated oscillation. Therefore, we elucidated the mechanism of SCN rhythmicity mechanisms by simultaneously measuring clock genes and Ca2+ under various temperature conditions.Whether the clock gene transcriptional oscillations in the SCN neurons continue under hypothermia, specifically in the case of hibernation and daily torpor, has long been debated.2+, Bmal1, and Per2 in the mouse SCN neurons were suspended at a cold temperature of 15°C. These results are consistent with those of previous reports using immunostaining or in situ hybridization in the brain samples of hibernating animals.,We discovered that the circadian rhythms of CaThis study primarily used the mouse SCN slice culture, which is an excellent model to study the cold response of circadian rhythms in mammals. However, the core body temperature of mice is not expected to drop to 15°C under physiological conditions. Mice undergo daily torpor that lasts only hours, not days, and their core body temperature do not drop below 20°C, suggesting that their SCN continues to function normally during daily torpor.Interestingly, similar to the present results, the cyanobacterial circadian clock is lost at low temperatures around 19°C, and the self-sustained rhythm of cyanobacterial KaiC phosphorylation transformed to damped oscillations, as predicted by the Hopf bifurcation theory.2+ sources reportedly contribute to Ca2+ rhythm generation in the SCN neurons, including tetrodotoxin-sensitive mechanisms via action potential firing and neuronal coupling, and Gq-Ca2+ signaling.,,,,,2+ rhythms generation.,2+ and tension,2+ release from the internal stores. Although the origin of the cold-induced Ca2+ increase in SCN neurons remains unknown, Ca2+ release from ER might regulate the cold responses in the SCN.Multiple Ca2+ signaling at low temperatures could be a key factor for rhythm suspension and resetting. Reported estimates of Ca2+ rhythm amplitude and baseline level vary depending on the Ca2+ probes and experimental conditions.2+ probe properties , which are measured in vitro at a given temperature. For example, the basal levels and peaks of Ca2+ rhythms using genetically encoded FRET probes are 119–440 nM,2+ rhythm and magnitude of the Ca2+ increase was approximately 30% and 200%, respectively, in SCN neurons at 15°C. Given the temperature sensitivity of mApple protein, the base fluorescent protein of jRGECO1a, the Ca2+ level would be 4.3 times the Ca2+ rhythm amplitude. For example, using a recent estimate in SCN neurons,2+ levels under cold at 15°C would be approximately 370 nM. Future studies should aim at accurate measurement of the absolute Ca2+ concentration achieved through further biochemical and physiological experiments.Ca2+ rhythms in the SCN neurons. Upon rewarming, the Bmal1 phase was widely distributed on Day 1, and the Bmal1 rhythms gradually re-established the phase relation relative to the Ca2+ rhythms .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ryosuke Enoki  with newborn pups and pregnant Syrian hamsters from the animal breeder . The animals were housed in our animal quarters under controlled environmental conditions . The light intensity was adjusted to approximately 100–200 lx at the cage surface. The animals were fed commercial chow  and tap water. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Institute of Natural Sciences and performed according to the National Institute for Physiological Sciences guidelines (Approved No.22A044 and No.23A080).2, 0.5 mM CaCl2, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose, 10 mM HEPES, and 75 mM sucrose. A 200-μm coronal slice containing the mid-rostro-caudal region of the SCN was carefully prepared using a vibratome  and explanted onto a culture membrane  in a 35-mm Petri dish containing 1 mL of Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) and 5% fetal bovine serum (Sigma–Aldrich). Prior to recording, the culture membrane was transferred onto a glass-bottom dish  and the dishes were sealed with O2-permeable filters  using silicone grease compounds . We applied 500 μL of Mineral Oil (Sigma–Aldrich) onto the surface of the DMEM to prevent evaporation and condensation during the long recording in the temperature-changing environment. We added 10 μM α-tocopherol  to DMEM to prevent ferroptosis.2+ concentration, 10 μM ionomycin (Sigma–Aldrich) and 20 mM CaCl2 were added to the DMEM.The brains of neonatal mice and Syrian hamsters  were obtained in the middle of the light phase under hypothermic anesthesia, and rapidly dipped into an ice-cold balanced salt solution comprising 87 mM NaCl, 2.5 mM KCl, 7 mM MgClpAAV-Bmal-forward-intron336-Venus-NLS-D2 was generated by swapping the Cry1 promoter of pAAV-P(Cry1)-forward-intron336-Venus-NLS-D2 (Addgene) with a Bmal1 promoter.pAAV-Per2-intron2-Venus-NLS-D2 was generated by deleting the LoxP cassettes of pAAV-P(Per2)-DIO-intron2-Venus-NLS-D2 (Addgene). The plasmids of pAAV-hSyn-Venus and pAAV-hSyn-mApple were designed and created at vectorbuilder (vectorbuilder Inc). AAV vectors were packaged by using the AAV Helper Free Expression System  as previously described.syn-FLEX-jGCaMP8s-WPRE, AAV1.hSyn.Cre, AAV1-hSyn1.Twitch2B and AAV5-GFAP104-mCherry were purchased from Addgene. AAV aliquots (0.8 μL) were inoculated onto the surface of the SCN slices on Days 4–5 of culture. The infected slice was cultured for a further 9–12 days before imaging. To examine the cold response of rhythms at single-cell levels, we sparsely labeled SCN neurons using a mixture of AAV (hSyn-Flex-GCaMP8s and 1/1000 diluted hSyn-Cre). The titer of all AAV was over 1.0 × 1013 genome copies/mL.2+ and transcription rhythmicity. The low-temperature time-lapse imaging system was composed of an EM-CCD camera , an inverted microscope , dry objectives , a stage incubator , an XYZ controller, a filter wheel  and an LED driver . The microscopic system was placed in the low-temperature controller . The upper part of the inverted microscope was removed to fit into the low-temperature controller. The microscopic system was controlled by the MetaMorph software (Molecular Devices).Bioluminescence imaging using luciferase reporter is a generally used standard method to observe circadian rhythms.2 with a gas mixer . Tissue and cell conditions were monitored by cultured slice bright-field images and calcium signals in individual neurons. The SCN slices were prepared from pups of the same mother and multiple imaging data were obtained in a single imaging experiment using a multi-dish holder and controllable XYZ-stages.0 = (F(t) - F0)/F0, where F(t) is the fluorescence value at a given time and F0 is the minimum fluorescence value. One round of cooling was performed per experiment.The time-lapse wide-field imaging was conducted with an exposure time of 100 ms at 1-h intervals. Venus and jRGECO1a were excited by cyan  and yellow color (580/14 nm) with the LED light source  and the fluorescence was visualized with a dual-edge dichroic mirror  and emission filters . For FRET imaging using Twitch2B, mCerulean was excited by blue color (438/24 nm) with the LED light source, and mCerulean and cpVenus