FUS-ERG is a chimeric gene with a poor prognosis, found in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). It remains unclear whether DNA hypomethylating agents, including azacitidine (Aza), are effective in FUS-ERG-harbouring AML and how FUS-ERG induces chemoresistance. Stable Ba/F3 transfectants with FUS-ERG were repeatedly exposed to Aza for 7 days of treatment and at 21-day intervals to investigate Aza sensitivity. Stable FUS-ERG transfectants acquired resistance acquired resistance after three courses of Aza exposure. RNA sequencing (RNA-seq) was performed when Aza susceptibility began to change; genes with altered expression or transcript variants were identified. Molecular signatures of these genes were analysed using gene ontology. RNA-seq analyses identified 74 upregulated and 320 downregulated genes involved in cell motility, cytokine production, and kinase activity. Additionally, 1321 genes with altered transcript variants were identified, revealing their involvement in chromatin organisation. In a clinical case of AML with FUS-ERG, we compared whole-genome alterations between the initial MDS diagnosis and AML recurrence after Aza treatment. Genes with non-synonymous or near mutations in transcription regulatory areas (TRAs), additionally detected in AML recurrence, were collated with the gene list from RNA-seq to identify genes involved in acquiring Aza resistance in the presence of FUS-ERG. Whole-genome sequencing of clinical specimens identified 29 genes with non-synonymous mutations, including BCOR, and 48 genes located within 20 kb of 54 TRA mutations in AML recurrence. These genes were involved in chromatin organisation and included NCOR2 as an overlapping gene with RNA-seq data. Transcription regulators involved in mutated TRAs were skewed and included RCOR1 in AML recurrence. We tested the efficacy of BH3 mimetics, including venetoclax and S63845, in primary Aza-resistant AML cells treated with FUS-ERG. Primary FUS-ERG-harbouring AML cells acquiring Aza resistance affected the myeloid cell leukaemia-1 (MCL1) inhibitor S63845 but not while using venetoclax, despite no mutations in BCL2. FUS-ERG promoted Aza resistance after several treatments. The disturbance of chromatin organisation might induce this by co-repressors, including BCOR, NCOR2, and RCOR1. MCL1 inhibition could partially overcome Aza resistance in FUS-ERG-harbouring AML cells. FUS-ERG  is a rare chimeric gene generated by t  in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS)1. AML or MDS with FUS-ERG has been reported to have a poor prognosis even with allogeneic haematopoietic stem cell transplantation3. FUS-ERG is neither specified in the latest World Health Organization classification nor listed as an adverse risk in the European LeukemiaNet classification5. However, it is listed as a poor prognostic chromosomal change in children with AML6.7; this is presumably linked to the dysfunction of ERG, which is a key transcription factor in the maintenance of haematopoietic stem cells and haematopoiesis of erythrocytes and megakaryocytes9. In addition, FUS-ERG reportedly colocalises with other key transcription factors in specific genomic areas responsible for transcriptional regulation and interferes with haematopoietic differentiation10. Furthermore, FUS-ERG binds to RNA polymerase II and interferes with RNA splicing11; this results from the dysregulation of FUS, which belongs to the FUS, EWSR1, and TAF15 family and is related to gene expression via transcription regulation and alternative splicing regulation12. However, the mechanisms underlying chemoresistance and poor prognosis in AML or MDS with FUS-ERG remain unclear.The molecular mechanisms underlying the leukemogenesis of FUS-ERG have been investigated in several studies. For instance, FUS-ERG alters myeloid and erythroid differentiation and enhances the proliferative and self-renewal capacities of myeloid progenitor cells14. Most studies have demonstrated that AML with FUS-ERG is refractory to traditional cytarabine-based regimens, but the efficacy of Aza has not been investigated. Although there is a case report that Aza is effective as a salvage therapy for paediatric AML with FUS-ERG and early relapse after allogeneic haematopoietic stem cell transplantation15, the direct effect of Aza on AML cells with FUS-ERG has not been demonstrated. In this study, we investigated whether Aza was effective against FUS-ERG.DNA hypomethylating agents (HMA) , including azacitidine (Aza), have recently been used as first-line drugs in high-risk MDS and elderly patients or those with refractory AMLIn this study conducted between January 2017 and October 2022, using haematopoietic cells stably expressing FUS-ERG, we investigated Aza sensitivity in cells with FUS-ERG and described gene expression profiles. Additionally, using samples from the clinical case patient, we compared whole-genome alterations between the initial diagnosis of MDS and the recurrence of AML after Aza treatment. We tested the efficacy of BH3 mimetics against primary FUS-ERG-bearing AML cells that acquired Aza resistance.–5 M β-mercaptoethanol, and 1% penicillin/streptomycin. The supernatant was collected after centrifugation and 0.22 µm filtration for cytokine supplementation. The full length of FUS-ERG was amplified from the cDNA of the patient sample using the primers GGTACTCAGCGGTGTTGGAA and CTTCCCCAGCCCCAGTAAAG and constructed into pcDNA3.1. cDNA was synthesised from the total RNA using SuperScript III Reverse Transcriptase . Total RNA was isolated using an RNeasy Mini Kit . FUS-ERG-transduced Ba/F3 cells were established using Nucleofector by Amaxa  and selected with 1 mg/ml G418 .To investigate the association between FUS-ERG and Aza sensitivity, we established haematopoietic cells stably expressing FUS-ERG  on days two, four, and seven. The surviving cells in 0.5 µM Aza of the initial course and the second course and 1 µM of the third course were used for the second, third, and fourth course Aza treatment, respectively.http://daehwankimlab.github.io/hisat2/). Transcripts were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/index.shtml) with aligned reads. Finally, expression profiles were represented as read counts and normalisation values, which were calculated based on transcript length and depth of coverage. Normalisation values were provided as fragments per kilobase of transcript per million fragments mapped. Gene Ontology (GO) analysis was performed using Metascape (https://metascape.org/gp/index.html#/main/step1). The alignment analysis was performed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Total RNA of FUS-ERG- or empty vector-transduced Ba/F3 cells was isolated before the fourth Aza treatment. The TruSeq stranded mRNA LT Sample Prep Kit  was used to prepare the libraries. Paired-end RNA sequencing (RNA-seq) was performed using Novaseq6000 with 40–60 million reads per sample. The trimmed reads were mapped to the reference genome using HISAT2 (https://genome.ucsc.edu/index.html). This study was approved by the ethics committee of our institution (permission number G-150).This study was approved by the ethics committee of Shiga University of Medical Science (permission number G-150). Bone marrow mononuclear cells were purified using Ficoll-Paque  from a patient initially diagnosed with MDS with FUS-ERG and relapsed as AML with FUS-ERG after obtaining written informed consent. Buccal swabs were used to subtract germline mutations. Genomic DNA extraction and library preparation were performed using a QIAmp DNA mini kit (Qiagen) and TruSeq Nano DNA Library Prep Kit (Illumina). Whole-genome sequencing (WGS) was performed using NovaSeq6000 (Illumina). Total reads were 1.2–1.5 billion per sample. Sequencing was performed by paired-end and multiplex methods using Illumina-specified flow cells with an average depth greater than 50 × . Uniformity (Pct > 0.2*mean) was over 97%. FASTQ files were mapped to the hg19 human reference genome using the DRAGEN: 3.9.5 software. The identification conditions of genome alterations were set at more than 20% of at least five reads after the subtraction of germline alterations. Genome areas involved in transcription regulators were visualised by reconstructing chromatin immunoprecipitation sequencing data from the ENCODE transcription factor binding site (TFBS) cluster (v3) dataset (wgEncodeRegTfbsClusteredV3.bed.gz) in the University of California Santa Cruz Genome Browser  and 4′,6-diamidino-2-phenylindole  and analysed with BD LSRFortessa X-20 and Flowjo v10 software .Primary AML cells from the patient were separated using Ficoll-Paque and maintained at 3 × 10Where experiments called for their use, 5-Aza , venetoclax , and S63845  were dissolved in dimethyl sulfoxide (DMSO).P value < 0.05.Analysis of variance was used for the comparative analysis of viability in the cell growth inhibition assay. Statistical significance was set at a https://www.ddbj.nig.ac.jp/jga), which is hosted by the Bioinformation and DDBJ Center, under accession number JGAS000587.RNA-seq data are available at GEO under accession number GSE217967. WGS genotype data have been deposited at the Japanese Genotype–phenotype Archive . This study was performed in accordance with the Declaration of HelsinkiPatient's samples were collected after obtaining written informed consent for this study and publication.Aza sensitivity was comparable between empty vector-transduced (hereafter referred to as control) and FUS-ERG cells in 0.25, 0.5, and 1  µM Aza conditions of the first course  production, such as Cx3cl1 and Lbp, were upregulated, whereas those linked to the positive regulation of TNF production such as Fcer1g and Tlr1 were downregulated  including three indels in CDS, including KRAS c.504delG (p.L168fs)　, 2,191 SNVs in non-CDS, and 139 indels including none in CDS , generating the FUS-ERG chimeric gene, and finally transformed to AML despite achieving complete remission once by Aza , defined as genomic areas where five or more transcription regulators can be recruited according to the available dataset of chromatin immunoprecipitation sequencing [ENCODE TFBS cluster (v3) data]. As a result, 32 common alterations and 54 additional alterations were located in the TRAs. Within 20 kb of common or additional TRA alterations, we found 32 genes, including PHF20 and SP100, and 48 genes, including NCOR2 and CCND1  were identified in FUS-ERG cells and did not completely match all reported isoforms according to the alignment analysis with BLAST , were identified as overlapping genes. NCOR2 is reported to be linked to leukemogenesis via functional dysregulation by major genetic alterations in AML, such as FLT3 internal tandem duplication and RUNX1-RUNX1T1 chimaera genes34. NCOR2 interacts with various nuclear receptors, including retinoic acid, and forms a complex with HDAC3 to repress its target genes36. Several NCOR2 isoforms have been reported, and aberrant splicing could lead to non-optimisation of the nuclear receptor corepressor family39. The current variant B has alterations in RD2 and ID2 and might, therefore, disturb NCOR2 function. The NCOR2/HDAC3 axis is critical for maintaining chromosomal structure and genomic stability40. The current GO analyses revealed chromatin organisation from RNA-seq data in FUS-ERG transfectants and WGS data in primary FUS-ERG-harbouring AML cells acquiring Aza resistance. Thus, genomic instability due to dysregulation of NCOR2 might cause the development of HMA-resistant clones in FUS-ERG-harbouring MDS and AML.Our WGS data revealed alterations in TRAs and specific genomic areas that multiple transcription regulators can bind. TRAs, including promoters and enhancers, influence genome structure and regulate gene expression11 and regulate the histone acetyltransferase activity of EP30041. In contrast, TRAs involved in RXRA, KDM5B, STAT1, GATA1, HDAC1, RCOR1, PHF8, and CHD2 appeared to be selectively mutated in transformed AML cells during Aza treatment. KDM5B, HDAC1, and PHF8 are histone modifiers43, and RCOR1 cooperates with histone modifiers such as HDAC144. CHD2, a modifier of chromatin structure, is reported to be somatically mutated in 8% of patients with chronic lymphocytic leukaemia45 and is less expressed in AML cells than in normal haematopoietic cells46. Considering recent reports that TRAs are hypermutated in haematological malignancies47, selective dysregulations of TRAs may be a part of the causes of HMA resistance.Our findings on TRA alterations also suggest that the target areas of several transcription regulators might be selectively altered. Alterations of TRAs that POLR2A and EP300 can recruit according to the available comprehensive ChIP-seq database were comparably observed in common alterations and additional alterations; this may be because FUS can bind to RNA polymerase II20, had no in vitro effect on primary AML cells or clinical effect on our AML patient with FUS-ERG. This mechanism remains unclear because our WGS data showed no evidence of mutations in or around the gene body of BCL-2. In contrast, S63845, an MCL1 inhibitor, was partially effective in primary AML cells. S63845 reportedly induces apoptosis of AML cells, and AML cells resistant to the BCL-2 inhibitor Aza are highly sensitive to S6384522. Further studies with larger cohorts are required to confirm whether MCL inhibitors can overcome HMA resistance. In this study, we proposed drug selection and anticancer drug resistance mechanisms for AML with FUS-ERG, despite the rarity of cases, which makes conducting clinical research with a large number of cases difficult. However, whether it can be used for other leukaemias remains unknown because the analysis only included AML with FUS-ERG.Venetoclax, combined with Aza, currently used as a preferred regimen for AMLIn conclusion, our findings suggest that dysregulation of BCOR and NCOR2 could cause the development of HMA-resistant clones from MDS or AML cells with FUS-ERG. In addition, selective mutations in TRAs involved in specific transcription regulators, including RCOR1, may contribute to the acquisition of HMA resistance. As these co-repressors are essential for regulating gene expression, further coordinated studies of mutations, DNA methylation levels, and histone modifications in TRAs are required to understand HMA resistance mechanisms.Supplementary Information.


