Such highly efficient SHG from a metal nanocavity also constitutes an ultrasensitive nonlinear nanoprobe to map the distribution of longitudinal vectorial light fields in nanophotonic systems.Efficient frequency up-conversion of coherent light at the nanoscale is highly demanded for a variety of modern photonic applications, but it remains challenging in nanophotonics. Surface second-order nonlinearity of noble metals can be significantly boosted up by plasmon-induced field enhancement, however the related far-field second-harmonic generation (SHG) may also be quenched in highly symmetric plasmonic nanostructures despite huge near-field amplification. Here, we demonstrate that the SHG from a single gold nanosphere is significantly enhanced when tightly coupled to a metal film, even in the absence of a plasmon resonance at the SH frequency. The light-induced electromagnetic asymmetry in the nanogap junction efficiently suppresses the cancelling of locally generated SHG fields and the SH emission is further amplified through preferential coupling to the bright, bonding dipolar resonance mode of the nanocavity. The far-field SHG conversion efficiency of up to  In nanoplasmonic systems symmetry can often suppress the second harmonic generation (SHG) is spite of the highly enhanced fields. Here, the authors demonstrate light-induced symmetry breaking that enables strong SHG enhancement from symmetric nanophotonic systems with certain properties. Both dielectric5 and metallic nanoparticles7 enable strong field enhancement inside and at the surface of a particle, respectively, and offer promising ways to enhance the nonlinear optical processes, including second-harmonic generation (SHG). High-index dielectric nanoparticles support Mie-type resonances with much larger mode volume than plasmonic nanoparticles and exhibit high SH conversion efficiencies9, compared to unstructured materials, capitalizing on a bulk second-order nonlinearity related to their non-centrosymmetric crystal lattice15. For plasmonic materials , the intrinsic inversion symmetry of the crystal lattice forbids the electric-dipole allowed SHG in their bulk, and the second-order nonlinear response mainly originates from the surface where the centrosymmetry is broken16. Such surface SHG is typically weak but much stronger localized than the SHG from dielectric nanoparticles, and very sensitive to the modification of the surface states and used for probing biochemical events, such as chemical reactions17, photocatalytic transformation18, and chemical mapping, potentially at the single-molecule level.Nonlinear nanophotonics has gained rapid appreciation as a route to control light with light at the nanoscale and realize many applications ranging from optical frequency conversion to imaging and biosensing as well as quantum technologies19. This strategy has been extensively implemented in various plasmonic entities to amplify the nonlinear conversion by exploiting mode resonances with stronger near-field localization of fundamental fields, such as the Fano-type resonances21. The antenna efficiency of a plasmonic nanostructure at the SH frequency also plays a significant role in the emission of the locally generated SH light22. Therefore, further improvement of the SHG efficiency resorted to the development of double-resonant plasmonic nanostructures that provide the fundamental field enhancement and the SH emission amplification simultaneously26, demonstrating SHG conversion efficiencies as high as 27, obtaining bright nonlinear nanoscale sources in the ultraviolet-visible spectral range remains a challenge in the absence of suitable plasmonic resonances at the SH frequencies where some metals, such as Au or TiN important for practical applications, do not have plasmonic behavior at all.In the absence of the phase-matching condition important in conventional macroscale nonlinear crystals, efficient SHG in plasmonic nanostructures heavily relies on the enhancement of the fundamental field: 8. At the surface of a symmetric metallic nanostructure, SHG is always associated with an anti-symmetric mode under either symmetric or anti-symmetric plasmonic excitation at the fundamental frequency. Within the electric-dipole approximation, SH emission is, therefore, available only for metallic structures with asymmetric geometries, for example, a small metallic particle with the shape deviating from a perfect sphere28. Beyond the electric-dipole approximation, the nonlocal, bulk electric quadrupole, and bulk magnetic dipole interactions can contribute to SHG by breaking the lattice centrosymmetry, but they are weaker than the contributions of the surface SHG19. Plasmonic excitations, which provide resonantly enhanced surface electric fields, can significantly amplify the SH conversion near a nanoparticle surface. Although composite plasmonic nanoantennas with a nanogap, such as a nanosphere or nanoparticle dimers or metal film-couped nanoparticles, are able to provide stronger near-fields to enhance the nonlinear polarization33, the far-field SHG efficiencies are limited by the degree of their structural symmetry36.The effective second-order nonlinear susceptibility of metal nanoparticles, which determines the far-field radiated SHG, strongly depends on the geometric symmetry of a nanoparticleHere, we demonstrate that a nanostructured plasmonic system, featuring extremely narrow gaps with an asymmetric geometry, exhibits exceptionally strong SHG in the ultraviolet-visible band. This nanostructure having a plasmonic response at a fundamental frequency consists of a single gold nanosphere closely separated from a gold film Fig. , forming37. The vertical dipolar mode of interest in this work features a strong electromagnetic field enhancement in the gap region with the dominant electric field component oriented in the vertical direction  the local SHG polarization , which produces the focal field distributions as presented in Fig. To validate the advantage of the VND configuration for SHG superior to a RND, we performed nonlinear confocal scanning measurements on both the single gold NPoMs and the nanodimers  is produced to illuminate the nanoparticles. The scattered light from the individual nanostructures is collected by the same objective with a smaller collection angle (53°) and then delivered to either the spectrometer for spectral analysis or the camera for wide-field imaging.The SHG spectroscopy of the plasmonic nanostructures is implemented on a commercial laser scanning confocal microscope system  combined with a Ti: sapphire femtosecond laser . The laser beam  is tightly focused on the sample with a high NA 0.95) objective. The emitted second harmonic photons from the plasmonic nanostructures are collected by the same objective in an epi-reflection configuration and then sent to high-sensitivity hybrid detectors (HyDs) for photon counting. The confocal image reconstruction within the SH band and spectral analysis are performed by a prism-based light dispersion module inserted in the detection path. To minimize the laser damage, the laser power used is 5 objectiThe SH near-field polarization fields of the nanodimer structures were simulated numerically using a commercial finite-element solver (COMSOL Multiphysics) in the frequency domain. A perturbative approach within the undepleted-pump approximation  was employed to run the simulations. The SH responses from plasmonic metals originate from the free-electron currents induced by both bulk and surface effects, but here we only consider the dominant ones normal to the metal surface in the simulations. The far-field SH radiation patterns of individual plasmonic nanostructures were derived by performing a near-to-far-field transformation of the simulated fields at the SH wavelength (Supplementary Note Supplementary Information


We emphasize how heterochromatin decompaction and mobility further support DSB repair, focusing on recent mechanistic insights into these processes. Finally, we speculate about potential molecular players involved in the maintenance or the erasure of heterochromatin marks following DSB repair, and their implications for restoring epigenome function and integrity.In response to DNA double-strand breaks (DSBs), chromatin modifications orchestrate DNA repair pathways thus safeguarding genome integrity. Recent studies have uncovered a key role for heterochromatin marks and associated factors in shaping DSB repair within the nucleus. In this review, we present our current knowledge of the interplay between heterochromatin marks and DSB repair. We discuss the impact of heterochromatin features, either pre-existing in heterochromatin domains or  The organization of the genome into chromatin in the nuclear space serves to precisely orchestrate cellular functions by controlling gene expression. While the euchromatin compartment is generally accessible and associated with active gene transcription, heterochromatin is more condensed and mostly transcriptionally silent . Beyond The establishment and maintenance of the silent state in heterochromatin domains involve DNA methylation and repressive histone post-translational modifications (PTMs). While constitutive and facultative heterochromatin are both enriched in DNA methylation, they show specific histone PTMs. Constitutive heterochromatin is enriched in H3K9me2/3, which is bound by heterochromatin protein 1 (HP1), a factor that plays a crucial role in heterochromatin assembly . FacultaOne of the major functions of heterochromatin is to ensure a tight control of transcriptional states, which is key for maintaining genome integrity and cell fate . NeverthAmong the many types of DNA lesions, highly cytotoxic DNA double-strand breaks (DSBs) are repaired by multiple pathways with different levels of fidelity. Non-homologous end joining (NHEJ) is predominant and proceeds by direct ligation of DNA ends, while homologous recombination (HR) requires an initial resection of the DNA ends followed by recombination with a homologous template, usually the sister chromatid, which restricts HR to the S and G2 phases of the cell cycle . Single-Here, by focusing on recent discoveries in the field, we provide an overview of our current knowledge of the interplay between heterochromatin marks and DSB repair and discuss potential mechanisms that preserve the integrity of heterochromatin domains.Heterochromatin marks and associated factors not only play critical roles in transcriptional silencing but also contribute to regulate DSB repair pathway choice, in part by controlling the recruitment of DSB repair factors. This regulation has been observed both in heterochromatin domains where heterochromatin marks are present before damage infliction and in euchromatin domains where DSBs trigger the deposition of specific heterochromatin marks. In this section, we discuss recent studies that provided new mechanistic insights into the regulation exerted by heterochromatin marks on DSB repair.Histone PTMs constitute an important layer of epigenomic information with a broad impact on chromatin organization and function; some of these marks define heterochromatin domains and have been shown to regulate DSB repair responses.For instance, a well-known PTM enriched in constitutive heterochromatin is H3K9me3. As previously described, an increase of this mark was observed at break sites in mammalian cells, both in heterochromatin