In heart failure (HF) myocardial β-ARs undergo desensitization/down-regulation due to sustained sympathetic adrenergic activation. β-AR desensitization/down-regulation diminishes adrenergic signaling and cardiac contractile reserve, and is conventionally considered to be detrimental in HF progression. Abnormal Ca2+ handling, manifested as cardiac ryanodine receptor (RyR2) dysfunction and diastolic Ca2+ leak (due to sustained adrenergic activation) also occur in HF. RyR2 dysfunction and Ca2+ leak deplete SR Ca2+ store, diminish Ca2+ release in systole and elevate Ca2+ levels in diastole, impairing both systolic and diastolic ventricular function. Moreover, elevated Ca2+ levels in diastole promote triggered activity and arrhythmogenesis. In the presence of RyR2 dysfunction and Ca2+ leak, further activation of the β-AR signaling in HF would worsen the existing abnormal Ca2+ handling, exacerbating not only cardiac dysfunction, but also ventricular arrhythmogenesis and sudden cardiac death. Thus, we conclude that β-AR desensitization/down-regulation may be a self-preserving, adaptive process (acting like an intrinsic β-AR blocker) protecting the failing heart from developing lethal ventricular arrhythmias under conditions of elevated sympathetic drive and catecholamine levels in HF, rather than a conventionally considered detrimental process. This also implies that medications simply enhancing β-AR signaling (like β-AR agonists) may not be so beneficial unless they can also correct dysfunctional Ca2+ handling in HF.Cardiac sympathetic activation, mediated by β-adrenergic receptors (β-ARs), normally increases cardiac contraction and relaxation. Accomplishing this task requires a physiological, concerted Ca Beta-adrenergic receptors (β-ARs) are members of a large family of cell surface receptors known as G-protein-coupled receptors (GPCRs) . β-ARs mHeart failure (HF) is a global epidemic problem, affecting more than 37.7 million individuals globally and imposes substantial economic burden to the health care system . HF occu2+ signaling has been identified as another form of pathological remodeling in HF  cardiac ryanodine receptor  dysfunction and diastolic Ca2+ leak, reduced Ca2+ pump  activity, etc.  current , further enhancing β-AR signaling would worsen the existing RyR2 dysfunction and Ca2+ leak, leading to exacerbation of cardiac dysfunction and arrhythmogenesis  rather than a conventionally considered detrimental/pathological process (a foe). This viewpoint is supported by the following several lines of evidence.In HF cardiomyocytes develop 2 distinct yet related abnormalities: β-AR desensitization/down-regulation and RyR2 dysfunction/Ca2+ leak . Both ab2+ leak , 14. β-A2+ leak , 11, 12.ogenesis . Accordi2+ channels (LTCCs) on the T-tubules and allows Ca2+ to enter the cell. The entering Ca2+ then binds to and activates Ca2+ release channels (RyR2) on SR, releasing Ca2+ to cytoplasm from the SR (where Ca2+ is stored at high concentrations). This process by which Ca2+ entering cardiomyocyte through LTCCs activates RyR2 is known as Ca2+ induced Ca2+ release . Cardiac relaxation (diastole) is initiated when RyR2 close (terminating SR Ca2+ release) and Ca2+ are pumped back into the SR by the SERCA or extruded from the cell by the Na+/Ca2+ exchanger (NCX). This coordinated Ca2+ release and reuptake in cardiomyocytes are essential for ECC.Cardiac excitation-contraction coupling (ECC) refers to the conversion from electrical excitation  into mechanical contraction. Briefly, cardiac action potential propagates along the cell membrane, including the specialized invaginations (T-tubules) . Depolar release . The spi2+ signaling is required in mediating physiological sympathetic response. Sympathetic activation, mediated by the β-ARs-Gs-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling pathway, activates and phosphorylates LTCCs, increasing inward Ca2+ current  . GRK act2+ leak in HF are caused by sustained adrenergic signaling  rather than a detrimental, pathological process (a foe), as conventionally considered.Thus, in the presence of RyR2 dysfunction and Ca2+ handling to be beneficial. Otherwise, simply enhancing adrenergic signaling without correcting RyR2 dysfunction (like β-AR agonists) may exacerbate RyR2 dysfunction and Ca2+ leak, increasing the arrhythmia risk and sudden cardiac death in HF. It should be noted that β-AR blocker treatment in HF is associated with normalization of RyR2 function and Ca2+ signaling (β-AR desensitization/down-regulation is one of the many characteristic abnormalities seen in HF that is conventionally considered to be detrimental in HF progression , 11, 12.ignaling , besidesThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.YZ contributed to conception of the manuscript. AM and KA wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Faecalibacterium) significantly decreases in faeces of AML patients, while gavage with butyrate or Faecalibacterium postpones murine AML progression. Furthermore, we find the intestinal barrier is damaged in mice with AML, which accelerates lipopolysaccharide (LPS) leakage into the blood. The increased LPS exacerbates leukaemia progression in vitro and in vivo. Butyrate can repair intestinal barrier damage and inhibit LPS absorption in AML mice. Collectively, we demonstrate that the gut microbiota promotes AML progression in a metabolite-dependent manner and that targeting the gut microbiota might provide a therapeutic option for AML.The gut microbiota has been linked to many cancers, yet its role in acute myeloid leukaemia (AML) progression remains unclear. Here, we show decreased diversity in the gut microbiota of AML patients or murine models. Gut microbiota dysbiosis induced by antibiotic treatment accelerates murine AML progression while faecal microbiota transplantation reverses this process. Butyrate produced by the gut microbiota  remains unclear. Here, the authors show disordered gut microbiota and reduced butyrate cause intestinal barrier damage in AML mice, with increased plasma LPS that accelerates AML progression. The long-term survival and prognosis of AML are closely related to many factors in AML progression. Thus, improved outcomes may depend on novel treatment strategies derived from a better understanding of the mechanism of AML progression.Acute myeloid leukaemia (AML) is a haematological malignancy characterised by excessive proliferation of immature myeloid cells. Although chemotherapy has been proven to be effective against this malignancy6. With further research, the relationship between gut microbiota and haematological diseases has gradually been reported. In a typical and stable environment, the gut microbiota regulates and sustains normal steady-state haematopoiesis7. Recently, it was reported that structural changes in the gut microbiota can occur in acute lymphoblastic leukaemia patients and that the microbiota diversity is significantly reduced8. Shifts in the intestinal microbiota following allogeneic haematopoietic cell transplantation were found to be associated with the development of gastrointestinal graft-versus-host disease (GVHD), suggesting that the intestinal microbiota may be an essential factor in GVHD9. However, the role played by the gut microbiota in AML progression has not been studied.The progression of AML is governed by many factors, including genetic and homoeostatic environmental changes. Accumulating evidence suggests that the human gut microbiota can affect the homoeostatic environment and is linked to the development of inflammation, autoimmune diseases, and even cancers10. In addition, butyrate, as an important SCFA13, has been reported as a chief energy source for intestinal epithelial cells, and administration of butyrate can effectively alleviate GVHD16. This finding suggests that the gut microbiota may play an important role in diseases through metabolic pathways, especially in GVHD. However, related research on SCFAs in AML is still lacking.Some microbial metabolites are exclusively derived from the gut microbiota and are not host derived, which mediates the biological effects of the gut microbiota. Microbial metabolites such as short-chain fatty acids (SCFAs) have been implicated in haematological diseases. Payen et al. revealed a dramatic decrease in the levels of the main SCFAs, acetate, propionate, and butyrate, in severe GVHD patients19. The intestinal barrier is the sole gate where entry of the gut microbiota or metabolites into the blood can be prevented. Its integrity and whether it can function normally are directly linked to fluctuations in the amounts of bacteria and their metabolites in the blood. Cui et al. found that the physical, chemical, immunological, and microbiological barriers in the intestinal tract constitute the complete intestinal barrier, which plays an important defensive role against the invasion of harmful substances from the intestines20. Another study confirmed that in haematological diseases, patients with GVHD showed significant incapacitation of gut barrier functionality compared with healthy volunteers21. The extent of integrity and permeability of the intestinal barrier often determine how the gut microbiota and their metabolites affect human systemic immunity22. The intestinal barrier tends to be disregarded in AML, and focusing on the intestinal barrier