This enhancement has been ascribed to the all-optical poling induced by the coherent photogalvanic effect. Yet, an analogous process for Si3N4 thin films has not been reported. Our article reports on the observation of laser-induced threefold SHG enhancement in Si3N4 thin films. The observed enhancement has many features similar to all-optical poling, such as highly nonlinear power dependence, cumulative effect, or connection to the Si3N4–Si interface. However, identical experiments for low-oxygen silicon oxynitride thin films lead to complex behavior, including laser-induced SHG reduction. Following a thorough experimental study, including the effects of repetition rate or pulse length, the observed results were ascribed to heat-induced SHG variation. In addition to revealing a new mechanism of laser-induced SHG variation, our results also provide a means to identify this mechanism.Strong second harmonic generation (SHG) in silicon nitride has been extensively studied—among others, in terms of laser-induced SHG enhancement in Si These materials are used for optical coating as a means of creating layers with a graded refractive index1. Nevertheless, recently the research into silicon nitride has been motivated predominantly by its nonlinear optical properties, including strong second harmonic generation (SHG)2. These properties can be utilized in waveguide structures, photonic crystal nanocavities, plasmonic structures, or optical modulators5.Silicon nitride  SHG generation on the Si3N4–Si interface11, and (ii) bulk-like SHG, which features dipolar character13. The bulk SHG with dipolar character has been assigned to breaking the material symmetry via strain or local inhomogeneities in these articles14.Many studies have investigated the nonlinear optical characteristics of Si17 and microresonators19. This enhancement has been attributed to the effect of all-optical poling, where the driving mechanism was the so-called coherent photogalvanic effect. This effect induces internal local electric fields in the material and, therefore, allows efficient frequency doubling in centrosymmetric materials via third-order nonlinearity (EFISH)16. The photogalvanic effect occurs when a sample is exposed to the fundamental laser beam and its second harmonic, which can either originate from an external source or be generated in the sample itself.In recent years, a number of workgroups have reported on the strong laser-induced enhancement of SHG in silicon nitride waveguides2 interface. However, this time-dependent SHG is restricted only to oxide layers of less than 10 nm and disappears for thicker layers20.The optically-induced SHG variation has also been reported for oxidized Si surfaces. The variation has been ascribed to the multiphoton electron and hole injection across the Si-SiOxNy optical thin films exceeding 10 nm in thickness, it has been accepted that SHG efficiency is determined by the deposition process and thin film structuring. For instance, SHG intensity has been shown to be promoted by a stoichiometry of Si3N47, a targeted deposition of Si3N4 and SiOxNy structures with enhanced residual stress21, or accumulated fixed charges on layer interfaces21. In contrast to the waveguides and microresonators, optically induced SHG enhancement in optical thin films has not been previously reported.For SiO3N4 layers. Some characteristics of the enhancement closely resemble the coherent photogalvanic effect, including highly nonlinear power dependence or the cumulative character of the SHG enhancement16. Our measurements also revealed that the SHG variation is not linked to any notable change in the layer refractive index or chemical composition.In this article, we report on our observations of optically-induced SHG variation in silicon nitride and oxynitride thin films on a silicon substrate. In particular, we observed a strong threefold SHG enhancement on SiHowever, as our study extended to silicon oxynitride layers, we noted a more complex behavior depending on the layer stoichiometry. In some cases, the illumination by IR femtosecond pulses actually reduced the SHG intensity. The observed behavior was incompatible with the coherent photogalvanic effect.Therefore, we carried out a set of experiments testing the presence of heat-induced changes in the sample, such as the effect of irradiation laser repetition rate, pulse length variation or ex-situ sample annealing. The acquired experimental results fully confirmed our hypothesis. Our results show that the all-optical SHG enhancement in silicon nitride and oxynitride can originate not only from optical poling but also from heat-induced material changes. The SHG variation can, therefore, be a complex process originating from an interplay of multiple phenomena.3N4 thin films deposited via dual ion beam sputtering on the Si substrate22. Throughout the study, we used p-polarized IR pulses at 1028 nm both for the sample irradiation and SHG measurement; the incident angle of the IR beam was 70 deg and we measured p-polarized SHG—see “6.As the first step, we studied laser-induced SHG enhancement in SiLaser-induced SHG variation measurements were implemented by placing a sample on an XY stage, which allowed us to irradiate the sample point-by-point and also to map the SHG intensity, as well as IR reflectivity. The laser-induced SH intensity variation was measured by illuminating several rectangular segments of the sample, using a different IR laser intensity for each segment—see Fig. 6, which had been previously reported also for optical poling via the coherent photogalvanic effect16. The SHG enhancement was homogeneous across the illuminated area, which implies that it was not connected to any local contamination of the sample. The enhancement also did not alter the dependence of the SHG on the polarization of the input beam—see Supplementary Fig. We observed significantly enhanced SHG in the regions previously illuminated by high-intensity IR pulses—see Fig. 2) = 0 sccm corresponds to the pure Si3N4, the flow of ϕ(O2) = 3 sccm leads to the formation of nearly SiO2 layers23. The estimate of stoichiometric factors for each sample is provided in Supplementary information, Sect. In parallel to the SHG measurement, we also measured the IR reflectivity of the illuminated area see Fig. A,B, whicWe also studied the case where a segment of a sample is irradiated multiple times—see Fig. xNy thin films deposited within the same batch, which differed only by their substrate. Therefore, in the following text, we restricted ourselves to layers deposited on the Si substrate. We discuss the implications of the difference between the substrates in the “Interestingly, we observed the laser-induced SHG variation only for the layers on the Si substrate, while the same layers deposited on the BK7 substrate did not show any sign of enhancement—see Fig. To exclude the possibility that the laser-induced changes occurred in the substrate only, we carried out measurements close to the layer edge, where we irradiated the layer and the bare substrate within a single experiment. The Si substrate without a layer showed only minor SHG changes within the statistical error of the measurement—see Fig. 3N4 waveguides. The similarities include the I6 nonlinear behavior, cumulative SHG enhancement, SHG enhancement without apparent change in the linear optical response of the layer, and connection to the Si–SiOxNy interface.The observed properties of SHG enhancement presented in Figs. xNy layers with an oxygen flow of 0.25–0.5 sccm, the SH intensity decreased with the increasing IR illumination intensity—see Fig. The photoinduced SHG variation became more complex for the low-oxygen oxynitride thin film samples. We observed that for SiO15. Moreover, previously reported methods of erasing the built-in internal electric field by illuminating the sample with an externally generated SH light have been proven not to affect the observed changes in our samples15.The SHG reduction on the low-oxygen oxynitride thin films in Fig. 24.We propose a different phenomenon consistent with the presented experimental data as the source of the SHG variation: heat-induced changes. By measuring the transmitted and reflected pulse energy, we verified that the vast majority of the pulse energy was absorbed within the layer and Si substrate. The linear absorption was dominated by the 1 mm thick Si substrate, while the losses within the layer are negligible—see Supplementary Information, Sect. To evaluate the possibility of a heat-induced sample transformation, we carried out a simple calculation, which provided us with a gross estimate of the temperature change induced by a single pulse—see Supplementary information, Sect. Therefore, we carried out sets of measurements where we varied the laser repetition rate. If a coherent photogalvanic effect or an analogous nonlinear process is responsible for the SHG enhancement, we should observe the same SHG enhancement when we keep a constant irradiation peak power and the same number of incident pulses on the spot. This would hold irrespective of the laser repetition rate. On the contrary, the heat-induced changes in a sample should strongly depend on the laser repetition rate, even if the total number of incident pulses is the same. This is because the long delay between the pulses allows for better heat dissipation between the pulses.xNy interface.By carrying out the SHG enhancement measurement for 100 kHz and 10 kHz repetition rates where we maintained the same number of incident pulses, we could see that the enhancement is highly dependent on the laser repetition rate—see Fig. 3N4 layer into two parts. One of those parts was uniformly heated to 400 °C for 30 min and then left to cool down at room temperature. Both parts of the sample were then placed into the setup and scanned as a single measurement. The results can be seen in Fig. To confirm this conclusion, we conducted an experiment with ex-situ sample annealing. We cut an