fluorescence was visualized with a dichroic mirror  and emission filters . We continuously perfused 5% COE. coli (JM109(DE3)) was transformed and cultured in 200 mL of liquid LB medium at 23°C for 72 h with 140 rpm rotation. E. coli was collected by centrifugation, resuspended in PBS, and crushed using a sonicator. The recombinant protein was purified from the supernatant using Ni-NTA agarose (QIAGEN) and a PD-10 column (Cytiva), followed by exchange with TN buffer  using the illustra NAP-5 column . Purified nes-jRGECO1a was characterized in 30 mM MOPS, 100 mM KCl, pH 7.2  containing either 10 mM CaEGTA  or 10 mM EGTA (EGTA buffer). Fluorescence spectra were measured with excitation at 400–600 nm (5 nm bandpass) and emission at 500–700 nm (5 nm bandpass) at temperatures of 5°C–35°C with 5°C increments using a fluorescence and absorbance spectrometer . The pH of the solutions at each temperature was measured using a pH meter (Horiba). The obtained data (excitation/emission wavelengths of 555 nm/600 nm) was plotted as the log of the [Ca2+]free (x axis) versus the log {(F − Fmin)/(Fmax − F)} (y axis) and the KD and Hill coefficients were calculated from the linear fitting. The [Ca2+]free was calculated from the Kd of EGTA for Ca2+ using the following equation.nes-jRGECO1a was cloned into a pRSETb vector. Using this vector, dEGTA for pH was calculated by the following equation.Correction of K1 and K2 are 109.58 M−1 and 108.96 M−1, respectively, and the primary Ca association constant KCa is 1010.97 M−1 at 20°C. The gas constant R is 1.9872 × 10−3 kcal·K−1mol−1 T, ΔH of KCa is −8.0 kcal mol−1, ΔH of K1 is −5.8 kcal mol−1, ΔH of K2 is −5.8 kcal mol−1, and T represents absolute temperature in kelvin. These values are put into the following Van’t Hoff isochore and the ponton and Ca association constants K′ for each temperature were calculated.,Ponton association constant KBmal1/Per2 immediately after rewarming might reflect an acute response of the reporter proteins, we thus analyzed the second peak of the rhythms. The group mean was presented as the mean ± SEM. Circular plots were generated using Oriana (Kovach Computing Services). One-way ANOVA with a post-hoc Dunnett’s post-test was used to validate the temperature effects when paired multiple group data were compared within the same groups. Paired or unpaired t-tests were used when comparing two dependent and independent group means.Statistical analyses were performed using Excel (Microsoft) and Prism GraphPad (GraphPad Software). The imaging data were detrended by a 24-h running average subtraction method, and the peak phases of the rhythms were estimated by the peak phase of the rhythms. The first peak of


Therapy-related acute myeloid leukemia (t-AML) is one of the most serious long-term complications of cancer chemotherapy. Various cytotoxic agents and exposure to ionizing radiation can lead to the development of t-AML, which is usually associated with adverse genetic changes and a poor prognosis. Over the past decade, insights into leukemogenesis have generated significant advances in the risk stratification of t-AML, and have offered us the opportunity to develop individualized options for treatment that target disease biology. In this article, we review current knowledge on the biology of t-AML, putting emphasis on its molecular origin; we also discuss recent advances in its treatment including CPX-351, the use of less intensive regimens , and novel, molecularly targeted and antibody-based therapies that promise to increase the cure rate.Therapy-related acute myeloid leukemia (t-AML) comprises 10–20% of all newly diagnosed cases of AML and is related to previous use of chemotherapy or ionizing radiotherapy for an unrelated malignant non-myeloid disorder or autoimmune disease. Classic examples include alkylating agents and topoisomerase II inhibitors, whereas newer targeted therapies such as poly (adenosine diphosphate–ribose) polymerase (PARP) inhibitors have emerged as causative agents. Typically, t-AML is characterized by adverse karyotypic abnormalities and molecular lesions that confer a poor prognosis. Nevertheless, there are also cases of t-AML without poor-risk features. The management of these patients remains controversial. We describe the causes and pathophysiology of t-AML, putting emphasis on its mutational heterogeneity, and present recent advances in its treatment including CPX-351, hypomethylating agent plus venetoclax combination, and novel, molecularly targeted agents that promise to improve the cure rates. Evidence supporting personalized medicine for patients with t-AML is presented, as well as the authors’ clinical recommendations. According to the 2017 World Health Organization (WHO) classification system for tumors of hematopoietic and lymphoid tissues, therapy-related myeloid neoplasms include cases of acute myeloid leukemia (t-AML), myelodysplastic syndromes (t-MDS), and myelodysplastic/myeloproliferative neoplasms (t-MDS/MPN), which arise as a complication of cytotoxic chemotherapy and/or radiation therapy administered for a prior neoplastic or non-neoplastic disorder .,35.TP53 TP53 is mutated in a large proportion of tumors ; this is generally attributed to the accumulation of over-stabilized, mutant protein). Less frequent mutations include deletions, truncations, insertions, nonsense, and splice-site mutations [TP53 mutation requires the presence of a somatic TP53 mutation with a variant-allele frequency [VAF] of >10%. Monoallelic TP53-mutated t-AML has a poor prognosis. The term “multi-hit” mutations refers to the presence of two distinct TP53 mutations (each with a VAF of >10%) or a single TP53 mutation with either (i) 17/17p deletion on cytogenetics; (ii) copy-neutral loss of heterozygosity (LOH) at the 17p (TP53 locus); or (iii) VAF of >50%, which is 75% concordant with copy-neutral LOH variants . Ac. Ac15].  unclear .TP53 mutations), but also due to the sequelae of prior chemotherapy and sometimes persistent primary disease, particularly metastatic cancer or lymphoma.Historically, patients with t-AML have been under-represented within clinical trials and excluded from large-scale studies. Excluding the rare patient with t-AML who belongs to the 2022 ELN favorable-risk group, the overall prognosis of t-AML remains very poor ,9,22,78.There have been conflicting reports regarding the incidence and prognosis of t-AML with favorable-risk lesions. In 2004, Schoch and colleagues showed that the prognosis of patients with t-AML was more favorable than s-AML, and that patients with t-AML and intermediate or unfavorable cytogenetics had similar OS with their de novo counterparts, whereas age and white-cell count had no impact on OS in t-AML . HoweverGemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody conjugated with the cytotoxic agent calicheamicin, has shown positive results in newly diagnosed CBF AML. A meta-analysis of five randomized trials confirmed that although adding GO to standard chemotherapy did not increase response rates, it reduced the risk of relapse and significantly improved OS among younger and older adults with CBF AML ,82. ThusNPM1 mutations in t-AML. Among 140 patients with t-AML, they identified 10 patients harboring NPM1 mutations, of whom four had a normal karyotype and raised the question whether these four patients actually represented “de novo AML with medical history” rather that true t-AML [NPM1-mutated t-AML without karyotypic lesions does exist [p = 0.95). They concluded that the best combination for survival was NPM1-mutated/FLT3 wild-type, and that such patients could be approached therapeutically similarly to patients with de novo AML. Notably, the presence of FLT3-ITD did not seem to have a negative impact on survival in t-AML (perhaps due to their low frequency), while NPM1-mutated t-AML carried significantly better survival rates than NPM1 wild-type t-AML. This study also revealed that patients with t-AML were less likely to receive intensive chemotherapy and undergo HCT than their de novo counterparts. The study by Nilsson and colleagues, unlike the study by Schoch, demonstrated worse OS in t-AML with intermediate-risk and adverse cytogenetics and similar OS in t-AML with favorable-risk cytogenetics, compared to de novo AML [Falini and colleagues first demonstrated that the abnormal cytoplasmic localization of nucleophosmin resulted in increased responsiveness to induction chemotherapy in patients with normal-karyotype AML . In 2008ue t-AML ,85. Anotes exist . In a Swnovo AML ,87.As a rule, patients with t-AML should be managed according to the same general therapeutic principles  as their de novo counterparts.In practice, the patient’s age, general health/performance status, nutritional status, specific coexisting conditions/comorbidities, possible complications from the original disease or previous treatments, sequelae of prior chemotherapy , cumulative dose of anthracyclines, chronic immunosuppression from prior disease or ongoing therapy, history of severe infections, and status and prognosis of the underlying primary malignant or autoimmune disorder, as well as the disease features, the patient’s wishes  and emotional status, and the physician’s attitude and interest all influence clinical decision making . It is iPatients considered fit for intensive chemotherapy should be treated with standard anthracycline plus cytarabine induction chemotherapy (“3 + 7”), followed by standard consolidation and/or allogeneic HCT, depending on their 2022 ELN genetic risk  are usually treated―outside of the context of a clinical trial―with the hypomethylating agents (HMA) azacitidine or decitabine, either alone or in combination with venetoclax, an oral selective inhibitor of the anti-apoptotic protein BCL-2.NPM1, IDH1/2, or TET2 (and possibly RUNX1 and STAG2 in the relapsed/refractory setting) respond favorably to azacitidine–venetoclax (or decitabine–venetoclax), independently of the underlying karyotype. In particular, the 1-year survival rate of NPM1-mutated AML receiving venetoclax with azacitidine was 80% versus only 12% for azacitidine alone and 56% for induction chemotherapy [IDH-mutated AML, particularly IDH2-mutated AML [TET2 mutations are also very sensitive to azacitidine–venetoclax, with a CR/CRi rate of ~86% (versus 39% for TET2 wild-type AML) [RUNX1-mutated AML may also respond favorably to the venetoclax with azacitidine or decitabine with an expected CR/CRi rate of 35–75% and prolonged OS. However, newly diagnosed RUNX1-mutated AML is less sensitive to azacitidine–venetoclax than RUNX1 wild-type AML (CR/CRi rate 50% versus 64%) [STAG2 mutations and improved survival after azacitidine–venetoclax combination was reported, albeit only when azacitidine–venetoclax was administered for relapsed/refractory AML [It has been shown that patients with AML harboring mutations in otherapy . Similarated AML ,94. TET2ype AML) ,94. Patisus 64%) . In a retory AML .TP53, PTPN11, N-RAS/K-RAS, U2AF1, and perhaps FLT3-ITD mutations have been associated with poor outcomes and, possibly, reduced sensitivity to azacitidine–venetoclax. The co-occurrence of high-allelic-ratio FLT3-ITD with NPM1 mutations adversely affected outcomes compared to NPM1 mutations alone or NPM1 mutations with low-allelic-ratio FLT3-ITD [FTL3-mutated AML treated with intensive chemotherapy was 5.8–26 months versus 12.5 months for azacitidine–venetoclax and 8.6 months for azacitidine alone [N-RAS, K-RAS, and PTPN11 mutations are prevalent in t-AML. When treated with HMA–venetoclax, N-RAS mutations are associated with poor response rates (CR/CRi 0–36%) and shorter median OS (3.8 months), compared with patients with wild-type N-RAS (7.4 months) [TP53 mutations respond poorly and relapse early after conventional chemotherapy (2-year OS 12.8–14%) [TP53-mutated AML) [In contrast, FLT3-ITD . The medne alone . N-RAS,  months) ,94,95. P2.8–14%) ; in addiSRSF2, ZRSR2, U2AF1, and SF3B1) have variable sensitivity to combination therapy, possibly due to the co-occurrence of other mutations. For example, SRSF2 mutations, which are often associated with IDH2 mutations, have demonstrated favorable response to azacitidine–venetoclax, with a 1-year and 2-year OS of 100% and 87%, respectively [IDH2 mutations preferentially co-occur with SRSF2 mutations rather than other mutations in RNA splicing machinery (88% versus 11%). By contrast, U2AF1 mutations co-occur with RAS mutations, suggesting that the resistance and early relapse of U2AF1-mutated AML after azacitidine–venetoclax may be the result of RAS co-mutation rather than of U2AF1 itself [SF3B1-mutated AML has been identified as a marker of venetoclax resistance [SF3B1-mutated AML appears to be particularly sensitive to decitabine-based therapy) [Spliceosome-complex mutations  , can ovetherapy) .ASXL1 mutations are associated with BCL-2 overexpression and increased levels of global cytosine hypermethylation, suggesting sensitivity to venetoclax–azacitidine [In vitro studies have shown that citidine ; howeverDNMT3A-mutated relapsed/refractory patients who were naive to HMAs achieved high response rates and prolonged survival with venetoclax combination therapy, whereas DNMT3A-mutated patients who had previously received HMAs responded poorly to HMA–venetoclax, with poor survival outcomes [DNMT3A mutations to epigenetic modification. Many studies to date have focused on the metabolic and apoptotic effects of these combinations , but it is also important to investigate the mechanism by which venetoclax may act synergistically with HMAs to modify epigenetic targets in DNMT3A-mutated AML. It is not expected that clonal hematopoiesis (particularly DNMT3A mutations) will be eradicated after first-line HMA-based treatment.In one study, outcomes . This maTP53 mutations and/or complex karyotype. In the phase III randomized control trial VIALE-A, the addition of venetoclax to azacitidine for patients with AML who were ineligible for “3 + 7” because of age (≥75 years), coexisting conditions, or both led to remarkable improvements in CR/CRi rates (66.4% versus 28.3%) and median OS (14.7 months versus 9.6 months) compared to azacitidine alone [TP53 mutations or complex karyotype . A phase II study found that a 10-day course of decitabine