may provide novel insight for improving the therapeutic effect of AML.The existence of the gut microbiota, along with its nutritious SCFA metabolites, can facilitate the growth of intestinal epithelial cells and regulate their differentiation and repairIn this study, we found that the gut microbes of AML patients and mice were significantly disordered and that gut microbiota dysbiosis further aggravated the progression of AML. SCFA metabolome analyses showed that the concentration of butyrate was significantly decreased in the intestinal contents of AML patients, which resulted in reduced intestinal barrier function and increased absorption of LPS into the blood. Moreover, the increased LPS content promoted AML progression, while the addition of intestinal butyrate protected intestinal epithelial cells and decreased AML severity. Our study on the relationship between AML and intestinal barrier function has confirmed that intestinal health is also worthy of attention in the process of AML progression, which might be useful for the diagnosis and treatment of AML.P = 0.0275), which demonstrated a significantly lower diversity in the faeces of AML patients than in those of healthy controls , suggesting that the microbial composition in AML patients was significantly shifted regarding OTUs  was enriched, while Faecalibacterium (P = 0.0005214), Roseburia (P = 0.0002254), Subdoligranulum (P = 0.00212), and Bifidobacterium (P = 0.02883) were significantly depleted in AML patients   and Roseburia (LDA = 4.107) were lower in the faeces of AML patients compared to the control group   included in this study were obtained from 31 newly diagnosed (ND) and untreated AML patients and 30 healthy controls  Fig. . The resoup Fig. . To furt+ MLL-AF9 leukaemia cells in the BM, spleen, or peripheral blood was much higher in antibiotic-treated AML mice than in control PBS AML mice  sequencing of mouse stool. Our results showed that the gut microbiota diversity of AML mice decreased significantly with the progression of disease compared with that of healthy control mice Fig. . Consistice Fig. . Furtherice Fig. . Importa28] Fig. . Further28] Fig. . The dec28] Fig. . These dFaecalibacterium correlated with a favourable prognosis and might be considered a beneficial bacteria in AML patients. At the species level, the abundance of F. prausnitzii was TOP 1 in control, and significantly decreased in AML  experiment with faeces from AML patients supplemented with or without Faecalibacterium. Our results showed that spleen sizes and the leukaemia cell frequencies in the BM and spleen were significantly reduced in AML-FMT mice supplemented with Faecalibacterium compared with AML-FMT mice without Faecalibacterium. The leukaemia cell frequencies in peripheral blood also showed a decreasing trend but did not reach a significant difference ] ] Fig. . Similar8)] Fig. . Taken tP = 0.0196) and butyric acid (P = 0.0065) levels were significantly decreased in the stools of AML patients compared with those in the stools of healthy controls  was positively correlated with butyric acid  and negative ion mode (ES–), respectively. These peak features were clustered by orthogonal partial least squares discriminant analysis (OPLS-DA) and cross-validated scores plot, showing that the samples from the AML and control groups were clearly separated from each other Fig. . We furtols Fig. . Importacid Fig. . These d+ leukaemia cells in the BM, peripheral blood, or spleen of butyrate-treated AML mice was lower than that in the BM, peripheral blood, or spleen of the control AML mice ] ] Fig. . Moreove9)] Fig. , which sAfter butyrate is produced in the intestinal tract, it is mainly absorbed by intestinal epithelial cells as an energy source, which promotes intestinal barrier integrity. Therefore, we tested the intestinal barrier function in AML mice and experimentally verified its relationship with AML progression. Our results showed that the colon length of AML mice was significantly shorter than that of the control group  to detect the LPS concentration in the plasma of peripheral blood, and the results showed that the LPS concentration in the plasma was significantly higher in the AML group than in the control group in humans Fig. . Moreove+ leukaemia cells in the peripheral blood, spleen, and BM were higher in LPS-treated than in control AML mice ] ] Fig. , which s25. It has been reported that an imbalance in gut microbial diversity can accelerate the development of ovarian cancer26, while a diversified gut microbiota is beneficial for the prognosis and chemotherapy sensitivity of colon cancer27. As the most important haematological tumour, increasing attention has been given to the relationship between leukaemia and gut microbiota. Although some progress was made recently regarding the gut microbiota in several leukaemia types29, it remains unclear whether gut microbes are mechanistically involved in AML progression. In this study, we found that AML could induce intestinal barrier damage, which is associated with altered gut microbiota and SCFAs in patients. The injured intestinal barrier and increased permeability in AML further promote the release of LPS into the blood circulation, which accelerates AML cell proliferation and disease progression. Our results might provide an insight into developing new targets for AML treatment.The gut microbiota and its metabolism have been reported to be associated with many cancersFaecalibacterium decreased significantly in treatment-naive AML patients. Faecalibacterium is the best-characterised microbiota constituent that is involved in the immune microenvironment31. According to reports, the role of Faecalibacterium in tumours is gradually becoming appreciated. Faecalibacterium also suppresses the proliferation and promotes the apoptosis of breast cancer cells, although these effects disappear after adding recombinant human IL-632. However, in our study, after administration of Faecalibacterium, AML mice showed no significant difference in survival or leukaemia infiltration, which indicated that the antitumour effect of Faecalibacterium was limited. Therefore, we further performed FMT in AML mice and found that AML-FMT aggravated the progression of AML compared with that in Con-FMT mice. A recent study by Yu et al.33 reported that the bacterial diversity was increased and no depletion of Faecalibacterium was found in AML, which is different from our results. These differences may be due to the regional and gender diversities which have been reported by other studies37.We found significant alterations in gut microbiota diversity in AML, and this change was related to the risk stratification of the disease. This finding indicated that while AML caused an imbalance in the gut microbiota, the disordered microbiota also affected the progression of the disease. To better serve clinical treatment, it is particularly important to study the mechanism of the flora in the progression of AML. Blocking the positive feedback between AML and bacterial dysbiosis mechanism will help to solve the unexplained infection in patients with AML and improve the clinical treatment effect. Regarding the species of bacteria, the abundance of 39, and it is well known that butyrate is a common metabolite of Faecalibacterium and Roseburia40. In our experiment, AML patients had a significantly lower faecal butyric acid content than healthy people, but no significant difference was found in the peripheral blood and BM. Consistent with our results, other reports have shown that butyric acid is mainly metabolised in the intestinal tract, and the change in intestinal concentration cannot cause corresponding changes in peripheral blood41. Therefore, the effect of intestinal butyric acid on AML did not occur via direct contact to kill leukaemia cells in the blood or BM. Conversely, butyric acid, as the main energy source of intestinal epithelial cells, plays an important role in the regulation and function of the intestinal barrier44, and its deficiency could lead to disruption of the gut barrier and cause multiple diseases46. Zheng et al. showed that butyrate has received particular attention for its beneficial effects on intestinal homoeostasis and energy metabolism. With anti-inflammatory properties, butyrate enhances intestinal barrier functions and mucosal immunity47. Butyrate plays key roles not only in the local intestinal barrier but also at the systemic level. At both sites, the mode of action is through modulating signalling pathways involving nuclear NF-kB and inhibition of histone deacetylase48. Although it has been reported that chemotherapy drugs can affect intestinal barrier function in AML patients49 and blast crisis chronic myeloid leukaemia murine model has intestinal barrier function alteration50, its role and mechanism in AML progression have not been reported. Our results showed that the intestinal barrier function of AML mice was decreased and that the expression of TJPs (claudin-1 and ZO-1) in intestinal epithelial cells was decreased and that of claudin-2 was increased in AML mice. Furthermore, administration of butyrate to AML mice effectively reversed intestinal barrier damage. Here, our research confirmed the role of butyrate in protecting the intestinal barrier in the progression of AML. This finding suggests that supplementation