Si substrate with an SixNy sample (0.5 sccm) for which we had previously observed a decrease in SHG due to irradiation with a laser beam—see Fig. Moreover, we conducted the same experiment on the SiO3N4, while all other properties, including energy per pulse and repetition rate, were kept constant. We observed that the relative SHG enhancement increased by 50% by prolonging the pulse length from 225 fs up to 2 ps—see Fig. 15. The peak power was almost ninefold decreased in the 2-ps pulses, yet the SHG enhancement increased.To elucidate the mechanism of nonlinear light absorption, we studied the effect of laser peak power on SHG variation. Namely, we tuned the incident pulse length between 225 fs and 4 ps and measured SHG enhancement in Si3N4 layers on an Si substrate, which underwent an IR irradiation leading to an SHG enhancement. We used samples with different layer thicknesses and irradiation intensities. The enhancement scale was measured immediately following irradiation via multiple scans to verify that the scanning itself did not alter the measured value. Subsequently, the samples were repeatedly measured for several days—see Fig. 25.Finally, we also studied the relaxation of the SHG variation in time. In particular, we measured SiIn light of our results, we can safely ascribe the observed laser-induced SHG enhancement/reduction to localized layer heating. From the observed absence of the SHG enhancement for the BK7 substrate, we can conclude that the induced heat must be generated either on the layer-Si interface or in the adjacent Si substrate itself. This interpretation is also consistent with other experiments carried on the same samples, such a pump-probe study of the initial dynamics on the picosecond timescale—see Supplementary information, Sect. 6 dependence) was combined with a more pronounced SHG enhancement for an increased pulse length. Such behavior is consistent with the observation of free-carrier absorption, i.e., induced absorption on excited charge carriers, which is followed for high laser intensities by impact ionization, where the excess energy of excited charge carriers can lead to the excitation of new ones. The intraband free-carrier absorption of excited carriers enables a rapid transformation of excess energy into heat via interaction with optical phonons. Nevertheless, our experimental results do not allow us to discern the free-carrier absorption at the layer-substrate interface from the same process in the adjacent substrate.A highly nonlinear character of the SHG enhancement  formation of a new heat-induced sublayer at the layer-substrate interface leading to the change in the interface SHG, (ii) heat-induced restructuring of the layer bulk alternating the efficiency of the bulk SHG.3N4 layers deposited via IBS, the SHG efficiency scales with the layer thickness, and, also, its angular dependence suggests that the bulk-like SHG dominates for layers with a thickness exceeding 1 μm6. If the enhancement originated from the SHG on the layer-Si interface, we would expect to observe a lower SHG enhancement for higher Si3N4 layer thickness because the dominating bulk-like SHG would remain unchanged. However, this is contrary to the results, where we have observed analogous levels of SHG enhancements for layers with thicknesses varying more than threefold.We have previously observed that for the studied SiWe can also discuss the results with respect to the IR reflectance of the samples, which showed only negligible changes following high-intensity IR irradiation. To do this, we created an optical model of the sample—see Supplementary Information, Sect. 3N4 and the corresponding diffusion length during the short illumination period of 2 s would reach a notable effect only when the local temperature in the layer was significantly above 1700 °C26, i.e., exceeding the melting point of Si and approaching the melting point of Si3N4. This temperature level is not viable since we have observed that the layer can resist a significantly higher irradiation level and the related temperature without being destroyed. Diffusion of atoms  across the layer-air interface cannot be responsible for the enhancement, as this process would be active for both Si and BK7 substrates.Heat can, in principle, induce the formation of a very thin sublayer at the layer-substrate interface via atom diffusion across the interface. However, the diffusion coefficients of Si and N in Si21; (ii) leads to a low refractive index change27; (iii) its hysteresis is highly different for Si3N4 and SiOxNy layers, as the character of the stress changes from compressive to tensile28. Our experiments with layer annealing at 400 °C, which is sufficient to change the stress in the layer29, led to SHG modification in line with laser-induced changes. Finally, the partial relaxation of the SHG enhancement might be explained by the relaxation of residual stress in the layers over the range of several days, which has been previously reported in the literature30.Therefore, we propose heat-induced restructuring of the layer bulk as the most feasible explanation. We can speculate that the mechanical stress in the layer might be responsible for the SHG variation, as the stress: (i) highly affects SHG efficiency due to symmetry breakingIt is worth stressing again that the discussion of the physical origin of the SHG enhancement is based only on indirect evidence, where we discuss the consistency of the proposed mechanisms with the observed results. While we can safely assign the SHG enhancement to heat induced in the samples by the focused laser beam, the actual heat-induced mechanisms cannot be confirmed or excluded with certainty.In summary, we carried out a thorough investigation of light-induced SHG modification, which we observed, unlike other reports, on thin-film samples. While the nonlinear behavior and other aspects might suggest that we witnessed the coherent photogalvanic effect, we carried out a set of measurements that safely ascribed the SHG modification to localized heating of the layer. We propose that the heat-induced restructuring of the layer bulk is the most viable source of the observed SHG enhancement; however, we cannot exclude other options.3N4 and SiOxNy thin-film layers on a Si substrate. This mechanism is entirely different from the previously reported optical poling. Therefore, it is of great interest to distinguish the two effects in future experiments and to take SHG enhancement into account in layer characterization.First of all, our results provide information on a new mechanism of laser-induced SHG variation in Si3N4 layers, it is highly beneficial to use a reduced laser repetition rate as a simple means of minimizing laser-induced layer modification6.In particular, we propose measurements of SHG under various laser repetition rates and varying laser pulse length as a simple way to verify the presence of heat-induced modification. In general, for studying SiThe SHG setup used in this study was described in detail in Ref. 6. Amplified Yb:YAG fs pulses (4 µJ/pulse) at 1028 nm were directed into the SHG setup, where their intensity was modulated to the desired level using a λ/2 waveplate and a polarizing cube beam splitter. The IR intensity level at the sample was calibrated by a thermal power meter (thermopile). The IR pulses were focused onto the sample into a spot 20 µm in diameter, which was determined using the knife-edge technique. The focused IR beam was used to generate SHG in the reflective geometry.Throughout the article, we used p-polarization IR light, incident angle 70 deg, and we detected p-polarization SHG radiation. Using a series of dichroic optics and color filters, we simultaneously detected the intensity of the reflected IR light and the generated SH radiation. Unless stated otherwise, the experiments were carried out at the laser repetition rate of 100 kHz.Unless stated otherwise, the IR pulses were compressed into the pulse length of 225 fs. By using the grating compressor integrated in the laser, we were able to increase the pulse length up to several picoseconds by inducing a chirp in the pulses. The chirped pulse length was determined based on the calibration provided by the manufacturer (Light Conversion). The compression of pulses was optimized by supercontinuum generation in sapphire.6. A beam of Ar+ ions  sputtered Si atoms from a target onto substrates (Silicon or BK7), where the deposited atoms interacted with nitrogen and oxygen ions generated by an assistance ion beam . By changing the ratio between the oxygen and nitrogen ion flux, we were able to vary the stoichiometry of the layers from the pure Si3N4 (ϕ(O2) = 0 sccm) through SiOxNy to nearly SiO2layers (ϕ(O2) = 3 sccm). The estimate of stoichiometric factors is provided in Supplementary information  on the scale of years.Laser-induced SHG variation measurements were implemented by placing a sample on an XY stage, which allowed us to scan the sample and irradiate it point by point. The laser-induced SH intensity variation was measured by illuminating several rectangular segments of the sample, using a different IR laser intensity for each segment—see Fig. 2, corresponding to 35–100 mJ/cm2/pulse. Subsequently, we performed one overall XY scan recording the SH and reflected IR intensity—see Fig. 2, 35 mJ/cm2/pulse) to avoid additional sample changes. The data gained from the overall scan were then evaluated independently for each segment by calculating the average intensity and the standard deviation as an error estimation—see Fig. During the irradiation, the segment was scanned spot-by-spot by the IR laser 1028 nm), irradiating each spot for 2 s unless stated otherwise. The irradiation intensity ranged from 170 to 430 GW/cm028 nm, iThe long-term reproducibility of the measurements was assured by using a laser with a stabilized output power. The pulse compression stability was verified by a supercontinuum generation in a sapphire plate.Supplementary Information.