improved the outcome of patients with TP53-mutated AML enough to resemble that of intermediate-risk AML [TP53, leading to venetoclax resistance). Clearly, there is an urgent need for new treatments for these patients.Despite their widespread use, it is important to note that HMAs have not significantly improved the outcome of patients with t-AML; the median OS of t-AML treated with first-line HMA is approximately 7 months. A recent study confirmed the poor outcome of t-AML treated with HMAs . The outne alone . This strisk AML ,107. Howrisk AML . DespiteFor patients with t-AML aged ≤60 years who are deemed fit for intensive chemotherapy, National Comprehensive Cancer Network (NCCN) guidelines recommend either standard induction chemotherapy (“3 + 7”) or CPX-351, whereas for patients aged ≥60 years who are eligible for chemotherapy, NCCN guidelines recommend CPX-351 . SimilarAlthough CPX-351 offers something new in the landscape of t-AML therapy, most patients with t-AML are less chemosensitive than those with primary AML and, therefore, do not benefit from either CPX-351 or “3 + 7”. The efforts made by the researchers over the previous decades to improve chemotherapy regimens have not led to major improvements, until the recent evolution seen with new molecularly targeted therapies directed at specific mutations. Ongoing research and clinical trials are actively seeking ways to personalize therapy in t-AML by identifying new targets, discovering patient-specific and disease-specific risk factors, and finding effective combinations of new agents targeting multiple cellular processes .TP53-mutated AML treated in this trial. The median duration of response was 8.7 months and the median OS was 10.8 months. In another study, patients with newly diagnosed TP53-mutated AML (n = 14) were treated with magrolimab in combination with venetoclax and azacitidine. The ORR was 86%, with a CR rate of 64%, MRD-negativity of 55%, and substantial clearance of TP53-mutated clones in eight out of nine patients who achieved CR/CRi [Magrolimab (Hu5F9-G4) is a first-in-class humanized IgG4 monoclonal antibody against CD47 and prompts cancer cell phagocytosis by macrophages through the disruption of the CD47–signal-regulatory-protein-alpha (SIRPα) inhibitory checkpoint, thereby blocking the “don’t eat me signal”. CD47 is also a marker of LSC, and targeting CD47 can potentially eliminate LSC while sparing normal HSC . Magrolid CR/CRi .TP53-mutated AML with a median duration-of-response of 6.4 months [TP53-mutated AML, the overall response rate was 47% with an encouraging median OS of 10.3 months in responding patients.Sabatolimab is a humanized, high-affinity IgG4 monoclonal antibody targeting TIM3, a myeloid checkpoint that forms part of a co-inhibitory receptor complex overexpressed on exhausted T-cells and also on LSCs in patients with AML/MDS. A phase Ib trial evaluated sabatolimab in combination with HMAs in newly diagnosed patients with AML (n = 48) and higher-risk MDS (n = 53) unfit for intensive chemotherapy. The CR/CRi rate was 40% in patients with 4 months . Another4 months . Among pTP53 mutations in t-AML and their poor prognosis, particular reference is required for novel agents that target the TP53 pathway. The frequency of TP53 abnormalities increases to 70–80% in patients with complex karyotype, -17/17p, -5/5q, and -7 [TP53 mutations in t-AML may be explained by the observation that pre-existing progenitor cells that carry a TP53 mutation are resistant to DNA damage and, therefore, can expand under selective pressure from chemotherapy administered for a non-myeloid disorder [Given the frequency of , and -7 ,116. As disorder ,117.TP53 mutations is inherently problematic for patients receiving cytotoxic chemotherapy since the mutated p53 protein causes dysregulation of the apoptotic pathway, decreasing the efficacy of DNA-damaging chemotherapy agents. TP53 mutations, especially multi-hit, result in poor clinical outcomes regardless of whether the underlying disorder is classified as MDS or AML on the basis of blast-cell count. Multi-hit t-MDS/AML often represents a distinct stem-cell disorder with a paucity of co-mutations in other myeloid malignancy-related genes (co-mutations occur in <20% of cases). As mentioned, another adverse-risk biological characteristic associated with TP53 mutation and complex karyotype in t-AML is chromothripsis, which is a catastrophic event leading to extensive chromosomal rearrangement and defines a subset of complex-karyotype AML with an extremely poor outcome [The presence of  outcome .TP53 mutations only (52% versus 30% in patients with co-mutations), including those with biallelic TP53 mutations or complex karyotype (CR 49%) [Eprenetapopt (APR-246) is a first-in-class drug that binds covalently to cysteine residues in the DBD domain of the mutant p53 protein. It has been suggested that this binding would induce mutant p53 protein to fold back into its active, wild-type conformation and function. Two studies evaluated eprenetapopt-azacitidine in patients with newly diagnosed MDS, AML, and MDS/MPN; they found a significantly higher CR rate in patients with (CR 49%) .TP53-mutated AML. TP53 mutations are associated with an accumulation of immune-inhibitory checkpoints including PD-L1 on LSCs, TIM-3 on myeloid-derived suppressor cells (MDSCs), and LAG3 and TIGIT on leukemic blasts. Furthermore, TP53-mutated MDS and AML is characteristically associated with an immunosuppressive marrow environment with FOXP3 overexpression, increased numbers of activated (ICOShigh) T-regs and PD1+ MDSCs, decreased numbers of OX40+ cytotoxic T-cells and NK cells, and a marked impairment of CD28+ T-cells to secrete immune-effector T-helper-1 cytokines [TP53-mutated t-AML [In search of potential therapies, studies have also evaluated the immune microenvironment and cytokine milieu of ytokines ,119. Suced t-AML .The occurrence of t-AML is an important complication of the treatment of a variety of tumors and autoimmune disorders. More than two thirds of these patients have adverse prognostic features. Over the past decade, an understanding of the molecular pathways leading to the development of AML has resulted in significant improvements in the risk stratification of patients and offered the opportunity to develop individualized therapeutic approaches that target disease biology. The development of new targeted drugs and treatment strategies have improved the outlook of patients with AML, including t-AML. Despite these improvements, however, survival rates for most patients with t-AML remain poor. Clearly, new approaches to therapy are needed in this large cohort of patients. In general, the trend in the treatment of t-AML is toward the modification of therapy to treat specific subtypes of the disease (“biomarker-driven” strategy) and, more specifically, the targeting of the leukemic cells with highly efficacious molecular-targeting agents and/or immunologic (antibody-based) therapeutic strategies . Current