with butyrate may be of great significance in AML treatment or post-chemotherapy care. Interestingly, in addition to our research, G protein-coupled receptors for butyrate and their role in modulating the immune system have been reported, which is worthy of further study.A large number of reports have shown that most SCFAs in the human body, including butyrate, are derived from the metabolism of intestinal bacteria51. The intestinal barrier is the main way through which LPS can enter blood circulation. In this study, we found that intestinal barrier damage in primary AML patients caused more LPS to enter the blood and higher LPS concentrations. In vitro experiments showed that LPS increased leukaemia cell proliferation, and in vivo studies revealed that LPS exacerbated the leukaemia burden and shortened OS in AML mice. We also clarified that its leukaemia-promoting effects might function through decreased BAX and cleaved caspase-3 and upregulated bcl-2 levels. These results suggest that gut metabolites are another independent contributing source of elevated plasma LPS levels and that the absence of butyrate and elevated LPS play important roles in AML progression. Though our results demonstrated that LPS promoted AML progression in the experiments of in vitro cell culture and murine model, whether the small increase in LPS concentration plays a vital effect in AML patients is unknown. In addition, leukaemia patients need to take ABX to prevent infection, especially during chemotherapy. During this period, whether the use of antibiotics will cause the disorder of intestinal flora and the content of LPS in the serum of AML patients, and then have adverse effects on the disease, is an interesting question worthy of our further research.As the main component of intestinal bacteria, LPS affects the immune microenvironment in the human bodyImportantly, while our findings relate to treatment-naive AML patients, they cannot yet be generalised as an approach for patients with relapsed and refractory disease. Future studies are needed to elucidate how the gut microbiota and other SCFAs affect recurrent or refractory leukaemia. In addition, our research is currently limited to horizontal clinical studies, and more clinical samples are needed for longitudinal clinical studies. Finally, our study can only prove that gut microbiota dysbiosis and GI damage can promote the progression of existing AML, but there is no evidence to prove whether GI damage can cause the occurrence of AML. Therefore, we intend to gradually address these problems and provide further insight into the relationship between AML and the gut microbiota.In conclusion, our results suggest that AML caused compromised intestine integrity, disordered bacteria diversity and the change of bacterial-derived factors. Decreased barrier function, loss of butyrate and higher blood LPS are cancer-promoting factors in AML and butyrate regulates the concentration of LPS in peripheral blood by affecting the intestinal barrier Fig. . The regn = 31) and a healthy control (Con) group (n = 30). Their characteristics are summarised in Supplementary Table n = 5 per group). Among the 61 individuals, all were chosen for stool 16S rRNA sequencing. Our study was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University (KYLL-2018-137) and informed consent was obtained from all participants . Our study did not involve any interventional clinical trial.Sixty-one adults were enrolled from 2018 to 2020. Stool, peripheral blood and BM samples included in this study were obtained from 31 ND AML patients and 30 healthy controls. These specimens were collected at Qilu Hospital of Shandong University. The exclusion criteria were as follows: age <15 and >70 years, diarrhoea, receiving antibiotics or hormone therapy within the last 10 weeks, and blood pressure anomalies. The patients were divided into an AML patient group . Adriamycin (ADR), daunorubicin (DNR) and cytosine arabinoside (Ara-C) were dissolved in normal saline and diluted in RPMI 1640 medium immediately before use. Primary antibodies against cleaved caspase-3 (#9661), claudin-2 (#48120) and Bcl-2 (#3498) used for western blot were obtained from Cell Signaling Technology , and antibodies against ZO-1 (ab221547), occludin (ab167161), claudin-1 (ab180158), BAX (ab199677), GAPDH (ab8245), β-tubulin (ab6046) and β-actin (ab227387) were purchased from Abcam . Anti-Ki67 antibody  used for immunohistochemistry. ZO-1 , claudin-1  and claudin-2  used for immunofluorescence. Secondary antibodies were obtained from ZSGB-BIO(China). All reagents were dissolved and preserved following the manual’s instructions.MLL-AF9, a murine AML cell line labelled with a GFP reporter, was obtained from the spleen of the successfully established MLL-AF9 murine AML model. Primary leukaemia cells were cultured in IMDM culture medium  supplemented with 10% foetal bovine serum  and 1% penicillin/streptomycin . MLL-AF9 cells were cultured in DMEM containing 10% FBS, cytokines  and 1% penicillin/streptomycin. C1498 cells were obtained from ATCC (Cat. TIB-49) and cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin. Cells were incubated at 37 °C in a humidified atmosphere with 5% COFaecalibacterium  was grown at 37 °C in LYHBHI medium (brain–heart infusion medium supplemented with 0.5% yeast extract (Difco) and 5 mg/L) supplemented with cellobiose , maltose , and cysteine  in an anaerobic chamber.+ cells from the BM of WT mice transfected with MSCV-MLL-AF9-IRES-GFP (107 cells/host), together with BM mononuclear cells from C57BL/6J mice (107 cells/host), were transplanted into lethally irradiated C57BL/6J recipients. On day 30 after transplantation, AML mice were verified by detecting the GFP frequency in peripheral blood from the lateral tail vein with a FACS Aria II sorter (BD Biosciences). Then, we collected spleen cells from successfully established MLL-AF9 AML mice and froze them as murine MLL-AF9 AML cells at −80 °C. For subsequent murine experiments, we injected MLL-AF9 AML cells into WT mice through the tail vein without irradiation, which was used as a murine AML model. The MLL-AF9 murine AML model is commonly applied in many studies as it relates to human AML53. AML1-ETO murine model was established in the same way as the MLL-AF9 murine model and AML1-ETO cells were kindly provided by Dr Jianxiang Wang . All mice were 6–8 weeks old and were maintained in an SPF environment. Animal protocols were approved by the Animal Ethics Committee of Qilu Hospital, Shandong University.Specific pathogen-free (SPF) mice on a C57BL/6J background were purchased from the Laboratory Animal Center of Shandong University. Female mice (6–8 weeks) were maintained on a 12-h dark/light cycle at ambient temperature (72 ± 2 F) with controlled humidity (~45%). A retrovirus vector containing the intracellular domain of MLL-AF9 (MSCV-MLL-AF9-IRES-GFP) was kindly provided by Dr Hui Cheng . To establish a murine AML model, C-kitFresh faeces from patients or healthy controls were weighed and diluted in normal saline to achieve a final concentration of 150 mg/mL. The patients were selected based on their adequate faeces, balanced sex and risk stratification. Then, the faecal suspension was filtered with a 40-μm filter sieve to remove large particles, the filtrate was collected, glycerol was added to a concentration of 20%, and the samples were stored at −80 °C. The faecal hydration liquid was thawed for oral gavage when FMT was performed.For FMT, mice were randomly grouped according to the experimental requirements. First, an ABX was administered by oral gavage. Briefly, the antibiotic was used as follows: on days −21 to −7 before MLL-AF9 cell injection, mice received a daily gavage of metronidazole (100 mg/kg), while the ABX (ampicillin (1 g/L), vancomycin (0.5 g/L) and neomycin (1 g/L)) was added to the drinking water. Then, the frozen faecal hydration liquid was thawed and mixed for the same group of AML patients or controls. FMT was carried out via oral gavage in a final volume of 250 µL. In order to keep the time consistent, we performed faecal bacteria transplantation at 8 am every day. FMT was performed daily from 14 to 35 days after the start of the antibiotic regimen. We also collected stool and faecal hydration liquid on day 0 and day 7 after transplantation for 16S rRNA analysis to prove the success of transplantation. See the Supplementary materials for detailed patient information  or carrier (sterile PBS) through a 20G-1.5-inch flexible intragastric gavage needle  every day. Continue until the mouse is sacrificed. LPS was injected into mice at a concentration of 1 μg/mL in 200 μL via the tail vein together with MLL-AF9 cells. Next, it will be injected every 3 days during the experiment. Mice were given 1.9% DSS  in autoclaved drinking water (w/v) for 3 days, followed by other days of DSS free drinking water.MLL-AF9 cells were transfected with a luciferase (MCS-Luciferase-IRES-Puror) retrovirus vector. Firefly and Renilla luciferase activities were measured consecutively 24 h following transfection using a Dual-Luciferase Reporter Assay  according to the manufacturer’s instructions. Moreover, to monitor tumour progression and metastasis in vivo, bioluminescent imaging was performed with an IVIS Lumina Series III  on days 10 and 14 after injection and was used to confirm