We investigated the effects of a KD on Ca2+ homeostasis and electrophysiology in diabetic cardiomyopathy. Male Wistar rats were created to have diabetes mellitus (DM) using streptozotocin , and subsequently treated for 6 weeks with either a normal diet (ND) or a KD. Our electrophysiological and Western blot analyses assessed myocardial Ca2+ homeostasis in ventricular preparations in vivo. Unlike those on the KD, DM rats treated with an ND exhibited a prolonged QTc interval and action potential duration. Compared to the control and DM rats on the KD, DM rats treated with an ND also showed lower intracellular Ca2+ transients, sarcoplasmic reticular Ca2+ content, sodium (Na+)-Ca2+ exchanger currents (reverse mode), L-type Ca2+ contents, sarcoplasmic reticulum ATPase contents, Cav1.2 contents. Furthermore, these rats exhibited elevated ratios of phosphorylated to total proteins across multiple Ca2+ handling proteins, including ryanodine receptor 2 (RyR2) at serine 2808, phospholamban (PLB)-Ser16, and calmodulin-dependent protein kinase II (CaMKII). Additionally, DM rats treated with an ND demonstrated a higher frequency and incidence of Ca2+ leak, cytosolic reactive oxygen species, Na+/hydrogen-exchanger currents, and late Na+ currents than the control and DM rats on the KD. KD treatment may attenuate the effects of DM-dysregulated Na+ and Ca2+ homeostasis, contributing to its cardioprotection in DM.A ketogenic diet (KD) might alleviate patients with diabetic cardiomyopathy. However, the underlying mechanism remains unclear. Myocardial function and arrhythmogenesis are closely linked to calcium (Ca Low-carbohydrate diets have gained popularity recently because of the increased awareness regarding the harmful metabolic effects of processed carbohydrates. The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that serves around 60% of fats for daily energy needs. It has been linked to mitigating epileptic seizures in children  and seveDiabetic cardiomyopathy is clinically asymptomatic in its early stage. It is marked by increased fibrosis, reduced early diastolic filling, elevated atrial filling, and enlargement, and raised left ventricular (LV) end-diastolic pressure . The sec+) or calcium (Ca2+) homeostasis through its potential consequences on the myocardial electrical and structural remodeling in DM hearts. Our earlier investigation demonstrated that sodium-glucose co-transporter-2 inhibitor (SLGT2i) modulated Na+ or Ca2+ homeostasis by decreasing oxidative stress in diabetic cardiomyopathy i transients  than that in the ventricular myocytes of the control rats  and DM rats on the KD . Nevertheless, the control and DM rats on KD ventricular myocytes had similar decay times of [Ca2+]i transients  compared with that in the ventricular myocytes of the control rats  and of DM rats on the KD . By contrast, the control and DM rats on the KD ventricular myocytes had similar [Ca2+]i leak (As illustrated in +]i leak .Ca-L density (in the peak ICa-L elicited from −50 to 10 mV) in the ventricular myocytes of DM rats on an ND was higher  than that in the control rats  and DM rats on the KD .INa-late density of DM rats on ND ventricular myocytes was higher  than that in the ventricular myocytes of the control rats  and of DM rats on the KD  than that in the ventricular myocytes of the control rats  and of DM rats on the KD  than in the control rats  and DM rats on the KD . The cytosolic ROS levels of the control and DM rats on KD ventricular myocytes were similar (+ ([Na+]i) levels in ventricular myocytes were 1.5-fold higher in DM rats on an ND  than in the control rats  and DM rats on the KD , NCX, and voltage-dependent L-type alpha 1C subunit (Cav1.2) /CaMKII-δ compared to both the control and DM rats on the KD (The expression of Cablotting . Compare(Cav1.2) . Furthern the KD .2+]i signaling [2+]i transients, and INa-late, which may be electromechanical mechanisms underlying the antidiabetic cardiomyopathy effects of a KD.A KD may have cardiovascular benefits in DM hearts ,16,17; hignaling , SERCA2aignaling , RyR2 2+i signalignaling . HoweverDM patients generally demonstrate a high incidence of prolonged QT and QTc interval because of an increment in the APD and repolarization, making them liable to have an increased incidence of cardiac arrhythmia ,25. Seve2+ is responsible for the connection between mechanical contraction and electrical activation and is crucial in excitation–contraction coupling. Ca2+ from its sarcoplasmic reticulum (SR) stores through RyR2 triggers the release of ionic Ca2+. The ionic Ca2+ further facilitates Ca2+ binding to the myofilaments, enhances [Ca2+]i content, and activates myocardium contractions [2+ content associated with prolonged [Ca2+]i decay and relatively diminished [Ca2+]i transients in DM hearts. According to a previous study, a reduction in SR Ca2+ uptake is one of the primary mechanisms causing a diminution in the myocardial contractility of diabetic cardiomyopathy [2+]i transient amplitudes [2+]i transients may have originated from the reduced SR Ca2+ content, impairing excitation–contraction coupling efficiency. The impaired reuptake of Ca2+ during the diastolic phase may have caused SERCA function impairment, causing a decline in SR Ca2+ stores, which might have worsened the condition of our DM rat hearts due to lowered SERCA2a levels. The decrease in SERCA2a levels might have reduced SR Ca2+ stores in our DM rat hearts. The prolonged [Ca2+]i transient decay in our DM rat hearts might also have been due to SR Ca2+ depletion. The rise in the transient decay of [Ca2+]i might be attributed to the dysfunction of the SERCA pump, generating a defect in cardiomyocyte relaxation and a slower removal rate of cytoplasmic Ca2+ i transient decay may also result from prolonging APD in DM ventricular myocytes. We also discovered that a KD reversed the decreased [Ca2+]i transients and delayed [Ca2+]i decay in DM hearts, which diminished the Ca2+ storage in DM hearts. An augmented SERCA2a function in the myocardium could have caused these effects. Furthermore, as assessed through echocardiography, the modulation of Ca2+ by the KD in DM hearts likely significantly mitigates diabetic cardiomyopathy. In our study, a KD alleviated Ca2+ dysregulation caused by DM, as evidenced by reduced phosphorylation of both RyR2 at serine 2808 and CAMKII-δ and increased SERCA2a expression. These findings suggest that a KD improves Ca2+ homeostasis, increasing EF in DM hearts.An increase in i, similar to our previous results i transients and suggesting the cardioprotective effect of a KD in diabetic cardiomyopathy. Similarly, we found that NCX function is reduced in DM cardiomyocytes. Inhibition of NCX in its reverse mode, which mediates Ca2+ influx, might have restricted the cellular Ca2+ content, subsequently contributing to a reduction in [Ca2+]i and leading to electrical dysfunction. The KD effectively improved the reduced NCX current in the ventricular myocytes of DM hearts.Comparable with the outcomes from other investigations ,41,42, o+/H+ exchanger current and INa-late in our DM rats cardiomyocytes may have contributed to the rise in [Na+]i, causing Ca2+ overload and increasing the risks of oxidative stress and arrhythmia i transient amplitude, improved myocardial contractility, and reversed RyR2 hyperphosphorylation in our DM hearts. As observed in our DM rats’ ventricular myocytes, a decrease in RyR2 function prolongs the time to peak Ca2+ transients and slows down Ca2+ release i leak from cardiomyocytes causes an increase in endoplasmic reticulum stress and mitochondrial dysfunction. Mitochondrial dysfunction also increases ROS production and provokes redox modifications of RyR2, thereby exacerbating the leakage of Ca2+ [2+]i leak inhibition is a critical mechanism of action of the KD in DM rats. Thus, a KD may help reduce excessive Ca2+ leakage in the SR, resulting in a Ca2+ shortage in diabetic cardiomyopathy.In the heart’s excitation–contraction coupling, RyR2, a macromolecular homotetrameric protein complex, controls the release of Cam the SR . CaMKII,a2+ leak . Similara2+ leak ,46. The  release . Becauseotential . Increas of Ca2+ . Consequ2+ regulatory proteins [2+]i signaling, causing impairments in transcriptional regulation, cell mechanics, energetics, and excitation–contraction coupling i level and CaMKII activity, contributing to reductions in cardiac remodeling related to diabetic cardiomyopathy.Heart disorders, including cardiac hypertrophy, arrhythmias, and heart failure, are linked to CaMKII overexpression . CaMKII proteins . CaMKII coupling ,51. CaMKlic Ca2+ , leadinglic Ca2+ . CaMKII-2+ release events, enhancing the frequency of sparks and Ca2+ leaks in the cardiomyocytes. The reduced ROS generation following KD ingestion may have helped to decrease the Ca2+ leak in DM cardiomyopathy. Additional assessment of the mechanical functions to comprehend the cardioprotective impact of a KD is necessary for DM cardiomyopathy. The decrement of Ca2+ leak, ROS, INa-late, and Na+/H+ exchanger current in the DM cardiomyocytes on the KD suggests that a KD may have antiarrhythmic potential. Therefore, further investigation to establish the antiarrhythmic effect of a KD is necessary.We also investigated ROS generation in DM hearts and noted that a KD reduces cytosolic ROS production. ROS can cause local endoplasmic reticulum Ca2+, which are dysregulated in diabetic cardiomyopathy, including altered expressions of RyR2, phospholamban, SERCA2a, NCX, and CaMKII-δ, and levels of ICa-L channel activity. High cytosolic concentrations of Ca2+ have been demonstrated to induce early cell death. Additionally, changes in mitochondrial Ca2+ handling may lead to mitochondrial respiratory dysfunction and subsequent cell death i was evaluated based on the fluorometric ratio (fluo-3 fluorescence). Briefly, the freshly isolated single LV myocytes were plated on glass coverslips (diameter = 25 mm), which were pre-coated with poly-L-lysine solution (0.01% w/v) for one hour. No Ca2+ agonist was used. Cells (around 50–60% confluent) were loaded with a fluorescent Ca2+ indicator fluo-3/AM (10 μM) for 30 min in standard Tyrode’s solution at room temperature. Excess extracellular dye was removed by changing the bath solution and allowing the intracellular hydrolysis of fluo-3/AM after 30 min. At >515 nm, emission was confirmed with an argon-ion laser 3 fluorescence stimulated at 488 nm. The cells were repeatedly scanned at 2 ms intervals using a line scan (8-bit) imaging. Fluorescent cells were monitored using Zeiss LSM 510  laser scanning confocal microscope and an Axiovert 100 inverted microscope . The transient [Ca2+]i changes were calculated by measuring the fluorescent signals and baseline values (F/F0). The fluorescence of fluorescent signals (F) was compared to that of the baseline fluorescence (F0), variations in dye concentrations were corrected, and fluorescence intensity variations generated by various dye injection volumes were excluded [2+]i transients, diastolic [Ca2+]i, peak systolic [Ca2+]i, and decaying fraction [Ca2+]i transients were monitored using 10 ms twice-threshold strength square-wave pulses after 1-Hz field stimulation. Moreover, the monoexponential least-squares fit was used to calculate [Ca2+]i. After a steady-state [Ca2+]i transient was achieved with multiple pulses, the superfusate containing tetracaine (1 mmol/L) for 20 s was changed to 0 Na+/0 Ca2+ solutions (1 Hz for 15 s). The 1 mmol/L tetracaine-reduced [Ca2+]i was used to quantify the SR Ca2+ leak as previously described [The [Caexcluded . [Ca2+]iescribed .2+ reserves were measured using a 30 s pulse stimulation train at 1 Hz followed by the addition of 20 mmol/L of caffeine. The SR Ca2+ reserve was calculated from the peak amplitude of the caffeine-induced Ca2+ transient. An amount of 20 mmol/L of caffeine voltage-clamped at −40 mV was quickly injected into cells. As previously mentioned, /(Cm × 8.31 × 8.44). In addition to the cell surface-to-volume ratio being 8.44 pF/pL, Ccaff is the integral of the inward NCX current caused by caffeine, F is Faraday’s number, and Cm is the capacitance of the