The impacts of ear disease on animal welfare and behaviour are little documented. Ear disease may be common in rabbits, but difficult to recognise, and lop-ears have previously been indicated as a risk factor for ear disease. We aimed to better understand the range of ear conditions in pet rabbits, signalment risk factors, and impacts on welfare and behaviour. Through an online questionnaire, we investigated owner-reported signalment, veterinary diagnosis of ear conditions, impaired hearing, and ear pain for UK pet rabbits. Relationships between ear condition measures and ear conformation, quality of life, and behaviour were analysed using logistic regression. Of 551 valid responses, 28.5% of rabbits reportedly had experienced ear conditions; 21.2% diagnosed or mentioned by vets, with otitis and excess cerumen most common. Approximately 25% of lop-eared rabbits had ear conditions indicated by a vet versus 10% of erect-eared rabbits. Lop-eared, half-lop, and older rabbits were most at risk (P<0.050). Rabbits reported as showing ear pain responses had reduced owner-reported quality of life compared with other rabbits (P<0.050). Rabbits with ear problems were less likely to be responsive to relevant sounds, and performed binky behaviour (joy jumps) less frequently, than rabbits without such issues. Understanding prevalence and risk factors for ear conditions is critical to improving welfare standards across this widely owned pet species. The findings suggest that improved recognition and treatment of ear conditions, and avoiding breeding from rabbits with early signs, or a family history, of ear disease are necessary to help combat this animal welfare issue. Ear conditions could represent considerable animal welfare issues, because they can variously cause pain, hearing impairment, loss of balance, or a combination of these. We aimed to explore the nature and scale of ear conditions as a welfare issue in pet rabbits, via an owner questionnaire. In particular, we were interested in the relative prevalence of different ear conditions in a pet population, effects of ear conformation and other signalment factors on ear disease prevalence, and effects of ear conditions on rabbit welfare and behaviour.Textbooks often describe ear diseases as ‘common’ in rabbits e.g. –3, but pHowever, the true prevalence could be higher, because many ear conditions are difficult to recognise in rabbits , 7. ThisEar conditions have been reported as especially common in rabbits with ‘lop’ ears . This is mainly because the ear canal in lop-eared rabbits is usually stenotic (narrowed) and the pinna folds over the canal, blocking cerumen (ear wax) and other exudates from being expelled , 16, 17.The effects of ear conditions on quality of life (QoL) in animals generally are not well documented, but vets and animal welfare experts have highlighted ear disease as a potential welfare concern for pet rabbits . Vets ofMammals share many of the ear pathologies observed in humans , 31 and,Effective owner identification of problems to then enact interventions is critical to improving welfare for pet species. Some ear problems do cause distinct signs that may be observed by owners, according to veterinary texts. For example, moderate–severe otitis externa, including ear mites, can cause head shaking and ear-scratching by rabbits, and excess white exudate, brown crusting, and erythema (redness) may be seen inside the ear , 41 and It will be important to better understand signs of ear conditions, how owners in everyday situations perceive ear pain (e.g. via rabbits’ responses to the face and ears being touched), and hearing loss (e.g. via responsiveness to significant positive and negative sounds), as well as what they notice when looking inside their pets’ ears. Similarly, understanding of whether ear conditions reduce positive behaviours, specifically playing, binkying (‘joy jumps’ or ‘freudensprung’), and exploration, will help characterise the potential welfare outcome of ear conditions. For example, reduction of play is included within an otitis media pain scoring system for children , and becWe aimed to describe the range of ear conditions reported by owners in a population of UK pet rabbits, and to investigate whether the lop-eared phenotype increases the risk of ear problems in these rabbits. We hypothesised that, if lop-eared conformations predispose rabbits to ear conditions, then owners of lop-eared rabbits would be more likely than owners of erect eared rabbits to report (a) that a veterinarian had indicated an ear condition in their rabbit; (b) that their rabbit showed ear pain responses (behaving as if it was painful when owners looked inside their ears); (c) that their rabbit had impaired hearing or deafness; and (d) that their rabbit had an ear problem that reduced its QoL.We also aimed to understand the potential welfare impact of ear conditions on rabbits (regardless of their ear conformation). Our hypothesis was that, if ear problems impair QoL in rabbits and if owners are able to perceive this, then rabbits reported to have veterinary indicated ear conditions, ear pain responses, or impaired hearing/deafness, would have significantly worse owner reported QoL ratings than rabbits without those problems.Finally, we aimed to elucidate what owner-observed rabbit behaviours may be associated with ear problems including hearing impairment and ear pain responses. Awareness of these behaviours could aid owners, vets and breeders to recognise ear problems and their impact, potentially increasing treatment of these conditions in rabbits. We hypothesised that, if hearing impairment and pain affects behaviour that owners can perceive, then, compared with healthy rabbits, affected rabbits would be less responsive to sounds of a treat being prepared, less responsive to loud or threatening sounds, and less likely to perform binky behaviour, show reduced play behaviour, reduced exploration of new areas, and show avoidance of being stroked on the face (risking touching the ears).Taken together, this information could be used to improve rabbit welfare by raising awareness of ear conditions, which rabbits may be most susceptible to ear problems, and the extent of their impact on rabbit welfare and behaviour. Increased awareness of these factors should help owners and veterinarians to identify which rabbits need treatment, and help owners and breeders to select healthy rabbits that may be less prone to debilitating ear conditions.