the dynamic process of tumour cell load in vivo.After different treatments, the cells were incubated with 10 μL of CCK-8  for 3 h. The absorbance was measured at 450 nm. Each sample was measured in triplicate. Details of the gating strategy are described in Supplementary Fig. Apoptosis was assessed with an Annexin V/propidium iodide (PI) apoptosis detection kit  according to the manufacturer’s protocol. Cells were harvested after different treatments and washed twice with PBS. Then, the cells were resuspended in 400 μL of binding buffer and stained with 5 μL of Annexin V for 15 min and 10 μL of PI for another 5 min in the dark at 4 °C. The percentages of apoptotic cells were analysed immediately using a Galios flow cytometer .Blood plasma samples were collected from EDTA-stabilised peripheral blood of 20 ND AML patients and 18 controls, and supernatants of mouse peripheral blood were collected and stored at −80 °C for determination of the LPS concentration. Human and mouse LPS ELISA kits were purchased from R&D Systems . ELISAs were performed in accordance with the manufacturer’s instructions.Cells were collected, washed twice with PBS, and then lysed with RIPA buffer  containing protease inhibitor compound  on ice. A bicinchoninic acid protein assay kit  was applied to measure protein concentrations. Protein extracts (30 μg) were loaded into 10% SDS-PAGE gels and then electrotransferred onto nitrocellulose membranes . After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated overnight with specific primary antibodies at 4 °C followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were detected by a FluorChem E Chemiluminescent imaging system  after washing. Uncropped and unprocessed scans of western blots are available in the For SCFA determination in faeces or blood plasma, we first screened out significantly different microbial metabolites by LC-MS. Briefly, for untargeted metabolomics of polar metabolites, extracts were analysed using a quadrupole time-of-flight mass spectrometer (Sciex TripleTOF 6600) coupled to hydrophilic interaction chromatography via electrospray ionisation at Shanghai Applied Protein Technology Co., Ltd. LC separation was performed on an ACQUIY UPLC BEH Amide column  using a gradient of solvent A (25 mM ammonium acetate and 25 mM ammonium hydroxide in water) and solvent B (acetonitrile). The gradient was 85% solvent B for 1 min and was linearly reduced to 65% in 11 min, reduced to 40% in 0.1 min and maintained for 4 min, and then increased to 85% in 0.1 min, with a 5 min re-equilibration period. The flow rate was 0.4 mL/min, the column temperature was 25 °C, the autosampler temperature was 5 °C, and the injection volume was 2 µL. The mass spectrometer was operated in both negative ion and positive ionisation modes. The ESI source conditions were set as follows: ion source gas 1 (Gas1) as 60, ion source gas 2 (Gas2) as 60, curtain gas (CUR) as 30, source temperature of 600 °C, and ion spray voltage floating of ±5500 V.For targeted SCFA metabolism, we quantitatively determined the content of SCFAs by gas chromatography-mass spectrometry GC-MS. Samples were thawed on ice, and 100-µL aliquots were added to a 2-mL glass centrifuge tube and mixed with 50 μL of water with 15% phosphoric acid and 150 µL of 5 µg/mL 4-methyl valeric acid. The suspensions were homogenised with a vortex for approximately 1 min and centrifuged for 10 min at 12,000× g. Then, 1 μL of supernatant was collected for GC-MS analysis using an Agilent Model 7890A/5975C GC-MS system. To quantify SCFAs, a calibration curve for the concentration ranges from 0.1 to 100 µg/mL was constructed. The internal standard was used to correct for injection variability between samples and minor changes in the instrument response.The samples were separated with an Agilent HP-INNOWAX capillary GC column (30 m × 0.25 mm ID × 0.25 µm). The initial temperature was 90 °C, which was increased to 120 °C at 10 °C/min and then to 150 °C at 5 °C/min and finally to 250 °C at 25 °C/min, where it remained for 2 min. The carrier gas was helium (1.0 mL/min). The temperatures of the injection port and transmission line were 250 and 230 °C, respectively. An electron bombardment ionisation source was used, selected ion monitoring scanning mode was applied, and the electron energy was 70 eV .® soil DNA Kit  according to the manufacturer’s protocols. The final DNA concentration and purification were determined using a NanoDrop 2000 UV-vis spectrophotometer , and the DNA quality was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with primers 338 F (5’-ACT CCT ACG GGA GGC AGC AG-3’) and 806 R (5’-GGA CTA CHV GGG TWT CTA AT-3’) using a thermocycler PCR system . PCR was conducted using the following programme: 3 min of denaturation at 95 °C; 27 cycles of 30 s at 95 °C, 30 s of annealing at 55 °C, and 45 s of elongation at 72 °C; and a final extension at 72 °C for 10 min. PCR was performed in triplicate in a 20-μL mixture containing 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel and further purified using an AxyPrep DNA Gel Extraction Kit  and quantified using QuantiFluor™-ST  according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 300) on an Illumina MiSeq platform  according to standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. . OTUs were clustered with a 97% similarity cut-off using UPARSE (version 7.1 http://drive5.com/uparse/), and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analysed by the RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using a confidence threshold of 70%.Microbial DNA was extracted from faecal samples using an E.Z.N.A.Raw fastq files were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH with the following criteria: (i) reads were truncated at any site receiving an average quality score <20 over a 50-bp sliding window; (ii) primers were exactly matched, allowing two nucleotide mismatches, and reads containing ambiguous bases were removed; (iii) sequences overlapping more than 10 bp were merged according to their overlap sequence. Alpha diversity was measured with Shannon metrics, and beta diversity was calculated using UniFrac.2O, 5 μL of 2×SYBR Green Real-time PCR Master Mix, 0.4 μL of forward and reverse primers, and 1 μL of cDNA in a final volume of 10 μL. PCR conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min. The primer sequences for the genes are shown in Supplementary Table –ΔCT method.Total RNA was extracted from cells using TRIzol . The total RNA concentration and purity were quantified using a spectrophotometer . Reverse transcription was performed at 37 °C for 15 min followed by 85 °C for 10 s using a Prime Script RT reagent Perfect Real Time Kit . Quantitative PCR was performed in duplicate on a Light Cycler 480II real-time PCR system  with a SYBR Green Real-time PCR Master Mix kit . The PCR contained 3.2 μL of ddHFood and water were withheld from all mice for 4 h. FITC-dextran was administered by a 20G-1.5-inch flexible intragastric gavage needle at a concentration of 50 mg/mL in PBS. Mice received FITC-dextran at 800 mg/kg (~16 mg/mouse). Four hours later, serum was collected from peripheral blood, diluted 1:1 with PBS, and analysed on a plate reader at excitation/emission wavelengths of 485 nm/535 nm. Concentrations of FITC-dextran in experimental samples were determined based on a standard curve.Briefly, the intestines from control mice or AML mice treated with butyrate or vehicle were harvested 14 days after injection of MLL-AF9. Cross-sections were immediately sliced (2 mm wide) from the colon. The tissue was then rinsed three times with Sorensen’s buffer containing 0.1% ruthenium red (RR) and postfixed for 1 h in 1% osmium tetroxide in the same buffer containing RR. The samples were again rinsed with Sorensen’s buffer containing RR. Next, the tissue was dehydrated in ascending concentrations of ethanol, treated with propylene oxide, and embedded in Epon epoxy resin. Semithin sections were stained with toluidine blue for tissue identification. Selected regions of interest were ultrathin sectioned (70 nm thick), mounted on copper grids, and poststained with uranyl acetate and lead citrate. The samples were examined using a Philips CM100 electron microscope at 60 kV. Images were recorded digitally using a Hamamatsu ORCA-HR digital camera system operated using AMT software .Faecalibacterium and WBC. The correlation between Faecalibacterium and WBC was performed by multiple linear regression. The Shapiro–Wilk test was used for normality tests. Normally distributed data were analysed by Student’s t-test or paired t-tests. Otherwise, comparisons between groups were performed using the Mann–Whitney U test for nonpaired data and the Wilcoxon signed-rank test for paired data. Results are expressed as the mean ± SEM, except where stated otherwise. *P < 0.05, **P < 0.01 and ***P < 0.0001 were considered statistically significant. All figures’ details of the statistical analysis and the exact P value are in the Supplementary Data.GraphPad Prism 8.0 was applied for graph development. ImageJ V1.50 was applied for western blot image processing and Kaluza 2.1.1 was applied for flow cytometry analysis. Statistical analysis was conducted with raw data using SPSS 20.0 software. Logistic regression was used to analyse the causal relationship between Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