membrane.SR CaCa-L was measured using micropipettes filled with a solution composed of 130 mM CsCl, 1 mM MgCl2, 5 mM MgATP, 10 mM HEPES, 5 mM Na2+ phosphocreatine, 10 mM EGTA, and 0.1 mM NaGTP. The NCX current was measured with 110 mM CsCl, 20 mM NaCl, 1.75 mM CaCl2, 20 mM tetraethylammonium, 0.4 mM MgCl2, 10 mM HEPES, 5 mM 1,2-bis (2-amino phenoxy) glucose, 75 mM 1,2-bis(2-amino phenoxy) ethane-N,N,N′,N′-tetraacetic acid, and 5 mM MgATP titrated to a pH of 7.25. The late sodium current (INa-Late) was measured using 130 mM CsCl, 10 mM NaCl, 5 mM HEPES, 5 mM EGTA, 5 mM MgATP, and 5 mM glucose. The action potential (AP) was measured using 110 mM K aspartate, 20 mM KCl, 5 mM MgATP, 1 mM MgCl2, 0.5 mM EGTA, 10 mM HEPES, 5 mM Na2+ phosphocreatine, and 0.1 mM LiGTP titrated with KOH to a pH of 7.2. A digital-to-analog converter (12-bit) was used to create voltage command pulses on a pCLAMP (Axon Instruments). A low-pass filtration was applied to the recordings during half the sampling frequency. The AP amplitude (APA) was assessed as the difference between the peak AP depolarization and resting membrane potential (RMP). The repolarization of APD90, APD50, and APD20 were calculated.An Axopatch 1D amplifier  at 35 ± 1 °C was used on the newly isolated rat ventricular myocytes to perform whole-cell patch-clamp. In the voltage-clamp mode, ionic currents were reported, and action potentials (APs) directed at 1 Hz in the current-clamp mode were identified. ICa-L was measured during depolarization from a holding potential of −50 mV at 0.1 Hz as an inward current to evaluate the possibilities of −40 to +60 mV in 10 mV increments for 300 ms. CsCl and tetraethylammonium chloride were used with the external solution instead of NaCl and KCl. We used pulses between −100 and +100 mV given over 300 ms at depolarizing of 0.1 Hz to produce the current of NCX. Nickel-sensitive currents with a 10 mM nickel Cl were used to quantify NCX current amplitudes. The external solution contained 5 mM HEPES, MgCl2, 10 mM glucose, and 140 mM NaCl. Additionally, the external solution had dihydropyridine antagonist, 10 μM nitrendipine, 10 μM strophantidin to inhibit Na+/K+ pump, and 100 μM niflumic acid to impede Ca2+-activated Cl currents, and the pH was adjusted to 7.4.INa-Late was documented with an external solution comprising 130 mM NaCl, 5 mM CsCl, 1 mM CaCl2, 10 mM HEPES, 1 mM MgCl2, and 10 mM glucose. INa-Late current was determined when the current was produced and when the voltage was ramped back to −100 mV and was sensitive to 30 μM tetrodotoxin as described previously [Using a step/ramp procedure (where −100 mV was stepped to +20 mV at ambient temperature for 100 ms and then ramped back to −100 mV over 100 ms), Ieviously .2(6 H2O), 0.005 mM EGTA, and 10 mM HEPES/KOH (pH 7.3) was used to evaluate the Na+/H+ exchanger current in a whole-cell model. The external solution contained 3.6 mM CaCl2, 150 mM NaCl, 1.2 mM MgCl2(6 H2O), 5.4 mM KCl, 20 mM glucose, and 5 mM HEPES/NaOH (pH 7.4). At 3 Hz, rat ventricular myocytes were isolated, and 40 ms depolarizing pulses from −45 to 0 mV were used [An internal solution comprising 130 mM K-aspartate, 20 mM KCl, 1 mM MgClere used .+ concentration in the ventricular myocytes was measured using Asante NaTRIUM Green-2 AM (Teflabs). Like our previous investigation [2, 10 mM HEPES, 1.8 mM CaCl2, and 5.6 glucose at pH 7.3. Then, these isolated single LV myocytes were incubated for 25 min at 23 °C with an appropriate fluorescent dye, either 10 μM CellROX Green, or 5 μM Asante NaTRIUM Green-2 AM (Teflabs).The ROS production in the mitochondria and cytosol of the isolated rat LV myocytes was evaluated using CellROX green (Life Technologies). Moreover, cytosolic Natigation , an expeSimilar to other investigations , CellROXThe LV tissues were first homogenized using a homogenizer and extracted with M-PER™ Mammalian Protein Extraction Reagent , supplemented with Protease and Phosphatase Inhibitor (Thermo Scientific). Subsequently, the proteins were centrifuged at 13,500 rpm for 30 min. Following protein extraction, their concentrations were determined using the QubitTM Protein Assay Kit (Invitrogen). These proteins were then boiled for 5 min, and each lane was loaded with 30 µg of protein. These proteins were separated using standard sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), utilizing 8% SDS-PAGE or 15% SDS-PAGE, depending on the size of the target protein. The running buffer  was placed at 95 v for about 90 min.2+ channel, Cav1.2 , NCX , SERCA2a , CaMKII-δ , pCaMKII-δ , PLB , and phosphorylated PLB (pPLB)-S16; . The blots were then exposed to the specific enzyme-conjugated secondary antibody in diluted 5% skim milk in PBST at room temperature for 60 min. Hereafter, washing with PBST 3 times (5 min per wash) was performed. Bound antibodies were detected using an electrochemiluminescence (ECL) system. Band intensities were measured using Alpha Innotech by AlphaEaseFC  Gbox Chemi-XL . Glyceraldehyde 3-phosphate dehydrogenase  was used to standardize and validate equal protein loaded from the targeted bands as described previously [After electrophoresis, the separated proteins were transferred to a polyvinylidene fluoride  membrane in a transfer buffer  at 70 v for 120 min . Blockineviously ,80,81.p-value of <0.05.All quantitative data are expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) with Tukey’s HSD test was conducted  to identify the significant between-group differences. Statistical significance was defined as a 2+ or Na+ dysregulation in the cardiomyocytes was restored in the hearts of KD-treated rats by reducing ionic channel modification and ROS, resulting in decreased ventricular hypertrophy and prolonged QT interval correction in diabetic cardiomyopathy.In conclusion, as summarized in


In the present study, we employed a novel drug, carbonyl cyanide chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, to explore the role of mitochondria in cultured neuronal morphology. CCCP caused a sustained elevation of [Ca2+]i and, quite surprisingly, a massive increase in the density of dendritic filopodia and spines in the affected neurons. This morphological change can be prevented in cultures exposed to a calcium-free medium, Orai1 antagonist 2APB, or cells transfected with a mutant Orai1 plasmid. It is suggested that CCCP activates mitochondria through the influx of calcium to cause rapid growth of dendritic processes.A major route for the influx of calcium ions into neurons uses the STIM-Orai1 voltage-independent channel. Once cytosolic calcium ([Ca This is controlled with voltage and ligand-gated calcium entry and with extrusion channels. Recent evidence assigned a pivotal role of stromal interaction molecule 1 (STIM1) in the regulation of [Ca2+]i [2+]i rise in an Orai1-dependent manner, conjugates mitochondrial calcium saturation and massive growth of dendritic processes, followed by apoptotic swelling and subsequent death of the cultured hippocampal neurons.Variations in cytosolic calcium concentrations (Ca+i) play  [Ca2+]i . Relativ [Ca2+]i ,7, in th [Ca2+]i  and in t [Ca2+]i , ischemi [Ca2+]i  and oxid [Ca2+]i . While S [Ca2+]i , the dyn [Ca2+]i . In the  [Ca2+]i . We now 2+]i and the formation of novel dendritic filopodia/spines. As can be seen in 2+]i, measured with the membrane permeant Fluo-2 AM, grows gradually over 30 min of exposure (2+]o) to change in [Ca2+]i, the cells were maintained in nominally 0 mM calcium in the imaging medium. In these conditions, there was no change in [Ca2+]i and no change in filopodia/spines density  by the use of 2-APB , we compared the level of i, long-term treatment with CCCP also led to an increase in mitochondrial calcium ([Ca2+]m). We detected an initial weak followed by a sharp increase in the fluorescence of the mitochondrial calcium sensor mtRCaMP against the background of a gradual increase in [Ca2+]i  + TTX (a sodium channels blocker) + DNQX (a competitive antagonist of AMPA receptors) with the help of which excitatory synaptic potentials and backpropagation action potentials, and activity-induced calcium transients were blocked. In addition, the application of nifedipine  and ω-c blocker  did not  of CCCP C. Howeve2+ entry , preventWe measured the number of new processes with CCCP in categories; filopodia/thin, mushroom and stubby spines. The majority of new processes were filopodia and a few stubby and mushroom spines . However2+]i. These results do not allow us to speculate at the present time if these processes are to be converted to functional dendritic spines since further treatment with CCCP leads to apoptotic swelling and cell death, as shown in the panels of The present results demonstrate that exposure to CCCP for 20–30 min can cause a dramatic effect on the formation of dendritic filopodia via a sustained rise in [Ca2+]m increased at different rates during the experiment  and imaged at a rate of frame/3, 5 or 10 s. No photobleaching was detected under these conditions. Standard recording medium contained (in mM); NaCl 129, KCl 4, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, pH was adjusted to 7.4 with NaOH and osmolality to 320 mOsm with sucrose. All measurements were conducted with identical laser parameters for all groups  at room temperature.Imaging: Fluo-2 AM  or Calcium Orange AM was incubated for 1 h at room temperature to image variations in  are acquired, where green (488 nm) and red (545 nm) fluorescent cells represent live and dead cells, respectively. Percentage of live and dead cells is calculated: live cells (cells stained green) or dead/dying cells (cells stained red) multiplied by 100% and divided by the total number of cells (green and red) in the field 200 × 200 micron at  as live . Standart-tests, using MATLAB , KaleidaGraph , and OriginPro 2021  software. Statistically significant differences were considered at p < 0.05. Dendritic spines/filopodia were identified in the eBFP or DsRed-transfected neurons and analyzed independently of the measurements of calcium.Fluorescence and statistical analysis: High-resolution fluorescent images were analyzed using Image-J  and MATLAB -based programs. Statistical comparisons were made with post hoc tests using ANOVA and


Force sensors on climbing robots give important information to the robot control system, however, off-the-shelf sensors can be both heavy and bulky. We investigate the optimisation of a lightweight integrated force sensor made of piezoelectric material for the multi-limbed climbing robot MAGNETO. We focus on three design objectives for this piezoelectric component. The first is to develop a lightweight component with minimal compliance that can be embedded in the foot of the climbing robot. The second objective is to ensure that the component has sensing capability to replace the off-the-shelf force sensor. Finally, the component should be robust for a range of climbing configurations. To this end, we focus on a compliance minimisation problem with constrained voltage and volume fraction. We present structurally optimised designs that satisfy the three main design criteria and improve upon baseline results from a reference component. Our computational study demonstrates that the optimisation of embedded robotic components with piezoelectric sensing is worthy of future investigation. Topology optimisation of macroscopic components has been applied to a wide array of industries including mechanical, civil, aerospace and biomedical engineering ,2. RecenThe ability to sense the robot’s state and surrounding environment is also key to the design and control of robots. The use of piezoelectric materials as sensors is common . The problem is therefore self-adjoint with In FE notation this is written asNext we consider the single inclination voltage functionalq: Suppose we let Differentiating givesWeak Form 3. Find Setting the first two lines to zero gives a weak form for the adjoint functions Solving this weak form for the adjoint functions In FE notation this is written as matches  up to noD to be 25 mm, 10 mm, and 25 mm for the x, y, and z directions, respectively. This is approximately the size of the existing