and in euchromatin regions , and sevRecent studies have elucidated the mechanism underlying the deposition of H3K9me3 around DSBs and described new interactions between this histone modification and the regulation of DSB repair. It was shown that H3K9me3 actually depends on another damage-induced PTM on histone H4. The DSB sensor complex MRE11-RAD50-NBS1 indeed recruits UFM1-specific ligase 1, leading to the conjugation of a ubiquitin-like protein to histone H4 lysine 31, a process known as ufmylation. This histone PTM is bound by the serine/threonine-protein kinase 38 (STK38), which in turn recruits the H3K9 methyltransferase suppressor of variegation 3–9 homolog 1 (SUV39H1) leading to the trimethylation of H3K9 around DSBs , 2020. TDrosophila melanogaster in contrast, DSBs in pericentromeric heterochromatin trigger the recruitment of Drosophila lysine demethylase 4a, which demethylates H3K9me3 and H3K56me3, another conserved pericentromeric heterochromatin mark , promote NHEJ at deprotected telomeres in mouse cells. Interestingly, the involvement of H3K36me2 seems to occur downstream of DSB recognition and repair pathway choice .While the mechanisms through which H3K9me3 impacts DSB repair are now quite well characterized, how other histone marks, like H3K27me3 and H3K36me2, influence this process is still unknown. Further studies are necessary to determine whether those marks modulate the recruitment of specific DSB repair factors to chromatin. In addition, it will be interesting to investigate whether histone PTMs also underlie the differential regulation of HR between centromeric and pericentromeric heterochromatin observed in mouse cells . HomologBesides histone modifications, another layer of chromatin regulation builds upon the incorporation of histone variants , some ofThe histone variant MacroH2A1, for instance, is enriched in facultative heterochromatin domains on autosomes and on the inactive X chromosome . RemarkaAnother histone variant that may regulate DSB repair is CENP-A, which defines centromeric heterochromatin. There is conflicting evidence regarding CENP-A accumulation post DSBs , and theIn addition to histone variants and modifications, several heterochromatin-associated factors play a central role in DSB repair, including histone readers, architectural factors, and chromatin remodelers.Chromatin remodelers were shown to regulate chromatin relaxation at heterochromatic DSBs in mammalian cells. In this respect, imitation switch (ISWI)-class and chromodomain-helicase-DNA binding (CHD)-class chromatin remodelers play antagonistic roles. CHD3 promotes heterochromatin compaction, but is released from chromatin following ATM activation, while ACF1 and SNF2H (ISWI class) are recruited to the damage site and lead to heterochromatin decompaction, which allows Artemis-dependent NHEJ . A recenAmong readers of heterochromatin-specific modifications, HP1 is recruited to DSBs arising in euchromatin and heterochromatin domains, and a major regulator of DSB repair, with HP1 isoforms having different effects on DSB repair pathways: HP1α and β stimulate HR at the resection step, while HP1γ inhibits this pathway . In lineFinally, the heterochromatin-enriched architectural factor structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) contributes both to transcriptional silencing and to DSB repair. It is still unknown if both functions of SMCHD1 are mechanistically connected. This protein indeed plays a crucial role in mammalian X chromosome inactivation  and is iTogether, these studies illustrate that several heterochromatin marks, including histone trimethylation, histone variants, and non-histone proteins, regulate DSB repair pathway choice . InteresWhile the choice of repair pathway is influenced by the chromatin context, DSB repair itself leads to changes in heterochromatin organization, which can have a profound impact on its function and thus on the maintenance of genomic integrity.D. melanogaster, it was observed that DSB repair kinetics were comparable between heterochromatin and euchromatin (Drosophila and mouse cells (Heterochromatin is a highly compacted nucleoprotein structure that can be seen as an obstacle for the detection of DNA lesions and their repair. However, in mammals and hromatin . This ishromatin . Heterochromatin  and perise cells . Of notese cells . H1 evicse cells , but wheDrosophila and mouse cells (Drosophila cells, where RAD51-mediated recombination takes place (Mechanistically, chromatin decompaction is accompanied by DSB repositioning outside of pericentromeric heterochromatin domains, favoring repair completion through HR, as shown in both se cells . In bothes place . A similes place . FunctioDrosophila for instance, nuclear actin filaments and myosins promote the relocalization of pericentromeric DSBs to the nuclear periphery (A role for the nucleoskeleton and molecular motors in the relocalization of heterochromatic breaks was put forward in several species . In Droseriphery , with a eriphery . Mechanieriphery . Nucleareriphery , but wheeriphery . A receneriphery . The lineriphery . Similareriphery . This mieriphery . Togetheeriphery . Consideeriphery , we can Heterochromatin regions play a crucial role in silencing transposable elements and in regulating the segregation and stability of chromosomes . Thus, tDrosophila, where H3K9me3 levels decrease post DSB (Despite dramatic changes in heterochromatin organization following DNA breaks, some heterochromatin marks are maintained during the DSB repair process, as shown for H3K9me3 in mouse pericentromeric heterochromatin  but not post DSB . The mecpost DSB .DNA double-strand breaks within transcribed genes trigger the incorporation of heterochromatin-specific histone variants and histone post-translational modifications, leading to a transient heterochromatinization of the damaged locus, which contributes to transcriptional silencing . Among tde novo established in euchromatin, indeed play a central role in regulating DSB repair pathway choice. Thus, heterochromatin features should not be considered as barriers to DSB repair but as fine-tuners of the DSB response. While our knowledge of the DSB repair pathways that operate in different heterochromatin domains is increasing, the players involved in restoring heterochromatin organization and in erasing heterochromatin marks from euchromatin regions after DSB repair are still unknown. Beyond histone variants and modifications, another crucial epigenetic mark enriched in heterochromatin domains is DNA methylation. Interestingly, DSB repair alters DNA methylation patterns (During the last decade, a growing number of studies have highlighted the key contribution of histone post-translational modifications and factors implicated in heterochromatin formation in the response to DSBs. Heterochromatin marks, either pre-existing in heterochromatin domains or patterns , but litpatterns , 2021. TAll authors contributed to write this review and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate− were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate− (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate− stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).The ruminal epithelium absorbs large quantities of NHThe online version contains supplementary material available at 10.1007/s00424-021-02647-7. Arguably, the era of modern transport physiology began with Ussing’s famous discovery of active transport across amphibian skin . Over 70A classic example for a transporting cornified stratified epithelium in mammals is the ruminal epithelium of cattle and sheep , 93. Int4+ or NH3) are continuously absorbed across the ruminal wall into the hepatic circulation i) levels i was calculated from the emission ratio (f340 nm/f380 nm) according to Grynkiewicz et al. . T. T107]. −1 at a pipette potential of + 100 mV, and the inward current was − 29 ± 14 pA ∙ pF−1 at − 120 mV, while the reversal potential (Vrev) was 8 ± 4 mV (n = 12). Mean capacitance was 10.5 ± 2.1 pF and the mean series resistance 8.1 ± 1.5 MΩ. The positive Vrev argues for a contribution of Ca2+ to total permeability. The application of GSK1016790A (50 nmol · L−1) led to a significant rise in inward and outward currents. Outward current rose to 226 ± 56 pA ∙ pF−1 at + 100 mV (p ≤ 0.001), and inward current rose to − 220 ± 49 pA ∙ pF−1 (p ≤ 0.001) at − 120 mV, reflecting increases in the efflux and influx of Na+. The Vrev dropped slightly to 0.8 ± 2.3 mV (p = 0.042), close to the Veq for Na+. Addition of the TRPV4 antagonist GSK2193874 (1 µmol · L−1) partially reversed the current effects. At + 100 mV, the current amplitude decreased significantly , while at − 120 mV, the decrease was just numerical . Vrev did not change (p = 0.3). Washout in NaCl was partial, with currents at 148 ± 30 pA ∙ pF−1 (at + 100 mV) and − 159 ± 34 pA ∙ pF−1 (at − 120 mV), while Vrev remained at 1 ± 4 mV (p = 0.6).In NaCl, the outward current of bTRPV4 cells was 24 ± 12 pA ∙ pF4+, HEK-293 cells expressing bTRPV4, bTRPV3, and controls were investigated in parallel  should reflect efflux of Na+ and/or influx of Cl− induced by swelling, pH changes, or the classical stimulatory effect of a permeant ion on channel conductance . Ap. Ap46]. he rumen , this le2+ transport in Ussing chambers [−1, however, menthol should inhibit bTRPA1 [−1 of menthol showed an Isc and Gt response that mirrored the effects of 2-APB in the current study [It is certainly tempting to speculate that bTRPA1 is involved in the previously observed stimulatory effects of menthol on ruminal Cachambers , 78. Micchambers . At a hit bTRPA1 . Conversnt study , 78. Sinnt study , 78, theIn this context, it should also be noted that while mRNA signals for TRPV3 and TRPV4 are robust, mRNA encoding for bTRPA1 was frequently weak  or even −, in particular, stimulate Ca2+ uptake by the rumen [− have been shown to be stronger than those of acetate− or propionate− [2+ imaging.Our interest was triggered by the fact that SCFA, in general, and butyratehe rumen , 117 andhe rumen  and humahe rumen , all of pionate− , we test2+ absorption is the duodenum and the upper jejunum, where the bulk of Ca2+ is apically taken up via TRPV6, crosses the cytosol bound to calbindin-D9K, and is basolaterally extruded via a Ca2+-ATPase (PMCA1b), all under the control of calcitriol [2+ absorption takes place pre-intestinally [+/Ca2+ exchange [9K was detected in goat rumen [2+ involves an electrogenic component [2+ absorption by calcitriol [In monogastric species, it has been solidly established that the major site of Calcitriol , 80. Constinally . Ruminalexchange , 55, 82 exchange . Expressat rumen  but not at rumen , 117. Alomponent , 54, 118omponent , 117. Fulcitriol .− (butyric acid) is a weak acid with a pKa ~ 4.8. At pH 7.4, 0.25% is present in butyric acid, rising to 2.45% at pH 6.4. In the classical model, the uncharged butyric acid diffuses into the cytosol, where it dissociates, releasing butyrate− and a proton [2+ transport. In similar fashion, elevation of mucosal chloride–which acidifies the cytosol via stimulation of Cl−/HCO3− exchange–enhances ruminal Ca2+ transport [2+ transport. Furthermore, acidification of the cytosol by application of amiloride blocks extrusion of protons via Na+/H+ exchange (NHE) and stimulates Ca2+ transport. These findings led to the postulation of a Ca2+/H+ exchanger [+ with NMDG+, leading to a drop in net Ca2+ flux [+ can enter the pore in response to triggers that include membrane stretch, interfering with the passage of other cations [+ suggest that the stimulatory effects of SCFA on ruminal Ca2+ transport may involve TRP channels. The whole-cell patch-clamp data suggest that intracellular protons activate bTRPV3  . Furtherstratum granulosum of the ruminal epithelium, with cytosolic staining in the epithelial layers below ESM 2(MP4 7.57 mb)ESM 3(MP4 2.22 mb)