® , as well as via relevant organisations including the Rabbit Welfare Association and Fund, RSPCA, the Rabbit Residence Rescue, and the Royal Veterinary College External Relations Team. It was also posted to a number of rabbit-related Facebook™ groups.An online questionnaire was hosted by Survey MonkeyThe anonymous respondents had to be at least 18 years old, and to currently own a rabbit. If they owned more than one rabbit, they were asked to answer the questionnaire about only one of their rabbits. To help minimise sampling bias towards or against owners of rabbits with ear disease or with particular ear conformations, the only stated aim of the project was ‘to understand how both good and bad ear health and hearing impairments affect rabbit welfare and overall quality of life’. We called for ‘owners of rabbits, whether the rabbit has excellent hearing or seems to have sore ears or difficulty hearing’. Ear conformation was not mentioned in the survey introduction, and only appeared as a signalment question in the ‘About your rabbit section’, to try to avoid leading owners to think about any connection between ear conformation and disease.The questionnaire took approximately 10 minutes to complete, with most questions being optional. There were 32 questions, comprising multiple choice options where respondents could either tick one answer only, or all that applied, as appropriate. There were also optional free-text comment boxes for many questions. For answer options with no natural order, e.g. lists of rabbit breeds or ear diseases, the order in which the options appeared was randomised for each respondent. This was done to help minimise order effects arising from respondents selecting early answer options more frequently than later options.The survey was structured in sections as follows.Introduction. This explained the broad aim of the project as being to understand the rabbit welfare impact of ear health, asked for rabbit owners aged 18 years or over to respond, and asked for consent.About you. This contained demographic questions, including respondent age, gender, country of residence, and whether they worked with rabbits or in the veterinary profession.About your Rabbit. This contained demographic questions about the rabbit, specifying that owners must only answer about one rabbit. Questions covered the number of rabbits owned, sex and neuter status of the focal rabbit, age, breed type, and ear conformation , or unsure).Your rabbit’s quality of life. In this section, owners were asked to rate their rabbit’s QoL (Very good–Very Bad) and whether the rabbit had an ear problem or other health problem that affected this. They were also asked about their rabbit’s behaviour, including response to being stroked on the face, frequency of play behaviour, binkying, response to new environments, and a subjective description of the emotional character of the rabbit .Your rabbit’s hearing. This included questions about the owner-perceived extent of hearing ability in the focal rabbits, why owners believed their rabbit could/could not hear well, and how their rabbit responded to specific sounds .Your rabbit’s health. Questions in this section included frequency of ear cleaning by a vet, questions about what was seen in the rabbit’s ears and how the rabbit responded to the owner looking into its ears, ear conditions indicated by a vet, and age of diagnosis. A final invitation for any other comments as free-text was also provided.Respondents were excluded if they reported being under 18 years old or did not provide their age, or who did not live in the UK (due to the fact that insufficient responses were received from outside the UK). Any respondents who dropped out without answering the ‘about your rabbit’ section, or before answering any questions in the subsequent sections, were excluded. Respondents reporting not owning a rabbit, or having only owned a rabbit in the past, were also excluded. Respondents who reported current fostering of rabbits were included.Following manual data check and cleaning, analyses were performed using SPSS . Some respondents did not answer all the questions, so the sample size varied between analyses. Categorical data were described as percentages  and non-normally distributed scales were summarised using median and interquartile range (IQR). Some variables could not be analysed due to insufficient variation in responses . A Kruskall-Wallis H test was used to compare the distribution of rabbit age across the ear conformations. Pearson’s chi-square tests with pairwise Z-tests were used to compare the distribution of sex/neuter status, owner age, and owner gender between erect and lop-eared rabbits. To test the convergent validity of our four measures of ear problems, Mann-Whitney U tests were performed to understand relationships between each pair of ordinal and binary variables.P < 0.05.To investigate risk-factors for ear or QoL outcomes, multivariate binary logistic regressions were performed. Individual models were produced for each outcome variable and included other background predictor variables, as detailed below. Outcome variables with more than two categories were collapsed to form binary variables ?’); hearing impairment; ear pain response (from the question: ‘Do you think your rabbit behaves as if it is painful when you try to look into their ears?’); and an ear-related problem which affected rabbit QoL. Predictors used in these models were ear conformation, rabbit age, sex/neuter status, owner age, and owner sex. The two-way interaction between age and ear conformation was also initially included in the models and removed if non-significant.To test whether owners perceived ear conditions to affect rabbit QoL, reported overall QoL was the outcome variable and was collapsed into two categories: ‘very good’, and ‘reduced’, which incorporated all other responses (which comprised ‘good’ or ‘moderate’). Further output variables tested were the owner reports of their rabbits being ‘happy’, ‘relaxed’, ‘curious’, or ‘nervous/fearful’. Predictor variables tested in separate models were non-collapsed reports of hearing ability, and the binary conditions of vet indication of an ear problem, and having ear pain response or not. Additional predictors used in this model were again rabbit age, sex/neuter status, owner age, and owner sex, plus whether the rabbit had a non-ear-related health condition that impaired their QoL.To investigate perceived behavioural predictors of ear problems, outcome variables of ‘does not respond, and may startle, when approached from behind’, ‘does not respond to the sound of a treat (unless they can see/smell the treat)’, ‘does not respond to loud or threatening sounds’ were each collapsed into binary variables indicating responsiveness or unresponsiveness to the respective sounds. Further binary outcome variables of a rabbit ‘does not enjoy being stroked around the face/ears’ (At least sometimes/Not particularly or no), ‘rarely performs binky behaviour’ (At least sometimes/Rarely), ‘plays with toys, other rabbits, or familiar people?’ (Sometimes/Not particularly), and ‘actively explores new environments’ (explorative/cautious) were also modelled. Predictor variables included non-collapsed vet indication of an ear problem and impaired hearing, and the binary condition of having ear pain response. Additional predictors used in these models were ear conformation, rabbit age, sex/neutered status, owner age, owner sex.Initially, 693 responses were received, with 142 responses being excluded by the defined criteria. Of the 551 included respondents, 95.1% were female and 4.0% were male. Four respondents were non-binary (0.72%) and a single person selected ‘prefer not to say’. The most common age category was 25–34 (37.0%), followed by 35–44 (22.7%). The younger, 18–24 (13.7%) and older age groups, 45–54 (15.6%), 55–64 (8.5%) and 65+ (2.4%), were less represented. The oldest two age categories were combined for analysis due to the low numbers. Regarding work type, 238 (75.6%) of respondents did not work with rabbits, and 35 (11.1%) reported working with rabbits . There were 15 (4.8%) vet students and 11 (3.5%) veterinarians. Almost half of all respondents (46.7%) owned two rabbits, with 31.8% owning a single rabbit and 21.0% owning three or more.P = 0.314). There was similarly no significant difference in the proportion of sex/neutered status (P = 0.880), owner age (P = 0.764), or owner gender (P = 0.692) between the erect and lop-eared rabbits. The most common owner reported breed types were dwarf/mini lops (which were a combined category in the survey) and crossbreeds.Of the 551 rabbits, 48.5% of rabbits were reported as being lop-eared, 42.5% were erect-eared, and the remainder were either asymmetrical , or had approximately horizontal ears  as having had a vet indicated ear problem . Taken tThe most common ear conditions indicated by a vet were otitis–particularly otitis media and interna–and excessive ear wax ; ear mitU = 21554, N = 445, P < 0.001). Of the rabbits with a vet indicated ear problem, and whose owners had looked into their ears, 11.1% were reported to flinch and pull away during ear examination compared with 2.6% of rabbits flinching during ear examination if they had no such veterinary indication. Similarly, lower hearing ability scores (indicating more impaired hearing) were more prevalent with vet indicated ear problems  than without . Finally, better QoL scores were more prevalent without vet indicated ear problems  than with such indications .Regarding convergent validity of our four measures of ear problems, ear pain responses, hearing ability, and QoL were all worse in rabbits with vet indicated ear problems. Higher ear pain response scores were more prevalent in rabbits with vet indicated ear problems  than without such vet indication  and ear pain responses (P = 0.024) increased with age, but only for the lop eared rabbits and not the other ear conformations (P < 0.001), with increasing age having an independent worsening effect (P <0.001). The interaction between ear conformation and age was not significant for hearing impairment or deafness. Also, compared with erect eared rabbits, lop eared rabbits were 5.5 times more likely to be reported as having impaired QoL due to an ear problem (P < 0.001), and asymmetrical-eared rabbits 4.1 times more likely (P = 0.019). Older age was also associated with reduced QoL across rabbits of all ear conformations  of the lop-eared rabbits in this survey population had a vet indicated ear problem, compared with 9.8% of erect-eared rabbits, and almost all specific conditions reported were seemingly more common in the lop-eared phenotype than in the other ear conformations . Logistirmations ; Fig 1. P = 0.010; P <0.001). Regarding owners’ subjective descriptions of their rabbits’ temperament, rabbits reported to have an ear problem indicated by a vet were significantly more likely to be reported as happy than those without a vet indication  respondents reported their rabbit as having an ear problem that reduced QoL; conversely 6.0% reported their rabbit to have an ear problem that did not affect their QoL. When controlling for cases where QoL was affected by a non-ear problem, rabbits reported to have ear pain responses were three times more likely to have a perceived reduction in overall QoL than those without ear pain responses , and loud or threatening sounds (P = 0.001) than rabbits who had had no such indication  and were ~3 times less likely to perform binky behaviour than those without reported ear pain responses . Similarly, rabbits reported to have impaired hearing were also more likely to be reported as being unresponsive to being approached from behind (P = < 0.001), to having a treat prepared within earshot (P = < 0.001), and to loud and threatening sounds (P = < 0.001) than rabbits who had ‘very good’ hearing. Lop-eared rabbits were twice as likely to be reported as unresponsive to being approached from behind than erect eared rabbits (P = 0.014). Rabbits being reported as having ‘good’ hearing were 2.3 times more likely to be reported as rarely binkying than rabbits who had ‘very good’ hearing (P = 0.011).Rabbits reported as deaf were more likely to be unresponsive to sound cues than rabbits who were not deaf , or their behaviour in an unfamiliar environment.Certain age groups of older owners were significantly more likely to report their rabbits to be unresponsive to an approach from behind and less likely to report their rabbits to respond to a treat being prepared than were 18–24 year olds . No otheWe aimed to describe a range of ear conditions reported to affect UK pet rabbits, ear conformational risk factors for ear problems, how ear problems affected rabbit QoL, and which owner-reported behaviours were predictive of ear problems. The results broadly indicate that approximately a quarter of pet rabbits in this sample were reportedly affected by ear conditions that are painful and/or impair their hearing, and that lop-eared and half-lop rabbits are more prone to these than erect-eared rabbits are. Owners perceived rabbits with ear pain responses to have worse QoL than rabbits who did not show ear pain, but they did not perceive this effect on QoL in hearing impaired rabbits or those who had had ear problems indicated by a vet. The behaviours most indicative of vet indicated ear problems, ear pain, and hearing impairment were lack of responsiveness to relevant sounds, and a reduction in the frequency of binkying. Each set of results will be discussed in more detail.In this sample, more than a quarter of rabbits 28.5%) had had at least one ear problem reported. Our four measures of rabbits having ear conditions showed convergent validity, with statistically significant pairwise associations between veterinary indication of ear disease, owner reported ear problems affecting QoL, ear pain responses and hearing impairment. The lifetime prevalence of vet indicated ear problems for rabbits with a median (IQR) age of 4 (2–6) years was 21.2%. Moreover, 13.6% of rabbits were reported to have an ear problem that affected their QoL at the time of the survey, 7.1% had ear pain response, and 15.8 had owner reported hearing impairment or deafness . The pre% had hadThe prevalences here do not represent the whole UK pet rabbit population, because the sample population was self-selected and thus not a random sample, and the owner reports cannot be verified. The true prevalence could therefore be higher or lower. We attempted to avoid over-sampling rabbits with ear problems by wording our introductory text to be as much about healthy ears and excellent hearing ability as about the opposite, and indeed most respondents reported their rabbits not to have any ear problems. However, an inflated prevalence is possible because, compared with owners of rabbits without ear problems, those whose rabbits were affected could have been more likely to participate in the questionnaire, e.g. if they