component on MAGNETO. We let the boundary z direction and span the domain in the y direction. Likewise, the boundary x direction and span the domain in the y direction.We take the size of the design domain 2. The contact stress For the Neumann boundary conditions on Equation  with m=5We use three loading angles er below . We consz-poled PZT-5A, which has been used for previous piezoelectric optimisation work [3. The piezoelectric coefficients for this material are given in Voigt notation by  method , where tHere ier work , the penEven with these appropriate penalisation exponents, optimised designs contain intermediate densities. This issue is addressed via a post-processing optimisation problem, where after 300 iterations of the optimisation algorithm the penalty termeviously  and effemove: 0.1, asyinit: 0.1 and asyincr: 1.0. These are relatively conservative choices that prevent the optimiser from too aggressively adding or removing material  [Filtering of sensitivities using the standard mesh-independence filter  is emploaterial  ,35.In the following we give computational results for a reference component and subsequently present our optimisation results.We briefly present and discuss the computational results for a reference component made of the two base materials described above  along wiABS has a density of 1.1 g/cmectively . ABS doetion see . Figure In this section we present our computational results for topology optimised components with PZT-5A and the open-cell optimised PZT-5A as base materials. As mentioned previously, we consider three configurations via the inclination angles In hed work . In parthed work .The four topology optimised designs with the two different base materials and two maximum volume fractions are presented in It is important to emphasise that in In this section we consider one additional optimisation problem where we minimise the volume of the piezoelectric component while constraining the average compliance and voltage for the three robot configurations. Such a volume minimised design would have low mass and provide a good voltage response. The optimisation problem we consider is: We solve this topology optimisation problem with a compliance constraint of Mechanical properties: lightweight and low compliance.Sensing capability: piezoelectric sensing capability.Robustness to climbing configurations: functional for inclination angles In this section we discuss the properties of our presented optimised piezoelectric components in comparison to the reference component and reflect on the design objectives described in the Introduction. We summarise the main design objectives as follows:Focussing first on the mass of the reference and optimised components, we see that the reference component with ABS as the base material is the lightest of the presented components with a mass of 3.218 g see . The opeThe relatively low Young’s modulus of ABS results in a high compliance for the ABS reference component. It is clear from the left plot of Piezoelectric sensing capability of the optimised components was achieved by imposing an inequality constraint on the average voltage The optimised piezoelectric components need to be robust to a range of climbing configurations, and particularly for the inclination angles We considered one additional topology optimisation problem where we minimised the volume of the component while constraining the average compliance and voltage to be similar to the PZT-5A reference component. The optimised design presented in The above discussion demonstrates that our optimised piezoelectric components satisfy all three design criteria. However, there are clear trade-offs that would need to be considered to choose a single best design. For example, using the optimised open-cell base material gives a significant improvement in potential sensing capability and would be lighter in weight compared to a design using solid PZT-5A as the base material. However, an open-cell design would also be more compliant, more difficult to manufacture, and may not have the mechanical strength needed for use in the robotic application. Experimental research including building of prototypes would be needed to evaluate these other aspects of each of the designs. It would also be possible to further enhance the component’s sensing capability by considering the poling direction of piezoelectric base material as a design variable. This idea is closely related to the consideration of material orientation when using anisotropic base materials, e.g., . MateriaFinally, while our work has focussed on the computational analysis of possible piezoelectric component designs, we briefly discuss manufacturability. Recent research has demonstrated the successful additive manufacture of piezoelectric materials . Such teIn this paper we have considered the computational design of a multi-functional, lightweight, integrated piezoelectric force sensor for the foot of a climbing robot. We consider the average compliance and measured voltage of the designed component for three robot configurations. The design problem is formulated in three dimensions, without restriction to two dimensions or a layered structure. Furthermore, we consider both a solid base material and the use of an architectured piezoelectric material as the base material in the optimisation problem. Our optimised designs provide improved compliance and piezoelectric sensing capability compared to a reference design. The designs would reduce robot weight and increase payload capacity if they can replace existing off-the-shelf force sensors. Overall, we have demonstrated the successful computational optimisation of three-dimensional integrated piezoelectric sensing components for a climbing robot. Such components are worthy of future experimental investigation.


UBTF gene have been recently described as a recurrent alteration in pediatric acute myeloid leukemia (AML). Here, by screening 1946 newly diagnosed adult AML, we found that UBTF-TDs occur in about 3% of patients aged 18–60 years, in a mutually exclusive pattern with other known AML subtype-defining alterations. The characteristics of 59 adults with UBTF-TD AML included young age (median 37 years), low bone marrow (BM) blast infiltration (median 25%), and high rates of WT1 mutations (61%), FLT3-ITDs (51%) and trisomy 8 (29%). BM morphology frequently demonstrates dysmyelopoiesis albeit modulated by the co-occurrence of FLT3-ITD. UBTF-TD patients have lower complete remission (CR) rates (57% after 1 course and 76% after 2 courses of intensive chemotherapy [ICT]) than UBTF-wild-type patients. In patients enrolled in the ALFA-0702 study (n = 614 patients including 21 with UBTF-TD AML), the 3-year disease-free survival (DFS) and overall survival of UBTF-TD patients were 42.9% (95%CI: 23.4–78.5%) and 57.1% (95%CI: 39.5–82.8%) and did not significantly differ from those of ELN 2022 intermediate/adverse risk patients. Finally, the study of paired diagnosis and relapsed/refractory AML samples suggests that WT1-mutated clones are frequently selected under ICT. This study supports the recognition of UBTF-TD AML as a new AML entity in adults.Tandem duplications (TDs) of the  UBTF) gene, located at 17q21, encodes a ubiquitously expressed nucleolar protein. UBTF is a key component of the pre-initiation complex mediating the recruitment of RNA polymerase I to ribosomal DNA promoter regions but is also enriched at polymerase II-transcribed genes throughout human genomes . Th. ThUBTF-l disomy . In mostWT1 mutations. All relapse AML cases (n = 10) harbored at least one WT1 mutation with high VAF compatible with clonal dominance  were found in 6/10 (60%) relapse samples. The WT1bi_alt clone driving relapse was already present at diagnosis at low levels in 2 cases (E and J), derived from a WT1 monoallelic clone  in 3 cases , or fully emerged at relapse (or was present below the sensitivity threshold [VAF 1%] at diagnosis) in 1 case (patient G). Other mutations could coexist within the WT1bi_alt clone at relapse, frequently involving the RAS pathway . In addition, patient K who was the only studied patient with a dominant WT1bi_alt clone (mutation + deletion) at AML diagnosis was refractory to induction, suggesting the resistance of this clone to intensive chemotherapy. Consistent with these observations, 21/35 (60%) WT1-mutated patients (regardless of the number or VAF of the mutation) receiving ICT were refractory to the first course of ICT, compared with 4/23 (17%) WT1 wild-type patients.Analysis of paired diagnostic-relapse cases revealed a preferential pattern of disease progression likely driven by mono_alt arising in a preexisting FLT3-ITD clone. While FLT3-ITD is generally associated with resistance and disease progression in AML [FLT3-ITDs did not seem to confer clonal advantage in UBTF-TD AML when not associated with WT1 mutation. Indeed, some FLT3-ITDs detected at diagnosis were frequently lost during progression . Similarly, patient L who was refractory to induction chemotherapy (without FLT3 inhibitor) demonstrated the rapid disappearance of FLT3-ITD and the selection of a preexisting WT1mono_alt/NF1-comutated clone.Another pattern of disease relapse was observed in patients A and B for whom the relapse appeared to be driven by a WT1n in AML , FLT3-ITFLT3-ITD and characterized by the emergence of 2 distinct FLT3-ITD/FLT3-TKD (D835H/Y) mutants, which have been described as a recurrent mechanism of resistance to FLT3 inhibitors  at relapse, one of whom (patient A) experienced a second relapse that was documented by sequencing. The post-quizartinib relapse was still positive for FLT3-ITD and/or WT1 mutation were known to lack a known initiating event, while being associated with a poor outcome in both adults and children [UBTF-TD AML, paves the way for their detection in routine practice and future development of new therapies. Overall, it is likely that UBTF-TDs have been underestimated in previous studies due to limitations in detecting this type of aberration with many of the current bioinformatic approaches used in genetics laboratories [UBTF sequence which includes repetitive motifs and frequent homopolymers complicating variant calling.Molecular and cytogenetic analyses are routinely used to identify AML subtype-defining structural variants and mutations and have become critical for risk stratification and treatment guidance. Despite 15 years of genomics research since the first AML genome publication and large studies like The Cancer Genome Atlas (TCGA), some AML cases remain genetically unclassifiable with current knowledge –31. Notachildren , 32. Theratories . This maUBTF with a common minimal duplicated region encoding the ELLTRLA motif of the HMG4 domain of the UBTF protein, as previously described in pediatric cases [FLT3-ITD [UBTF-TD expression in CD34 + cells was sufficient to induce a proliferative advantage, increases clonogenic activity and activates the HOXB gene cluster, recapitulating the transcriptional signature observed in UBTF-TD AML patients [All TDs identified in the present study led to in-frame insertions within exon 13 of FLT3-ITD . The queUBTF-TDs occur in about 3% of AML in patients aged 18–60 years. However, given the variability of TDs, we cannot exclude that some perfect and large duplications were missed by the screening method. Adult AML patients with UBTF-TDs were significantly younger and had lower BM blast infiltration with a BM morphology frequently demonstrating myelodysplastic features including severe dyserythropoiesis. It should be noted that UBTF-TD AML were associated with the M6 morphology subtype [FLT3-ITD or low ratio) and the diagnosis of AML could thus be made by counting the proportion of BM blasts among the non-erythroid cells according to the FAB criteria [Here we found that  subtype  accordincriteria . It is lcriteria , 36.UBTF-TDs were mutually exclusive with other known AML subtype-defining alterations. Sequencing data revealed UBTF-TDs were always clonal and stable during disease progression, in agreement with an early/initiating event. The most frequent co-occurring somatic aberrations were by