Core binding factor acute myelogenous leukemia (CBF-AML), characterized by the presence of either t  or inv(16) (p13q22)/t, is considered good-risk AML in the context of cytarabine based intensive chemotherapy. Still, outcome can be improved significantly through the effective implementation of available therapeutic measures and appropriate disease monitoring. The incorporation of gemtuzumab ozogamicin into frontline therapy should be standard. Cytarabine based induction/consolidation regimen may be combined with anthracycline (3 + 7 standard) or antimetabolite, fludarabine. Serial quantitative polymerase chain reaction (QPCR) monitoring of unique fusion transcripts allows monitoring for measurable residual disease clearance; this allows for better prognostication and well as treatment modifications. RUNX1/RUNX1T1 (AML1/ETO) . A post hoc analysis of the Medical research Council (MRC) AML15 randomized trial showed that patients with CBF AML who completed two courses of FLAG and idarubicin (FLAG-IDA) followed by 2 courses of HDAC had the best long term survival . After a median follow-up of 48 months, the 5-year probabilities of overall survival (OS) and leukemia-free survival (LFS) were 31% and 27%, respectively. The 5-year DFS rate was significantly longer with intensive consolidation compared with low intensity maintenance chemotherapy (p = 0.05), and most of the benefit was observed in patients with t (p = 0.007) but not in patients with inv(16)/t (p = 0.78) [Patients older than 60 years comprise about 5-15% of adult CBF AML patients , 17, 29  = 0.78) . TherefoP = 0.004) [P = 0.02). In an analysis from the French group, a first remission duration of over 1 year and treatment with regimens incorporating GO were associated with better DFS and OS. The CBF AML cytogenetic subset did not impact outcome [Salvage intensive chemotherapy has been the norm in relapsed CBF AML. In an analysis of the outcome of 92 patients with relapsed-refractory CBF AML at MD Anderson, the median survival for patients with inv(16) was 15.6 months (range 10.32–20.88 months) and for patients with t 9 months (range 3.68–14.32) (= 0.004) . A multi outcome .JAK2 mutation, a poor risk mutation in newly diagnosed CBF AML, is more common in therapy-related CBF AML. In a multi-center data analysis, the t-CBF-AML patients had shorter OS than de novo patients  [While most cases of CBF-AML are de novo, CBF AML can emerge as part of therapy-related AML. While the outcome of secondary or therapy-related acute promyelocytic leukemia (APL), another member of the good-prognosis AML subsets, is similar to de novo APL, the outcome of secondary or therapy-related CBF AML (tCBF-AML) is significantly worse than de novo CBF AML , 47. The= 0.038) .Although CBF AML is considered a good-risk AML subgroup, given the results from large co-operative groups and SEER data, improving our current therapeutic strategies with use of existing drugs, can potentially improve the outcome substantially. The incorporation of GO should be widely adopted. Fludarabine and cytarabine based frontline regimens provide effective alternatives to 3 + 7 Fig. . Monitor


Embodied interfaces are promising for virtual reality (VR) because they can improve immersion and reduce simulator sickness compared to more traditional handheld interfaces . We present a novel embodied interface called the Limbic Chair. The chair is composed of two separate shells that allow the user’s legs to move independently while sitting. We demonstrate the suitability of the Limbic Chair in two VR scenarios: city navigation and flight simulation. We compare the Limbic Chair to a gamepad using performance measures , head movements, body sway, and standard questionnaires for measuring presence, usability, workload, and simulator sickness. In the city navigation scenario, the gamepad was associated with better presence, usability, and workload scores. In the flight simulation scenario, the chair was associated with less body sway  and fewer head movements but also slower performance and higher workload. In all other comparisons, the Limbic Chair and gamepad were similar, showing the promise of the Chair for replacing some control functions traditionally executed using handheld devices. However, most current VR technologies do not provide a convincing sensation of self-motion in the absence of actual motion  , 2. Thisembodied interaction necessitates control interfaces that encourage a high level of engagement with the virtual environment during which the system reacts to users’ actions in a meaningful way embodied gful way . For exagful way . VR systgful way , 11 or ogful way , 13 and gful way , 15. Navgful way . Interfagful way . For exagful way . Such legful way  and presWe propose a new embodied interface, the Limbic Chair, that provides two degrees of freedom to each leg to simulate locomotion in VR. In two experiments, we aimed to answer the following research question: How suitable is the Limbic Chair for tasks in virtual environments that are typically performed with conventional control interfaces? We employ a different type of task in each experiment  and measure several variables related to locomotion performance and user experience see . In bothThis paper has two original contributions. First, we introduce a novel embodied interface for human-computer interaction. The Limbic Chair is an ergonomic apparatus with great potential for applications that make use of human movements as spatial inputs. Second, we perform a thorough user evaluation of the suitability of this chair for embodied interactions with virtual environments. The evaluation is composed of two user experiments that systematically compare this new interaction device with a more conventional gamepad.All virtual reality technologies require a control interface to mediate between the user’s actual, physical movements and the movements of the user’s avatar/camera through the virtual environment. Control interfaces can vary with respect to the types of physical movements performed by the user, the types of movements that are possible in the virtual environment, and the mapping between physical and virtual movements. Physical movements can range from fine-grained hand movements to full body movements, and virtual movements typically involve walking, riding in a vehicle, or flying through the air. Innovative control interfaces are also being explored for performing novel types of actions and tasks such as remotely controlling a robot , 20. HowSelf-location can be maintained with an egocentric visuospatial perspective that is supported by other vestibular and tactile feedback. During active control movements in a virtual environment, the simulated and actual consequences of a user’s movements must match so that the user can feel a Sense of Agency with those actions. Any discrepancy between the actual movements and feedback from the actions  might interfere with the subjective perception of agency. Finally, Body Ownership affects embodiment and is related to the level at which a user feels in possession of a virtual body through multi-sensory feedback .Immersive virtual environments allow for embodied interaction insofar as users can find a sense of self-location, agency, and body ownership in those environments , 25. ForFor navigation tasks in virtual environments, existing control interfaces include a rich set of body-aware input techniques that vary from hand-based interaction to whole-body interaction . AccordiHead-directed Interface that employs a head-mounted display for simulated translation and rotation movements and the Swivel Chair that combines a conventional chair with an external tracking system. Similarly, Probst and colleagues employ a commercially available flexible chair on which user’s leaning, rotational, and bouncing movements are tracked by the inertial measurement sensors of a smartphone that is attached to the backrest of the chair  and “the chair was difficult to figure out” [P18]. The gamepad seems to have a more positive effect on solving the Task because “it takes time to get used to the chair” [P4]. Participants reported their User experience with the gamepad rather negatively: “With the gamepad you actually do not move but you move virtually.” [P9]. On the other hand, there were twice as many positive statements than negative statements about the Limbic Chair for the User category. Many participants found the Limbic Chair to be “more natural” [P9], “more immersive” [P6], and “more real life-like” [P3], while only few found it “not natural” [P15] and “not intuitive” [P2].For both control interfaces, In summary, the gamepad seems to be the more suitable control interface for the city navigation task. This pattern may be due to familiarity, represented by the quote “I am more used to joysticks than to using my legs” [P5]. On the other hand, users’ VR experience was reported as “much more engaging with the chair” [P3]. “I had one moment with the chair where I felt immersed while I was moving forward and turning at the same time. It was cool. It did not happen with the gamepad” [P6]. However, “the chair requires more effort” . Thus, the comments on the Limbic Chair suggest that improved user experience comes with the cost of extra physical effort.Los Angeles 2000 at ACM SIGGRAPH ‘93 , “jerky” [P9], snapping back to the center and causing “fast jumps” [P11], or “discrete” [P22]. On the other hand, participants had a split opinion on the Limbic Chair’s usability as a control interface. Some participants found that the control and movements with the chair were much “smoother” , while others found that the chair’s “stiffness and height should be adjusted for the user” [P14]. For solving the Task, the Limbic Chair was associated with more positive comments than the gamepad. Overall, some participants found the chair “easier” for the task , while others favored the gamepad . The comments for both control interfaces in terms of User experience were approximately evenly distributed. For some participants, the Limbic Chair was more “natural” [P17] and “enjoyable”  to use, while others found the gamepad “natural” to use . Overall, participants provided more comments about the Limbic Chair than the gamepad.In summary, for the flight simulation scenario, the Limbic Chair seems to be the more suitable control interface than the gamepad: “Body control gives the feeling of VR” [P19], “I am actually sensitive to simulator sickness and my experience was very comfortable with the chair” [P7], “You feel like you are more in the game with the chair with respect to the gamepad” [P20].In this paper, we pursued two goals. First, we introduced a novel embodied interface called the Limbic Chair. Then, in two experimental scenarios, we studied the following research question: How suitable is the Limbic Chair for tasks in virtual environments that are typically performed with conventional control interfaces? Concretely, we compare