A fundamental task faced by the auditory system is the detection of events that are signaled by fluctuations in sound. Spiking in auditory cortical neurons is critical for sound detection, but the causal roles of specific cell types and circuits are still mostly unknown. Here we tested the role of a genetically identified population of layer 4 auditory cortical neurons in sound detection. We measured sound detection using a common variant of pre-pulse inhibition of the acoustic startle response, in which a silent gap in background noise acts as a cue that attenuates startle. We used a Gpr26-Cre driver line, which we found expressed predominantly in layer 4 of auditory cortex. Photostimulation of these cells, which were responsive to gaps in noise, was sufficient to attenuate the startle reflex. Photosuppression of these cells reduced neural responses to gaps throughout cortex, and impaired behavioral gap detection. These data demonstrate that cortical Gpr26 neurons are both necessary and sufficient for top–down modulation of the acoustic startle reflex, and are thus likely to be involved in sound detection. Understanding how cortical circuitry contributes to sensory perception and behavioral output is one of the central goals of systems neuroscience. Pursuit of this goal has benefited tremendously from advances in the ability to manipulate and record from identified cell types in behaving animals , Archaerhodopsin , or tdTomato (n = 6) was expressed in Cre-expressing, Gpr26 pyramidal neurons. The Gpr26 gene encodes G protein-coupled receptor 26 . We used Gpr26-tdTomato (n = 3) and Gpr26-Cre-negative (n = 8) littermates for fluorescent retrobead injections.All procedures were performed in accordance with National Institutes of Health guidelines, as approved by the University of Oregon Institutional Animal Care and Use Committee. We used + / + offspring (8–12 weeks of age) of crosses between hemizygous Tg(Gpr26-Cre)KO250Gsat  and homozygous CAG-ChR2-eYFP , or CAG-Arch-eGFP , or Rosa-CAG-LSL-tdTomato-WPRE  lines. In these offspring, Cre-dependent Channelrhodopsin-2 . We used a digoxygenin (DIG)-labeled riboprobe (1:500), visualized by Anti Fluor-POD  and Fluorescein , as described previously  and the medial geniculate body of the thalamus (MGB). To do this, we injected retrobeads into dIC or MGB as described below (see section “Retrobead injection”), waited 2 weeks, and then quantified retrobead signal in auditory cortex by sublamina as described above. We performed retrobead injections in 3 Gpr26-tdTomato mice (green retrobeads only) and 8 Gpr26-Cre-negative mice (red and green retrobeads). In Gpr26-tdTomato mice, we first identified labeled cells separately in the green (retrobead) and red (tdTomato) channels, and then merged the images to assess co-localization.Mice were perfused transcardially with 0.1 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS . Brains were removed and post-fixed for an additional 24 h in 4% paraformaldehyde, then sectioned at 50 μm in the coronal plane. We used six Gpr26-tdTomato mice (8–12 weeks of age), and took photomicrographs of alternating sections on a Zeiss microscope using Zen software (Carl Zeiss Microscopy GmbH 2011). Seven of these photomicrographed sections from each mouse were matched to the closest representative atlas section . We seleeviously . We wereWe administered atropine (0.03 mg/kg) pre-surgically to reduce respiratory irregularities. Mice were anesthetized with isoflurane (1.25–2.0%). We used fluorescent retrobeads (Lumafluor) and performed injections  through craniotomies in the skull into either the dIC  or the MGB . The injection needle (Hamilton Microliter #7000.5) was maintained at depth for 5 min following each injection, and then slowly withdrawn. The craniotomy was filled with anti-bacterial ointment and the skull covered with dental cement. We administered ketoprofen (4.0 mg/kg) post-operatively. Mice were housed communally following surgery.We administered atropine (0.03 mg/kg) pre-surgically to reduce respiratory irregularities. Mice were anesthetized with isoflurane (1.25–2.0%). One craniotomy was drilled in each hemisphere dorsal to auditory cortex  for the placement of 200 μm-diameter optic fibers . We used cyanoacrylate and dental cement to secure the fibers to the skull. We administered ketoprofen (4.0 mg/kg) post-operatively. Mice were housed communally following surgery and were given 7 days to recover.Methods are as described previously ,2020b. AWe inserted silent gaps into continuous 80 dB SPL background white noise, and measured how these gaps attenuated startle responses elicited by a 100 dB SPL, 25 ms white noise burst. Gaps were 2, 4, 8, 16, or 32 ms in duration, and were separated from the startle stimulus by a 50 ms post-gap interval. We also presented startle stimuli in isolation, not preceded by a gap  to provide a baseline startle response. Each combination of gap duration and light condition (see below) was presented 20 times per session, randomly interleaved and separated by a randomized inter-trial interval of 15 ± 5 s.2. For photosuppression we used mice expressing Archaerhodopsin (Gpr26-Arch) and 520 nm wavelength laser diodes with an output power of 300 mW/mm2. We chose these intensities based on previous characterization of their spatial spread in auditory cortex (We separately examined how photostimulation and photosuppression of Gpr26 cells affected behavior. For photostimulation we used mice expressing Channelrhodopsin-2 (Gpr26-ChR2) and 445 nm wavelength laser diodes set to an output power of 50, 100, 200, or 300 mW/mmy cortex ,b. Lighty cortex . For phoy cortex . To visut-test, p < 0.05). Behavioral curves were based on the median startle attenuation at each gap duration. We used non-parametric tests for group analyses because data for some gap durations were not normally distributed (Lilliefors test), and because statistical power is comparable even when the underlying assumptions for the corresponding parametric analysis is met  were included for analysis, based on a significance test comparing startle amplitudes associated with each gap duration to startle amplitudes on gap-free trials (paired s is met . We useds is met .Mice were anesthetized with isoflurane (1.25–2.0%). A headpost was secured to the skull and a mark was made on the skull over auditory cortex for a future craniotomy . Mice were housed individually following surgery and were allowed at least 5 days of post-operative recovery. On the day of recording, mice were anesthetized with isoflurane (1.25–2.0%), the head was fixed in position using the headpost, and a small craniotomy was made over auditory cortex (1 × 1 mm). Craniotomies were covered with a thin layer of agar and mice were allowed to recover for at least 1 h before recording.All electrophysiological recordings were performed while the animal was awake and head-fixed on a rotating, spindle-mounted styrofoam ball inside a double-walled acoustic isolation booth. We recorded neurons in auditory cortex with a 32-channel silicon probe , using an Intan RHD2132 headstage and RHD2000 acquisition board, and Open Ephys software . The silt-test) in spiking activity during the 50 ms post-gap interval  for at least two consecutive gap durations relative to the same interval during gap-free trials . We used the Kruskal–Wallis (K–W) test to assess within-group effects of illumination  across depths. We used the Wilcoxon rank-sum test for post hoc comparisons to the K–W test, as well as for group comparisons (transgenic versus control). We used a Bonferroni correction for multiple comparisons where appropriate. We report effect sizes as eta-squared (η2) , as well as to 445 nm light pulses for photoidentification of Gpr26 neurons (Gpr26-ChR2 only). The presentations of gap-in-noise stimuli differed from the behavioral protocols in two respects. First, no startle stimuli were presented. Second, a shorter inter-trial interval was used . Recording sessions included 20 presentations each of gaps 1, 2, 4, 8, 16, and 32 ms in duration, as well as 20 gap-free trials. Gap termination responses (GTRs) were defined as a significant increase  . η2 varip-value of a paired t-test comparing spiking activity during the 25 ms following onset of each light pulse to an equivalent light-free baseline interval. We measured response signal-to-noise as the z-value generated from a rank-sum test of the same data. We measured response latency as the time-to-peak of the Gaussian-smoothed (5 ms S.D.) trial-averaged firing rate following the onset of each light pulse. We measured response reliability as the proportion of trials on which light evoked 1 or more spikes in a 50 ms window following the onset of each light pulse. We classified cells as “Photoresponsive” if they met either of two sets of criteria: (1) significance p < 0.0001, peak latency < 20 ms, and reliability > 0.5, or (2) significance p < 0.0001 and peak latency < 15 ms.We used light pulses to attempt to identify putative Gpr26 neurons in Gpr26-ChR2 mice, as described previously . A trainFollowing behavioral and electrophysiological experiments, we coronally sectioned (50 μm) all brains and confirmed the presence or absence of transgene expression (based on eGFP or eYFP fluorescence) in auditory cortex.The detection of brief gaps in noise involves