far WT1 mutations, FLT3-ITD (high ratios being associated with increased WBC count and BM blast infiltration) and trisomy 8 (85% of cases carried at least one of these aberrations and 47% had at least two). Patients with UBTF-TDs displayed high rates of induction failure.UBTF-TD AML) showed that DFS and OS of UBTF-TD patients were not significantly different from those of UBTF-wt ELN 2022 intermediate or adverse risk patients. Although long-term remissions were observed after allo-HCT, post-allo-HCT relapse remained common and could occur after a significant delay. This shows that surveillance as well as MRD and chimerism monitoring should be repeated and would potentially allow for preemptive measures . However, considering the limited number of patients studied, additional data are required to confirm the prognosis of UBTF-TD AMLs and assess the benefits of allo-HCT in this context.Prognostic analyses in patients enrolled in the ALFA-0702 study (including 21 WT1 mutations are likely to play a major role in disease progression. Especially, WT1bi_alt or WT1mono_alt/FLT3-ITD co-mutations were observed in most relapses, whereas patients with these features at AML diagnosis were more likely to be refractory to induction chemotherapy. Together, these results suggest strong cooperation within the UBTF-TD/WT1/FLT3-ITD trio in establishing the leukemic phenotype and determining the clinical outcome. Also, these results emphasize the need to evaluate new therapies in combination to first-line treatment or in maintenance. FLT3 inhibitors have been shown to improve outcome in FLT3-ITD-positive AML but it should be noted that WT1 mutations have been identified as contributing to drug resistance [WT1 mutations in leukemogenesis remains elusive. WT1 has a functional duality since it may act, depending on the cellular context, as a transcriptional activator or repressor through its DNA-binding zinc-finger domain and interactions with Tet enzymatic proteins that regulate DNA hydroxymethylation [HOX cluster genes in UBTF-TD AML, the use of menin inhibitors is also a promising therapeutic approach that deserves to be studied in the future [Interestingly, the study of paired diagnosis and relapsed/refractory AML samples showed that sistance , 38. Thehylation , 40. Givhylation . Finallye future .UBTF-TDs can be found in about 3% of adult AML aged 18–60 years and define a distinct subtype of AML with specific biological and clinical features and lower CR rates. Together with pediatric data, this study supports the recognition of UBTF-TD AML as a new AML entity to be included in disease classifications, a prerequisite for the development of future guidelines and therapies, especially in young patients.Overall, our study revealed that Supplemental appendixSupplemental tables


TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in G12D/+Nras–/–p53 (NP–/–) mice, G12D/+NrasR172H/+p53 (NPmut) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an NPmut-AML transcriptome distinct from that of NP–/– cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-κB signaling. NF-κB was also upregulated in human NRASTP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NF-κB inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-κB–inhibitory protein IκBα, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and   TP53 mutations, as patients with t-AML who do not haveTP53 mutations have a median survival that approximates that of patients with de novo AML.Acute myeloid leukemia (AML) is an aggressive and devastating hematologic malignancy characterized by the accumulation of partially differentiated myeloid blast cells ≥20%) in bone marrow (BM) and/or other hematopoietic organs, leading to BM failure and death . Patient0% in bonNRAS and KRAS, are prevalent in all 3 categories of AML but differ in the disease stages at which they arise. In de novo AML, NRAS and KRAS mutations are usually acquired later in clonal evolution to drive AML progression , leading to a dismal overall AML survival of less than 5 months  mice developed a mixed AML and T cell lymphoma/leukemia. The NP–/– AML transcriptome is predominantly regulated by p53 loss. In this study, we investigate genetic interactions between oncogenic NRAS signaling and the p53 R172H mutant. Our data demonstrate that G12D/+Nrasmut/+p53 (NPmut) rapidly induced AML characterized by inflammation and cellular and molecular mechanisms distinct from those of NP–/–.We previously tested genetic interactions between d p53–/–  and founVav-Cre to activate both mutations since E11.5 (https://doi.org/10.1172/JCI173116DS1). We then switched to the inducible Mx1-Cre line and generated Mx1-Cre (control), LSL-R172H/+p53Mx1-Cre (p53mut), LSL-G12D/+NrasMx1-Cre (NrasG12D), and LSL-G12D/+NrasLSL-R172H/+p53Mx1-Cre (NPmut) mice. Six-week-old mice were administered polyinosinic-polycytidylic acid (pI-pC) to induce the expression of oncogenic Nras and mutant p53 from their respective endogenous loci. Unexpectedly, NPmut mice were either found dead or became moribund within a few days after the initial pI-pC injection  or osteosarcoma , whereas 100% of NrasG12D mice developed myeloid disorders as described previously  , and a reduction of neutrophils in BM  , charact80 days) . Flow anls in BM . By contenotypes . Unlike enotypes , AML blaecursors . Blood s  from control, p53mut, and NrasG12D mice 1 week after the last pI-pC injection and from age-matched NPmut mice. Long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) 2–4 were delineated as previously described (–Sca1+cKit+ (LSK) cells in p53mut mice were indistinguishable from those in control mice  were hyperproliferative. Surprisingly, the cell-cycle profiles of NPmut HSPCs were comparable to those of control cells  and subpopulations  in control, p53mut, NrasG12D, and NPmut mice . Surprisingly, in contrast to these modest NPmut HSPC phenotypes, phosphorylated ERK (p-ERK) levels in NPmut HSPCs were 2-fold higher than those in control, p53mut, and NrasG12D cells in the absence or presence of GM-CSF stimulation  were significantly increased in NrasG12D but not NPmut BM and SP compared with controls .Interestingly, despite the comparable expansion of lymphoid-primed MPP4 cells in Nrasmut mice , the numcontrols . Moreovephocytes  and inviphocytes , whereasartments  and a moartments . SimilarP and PB . Our dat–cKit+ BM HSPCs from moribund NPmut as well as pI-pC–treated, age-matched control, p53mut, and NrasG12D mice. RNA-Seq analysis of NPmut versus control HSPCs identified 716 differentially expressed genes (DEGs) , with 258 and 458 genes significantly up- and downregulated, respectively . Among the established positive regulators of the ERK1/2 signaling pathway , only Rasgrp4, encoding a RAS guanine nucleotide exchange factor, was moderately upregulated. However, Rasgrp4 levels were almost undetectable (reads per kilobase per million mapped reads [RPKM] <1)  revealed that Dab2ip, a RAS GTPase activating protein, was downregulated in NPmut HSPCs  , A and But HSPCs , C and Dut HSPCs . These fmut AML transcriptome is mainly driven by the synergistic activities of mutant p53 and oncogenic NRAS, it showed minimal overlap with the NP-/- AML transcriptome, which is predominantly driven by p53 loss (Junb), whereas common downregulated genes were enriched for NPM1-mutated or MLL1-driven AML-related gene sets   (Rela and Nfkb1) and the myeloid/B lineage transcriptional regulator (PU.1) were enriched in upregulated genes in NPmut HSPCs  comparing NP(MSigDB) . Severalol cells . In cont/– HSPCs , the TLRut cells . In addiqRT-PCR) . ConsistqRT-PCR) , 35, ideut HSPCs . Becauseut HSPCs –38, we eex ELISA . This anBM cells .mut HSPCs revealed enrichment for GATA1- and GATA2-linked transcriptional networks . Although this gene set was not included in the databases we previously used, it was significantly enriched in NPmut HSPCs  . By contrast, BM cells isolated from moribund p53mut mice (IC50: 1.8 μM) or NrasG12D mice (IC50: 3.6 μM) were less sensitive to IKK-16  complex is activated and phosphorylates the inhibitory protein IκBα, leading to its proteasome-mediated degradation and subsequent nuclear localization of NF-κB TFs  47). IK. IK47). ib (Tra)  and/or Iectively . Combineo IKK-16 .mut KY821 AML cells showed similar sensitivity to Tra (IC50: 25 nM), whereas K562 cells were resistant to Tra (IC50 >200 nM)   . To deteto Tra IC: 25 nM, lication . KY821 c 3.1 μM) . Our resmut HSPCs overexpressed CD74  than were K562 cells (IC50: 27.4 nM)  . Consist27.4 nM) , 56, Btz27.4 nM) . Our datmut leukemia cells were transplanted into sublethally irradiated mice. Upon establishment of AML, the recipient mice were divided into 4 groups with comparable leukemia cell burdens and treated with vehicle, Tra, Btz, or combined Tra and Btz , we believe that the reduced T cell compartment in NPmut-AML recipients did not result from a suppressive immune microenvironment, as we had previously described in G12D/+Nras–/–Asxl1 mice  downstream of hyperactivated ERK signaling to promote inflammation and NPmut AML. Given these results, we expect that p53R172H would display distinct properties in the context of other mutations found in AML.Compared with the prevalent p53titution  through 8W HSPCs  were comol cells . In addiut HSPCs , 81. TheLSL G12D/+Nras, LSL-R172H/+p53 , Mx1-Cre, and Vav-Cre mice was done as previously described (+ congenic C57BL/6J recipient mice were purchased from The Jackson Laboratory (stock 002014). To induce Mx1-Cre expression, approximately 6-week-old mice were injected i.p. with 100 μg pI-pC  every other day for 3 cycles. The day of the first pI-pC injection was defined as day 1. All experiments were performed on day 12 or at the moribund stage.Mouse lines were maintained on a pure C57BL/6J genetic background (>N10). Genotyping of escribed , 59, 82.t test was used to compare 2 data sets unless otherwise specified. A 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons was used to compare more than 2 data sets with 1 variable. A 2-way ANOVA followed by Tukey’s post hoc test for multiple comparisons was used to compare more than 2 data sets with 2 variables, whereas a 2-way ANOVA followed by Bonferroni’s multiple-comparison test was used to compare 2 data sets with 2 variables. P values and adjusted P values are indicated in the figure legends. A P value of 0.05 or less was considered significant. ImageJ software (NIH) was used to quantify protein levels by densitometry. Data were graphed and analyzed using GraphPad Prism 7.0 (GraphPad Software). The synergy score was calculated using the Combination Index to characterize the strength of synergistic interaction between 2 drugs.All results are presented in dot plots with the mean ± SD. All in vitro studies were performed at least 3 times, with 2–3 technical replicates for each condition. Results from 1 representative experiment are shown. A log-rank test followed by a Benjamini-Hochberg multiple-comparison analysis was used to compare Kaplan-Meier survival curves. An unpaired, 2-tailed Student’s Guide for the Care and Use of Laboratory Animals  and approved by the IACUC of the University of Wisconsin–Madison. The program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (protocol M005328). All human samples were obtained from the University of Wisconsin Hospitals and Clinics with IRB approval (protocol 2014-0904).All animal experiments were conducted in accordance with the NIH’s All data in this study are presented in the article and supplemental materials. All materials are available upon request through a material transfer agreement; inquiries should be directed to the corresponding author. Values for all data points in graphs are reported in the Additional methods are described in AR, YF, and MBG designed and performed experiments and wrote and reviewed the manuscript. EAR conducted histopathological analysis and reviewed the manuscript. TK, MHL, YZ, XG, and SM provided technical support and reviewed the manuscript. DRM, MMJ, KVGN, DY, VLT, EP, and EHB provided material support and reviewed the manuscript. JZ supervised the study, designed experiments, and wrote and reviewed the manuscript.