the Limbic Chair to a gamepad using five measures: presence, system usability, work load, simulator sickness, and performance . In the city navigation scenario, the gamepad was associated with better presence, usability, and workload scores. In the flight simulation scenario, the chair was associated with less simulator sickness but slower performance and higher workload. Below, we discuss the potential implications of our findings for each of these measures and the limitations of our study.According to Witmer and Singer, presence is defined as the subjective experience of being in a virtual environment despite being physically located in a different environment. Presence in a virtual environment can be affected by mediation between the user and the environment by the control interface . More “nIn our city navigation experiment, the gamepad led to higher presence scores than the Limbic Chair. This finding is consistent with Kitson and colleagues  but contOur findings suggest that the experimental design as well as the type of task have an impact on to what extent an embodied control interface can induce presence in a virtual environment. Although presence was significantly lower for the Limbic Chair compared to the gamepad in the city navigation task, in the flight simulation task, the average presence score with the Limbic Chair was similar to the gamepad (approximately 0.6). The content analysis revealed that, in the flight simulation, experiences with the Limbic Chair were reported as more “natural” than experiences with the gamepad. In this second experiment, the pitch movement of the controllers were directly mapped to the pitch movement of the hang-glider in 3D space. This difference in mappings between the two experiments was necessary because of the spatial dimensionality of the tasks. However, these findings indicate that the use of embodied interfaces may be task-dependent (cf. ).The usability ratings of the two control interfaces also differed between the two experimental scenarios. The usability scores for the gamepad were similar across experiments , but the usability scores for the Limbic Chair were substantially lower for the city scenario (62.5) compared to the flight simulation scenario (71.0). This finding suggests that the usability of the Limbic Chair was affected by the underlying experimental task, but future research should systematically vary experimental task for the same participants in order to verify this pattern. The order of control interfaces also affected usability ratings. In the city navigation scenario, the gamepad was rated as more usable when participants used the gamepad before the Limbic Chair. This finding further supports the claims that familiarity with a control interface influences usability (consistent with ) and thaIn both experimental scenarios, the gamepad led to lower overall workload. This finding might be attributable to the facts that the Limbic Chair required more body movement and was less familiar than the gamepad. Indeed, during the exit interviews, some participants reported that the Limbic Chair provided an improved VR experience but required more effort than the gamepad. Similar findings were reported in other user experiments , 22, 28 According to previous research , 39, posIn recent years, there is a critical discussion around the use of SSQ as an established and appropriate measure of discomfort in virtual environments. Several alternative simulator sickness questionnaires have been proposed .One limitation of the present user evaluation is that we only employed one particular mapping between users’ leg movements on the Limbic Chair and their corresponding movements through the virtual environment. This mapping may not be optimal for these particular tasks. As suggested by others , 9, 22, Another potential limitation of the present experiments is that participants were much more familiar with the gamepad than the Limbic Chair, which may have contributed to the differences we observed. Our evaluation may also be limited because the control interface mappings differed between city navigation (2D navigation on a planar surface) and flight simulation (3D navigation in the air). Although these mapping differences were necessitated by the spatial dimensionality of the tasks, future work could explicitly and systematically disentangle the effects of these mappings and types of tasks. In future work, researchers can also train participants to use an embodied interface such as the Limbic Chair until they reach a particular learning criterion  that matches performance with a handheld control interface. Such a training study would allow researchers to investigate the time course of training on a novel interface, as well as more directly compare an embodied and non-embodied interface while controlling for familiarity.The extent to which embodiment can explain differences in locomotion performance between two interfaces could also be improved with a standardized questionnaire that specifically measures embodiment in terms of sense of self-location, agency, and body ownership. Recently, Gonzalez-Franco and colleagues have developed an embodiment questionnaire based on these three factors , 61. WhiThe present paper contributes to our knowledge of embodied interfaces in at least two ways. We show that leg movements performed during sitting are promising in their usefulness for VR applications. Our research also suggests that embodied interfaces might benefit from customization for specific tasks and users. Unfortunately, an adoption of the control interface to the structure and function of the human body does not guarantee better performance and user experience by itself because users may need time to first become familiar with the control interface and the task at hand. We therefore suspect that the Limbic Chair may provide greater benefits in future VR applications when users receive extensive training beforehand. Improvements in performance could also be achieved by tailoring the Limbic Chair to individuals’ physical characteristics and previous experience, as well as the task they need to solve in the virtual environment.In this work, we aimed for a systematical assessment of the suitability of a new embodied control interface, the Limbic Chair, for navigation tasks that are performed in VR. Previous research has assessed similar interfaces or developed standardized questionnaires that consider a range of factors related to user experience  and performance . However, they tend not to emphasize the importance of the task at hand. Our findings suggest careful consideration of the impact of tasks for future assessments of embodied control interfaces.


CYBB knockdown, although hampering induced NOX2 activity, did not affect the proliferation and differentiation of THP-1 and HL-60 cells. In summary, our data suggest that NOX2 is a marker of AML blast differentiation, while AML cell lines lack any NOX2 endogenous activity.In acute myeloid leukemia (AML), a low level of reactive oxygen species (ROS) is associated with leukemic stem cell (LSC) quiescence, whereas a high level promotes blast proliferation. ROS homeostasis relies on a tightly-regulated balance between the antioxidant and oxidant systems. Among the oxidants, NADPH oxidases (NOX) generate ROS as a physiological function. Although it has been reported in AML initiation and development, the contribution of NOX to the ROS production in AML remains to be clarified. The aim of this study was to investigate the NOX expression and function in AML, and to examine the role of NOX in blast proliferation and differentiation. First, we interrogated the NOX expression in primary cells from public datasets, and investigated their association with prognostic markers. Next, we explored the NOX expression and activity in AML cell lines, and studied the impact of NOX knockdown on cell proliferation and differentiation. We found that NOX2 is ubiquitously expressed in AML blasts, and particularly in cells from the myelomonocytic (M4) and monocytic (M5) stages; however, it is less expressed in LSCs and in relapsed AML. This is consistent with an increased expression throughout normal hematopoietic differentiation, and is reflected in AML cell lines. Nevertheless, no endogenous NOX activity could be detected in the absence of PMA stimulation. Furthermore,  Over the last few decades, oxidative stress has been reported in solid tumors as well as in hematopoietic malignancies ,2 includ2), catalyzing its transformation into superoxide anion (O2−) or hydrogen peroxide (H2O2) [Under physiological conditions, the mitochondrial respiratory chain, NO synthases (NOS), xanthine dehydrogenase/oxidase (XDH), and NADPH oxidases (NOX) are major contributors to ROS production . Unlike e (H2O2) ,12. Indee (H2O2) ,16,17,18e (H2O2) ,20,21. Te (H2O2) ,23. Likee (H2O2) , prolifee (H2O2) , migratie (H2O2) , differee (H2O2) ,27, and e (H2O2) ,25,28. BIn this work, we performed an extensive transcriptional analysis of all of the NOX subunits in AML, using public transcriptomic datasets and a large panel of 24 AML cell lines. We also evaluated the prognostic value of NOX genes in three AML cohorts, and examined whether NOX activity contributes to AML proliferation and differentiation-blockage using a knockdown strategy in AML cell lines.https://www.ncbi.nlm.nih.gov/geo/ (last accessed on 18 March 2021)). The datasets were subjected to background-correction and Robust Multichip Average (RMA) normalization using BioConductor’s Affy package [+) and depleted (LSC−) AML populations (GSE76009) [https://www.cbioportal.org/) (last accessed on 18 March 2021).Transcriptomics: raw CEL files from the AML microarray datasets GSE6891 and GSE10358 (Affymetrix GeneChip Human Genome U133 Plus 2.0) were downloaded from the GEO Omnibus  plots were created in the R environment using the ggplot2 package . HierarcOverall survival (OS) was defined as the time from the AML diagnosis until death, and the patients who were alive at last follow-up were censored. Event-free survival (EFS) was defined as the time from the diagnosis until an event occurred , and the patients with no events at their last follow-up were censored. Cox proportional hazard (CPH) regression was used to perform the univariate and multivariate analyses . The vio2. The cells were harvested in their exponential growth phase. Cytarabine-resistant cell lines were established, as described previously [Twenty-Four human myeloid leukaemia cell lines were purchased from DSMZ , and cultured in the recommended media  at 37 °Ceviously . The cel® VILO TM cDNA Synthesis kit . For most of the genes, primer  validation was performed on Stratagene’s cDNA mix . For the NOX1, NOX3, and NOXO1 primers, the validation was performed on plasmids carrying the corresponding cDNA; the pcDNA3.1-hNox1  plasmid #58344), pcDNA3.1-hNoxo1 (Addgene plasmid #58530) and pcDNA3.1-hNox3 (Addgene plasmid #58341) were gifts from Botond Banfi and Karl-Heinz Krause. Universal ProbeLibrary probes were purchased from Roche . The sequences of primers and probes are documented in The cells were washed twice with cold Phosphate Buffer Saline (PBS) before pelleting. The total cellular RNA was extracted using a Maxwell RNA purification kit , and was quantified using a NanoDrop Lite spectrophotometer. The RNA purity was analyzed using Agilent 2100 Bioanalyzer . Five micrograms of RNA were