auditory cortex, and specifically relies on the GTR  ,2020b, bn = 6 mice), and then counted CaMKII-positive (excitatory) cells in the same sections labeled by in situ hybridization. We did not measure whether individual cells showed co-localization of tdTomato and CaMKII , and instead quantified penetrance as the percentage of labeled Gpr26 cells relative to the total number of excitatory neurons in each layer. In contrast to the original report, we found that the density of Gpr26 cells in A1 was highest in layer 4, and dropped off gradually across layers 3 and 5  to compare the laminar distribution of corticofugal cells with tdTomato-labeled Gpr26 cells. Retrobeads injected into the dIC of eight mice (3 Gpr26-Ai14 and 5 Gpr26-Cre-negative) labeled two bands of cells, one predominantly in layer 5a and thus partially overlapping with the population of Gpr26 cells, and the other in layer 6b . InjectiBecause the more superficial band of dIC-projecting neurons overlapped spatially with tdTomato-labeled Gpr26 cells, we looked for co-localization in three Gpr26-tdTomato mice with dIC green retrobead injections. As indicated in Because Gpr26 cells were primarily found in layer 4, the main input layer to auditory cortex, we hypothesized that they might contribute to gap responses throughout the cortical column, as well as to behavioral gap detection. To test this with photosuppression, we generated a Gpr26-Arch cross that expressed Archaerhodopsin in Gpr26 neurons, and illuminated auditory cortex during the post-gap interval, when GTRs of A1 neurons occur. First, we validated our photosuppression method by recording from 336 A1 neurons in 5 Gpr26-Arch mice using silicon probes. To accurately determine the depth of recorded neurons, we used current-source density analysis to identify a sound-evoked robust short-latency sink at the L3/L4 border . Our anaTo validate the effectiveness of our photosuppression method, we first tested whether it reduced baseline firing rates of cells at the expected cortical depth . To see how suppression was related to depth, we sorted cells into middle layers 3b-5a (the sublayers of greatest penetrance), superficial layers 1a-3a, and deep layers 5b-6b . Figure In Gpr26-mice, illumination during the post-gap interval suppressed GTRs in some cells, but not others, across superficial, middle, and deep layers . No effect of illumination was observed on behavior in Gpr26-negative littermate control mice . We tested these predictions in Gpr26-ChR2 mice that expressed Channelrhodopsin-2 in Gpr26-Cre + neurons.z-values) was greatest in the middle layers, followed by the superficial layers. Multi-unit activity recorded in control mice did not show light-evoked activity (data not shown). These results are consistent with the anatomical penetrance of the mouse line, indicating that ChR2 is indeed expressed by Gpr26 neurons in the expected depth range, and that light robustly photoactivates these neurons. These results are also consistent with the interpretation that spikes from a considerable fraction of Gpr26 single neurons were missed by spike-sorting during photoactivation.To attempt to photoidentify Gpr26 neurons, we recorded from 277 cells from 4 Gpr26-ChR2 mice. Our analyses focused on the 241 cells for which we could unambiguously determine laminar depth based on current source density analysis. For comparison, we analyzed activity from 156 cells with verified depths recorded from 4 Gpr26-Cre-negative littermates that did not express ChR2. We observed fewer photoresponsive cells than expected, with a markedly different laminar distribution than that expected from the anatomical expression pattern . We suspAlthough we were only able to photoidentify a subset of Gpr26 cells, we still wondered whether any of those cells responded to gaps. Of the 36 putative Gpr26 neurons described above, more than half (21/36 or 58%) responded to gaps with a GTR. The response of one such cell to both photostimulation and gaps in noise is illustrated in 2: p < 0.0001, η2 = 0.72; 100 mW/mm2: p = 0.002, η2 = 0.32; 200 mW/mm2: p < 0.0001, η2 = 0.54; 300 mW/mm2: p < 0.0001, η2 = 0.59, rank-sum). Startle attenuation at the lowest light intensity was stronger than that produced by the longest (most effective) gap duration tested. Illumination had no effect in control mice not expressing ChR2, ruling out artifactual attenuation due to visual or intracranial detection of the light pulses. Thus, photoactivation of Gpr26 cells alone was sufficient to modulate startle responses.To test whether photoactivation could influence the PPI pathway, we delivered 25 ms light pulses beginning 50 ms before the startle stimulus on gap-free trials see , inset. Here we examined the involvement of a layer-specific, genetically identified class of pyramidal neurons in auditory cortex in PPI of the acoustic startle response. We found that Gpr26 neurons were predominantly located in layer 4, and are likely recipients of thalamic input. Photosuppression of Gpr26 cells suppressed spiking activity across layers in A1 and impaired behavioral gap detection. Gpr26 cells were responsive to gap-in-noise stimuli, and photoactivation of these cells was sufficient to attenuate the startle reflex. This startle attenuation was robust even at the lowest light intensity tested, and closely resembled that evoked by acoustic gaps in noise. To our knowledge, this is the first characterization of this Gpr26 line in auditory cortex, and provides insights into the role of these layer 4 cells in sound processing.In rodents, Gpr26 mRNA expression has been detected in numerous structures throughout the brain . SeveralOur analyses of neuronal activity, performed using silicon probes that enabled verification of recording depth, were consistent with the anatomical penetrance we observed in Gpr26-tdTomato mice. Illumination in the Gpr26-Arch cross significantly suppressed spiking activity in the middle layers, corresponding to the depth of greatest penetrance, and superficial layers 1a-3a, immediately downstream in the canonical circuit. In the Gpr26-ChR2 cross, illumination elicited significant increases in more multi-unit activity sites in middle layers compared with those in superficial or deep layers 5b-6b. The Gpr26-ChR2 recordings also revealed higher reliability and signal-to-noise in middle layers than at adjacent depths, and both middle layers and superficial layers exhibited briefer peak response latencies versus those observed further downstream in the canonical circuit in deep layers.We found that photoidentification of individual Gpr26 neurons was only partially successful, providing an important cautionary note about this method. We expected, based on penetrance of the Gpr26 line  as well Our results are somewhat at odds with another recent study utilizing optogenetics to examine interlaminar connectivity. In that study , optogenper se. Nevertheless, behavior may be driven by electrical or optogenetic stimulation of remarkably small populations of sensory cortical neurons  . We rece neurons , and int neurons . It ther neurons .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the University of Oregon Institutional Animal Care and Use Committee.AW designed and performed the experiments, analyzed the data, and wrote the manuscript. IY performed the electrophysiology experiments and analyzed the data. AN performed the behavioral experiments. MW conceived, designed, and directed research, and helped write the manuscript. All authors contributed to the article and approved the submitted version.


FLT3 mutation in the past decade. The clustering of keywords suggested interest in cell-targeted therapy , signaling pathways (mTORC1), epigenetic therapies, and leukemic stem cells (LSCs). Drug resistance causing failure and relapse of treatment has raised increasing concern, and LSCs play a key role in relapsed AML, which represent an important direction for identifying targets for AML treatment in the future.Acute myeloid leukemia (AML) is a malignant proliferative clonal disease, characterized by a wide spectrum of molecular alterations. Although targeted therapy for AML has resulted in pronounced achievements in the past decade, clinical resistance caused by mutations in targeted oncogenes has been observed. A subset of patients with AML still requires the treatment of refractory and relapsed diseases. The field of targeted therapy in AML still presents many challenges. More studies are needed to overcome those obstacles. Through the CiteSpace tool, we can gain a systematic understanding of the current achievements in targeted therapy in AML, and we can predict hot research topics. Relevant publications from the Web of Science Core Collection were retrieved, and the acquired data were analyzed by CiteSpace to identify and predict trends and research hotspots in this field. We found that research on AML had focused on venetoclax resistance, novel therapies (such as targeting epigenetic modification), and Acute myeloid leukemia (AML) is a heterogeneous disease caused by various gene mutations and cytogenetic abnormalities that affect the differentiation and proliferation of myeloid lineage cells . AdvanceFLT3-ITD), and mutations in isocitrate dehydrogenase (IDH1/2), nucleophosmin (NPM1), and/or CCAAT/enhancer binding protein alpha (CEBPA). As predictive biomarkers, these