Functional enrichment analysis of differentially expressed genes (DEGs) implied that cell activation involved in immune response, leukocyte cell–cell adhesion and migration, and cytokine production were the main biological processes. Phagosome, antigen processing and presentation, cytokine–cytokine receptor interaction, B-cell receptor, and chemokine were identified as the major pathways. To seize the distinct landscape in C1 vs. C2 subtypes, a 5-gene prognostic signature  was developed using LASSO Cox stepwise regression analysis and further validated in independent AML cohorts. Patients were divided into high- and low-risk groups, and decreased survival rates were observed in high- vs. low-risk groups. The TIME between high- and low-risk groups has a similar scenery in C1 vs. C2 subtypes. Single-cell-level analysis verified that LSP1 and CRIP1 were upregulated in AML and exhausted CD8+ T cells. Dual targeting of these two markers might present a promising immunotherapeutic for AML. In addition, potential effective chemical drugs for AML were predicted. Thus, we concluded that molecular subtyping using ferroptosis signatures could characterize the TIME and provide implications for monitoring clinical outcomes and predicting novel therapies.Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancies with a low 5-year survival rate and high rate of relapse. Developing more efficient therapies is an urgent need for AML treatment. Accumulating evidence showed that ferroptosis, an iron-dependent form of programmed cell death, is closely correlated with cancer initiation and clinical outcome through reshaping the tumor microenvironment. However, understanding of AML heterogeneity based on extensive profiling of ferroptosis signatures remains to be investigated yet. Herein, five independent AML transcriptomic datasets  were obtained from the GEO and TCGA databases. Then, we identified two ferroptosis-related molecular subtypes (C1 and C2) with distinct prognosis and tumor immune microenvironment (TIME) by consensus clustering. Patients in the C1 subtype were associated with favorable clinical outcomes and increased cytotoxic immune cell infiltration, including CD8 Acute myeloid leukemia (AML) is a type of aggressive blood malignancy characterized by the stagnant transition of immature myeloid cells to mature cells in the bone marrow and peripheral blood . Despite+ T cells by the combination of PD-1 blockade and CTLA-4 therapy aggravate ferroptosis-specific lipid peroxidation in tumor cells, suggesting that enhanced ferroptosis contributes to immunotherapy efficacy. Most studies on FAGs focused on solid tumors, but their functions in AML were not well understood. Therefore, it might provide new prospects for developing anti-leukemia agents for treating AML through the understanding of the ferroptosis regulatory heterogeneity of the tumor microenvironment (TME).Ferroptosis was recognized as a non-apoptotic and iron-dependent lipid peroxidation-induced type of programmed cell death, which is distinct from other forms of cell deaths, including necroptosis, apoptosis, and autophagy, in morphology and mechanisms . The halIn this study, we systematically profiled FAGs expression datasets and clinical features in patients with AML obtained from The Cancer Genome Atlas (TCGA) and GEO  databases. The molecular diversity of AML was delineated by molecular subtyping using ferroptosis signatures. The tumor immune microenvironment (TIME) was characterized using multiple immune cell subset deconvolutions, and the immune status between classifications was compared. To further specify the TIME, we developed a prognostic signature based on the overall survival (OS)-related differentially expressed genes between molecular subtypes. External validation demonstrated that the signature could predict patient prognosis and reflect TIME, which might have implications for developing new therapies by targeting ferroptosis in AML.An AML gene expression dataset with clinical features was downloaded from The Cancer Genome Atlas (TCGA) database and regarded as the training set. The gene expression profile of 514 normal cases was acquired from the Genotype-Tissue Expression (GTEx) database. For external validation sets, five AML cohorts with clinical information  , including drivers, suppressors, and markers, were obtained from the FerrDb database (ferrdb/) . DiffereIn addition, protein–protein interaction network analysis of differentially expressed FAGs was retrieved from the STRING database and illustrated using Cytoscape (version 3.8).p-value and a q-value <0.05 were considered statistically significant.To delineate the difference in enriched biological activities and signaling pathways of the target gene list, Gene Ontology (GO), including biological processes (BP), molecular function (MF), and cellular components (CC), was conducted using the ‘‘clusterProfiler’’ package . In addip < 0.05 were identified through univariate Cox regression analysis.The expression matrix of these differentially expressed FAGs was extracted from the TCGA-AML dataset, and the clinical features were merged with the expression matrix. OS-related FAGs in AML with To assess the molecular heterogeneity within the AML dataset related to FAGs, cluster robustness was calculated through K-means consensus clustering using the ConsensusClusterPlus package , with clDifferential gene expression analysis was conducted to screen the differentially expressed genes between molecular subtypes, and functional enrichment analysis was used to investigate the biological processes and pathways as described previously.The tumor and its surrounding microenvironment, including immune cells, stromal cells, signaling molecules, and extracellular matrix, constitute the tumoral niche. The TIME between ferroptosis-related subtypes was deciphered from transcriptomes using multiple cell subset deconvolution algorithms including CIBERSORT , TIMER , and xCep-value <0.05 was considered statistically significant. The human leukocyte antigen (HLA) system is a complex of proteins that are closely involved in the regulation of immune responses such as antigen presentation and stimulation of T-helper cells  algorithm, following the multivariate Cox regression analysis. The minimum number of features that comprised the optimal signature was determined by the Akaike information criterion (AIC)  . The ferModel discrimination ability was predicted by the receiver operating characteristic (ROC) curve using the timeROC package . The risThe prognostic utility of the signature was independently validated by three external validation sets. Furthermore, the predictive performance was assessed by the ROC curve.dtp.cancer.gov). The expression profile based on Affymetrix U133 Plus 2.0 microarray was normalized using GC-robust multi-array averaging. FSAscore was derived and correlated with compound sensitivity using CellMiner (discover.nci.nih.gov/cellminer)  (llminer) .http://gepia.cancer-pku.cn/). Single-cell-level analysis was further conducted to verify their transcripts in the AML microenvironment in the Tumor Immune Single-cell Hub   .The differential gene expression analysis was performed to compare the FAG expression levels between AML and normal samples. A total of 3,093 DEGs were identified, of which 1,501 genes were upregulated, and 1,592 genes were downregulated in AML. We found 72 differentially expressed FAGs in AML vs. normal samples. These FAGs showed a distinct expression pattern , which wTo delineate the molecular heterogeneity of AML linked to ferroptosis, we attempted to determine the ferroptosis-related molecular subtypes using consensus clustering based on the transcriptome of OS-related FAGs. Two subtypes (C1 vs. C2) were identified with distinct ferroptosis gene expression patterns following K-means clustering . Decreas+ T cells, B-cell plasma cells, NK cells, and CD4+ memory resting cells showed higher infiltration in patients in the C1 subtype, while monocyte, activated CD4+ memory cells, and macrophages showed lower infiltration in those in the C2 subtype , such as CD8+ T cells with potent activation and functions, are critical to kill leukemia cells, while most of these cells are inclined to exhibit an exhausted phenotype, leading to leukemia growth in the tumor niche. In addition, increased regulatory T-cell infiltration in bone marrow during disease progression suppresses anti-leukemia activities. + T cells, which in turn influences the efficacy of immune checkpoint blockade. The role of ferroptosis in AML remains fully unexplored. Patients benefited from novel therapies including bispecific antibodies or molecular targeted therapies, while the long-term remission rate was quite low. Understanding ferroptosis within the AML immune microenvironment might have implications for identifying prognostic markers and developing novel therapies for AML.Therapies with high efficacy and less toxicity for AML treatment are still under clinical trials. The low 5-year survival and high relapse rate of AML pose an urgent need to identify new prognostic biomarkers or develop effective therapies. Most effective antitumor treatment strategies are pursued to selectively eradicate cancer cells by inducing minimized damage to normal tissues or cells . Cell de+ T cells. To characterize the immune landscape defined by the ferroptosis classification, we developed a 5-gene signature that showed performance efficiency in predicting prognosis, which was validated by multiple independent AML cohorts. This might provide a prognostic indicator for AML patients; however, it can be confirmed once it is verified in datasets from multi-centers and preferably even in clinical settings in the future. We predicted the potency of its association with TIME. The strong correlations of the signature with immune cells re-engraved the same trend of ferroptosis subtyping.Here, we demonstrated that the expression of FAGs is closely related to patient survival and TIME of AML. Two distinct molecular subtypes were identified by consensus clustering based on FAG expression. C1, with favorable clinical outcomes, was associated with enhanced infiltration of immune cells, especially CTL subsets that mainly function as tumor killers in the process of immune response such as CD8Ferroptosis has attracted significant attention in tumor biology and treatment strategy in recent years. The FAG expression in various solid tumors has been profiled, and many signatures based on the ferroptosis notion used to monitor outcomes and predict drug sensitivity have been proposed . Ruiming+ T cells and B cells were observed in the C2 subtype, while infiltrated suppressive subsets were increased, including neutrophils, macrophages, and Treg cells. Differential expression analysis found that many immune regulatory pathways are enriched that are representative of antigen processing and presentation, cytokine–cytokine receptor interaction, and B-cell receptor signaling. These results indicated hot immune activities in the C1 subtype. Accumulating studies have indicated that some crosstalk between autophagy and ferroptosis exists at the molecular level (+ T cells and lower Treg cells were observed in the low-risk group defined using an autophagy signature. Moreover, the correlations of these two types of cell death-related signatures with immune checkpoints showed overlap and difference similar to PD-1 (+ T cells, indicating that dual-targeting them may reverse the dysfunction of CD8+ T cells and inhibit leukemia growth to delay disease progression; however, further work is required to be carried out. In addition, the association analysis of the signature with NCI-60 tumor cell line growth inhibition screen data facilitated the identification of the potential compounds for AML. A total of 11 compounds that were under clinical or approved by the FDA for diverse tumors were screened, such as alkylating agents, particularly those at the N-7 position of guanine. Meanwhile, we should consider drug safety as it has been reported that they may increase the risk of AML, which needs to be confirmed in further experiments.TME has been demonstrated to assess patient outcomes and response to therapies. Immune cell subsets were quantified using CIBERSORTx, TIMER, and xCell algorithms. Decreased CD8ar level . The inf to PD-1 . This ma to PD-1 . TME-rel to PD-1 . Accumul to PD-1 . S100A8  to PD-1 , and S10 to PD-1 . Increas to PD-1 . It can  to PD-1 , while i to PD-1 . LST1 is to PD-1 , and its to PD-1 . We checin vitro and in vivo assays will consolidate the dual-targeting treatment strategy for AML. Finally, we found that the expression of most immune checkpoint molecules was elevated in the unfavorable C2 subtype, indicating that patients with the C2 subtype may be sensitive to ICI-based therapy, while the association of our molecular subtyping with treatment response, especially immunotherapy, was not explored due to a lack of available datasets.It is noteworthy that this study has potential limitations that inspire future validation and mechanistic studies. The molecular subtyping was established on the limited retrospective datasets; further validation in multi-centric AML sets may provide adjusted clues to make the classification to be more accurate. The utility of the proposed signature was not tested in a clinical setting, and quantification of immune subsets following applying the signature in AML patients will convince TIME predictive potential. Additionally, as CRIP1 and LSP1 are expressed on leukemia and cytotoxic immune cells, functional investigation of CRIP1 and LSP1 by AML cells or immune cells from TIME This study depicted the molecular heterogeneity of AML through molecular subtyping by consensus clustering based on ferroptosis signatures. Two subtypes with distinct outcomes and TIME landscape were identified. Then, a 5-gene signature was constructed and validated, which could predict patient prognosis and characterize the ferroptosis subtype-related TIME. However, these data are derived from retrospective public datasets, and further investigation on the roles of FAGs in AML progression and assessment of the clinical use of the signature are imperative.