reverse-transcribed using the SuperScript® 480 Probes Master (Roche), as described previously [® 480 software. The geometric Ct mean of human ACTB, YWHAZ and RPL13A genes was used as the endogenous control, in order to calculate the expression of the target genes: ΔCT = “Ct gene” − “Ct reference geomean”.The RT-qPCR reactions were performed on 20 ng of cDNA using a LightCyclereviously . The samRadioimmunoprecipitation assay (RIPA) buffer  was used for the extraction of the NCF1 , NCF2 , and NCF4  proteins, while Laemmli buffer  was used for the extraction of the CYBA protein . The RIPA buffer was supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail  according to the suppliers’ instructions, and 1 mM of phenylmethylsulfonyl fluoride (PMSF). The extracted proteins were quantified using the bicinchoninic acid method. The lysates were then diluted to 1 µg/µL in Laemmli loading buffer and heated at 95 °C for 5 min before loading. Up to 40 µg of the proteins were loaded per well, and SDS-page was performed using 4–15% gradient polyacrylamide gels . The proteins were transferred onto 0.2 µm nitrocellulose membranes (BioRad), and were blocked for 1 h at room temperature in Tris-buffered saline (TBS)-Tween (0.2%) with 5% milk. The membranes were then incubated overnight at 4 °C with the appropriate dilutions of the primary antibodies. The horseradish peroxidase (HRP)-conjugated secondary anti-mouse and anti-rabbit antibodies were purchased from Vectorlabs , and the band detection was performed using an Amersham enhanced chemiluminescence (ECL) detection kit . The protein quantification was performed using ImageJ software.5 cells per well in an isotonic glucose solution (300 mM) containing luminol (10 µM)  and horseradish peroxidase (10 U/well) (Sigma-Aldrich). Phorbol 12-myristate 13-acetate  was added at a final concentration of 100 nM prior to the measurement. The luminescence was measured every minute for 2 h at 25 °C using a ClarioStar microplate reader . The area under the curve was calculated via ClarioStar Data Analysis software from the average of replicates of blank-corrected data. Diphenyleneiodonium chloride  and VAS3947 (Merck Millipore), both at 20 µM, were used to block the NOX activity. Dimethyl Sulfoxide  was used as the vehicle control.The NOX activity was measured as previously described, with some modifications . The celshLUC: CACGTACGCGGAATACTTCGA) or CYBB (shCYBB: TGCCTGAATTTCAACTGCATG), and harboring puromycin N-acetyltransferase (PAC) as a reporter gene, were produced as previously described [Lentiviral particles expressing shRNA sequences against luciferase mRNA . A Phycoerythrin (PE)-coupled mouse IgG1κ was used as the isotype control (Becton Dickinson). The data acquisition was performed on a C6 Accuri® flow cytometer, and the analysis was performed using FlowJo® software (Becton Dickinson).The differentiation of THP-1 cells was induced by PMA (100 nM), while that of the HL-60 cells was induced by a mix of all-trans retinoic acid  and DMSO (1.25%), as previously described . In brie5 cells/well in 6-well plates, and were incubated at 37 °C for 72 h. The cell numbers were counted on day 3 using a trypan blue exclusion assay.Cell counting: transduced cells were seeded in 3 mL media at a density of 2 × 104 cells/well in 96 well-plates, and were incubated at 37 °C for 72 h. The cell proliferation was followed at days 0 and 3 using a resazurin reduction assay. At both timepoints, resazurin (0.1 mg/mL) (Sigma-Aldrich) was added at a volume of 20 µL per well, and the plates were incubated for 4 h at 37 °C. The fluorescence  was measured using a ClarioStar microplate reader. The blank corrected signals were calculated using ClarioStar Data Analysis software, and normalization was performed with respect to day 0 for each condition.Resazurin assay: transduced cells were seeded in 200 µL media at a density of 5 × 10For the cell lines, the results are expressed as mean ± SEM of three independent experiments. R software was used to construct the figures and perform the statistical analyses.CYBB, NCF1, NCF2, NCF4 and CYBA) were highly expressed in most AML samples, whereas genes coding for NOX1, NOX3-5, DUOX1-2, NOXA1 and NOXO1 were either weakly expressed or absent  coding for NOX2 complex subunits were constantly deregulated across the three datasets (p < 0.002). In addition, CYBB had the lowest expression in the t group (p < 0.001), and NCF1 had the lowest expression in the t and t groups (p < 0.001), whereas NCF2 had its lowest expression in the t group (p < 0.001). These findings indicate that NOX2 is the most expressed NOX complex in AML, but it shows lower expression within the t and t subgroups.In order to characterize the expression profile of NOX-coding genes in AML cells a, we exar absent . Besidesdatasets . Each ofNCF4 expression correlated with a bad prognosis for EFS in the GSE10358 and TCGA datasets  subtypes (M0–M7), according to the differentiation orientation and degree of maturation of the blasts ,52. We nThe transcriptomic findings were validated at the protein level in a proteomics dataset of 33 AML samples (PXD019785) . As expeTogether, the data indicate that the expression of NOX2 complex genes in AML blasts might be dependent on the stage of differentiation blockage, and that these genes are tightly co-regulated.The NOX2 complex was first discovered in the oxidative burst of phagocytes. We postulated that its expression would increase throughout normal myeloid differentiation. The transcriptomic exploration of a comprehensive dataset of normal hematopoiesis (GSE24759) showed that the expression of NOX2 complex subunits is weak in HSCs, but increases gradually during normal granulocytic and monocytic differentiation a,b. This+) and non-engrafting LSC-depleted (LSC−) cells [CYBB was the most expressed gene, compared to NOX1, NOX3-5 and DUOX1/2  of mouse-engrafting primary human AML stem cells (LSC−) cells . The exp DUOX1/2 . The exp8+ cells . In lineC− cells a, indicals  on two representative cell lines.The characterization of the AML cell lines led to the identification of two groups: (a) cell lines harboring a low expression of genes coding for the NOX2 complex and lacking NOX2-inducible activity, and (b) cell lines expressing NOX2 subunits along with an inducible NOX2 activity. We further speculated that low NOX activity, below the detection threshold of the enzymatic assay, could still exist and contribute to cell proliferation and differentiation. In order to verify this, we investigated the effect of + cells; CYBB knockdown, an efficient (69.9 ± 3.2%) decrease in CYBB expression was achieved in the differentiated HL-60 cells. Otherwise, it had no effect on genes coding for the other catalytic subunits, indicating an absence of compensation effects  and inv(16) subgroups are both associated with good prognosis, they showed opposite expression profiles of NOX2 genes, which could partly explain the lack of consistent global prognosis for NOX. We also noticed high heterogeneity in the NOX profiles within most cytogenetic subgroups, suggesting distinct cellular properties between patients. Our data further show that NOX2 complex subunits are increased in AML cells from the M4 and M5 FAB stages, both at the RNA and protein levels. This is in line with previous observations at the protein level ,56. We aIt is well established that ROS can promote the proliferation and apoptosis of cancer cells, which is dependent on its delicate regulation by antioxidant enzymes. While exogenous and metabolic factors can contribute to oxidative stress during AML progression, the role of NOX enzymes at steady states remains elusive. Our transcriptomic meta-analysis revealed that the NOX2 complex is depleted in LSCs and HSCs; however, it is higher in AML blasts, and increases progressively throughout normal granulocytic and monocytic differentiation. Indeed, AML blasts with high ROS levels also have high CYBB expression, whereas AML cells with low ROS levels display lower CYBB expression and higher stemness . BesidesCYBB in AML cell lines did not affect the cell proliferation or their differentiation potential. In contrast, Kiffin et al. have shown a decreased differentiation potential of AML cell lines following CYBB KD, but—although significant—the difference with normal cells remained quite low. Reddy et al., on the other hand, have shown that CYBB and NOX4 can promote the proliferation and migration of AML cell lines, although independently of their ROS production activity. More recently, Adane et al. demonstrated that CYBB KD in primary AML cells results in a slower proliferation, concomitant with an induced differentiation in vitro, and a reduced leukemia burden after xenotransplantation in vivo [Cybb KO has been shown to reduce the expansion of murine hematopoietic cells transformed with BCR-ABL1 [Cybb KO in vivo may originate from chronic inflammation due to the absence of functional phagocytes, rather than a function of endogenous Nox2 itself in LSC homeostasis. Indeed, a murine model of chronic granulomatosis disease lacking functional Nox2 had shown defective HSCs due to chronic inflammation [In our study, the KD of  in vivo . BesidesBCR-ABL1 , and to BCR-ABL1 . In factBCR-ABL1 . Howeverammation . Thus, tMany studies relied on small-molecule inhibitors—such as VAS3947 and DPI—to further elucidate the role of NOX in leukemogenesis, and to examine the therapeutic potential of their inhibition ,27,60,61CYBB KD on AML cell lines under steady-state conditions in vitro. Hence, additional studies are needed to investigate the role of the NOX2 complex under stress conditions, and in the context of primary AML blasts in their microenvironment, or even in animal models in vivo. Furthermore, and importantly, the weak expression of genes coding for the NOX2 complex in LSCs does not necessarily rule out the possibility of weak NOX activity in these cells. Therefore, the investigation of the role of CYBB KD in human LSCs is also worth considering in future studies.It is worth noting that some limitations need to be taken into account concerning this work. Although our proteomic and transcriptomic analyses show concordant protein and transcriptional profiles for NOX2 complex genes, it is important to examine the protein localization and surface expression of NOX2 subunits in primary blasts, and to further validate the absence of other NOX proteins at the protein level. Moreover, the functional data reported in this study highlight the effect of CYBB KD did not affect the proliferation and differentiation of AML cell lines in vitro. Altogether, our data suggest that NOX genes are differentiation markers with no intrinsic role in AML cells.In summary, this work shows that NOX2 is the only NOX complex expressed in AML cells with higher expression in AML committed in the myelomonocytic and monocytic differentiation stages, in contrast to the low expression in LSC and relapsed AML. NOX expression, although it is deregulated in favorable cytogenetic groups, did not correlate with AML prognosis. No endogenous NOX activity was observed in AML cell lines in vitro, although many are inducible upon stimulation. Besides this,