are indicators of prognosis and, in turn, can guide targeted therapeutic strategies, because patients harboring these specific mutations do not benefit from traditional chemotherapy  midostaurin with induction therapy for newly diagnosed FLT3mut AML, and a more specific and potent FLT3i gilteritinib as monotherapy for relapsed/refractory (R/R) FLT3mut AML, has improved outcomes in patients with FLT3-mutated AML  of FMS-like tyrosine kinase 3 . The strategy used during the search was Topic search #1 = (“acute myeloid leukemia” OR “AML”) AND Topic search #2 = (“targeted therapy*”), and then, the results were refined by [Document Types = (Articles or Review) Timespan: 2012-01-01 to 2021-12-31]. A total of 4,205 results were found. After data cleaning, duplicate publications were removed by CiteSpace v.5.8.R3; and ultimately, 4,150 total unique records were used in the final analysis. All records were then imported into CiteSpace v.5.8.R3.The time slices for analysis were set at 1 year, and the sources selected included all publication details—title, abstract, supplementary keywords and author keywords, selected keywords, author, institution, country for the node, selected g-index for the threshold, and the labels of clusters chosen by the log-likelihood ratio (LRR) test method algorithm—were used in the subsequent analysis. Pathfinder was selected for fine-tuning the setting to highlight key points. Betweenness centrality (BC) >0.1 was considered as pivot nodes. Modularity (Q) >0.3 was considered reasonable for the group. Silhouette >0.5 was considered homogeneity clusters. BC can be used to partially assess the influence of each node in the network . UsuallyThe R programming language (version 4.1.0) was used for data visualization .The annual number of published articles is a significant indicator for studying research trends in the field and reflects the pace of subject knowledge . We founVisualized knowledge maps and institutional networks can provide information on the cooperative relationship between different research teams and countries . An analThe network of institutions participating in publications is visualized in BC measures the extent to which a node plays a bridging role or influence in a network. Specifically, BC measures the extent that the node falls within the shortest path between other pairs of nodes in a network. If the author plays a bridge that makes connections with other authors, then author’s BC value will high. The more an A depends on B to make connections with other people, the higher that B’s BC value . In partAs a highly condensed summary of the paper’s content, keywords can be used to summarize the topics of the paper simply and directly. When the co-occurrence network of the keywords was dense, we applied Pruning functions to select important connections to highlight the key aspects of the network. The co-occurrence of targeted therapy keywords in AML and related research can be visualized in DNMT3A mutations) in AML; and targeted drugs for AML .The identification of core literature in a certain field depends on the frequency of citations, and references with the highest cited frequency, namely, high-impact publications, are usually the focus of researchers. The top 10 cited references with the highest number of citations are shown in FLT3 mutations (cluster #4) from 2013 to 2021. The largest cluster focused on venetoclax resistance. The information, which consisted of highly cited and representative terms, in each group is shown in As can be seen in Targeted therapy in AML has received increasing attention. In the past decade, the number of publications in this field has increased rapidly. Through the co-authored network analysis of countries and institutions, we found that the United States and Germany were the representative countries with the highest BC and were found in different clusters, surrounded by other countries. The MD Anderson Cancer Center of the University of Texas, the Memorial Sloan Kettering Cancer Center, the Dana-Farber Cancer Institute, and Harvard Medical School, and Harvard University ranked the top five in the number of articles published.FLT3 study. Of these, the year of CH clustering was the most recent. We also noticed that the following categories were involved in driving the development of the field: biochemistry and molecular biology, pharmacology and pharmacy, immunology, applied microbiology and biotechnology, and chemistry. There are various degrees of interrelatedness between targeted therapy in AML and different categories, namely, the field of targeted therapy in AML is innovative and interdisciplinary.Ross L. Levine, Farhad Ravandi, and Guillermo Garcia-Manero were the top three most cited authors. We observed that Lars Bullinger and Ross L. Levine had extensive contact with other researchers. Farhad Ravandi and Guillermo Garcia-Manero collaborated closely and specialized in state-of-the-art treatments for leukemia at The University of Texas MD Anderson Cancer Center . The resFLT3 mutation (cluster #4) over the past decade. The clustering of keywords focused on cell-targeted therapy [chimeric antigen receptor T cells (CAR-T) and natural kill cells], signaling pathways (mTORC1), epigenetic therapies, and leukemic stem cells (LSCs). Combined with clustering of keywords and cited references, it is obvious that LSCs appeared not only in clustering of keywords but also in clustering of cited references, such as the largest cluster representing venetoclax resistance.We can discover topic distribution in the field and trends of research topics over time. Through the co-citation timeline, we found that research on targeted therapy in AML had changed to venetoclax resistance (cluster #0), novel therapy (cluster #3), and FLT3 inhibitor” was present in a cluster of co-references and in the keywords having the strongest citation burst, respectively. Up to 2017, the European Leukemia-Net (ELN) adjusted and updated cytogenetic and genomic mutations to the classification based on relative evidence. ELN categories were developed to correlate with genetic abnormalities having a clinical prognostic impact. The categories of these guidelines are divided into favorable, intermediate, and adverse. The adverse categories included FLT3-ITD with a high allelic ratio  for FLT3-mutated AML in 2017  from which AML develops , a strong specific inhibitor of DNA methylation, is commonly used as a single agent to treat patients with MDS and older aged patients with AML  are present in 15%–30% of AML patients  mutations leads to DNA hypermethylation, contributing to leukemogenesis (IDH1/2 inhibitor interrupts epigenetic changes. Recently (IDH inhibitors have been approved by the FDA: the IDH1 inhibitor ivosidenib (AG-120) and the IDH2 inhibitor enasidenib (AG-221)  family, also named the human KMT2 family, due to the role of the first member KMT2A was found in this disease. Recent exome sequencing studies have revealed that the KMT2 genes are among the most frequently mutated genes in many types of human cancers  appears to promote leukemia cell differentiation through direct effects on the HOX cofactor MEIS1 [HOX expression has been shown to be essential to maintain the leukemic phenotype of MLLr  leukemic cells but also prevented long-term engraftment and subsequent transformation of NPM1c-GMPs into leukemic cells after secondary transplantation in mice. Furthermore, the inhibitor was identified to suppress growth and induced differentiation in human NPM1c AML cells in patient-derived xenograft models (A new menin-itor VTP-469 appeak in AML ), pavingk in AML . VTP-504t models .KMT2A-rearranged AML, KO-539, and SNDX-5613 (both are menin inhibitors) useful for KMT2Ar and NPM1-mutated AML  promote proliferation, impose a full differentiation block, and drive the development of AML  were also frequent  should be considered to have CH of indeterminate potential (CHIP) . The mosothesis” –75. To rothesis” , 77. Theothesis” .The research into CH may be a promising field and worth focusing on. Such research could provide a theory for screening for people at especially high risk of transformation in the future. Additional studies are needed to understand and discover CHIP. Most importantly, it is necessary to find ways to reverse the pathogenic effects of CHIP. Ideally, drugs that can suppress mutant clones in the future should be identified, which could block malignant transformation in the “first hit” state to some extent.Recently, with the development of immunotherapy, CAR-T therapy targeting AML cells is undergoing active development . The trein vitro cytotoxicity of FLT3 CAR Ts in AML cell lines, especially in cells harboring the FLT3-ITD mutation , signaling pathways (mTORC1), epigenomic and epigenetic targeted therapies, and LSCs.This review has revealed that targeted therapy for AML has developed rapidly over the last decade. Through analysis of clusters , we defined the topic distribution in the field and the trends in research topics over time. The research of targeted therapy in AML has shifted to venetoclax resistance, novel therapy (such as targeting epigenetic modification), and IDH1/2 inhibitors) have led to the development of many potential novel therapeutic agents, many of which are being investigated in ongoing clinical trials. With the development of immunotherapy, CAR-T