were concerned about the problem and wanted to communicate this concern to researchers. This would lead to an over-estimate of the prevalence. The opposite–an underestimate–is also possible, especially given that otitis media and hearing loss are difficult to recognise in rabbits , 7, meanEither way, the proportion of rabbits with ear conditions here is of concern, because the results indicate that such conditions are probably considerably more prevalent than the 1% of rabbits having auditory conditions per year as indicated by veterinary case notes from UK first opinion practices . Our finOtitis, especially otitis media and otitis interna, was the most common group of diagnosed conditions, according to the owners in this survey. Otitis of any type is of direct welfare concern, as it is likely to be very painful, can cause hearing loss and (in the case of otitis interna) vestibular disturbance such that rabbits develop head tilts or disorientation . Otitis In this study, lop-eared rabbits were predisposed to ear conditions, which is consistent with veterinary opinion , 16, 17 Rabbit ear conformation is not a true dichotomy, and our population included half-lop and rabbits with horizontal ears. It is worth noting that 32% of half-lops had a vet indicated ear problem, similar to the prevalence in full-lops. There were only 18 horizontal-eared rabbits in the study, so it is difficult to draw conclusions about them, but they seemed to have a relatively low prevalence of 7% with a vet indicated ear problem. Whilst the numbers of these rabbits with intermediate ear conformations were sometimes too few for statistical analysis, the results suggest that half-lop rabbits may be at increased risk of certain ear problems compared with erect-eared rabbits, to a similar extent as rabbits with fully lop ears . In partAll four measures of ear problems also increased with age, but for vet indicated problems and ear pain responses, this age effect was only seen in lop-eared rabbits. The likelihood of hearing impairment increases with age in some species, e.g. humans  and ratsThe increased risk of ear problems with age presents a practical challenge for rabbit breeders wanting to select for healthy ears. Female rabbits, in small breeds, tend to be bred between the ages of 4 months and 3 years, with larger breeds starting later around 10–12 months . Males oOver two thirds (69.4%) of respondents who reported an ear problem in their rabbits felt it worsened the rabbits’ QoL. Additionally, rabbits with ear pain responses had significantly worse QoL scores than those without, suggesting that many owners recognise this as indicating suffering. It would be worth conducting objective welfare assessments of rabbits with ear conditions, because owner-reported QoL ratings are subjective. Even so, the results suggest that, regardless of rabbit ear conformation, ear diseases in rabbits should be regarded as a welfare issue and veterinary treatment should be sought.An unexpected finding, which is at odds with the other results was that owners were slightly but statistically significantly more likely to describe their rabbit as ‘happy’ if it had a vet indicated ear problem than if it did not. This could be a Type I error , especially because almost all respondents selected ‘happy’ to describe their rabbit. Alternatively, it could mean that rabbits whose owners took them to a vet when they had an ear problem are perceived as ‘happier’ than other rabbits. This effect could occur directly through effective veterinary treatment of ear or other problems, or indirectly through some other features associated with having an owner who takes their rabbit to the vet, e.g. having a more attentive owner.Another slightly unexpected finding was that hearing impairment and vet indication of ear problems were not significantly associated with worse QoL as perceived by owners. As above, it could be that rabbits who have been seen by a vet for an ear problem have had that condition resolved and are subsequently reported to have good QoL. It is possible that ear problems only worsen QoL if accompanied by pain, and that rabbits cope well with hearing impairment. Alternatively, it could be the case that hearing impairment, or a history of ear disease without obvious pain responses, do worsen rabbit QoL, but that the signs are too subtle for owners to consider them as clinically significant, e.g. rabbits being inactive, needing to be more visually vigilant, being more frequently startled, and missing positive sound cues that predict food or other rewards. The behaviours reported to be associated with both vet indicated ear problems and hearing impairment, discussed in the next section, suggest that the latter may be likely in many cases.Owners of rabbits with vet indicated ear problems, ear pain responses, or hearing impairment were reported to be significantly less likely to respond to the sounds of treats being prepared, and significantly more likely to only rarely binky, compared with unaffected rabbits. This suggests that these ear problems blunt the rabbits’ abilities to experience positive welfare. Affected rabbits miss out on the pleasurable anticipation of a treat, and if socially housed, may be at a competitive disadvantage compared with conspecifics who can hear the treat being prepared and approach it earlier. Binkying is considered a play behaviour, so the reduced binkying frequency both reflects reduced welfare, because poor welfare reduces animals’ motivations to play, and causes poor welfare because play is pleasurable in itself . Play isIn addition to this, rabbits reported to have vet indicated ear problems or varying degrees of hearing impairment were also significantly less likely to respond to loud or threatening sounds, and to the sound of the owner approaching from behind. This could make them more vulnerable to attack or injury, and more likely to be startled by the sudden visual appearance of their owner, potential predators and other stimuli. Some rabbits may be able to habituate to this, but it might alternatively cause other rabbits to be almost constantly vigilant and potentially anxious, similar to the anxiety reported in hearing impaired humans . IncreasThese results offer insights into how ear problems affect rabbit behaviour and welfare beyond specific ear-directed behaviour reported in the literature, such as scratching of the ears or head shaking . They alEar problems in rabbits are associated with owner-reported reductions in QoL, reduced responsiveness to sounds, and reduced binkying behaviour. They may be painful and cause hearing impairments. Lop- and half-lop-eared rabbits are more at risk than erect-eared rabbits are, as are older rabbits. Breeders wanting to select for good ear health should avoid breeding from rabbits with early signs of ear disease or with a family history of it. Ear disease can be difficult to recognise in rabbits, so it may be under-diagnosed and under-treated, but increased recognition of it is needed because of its associated harms to rabbit welfare.S1 File(PDF)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.