Ferroptosis is a newly discovered form of regulated cell death dependent on iron and reactive oxygen species (ROS). It directly or indirectly affects the activity of glutathione peroxidases (GPXs) under the induction of small molecules, causing membrane lipid peroxidation due to redox imbalances and excessive ROS accumulation, damaging the integrity of cell membranes. Ferroptosis is mainly characterized by mitochondrial shrinkage, increased density of bilayer membranes, and the accumulation of lipid peroxidation. Myocardial ischemia-reperfusion injury (MIRI) is an unavoidable risk event for acute myocardial infarction. Ferroptosis is closely associated with MIRI, and this relationship is discussed in detail here. This review systematically summarizes the process of ferroptosis and the latest research progress on the role of ferroptosis in MIRI to provide new ideas for the prevention and treatment of MIRI. Cell death is the natural endpoint of typical cells, occurring in growth and development, division and differentiation, and homeostatic metabolism, ultimately resulting in the irreversible end of the cellular function. The mode of cell death in the process of myocardial ischemia-reperfusion injury (MIRI) has been garnering substantial attention. Although cell death primarily constitutes apoptosis and necrosis, ferroptosis, a new form of programmed cell death that is iron-dependent and distinct from apoptosis and necrosis, has been discovered in recent years –3. In reEarly studies demonstrated that erastin and RAS-selective lethal compound 3 (RSL3) could cause cell death in a manner that is different from apoptosis, which can be inhibited by iron chelators and antioxidants –11. In 22+ and Fe3+ in the body and can be considered a “double-edged sword.” The element is also the primary raw material for the synthesis of hemoglobin and myoglobin and is crucial for important processes, such as electron transport, cellular respiration, DNA synthesis, cell proliferation and differentiation, and gene regulation. However, the excessive accumulation of iron ions can increase ROS production through the Fenton reaction, which affects iron stability and promotes iron deposition in vital organs, thereby leading to severe organ damage.The maintenance and regulation of iron homeostasis are extremely complex processes. Iron is one of the most important essential trace elements in the human body and is involved in a series of important biological processes. Iron ions exist as Fe3+ bound to transferrin enters the cell via the membrane protein, transferrin receptor-1 (TFR-1), to form endosomes. Free Fe3+ is reduced to Fe2+ in the endosomes by the metal reductase, six-transmembrane epithelial antigen of the prostate-3 (STEAP-3). Fe2+ is then transported from the endosomes to the labile iron pool in the cytoplasm under the mediation of DMT-1. This is the process of iron recycling [2+ to Fe3+ and stores it in ferritin molecules, thereby reducing free iron levels, while FTL is directly involved in iron storage. In addition, heme oxygenase-1 (HO-1) was found to lead to ferroptosis by increasing intracellular iron and mediating lipid peroxidation reactions [In the process of iron metabolism, divalent metal transporter-1 (DMT-1) is a key protein in the intracellular transport of iron. Feecycling . Ferritieactions . When ineactions , 20.Various iron regulatory proteins are also involved in the process of iron metabolism. Iron-responsive element-binding protein-2 (IREB-2) also has an important function, which involves the enhanced expression of FTL and FTH-1, leading to decreased levels of intracellular iron and the inhibition of erastin-induced ferroptosis. This implies that the IREB-2 expression can indirectly interfere with iron adsorption and inhibit ferroptosis . In addiThe formation of lipid ROS is a key component in the onset and development of ferroptosis. ROS production depends on the action of polyunsaturated fatty acid-phosphatidyl ethanolamine (PUFA-PE). PUFA can be acylated under the catalysis of acyl-CoA synthetase long-chain family member-4 (ACSL-4) to produce PUFA acyl-CoA (PUFA-CoA), which then reacts with PE under the action of lysophosphatidylcholine acyltransferase-3 (LPCAT-3) to produce PUFA-PE , 23, 24.− pathway, and the VDAC pathway, which ultimately leads to ferroptosis.In this pathway, the action of PUFA-PE depends on two key regulatory points, ACSL-4 and LOX. Therefore, decreasing ACSL-4 and LOX can effectively inhibit the action of PUFA-PE and suppress the onset of ferroptosis . The kno− is an amino acid transporter expressed in the plasma membrane of mammalian cells. It is a heterodimer composed of SLC7A11 and SLC3A2, acting primarily through SLC7A11 (the primary active site of erastin). System Xc- exchanges extracellular cystine (Cys) for intracellular glutamate (Glu) at a 1 : 1 ratio, thus providing the raw material for intracellular GSH synthesis. Cellular uptake of cysteine is an important step in GSH production [System Xcoduction , 30. GPXoduction . When GPoduction . Furtheroduction . Anotheroduction . In addioduction . TherefoVDAC is a channel protein for transporting ions and metabolites located on the outer mitochondrial membrane and consists of VDAC-1, VDAC-2, and VDAC-3. It controls the exchange of metabolites in the mitochondria and with other organelles . In addiAt present, further indepth investigations have led to the gradual recognition of the roles of NADPH oxidase-4 (NOX-4), HSPB-1, and other related proteins and signaling pathways in the regulation of ferroptosis , 41. TheMyocardial ischemia caused by coronary artery obstruction is clinically manifested as persistent severe retrosternal pain and can lead to myocardial infarction, shock, arrhythmia, or heart failure. The most common treatment strategy is the early restoration of blood flow to the ischemic area using techniques, such as coronary angioplasty, percutaneous coronary intervention, and coronary artery bypass grafting (CABG), which can restore myocardial oxygen and nutrient supply, salvage the ischemic myocardium, and save the patient's life.MIRI is a phenomenon wherein cardiac function does not improve but worsens immediately after perfusion is restored to the ischemic myocardium. The pathogenic mechanism of MIRI is not fully understood. At present, it is known to primarily be involved in processes such as oxidative stress, calcium overload, and in inflammatory reactions . Oxidati2+ homeostasis in the ER, leading to mitochondrial calcium overload and elevated ROS production, while the accumulation of ROS will activate downstream caspase family proteins through cascade amplification, thereby initiating the process of cellular damage. In addition, the ERS process is induced by upstream signaling proteins, including inositol-requiring enzyme-1, activating transcription factor-6 (ATF-6), and PKR-like ER kinase (PERK) [α (eIF2α)-ATF 4-CHOP pathway. The dissociation of PERK from binding immunoglobulin protein BiP will trigger the phosphorylation and subsequent activation of PERK. Furthermore, eIF2α is activated, leading to ATF 4 mRNA translation and the induction of downstream CHOP molecules. The CHOP-mediated apoptosis in ERS plays an important role in the MIRI process in rats [Ferroptosis occurs with the production of ERS, and ERS-induced unfolded protein response plays an important role in the ferroptotic process. When changes in the calcium level and redox status of the endoplasmic reticulum (ER) lumen induce a decline in chaperone protein function, cells can activate the unfolded protein response and cause ERS. ERS disrupts Cae (PERK) . The ERSe (PERK) , mainly  in rats .α-ATF 4 pathway accompanying the ESR response regulates the target gene of the unfolded protein response, CHOP, while the binding of CHOP to the corresponding promoter inducesthe expressions of PUMA, endoplasmic reticulum redox protein-1α, and B-cell lymphoma-2 (Bcl-2) [Ferroptosis induces ERS-triggered apoptosis. Studies have found that ferroptosis can induce ERS activation by inhibiting the System Xc--mediated exchange of extracellular cystine for intracellular glutamate , 51. The (Bcl-2) , 53. Fur (Bcl-2) . In addi (Bcl-2) . The int (Bcl-2) .Apoptosis results from ferroptosis-induced ERS and its correlation with MIRI. In a rat MIRI model, it was found that erastin probably increases ERS, which further increases ferroptosis. Inhibition of ferroptosis can reduce myocardial cell injury, and the inhibition of ERS can alleviate ferroptosis and reduce MIRI. These findings suggest that ferroptosis is involved in ERS-associated MIRI. Furthermore, based on a MIRI model established by ligating the left anterior descending branch of the coronary artery in diabetic rats, the tail vein injection of the ferroptosis inhibitor, ferrostatin-1, could attenuate ERS-induced ferroptosis in cardiomyocytes, while the ERS inhibitor, salubrinal, could also attenuate ferroptosis in cardiomyocytes. This suggests that the activation of ERS may exacerbate the process of ferroptosis in cardiomyocytes, while the occurrence of ferroptosis can further exacerbate ERS in cardiomyocytes, which can form a vicious circle . TherefoGPX-4 participates in the regulation of ferroptosis. GPX-4 is an endogenous antioxidant for selenium-dependent enzymes that serves as a core regulator in the ferroptotic signaling pathway. Under physiological conditions, GPX-4 can confer cellular protection by scavenging lipid peroxides, thus preventing iron-mediated lipid peroxidation and elevated lipid ROS, whereas GPX inactivation induces ROS lipid peroxidation and ferroptosis . System GPX-4 mediates ferroptosis to regulate MIRI. A previous study demonstrated that the levels of iron and malondialdehyde (MDA) in reperfused rat hearts gradually increased with increasing reperfusion time, accompanied by a decrease in GPX-4 levels . NotablyPathogenic mechanism of ROS participation in MIRI. Oxidative stress, attributed to the enhanced production of ROS during MIRI, is the main cause of MIRI. Excessive ROS accumulation causes membrane lipid peroxidation and disrupts the barrier function of the cell membrane. Excessive oxidation of lipids, DNA, and proteins causes increased cardiomyocyte damage and ultimately cell death. The antioxidant system regulates redox homeostasis by controlling intracellular ROS levels and the interactions between normal cellular metabolism and pathophysiology. Increased expression of antioxidant enzymes protects tissues from oxidative stress and produces cardioprotection after myocardial reperfusion . During 3+ and hydroxyl radicals in the presence of Fe2+ and H2O2 through the Fenton reaction. Alternatively, Fe catalyzes the production of -OH through the Haber-Weiss reaction. The inhibition of GPX-4 causes an increase in ROS, whereas the overexpression of GPX-4 reduces ROS and thus cellular ferroptosis [Increasing ROS levels lead to ferroptosis. Ferroptosis occurs due to increased intracellular iron concentration and the depletion of the antioxidant GSH, which leads to increased levels of ROS and in turn causes lipid peroxidation and eventually cell death. It was found that lipid peroxidation may occur in the lysosomal membrane due to ROS accumulation and iron overload, while the permeabilization of the lysosomal membrane can lead to oxygen radical production, cell membrane degeneration, and increased GSH depletion . Moreoveroptosis , 68. Theroptosis . On the roptosis .ROS play a role in MIRI-mediated ferroptosis. Ferroptosis is highly correlated with cardiomyocyte death. During MIRI, iron accumulates in cardiomyocytes around the myocardial scar, and excess iron leads to cardiomyocyte death, while the inhibition of ROS production attenuates cardiomyocyte death . Based oAutophagy is an important mechanism of MIRI. Autophagy is a precisely regulated, dynamically developing process that cleans up damaged organelles and proteins via lysosomes, and is a monitoring mechanism that is relatively conserved. It recycles the basic nutrients produced and plays an important role in maintaining the normal structure and function of the heart. This involves not only cell survival but also cell death , 73. In α or VEGF-A) and is implicated in the processes of apoptosis and oxidative stress [Ferroptosis is closely related to autophagy. To a certain degree, ferroptosis is dependent on autophagy and involves the embryonic lethal abnormal vision-like protein-1 (ELAVL-1) and forkhead box C-1 (FOXC-1) . ELAVL-1e stress . ELAVL-1e stress , 80. FOXe stress . FOXC-1 Autophagy regulates ferroptosis to participate in the pathogenic mechanism of MIRI. Previous studies showed that ferroptosis is different from cellular autophagy and other modes of cell death, whether in terms of cell morphology, biochemical characteristics, or the regulatory factors involved. However, recent studies have revealed an interconnection between autophagy and ferroptosis in cardiomyocytes during the course of MIRI, wherein the activation of ferroptosis depends on the induction of autophagy, and the regulatory proteins of autophagy may also be involved in the regulation of ferroptosis. In another study, iron ion levels and ROS were significantly enhanced in the cardiomyocytes of MIRI rats, whereas GPX-4 and GSH protein expressions were significantly reduced, thus suggesting that ferroptosis may be involved in the pathogenesis of MIRI. Further studies revealed that myocardial ferroptosis may be regulated by autophagy-related signaling pathways during the progression of MIRI, and the ELAVL-1 protein is able to bind specifically with the autophagy-related protein beclin 1 to promote the decrease in the P62 protein expression and increase in LC 3 levels. This will induce an increase in the autophagy levels of cardiomyocytes and thus activate the ferroptotic pathway . At presThere is currently no effective treatment for MIRI. With the advancement of research, researchers began identifying the inhibition of ferroptosis cardiomyocytes as a potentially important target for the treatment of MIRI . PretreaA recent study found that dexrazoxane or ponatinib inhibited ferroptosis during MIRI, and a combined treatment with both drugs markedly reduced the scar area of myocardial infarction. However, for myocardial infarction patients with an elevated ST section, percutaneous coronary intervention before myocardial reperfusion could not significantly reduce the scar area of myocardial infarction . Based o2+ content, and improve MIRI. Therefore, C3G is a potential medicine to prevent myocardial cells from being affected by MIRI [Recently, it was proven that phosphatidylcholine oxide content increased dramatically during MIRI, leading to reduced GPX-4 activity and ferroptosis. However, it is noteworthy that Fer-1 can inhibit OxPC-triggered ferroptosis . Another by MIRI .Ferroptosis has been shown to be related to diabetic MIRI. Fer-1 alleviates ERS, reduces cellular injury in H9c2 cells, and mitigates myocardial cell injury during diabetic MIRI. In addition, diabetes patients can induce MIRI by activating NOX-2-related oxidative stress and apoptosis, and inhibition of nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX-2) can alleviate MIRI in diabetic rats , 86. FerAdditionally, further understanding of the association between ferroptosis and MIRI after heart transplantation has been obtained. Fer-1 reduces myocardial cell death and blocks the recruitment of neutrophil granulocytes to damaged myocardial cells by damage-associated molecular patterns (DAMPs) after heart transplantation . TherefoFerroptosis is an iron-dependent, nonapoptotic mode of cell death characterized by ROS accumulation. However, current research on ferroptosis is still in its infancy. Ferroptosis is closely associated with MIRI and regulates MIRI through ERS, ROS production, GPX-4, and the autophagy-dependent ferroptotic pathways. Ferroptosis can serve as an important target in MIRI, which may help in the process of reducing the occurrence of MIRI. Therefore, indepth studies on ferroptotic mechanisms and possible interventions have now become a focus of current research. In addition, the precise stage of MIRI at which ferroptosis mainly occurs have not been located, and some studies have shown that the incidence of ferroptosis is different at different stages of MIRI . Therefo


Acute myeloid leukemia (AML) is a type of blood cancer with an extremely grim prognosis. This is due to the fact that the majority of patients will relapse after frontline treatment. Overall survival of relapsed AML is very low, and treatment options are few. T lymphocytes harnessed with antitumor T-cell receptors (TCRs) can produce objective clinical responses in certain cancers, such as melanoma, but have not entered the main road for AML. In this review, we describe the current status of the field of TCR-T-cell therapies for AML.Despite the advent of novel therapies, acute myeloid leukemia (AML) remains associated with a grim prognosis. This is exemplified by 5-year overall survival rates not exceeding 30%. Even with frontline high-intensity chemotherapy regimens and allogeneic hematopoietic stem cell transplantation, the majority of patients with AML will relapse. For these patients, treatment options are few, and novel therapies are urgently needed. Adoptive T-cell therapies represent an attractive therapeutic avenue due to the intrinsic ability of T lymphocytes to recognize tumor cells with high specificity and efficiency. In particular, T-cell therapies focused on introducing T-cell receptors (TCRs) against tumor antigens have achieved objective clinical responses in solid tumors such as synovial sarcoma and melanoma. However, contrary to chimeric antigen receptor (CAR)-T cells with groundbreaking results in B-cell malignancies, the use of TCR-T cells for hematological malignancies is still in its infancy. In this review, we provide an overview of the status and clinical advances in adoptive TCR-T-cell therapy for the treatment of AML. Acute myeloid leukemia (AML) is a type of blood cancer that carries a grim prognosis, despite considerable therapeutic advances in the last decade. Current treatment of newly diagnosed AML generally consists of intensive chemotherapy (IC) followed by allogeneic hematopoietic stem cell transplantation  in younger and fit patients, and low-intensity chemotherapy  combined with—depending on the availability of the drug—the oral BCL-2 inhibitor venetoclax in older and/or less fit patients. Allo-HSCT, which is considered an immunotherapeutic strategy, since part of its mode of action involves the administration of T-lymphocytes that can recognize and eliminate the leukemic cells, is the gold standard post-remission treatment in AML. As discussed above, it is generally reserved for younger patients. Only a small population of elderly AML patients actually receives allo-HSCT . NeverthIt is within this context that immunotherapy comes to the fore. So far, most clinical trials with immunotherapies in AML have focused on monoclonal antibodies or checkpoint inhibitors. Cell-based immunotherapies for AML, except for allo-HSCT, are still experimental and have not yet surpassed the clinical trial stage . Among tAlthough intracellular antigens can also be targeted by CARs using antibodies recognizing peptide-MHC (pMHC) complexes , traditiCancer immunotherapies using TCR-T cells require TCRs that recognize tumor antigens in a specific manner . CurrentWT1 messenger RNA (mRNA) levels can be used as a marker for MRD and a prognostic factor for relapse in AML if no other markers are available . GV. GV235-2; TCR-C4  56]. On. On235-26 T cells/kg  [Some of the abovementioned clinical trials have used different strategies to optimize antigen recognition, TCR expression, and mechanisms to address potential safety concerns . Autolog2550535) ,53. Codo2550535) ). In the2550535) ,53. Self2550535) . On the 2550535) ,67. In t2550535) . Transge2550535) ,53. This2550535) . These t2550535) ,59; UMIN2550535) ,70; NCT02550535) ).126-134 peptide (NCT02550535) [235-243-specific TCR construct to silence the expression of native TCRs and prevent TCR mispairing between native and transgenic TCR chains [TCR affinity plays an essential role in TCR-T-cell avidity, which in turn is critical for the efficacy and clinical benefit of TCR-T-cell products . Compare2550535) ,53 and H2550535) . In anot2550535) ,62,63. A2550535) ,74. With2550535) . Short hR chains . TransgeR chains ,76,77. RR chains . HoweverTCR-encoding mRNA is transfected into T cells alone or in combination with siRNA-mediated downregulation of native TCR, represent a transient self-limiting approach with a potentially better safety profile [Other clinical studies benefit from donor-derived virus-specific T cells for TCR transduction, such as EBV and CMV-specific T cells . This pr profile ,82,83. NPreliminary results indicate that TCR-T therapies for AML are safe and TCR-T cells can persist in most patients. Promising clinical data suggest that this form of therapy may also be efficacious in preventing relapse in AML patients. However, completed and ongoing trials have faced some limitations . In two Multiple antigens for AML have been described over the years that have yet to be explored in the clinical setting as target antigens in TCR-T-cell therapy ,8. For sHMMR-encoding mRNA elicited HMMR-specific TCR responses [Greiner and colleagues defined HMMR, also known as RHAMM or CD168, as an immunogenic AML-associated antigen that could be targeted in immunotherapies . Initialesponses . Howeveresponses ,102,103,esponses ,105. Howesponses . The addesponses . Therefoesponses . Moreoveesponses . More imesponses . Other responses  or humanesponses . In thesNPM1) mutations, which occur in approximately 30% of AML patients [Due to difficulties in isolating high-affinity/high-avidity TCRs for self-antigens, such as those mentioned above, novel target antigens are required. Donor T cells in HSCT can mediate both GVHD and GVL effects. The capacity of these T cells to recognize mismatched HLA molecules makes them a valuable tool in TCR-T therapy for relapsed patients after allo-HSCT. Similar to HA-1 antigen, HLA-DPB1-reactive T cells have been described as potent GVL inducers ,112. To patients ,116,117.patients ). Thus, patients .In summary, results from current clinical trials using TCR-T-cell therapy for AML indicate no treatment-related toxicities. This is achieved thanks to the careful selection of high-affinity TCRs derived from natural repertoires that do not rely on artificially enhancing TCR affinity above physiological levels. In terms of efficacy, it is too soon to draw conclusions from these studies, given their early phase design and the results from most of them being pending. However, preliminary results indicate that this type of therapy may be efficacious for AML, especially in cases of relapse, where remaining leukemic blasts may not be eliminated with the standard of care, warranting the development of improved TCR-T-cell strategies to further increase their clinical benefit.


There was some evidence of a beneficial effect in adjusted analyses where above median levels of BIRC5 were shown to be associated with improved OS (p = 0.001) including in Core Binding Factor (CBF) patients (p = 0.03). Above median levels of BIRC5 transcript were associated with improved relapse free survival (p < 0.0001). Utilisation of a second large cDNA microarray dataset including 306 AML cases, again showed no correlation between BIRC5 levels and OS, but high expression levels of BIRC5 correlated with worse survival in inv(16) patients (p = 0.077) which was highly significant when datasets A and B were combined (p = 0.001). In addition, decreased BIRC5 expression was associated with better clinical outcome (p = 0.004) in AML patients exhibiting CBF mainly due to patients with inv(16) (p = 0.007). This study has shown that BIRC5 expression plays a role in the survival of AML patients, this association is not apparent when we examine CBF patients as a cohort, but when those with inv(16) independently indicating that those patients with inv(16) would provide interesting candidates for immunotherapies that target BIRC5.Despite recent advances in therapies including immunotherapy, patients with acute myeloid leukaemia (AML) still experience relatively poor survival rates. The Inhibition of Apoptosis (IAP) family member, survivin, also known by its gene and protein name, Baculoviral IAP Repeat Containing 5 (BIRC5), remains one of the most frequently expressed antigens across AML subtypes. To better understand its potential to act as a target for immunotherapy and a biomarker for AML survival, we examined the protein and pathways that BIRC5 interacts with using the Kyoto Encyclopedia of Genes and Genomes (KEGG), search tool for recurring instances of neighbouring genes (STRING), WEB-based Gene Set Analysis Toolkit, Bloodspot and performed a comprehensive literature review. We then analysed data from gene expression studies. These included 312 AML samples in the Microarray Innovations In Leukemia (MILE) dataset. We found a trend between above median levels of BIRC5 being associated with improved overall survival (OS) but this did not reach statistical significance ( Acute Myeloid Leukaemia (AML) is defined as a malignant disorder of the bone marrow (BM) characterised by the clonal expansion and differentiation arrest of myeloid progenitor cells . AML is Despite treatments such as maximally intensive chemotherapies and allogeneic stem cell transplantation, survival rates have remained mostly unchanged for AML patients until recent years when there has been a significant shift towards the use of novel and effective, targeted therapies including inhibitors of mutant FMS-like tyrosine kinase 3 (FLT3)  and isocBaculoviral IAP Repeat Containing 5 (BIRC5) is expressed in 60% of adult AML patient samples and is more frequently expressed than FLT-3 , PRAME  or WilmsBIRC5, WNT1) or the control of cell proliferation . On this basis, and its relatively high frequency of expression in AML, we examined BIRC5, its molecular interactions, its potential as a biomarker and target for therapy in AML, further. In 2020, Davis et al.  describeUsing BloodSpot  we foundWe examined the pathways that BIRC5 engages in using in silico searches and RNA-seq data based on our own previous studies . We foun2/M. During mitosis BIRC5 is involved in spindle assembly checkpoint and cytokinesis. BIRC5 is downregulated by p53 to allow apoptosis to occur and in this way regulates cell proliferation and cell death [BIRC5 is periodically expressed during the cell cycle, with weak expression in G1, multiplied by six in the S phase and by more than 40 in Gll death .BIRC5 probesets and the clinical features of adult AML  showed a significant difference in BIRC5 expression between genders, Nucleophosmin 1 (NPM1) mutation and wild type (WT), M6 and all other French American British (FAB) subtypes , and M7 and all other FAB subtypes (M0–M6)  which occur in immature leukocytes. In addition, patient numbers in the M6 and M7 FAB subgroups were very low (n = 3 each) (Examination of the relationship between each  (M0–M6) . Analysi 3 each) A making p = 0.02).BloodSpot data indicated that the highest expression of BIRC5 was in AML patients with complex cytogenetic abnormalities compared B with alBIRC5 correlated with a number of genes involved in cell cycle regulation (BIRC5 expression was with cyclin B2 (CCNB2) which showed elevated expression in AML patients with 11q23 and t (MPZL1), Never in mitosis gene a-related kinase 11 (NEK11) and protocadherin gamma subfamily B, 4/8 (PCDHGB4/A8)  [CDC20 [Expression of gulation A as demogulation A. The hit B. InversHGB4/A8) B. AlthouHGB4/A8) C expressmal (CN) , with ce) [CDC20  and down) [CDC20 .BIRC5 has previously been shown to mediate blast cell proliferation in mice with Flt3-ITD [BIRC5 expression was found to show a correlation with WT rather than mutated FLT3  p = 0.4 in the whole cohort or when examining Core Binding Factor (CBF) patients alone, 1.09 (range 0.05–22.45), p = 0.9.In adjusted analyses neither level nor present calls associated with complete remission (CR) rates. However, there was no difference in BIRC5 levels between those patients who relapsed and those who did not Ci even wp = 0.001 which was maintained when CBF patients were analysed alone HR 0.16 (0.03–0.90) p = 0.03. There was a beneficial effect, in adjusted analyses, between above median BIRC5 levels and relapse free survival HR 0.51 (0.37–0.70) p < 0.0001 which was not significant when the CBF patients were examined alone 0.30 (0.08–1.20) p = 0.08. There was some evidence of a beneficial effect, in adjusted analyses, where above median levels of BIRC5 were shown to be associated with OS 0.65 (0.51–0.84) p = 0.0005 but this was not observed maintained when the CBF group of AML patients were analysed alone HR 0.37 (0.09–1.49) p = 0.15.Below median levels of BIRC5 were associated with elevated relapse rates in AML patients in adjusted analyses HR 0.54 (0.38–0.76) BIRC5 expression on AML patient survival, we evaluated its expression in a second independent microarray data set (referred to as the cDNA/data set B). This microarray data derived from AML cases comprising all cytogenetic AML subgroups [BIRC5 expression levels and age, BM blasts, lactate dehydrogenase (LDH), preceding malignancy or OS. However, we observed a correlation with distinct cytogenetic groups with significantly higher expression levels of BIRC5 in AML cases with monosomy 7/loss of 7q or a t . In 138 CN-AML cases [NPM1-mutated/FLT3-ITD-negative. However, in Core Binding Factor (CBF)-AML cases [BIRC5 expression was associated with better clinical outcome  and with days in remission (p = 0.028). In dataset A/MILE, high expression levels of BIRC5 correlated with worse survival in inv(16) patients too , t, 11q23 or t. We have previously shown that BIRC5 was associated with different 11q23/MLL abnormalities in adults with B-cell acute lymphocytic leukaemia [BIRC5 has been shown to play essential roles in cell cycle progression and mitosis. It binds with the chromosomal passenger complex (CPC) and Aurora-B kinase in the nucleus, leading to correct mitotic spindle formation . Converseukaemia  and in tBIRC5 expression and AURKB, PLK1, TPX2, KIF2C and cyclin A2 expression [AURKB [BIRC5 has been shown to interact with are cell cycle related and for exemplification, the most significant association between BIRC5 and any other gene, in this study, was with Cyclin B2. Cyclin B2 has been shown to stimulate the proliferation of triple negative breast cancer cells [BIRC5 has been shown to increase the sensitivity of colon epithelial cells to CDK inhibitors suggesting a mechanistic basis for the preclinical development of future CDK inhibitor-based therapeutic strategies [Our findings support data already generated in solid tumours showing a strong correlation between pression ,44,45. Sn [AURKB  in an efer cells  and to aer cells . In addier cells . Using srategies . BIRC5 expression and FLT3 WT, a correlation that has not been identified in AML patient samples previously, with other studies of the interactions between BIRC5 and FLT3-internal tandem duplication (ITD) being made predominantly through cell line studies and mouse models. For example other investigators have shown that BIRC5 mediates acute leukaemia in mice induced by Flt3-ITD [We also found a correlation between increased Flt3-ITD  and thatFlt3-ITD . In addiFlt3-ITD . BIRC5 and the expression of LAAs such as Synovial Sarcoma X breakpoint 2 interacting protein (SSX2IP) and hyaluronan-mediated motility receptor  expression (p < 0.001) has been described [HMMR, CA9, PRAME and SSX2IP which are associated with cell proliferation in vitro [We have previously described the role of many leukaemia associated antigens (LAAs) in cell cycle  and the escribed ,54,55. Iin vitro ,56. BIRC5 expression and improved clinical outcomes due to inv(16) but this same association was not seen with t patients despite AML-ETO being a critical regulator of BIRC5 in AML [BIRC5 correlated with OS, we did find that above median expression of BIRC5 correlated with relapse-free survival (MILE dataset) and while inv(16) correlated with low BIRC5 levels in the cDNA/Dataset B, the correlation between inv(16) and above median levels of BIRC5 and poorer survival were more obvious following the combination of both datasets in this study. This may reflect the higher percentage of inv(16) patients in the CBF cohort in dataset B compared with dataset A.CBF is a heterodimeric protein complex involved in the transcriptional regulation of normal haematopoiesis. Mutations in CBF-encoding genes  and inv(16)) result in leukaemia-associated proliferative advantages. CBF-AML accounts for around 20% of all AML patients and is often associated with improved outcomes compared to other subtypes of AML. However, it should be noted that although modern therapies may improve remission rates, they often lead to relapse, meaning the development of targeted therapies is still needed for improved outcomes. There was an association between decreased 5 in AML . AlthougBIRC5 which when used with chemotherapeutic agents can increase drug efficacy on AML cells [BIRC5 has been targeted by immunotherapy in a number of ways (recently reviewed in ) includiML cells . AlthougML cells . AlternaML cells , especiaBIRC5 expression appears to be able to predict better outcomes at least in a subset of CBF-AML cases (those with inv(16)) suggesting that this LAA may provide an immunologically relevant personalised target for a sub-group of AML patients.In summary, The BloodSpot database  includeswww.kegg.jp), STRING (https://string-db.org/ accessed on 27 March 2021 [BIRC5 was determined using the MILE dataset (GSE13159). When multiple probe sets were available for BIRC5 the following were used: 202094, 208052 and 212399.Relationships between BIRC5 and other genes/proteins were established using Kyoto Encyclopedia of Genes and Genomes , WEB-bahttp://www.cancer.gov/tcga (accessed on 27 March 2021). The association between BIRC5 levels in samples from patients with NPM mutation and WT, FLT3-ITD and FLT-WT, and all FAB subtypes were examined.Examination of the relationship between each BIRC5 probeset and the clinical features of adult AML was performed using data generated by the TCGA research network: The MILE/Data set A comprised 312 AML samples including 180 CN-AML and 63 CBF samples including 31 cases with t and 32 with inv(16). Samples were analysed using Affymetrix human genome U133A 2.0 or human genome U133 Plus 2.0 microarrays . cDNA/DWith regard to both datasets, gene expression profiling (GEP) was performed as previously described  using AfFor the correlation with survival data, expression values were dichotomised by the median expression of the respective gene across all AML samples and statistical analyses were performed as described previously ,56.BIRC5 mRNA expression is strongly associated with the expression of AURKB, PLK1, TPX2, HMMR and SSX2IP as well as other important cell cycle associated genes. Downregulation of this complex system involved in tumorigenesis might provide important targets for tumour cell control in acute leukaemias. We also showed that patients with CBF AML, and particularly patients with inv(16), who have above median levels of BIRC5, have poorer survival outcomes. This indicates that those AML patients with inv(16) would provide interesting candidates for immunotherapies that target BIRC5.Analysis of independent AML datasets using different microarray platforms showed that in AML