therapy targeting AML cells is rapidly developing. Therapy combination may give us possibilities for various treatments.The advances in FLT3 inhibitors and the discovery of epigenetic therapies .The authors are deeply indebted to ZL and GS for their support and effort in this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Interlaminar failure caused by scratches is a common damage mode in automotive coatings and is considered the potential trigger for irreversible destruction, i.e., plowing. This work strives to numerically investigate the mechanisms responsible for the complex scratch behavior of an automotive coating system, considering the interfacial failure. A finite element model is developed by incorporating a large deformation cohesive zone model for scratch-induced debonding simulation, where the mass scaling technique is utilized to minimize computational burden while ensuring accuracy. The delamination phenomenon of the automotive coating is reproduced, and its effects on scratch damage behavior are analyzed. Accordingly, it is revealed that the interlaminar delamination would produce significant stress redistribution, which leads to brittle and ductile damage of the coating and consequently affects the formation of plowing. Eventually, parametric studies on the effects of interfacial properties are performed. They demonstrate that the shear strength and shear fracture energy dominate scratch-induced delamination. The pursuit of aesthetic appeal in the exterior of a car has been a long-standing demand of consumers. Of the vast majority, automotive coatings are composite polymer films with a multilayer structure of clearcoat, basecoat, primer, electro coat, etc., which mainly provide vehicles with brilliant color and a glossy appearance, ref.  and alsoRecently, a variety of instrumented tests were carried out to investigate fracture scratches in automotive coatings ,3. Feng Numerical calculation methods provide further insights into the mechanism of polymer scratch behavior beyond the experimental approaches. For instance, the effect of material properties and internal structure on the scratch behavior of bulk polymers was studied through finite element (FE) simulations ,18,19. HScratch-induced damage in automotive coatings involves failure at the basecoat/primer interface, as emphasized in ,16. ScraBy way of contrast, the cohesive zone model (CZM) is a powerful tool to simulate the gradual scratch-induced debonding process ,32. Shu A finite element model for the scratch behavior of automotive coatings is developed, which accounts for the interlaminar failure of the coating system. This model is not only computationally efficient with the use of the mass scaling technique but is also verified to be effective by the good agreement between numerical and experimental results.The scratch-induced delamination phenomenon of an automotive coating is numerically reproduced, which makes it possible to understand its effects on coating scratch damage behavior via stress evolution analysis.The effects of the interfacial properties, i.e., interfacial strength and fracture energy on the delamination process have been numerically investigated.This study aims at investigating interlaminar failure and its correlation to scratch damage in the automotive coating system through numerical simulation. The previous FE scratch model  is develThe outline of the paper is as follows. In this work, the numerical simulations were performed on an in-house explicit finite element code FEP-Fracture (A Finite Element Program for Fracture) based on Fortran 90/95 . The empThe mixed-mode damage initiation is expressed in terms of a quadratic stress criterion, given by:termined ,41,42; TA common bilinear mixed-mode traction-separation law (TSL) , as showWhen the equivalent displacement  is used , given bduced as :(4)δf=2, and nodes on the symmetric plane were restrained in the y-direction (U2 = 0). An element-based mortar contact algorithm [lgorithm  was utillgorithm . Owing tlgorithm . Eight-nlgorithm . The reflgorithm . It was lgorithm . The tielgorithm . The emplgorithm . The estN=40 MPa .A rate-dependent bilinear elastoplastic model  is used The E-coat and the substrate, are modeled as perfect elastoplastic. The test data for calibration of the constitutive model were obtained from our former work . In the As already stated, the cohesive interface stiffness 5 MPa/mm, the calculation terminates soon after starting due to unreasonable oscillation occurring on the cohesive interface. However, it can be determined that the convergence results have already been obtained when the tested stiffness values ranged from 3.5 × 105 MPa/mm to 4.2 × 105 MPa/mm (the delamination loads differ less than 1.04%). Ultimately, 4 × 105 MPa/mm was chosen as a satisfactory value for initial stiffness.To determine an appropriate In this study, the overall material density of the coating system was scaled up to increase the incremental time step, and thus save computational cost. To obtain an appropriate value, a set of simulations were carried out with the scaling factor values set as 1, 4, 9, 16, and 25. z-stress distribution predicted with these two methods at a specific moment is shown in There are two distinct approaches to avert numerical problems that may occur when using common CZE for simulating large deformation delamination, as stated in the introduction. To intuitively illustrate the superiority of the employed LDCZM in simulating large deformation debonding between soft materials, a comparison between the performance of a CZM strategy  that comBefore the simulation results are formally analyzed, the scratch test carried out to reproduce typical damage patterns on automotive coatings should be introduced. Scratch tests were performed according to the ASTM D7027/ISO 19252 standard ,9. A linAfter tests, the typical damage patterns and surface topography along the scratch path were recorded, and are presented in To confirm the validity of the developed FE scratch model, the scratch friction coefficient (SCOF) obtained through the experiment was compared with the simulation outcome. The characterization of the scratch behavior of polymeric materials is challenging due to the high nonlinearity of their mechanical response . The SCOThe above discussed the initiation and progress of the scratch-induced delamination process. However, no distinct correlation between interlaminar interface failure and material removal has been found through the above analysis. To more accurately represent the delamination and material removal process, the damage mechanism could be accounted for in the mechanical modeling of the polymeric coating. Although attempts were made in the scratch damage modeling of bulk polymers , it remaBased on the FE results, the mechanism of scratch-induced delamination and post-delamination damage incentives of the automotive coating system were investigated. It was revealed that the delamination initiation was led by shear stress on the interface. Subsequently, the coating may be additionally damaged due to changes in the stress field induced by delamination and thus suffer material removal. The effect of delamination on the plowing formation could be summarized as follows: (1) severe ductile failure of the basecoat may facilitate the scratch tip to penetrate the thinned upper coating, and (2) the brittle damage of the coating that may occur in front of the tip would allow exposure of the delamination area beneath.2 in 25 J/m2 increments. The results are shown in Understanding the influence of interface properties on the delamination process is of great significance for improving scratch resistance. These properties include the interfacial strength and fracture energy in both the normal and tangential directions, which are parametrically investigated through a series of numerical simulations in this subsection. The basic concept is to study the influence of each parameter on the evolution of the maximum interface damage variable and delamination load. Specifically, the effect of strength is determined by varying the strength of one mode from 10 to 60 MPa in 10 MPa increments; consequently, the fracture energy was varied from 50 to 225 J/mN and N = 10 MPa, the trend of the curve is quite different from the other curves, as shown in It is revealed by Based on the above results, we may conclude that the scratch delamination of automotive coatings is shear-dominated, merely due to the enhancement of properties in the tangential direction contributing to the delamination resistance of the coating. In fact, this conclusion is closely relevant to the features of scratch deformation: indentation in the normal direction and tangential movement. Especially in thin coatings, the debonding interface is mainly compressed, making shear the only possible mode of delamination.With the aid of LDCZM, the developed FE model is capable of reproducing the delamination phenomenon when an automotive coating system is scratched. Although LDCZM enables the simulation of interfacial debonding between soft materials, nonphysical results appear at low interface strength. As a result, further development of the interface model is required to expand its application.The severe scratch damage of the automotive coating system was closely related to the interlaminar failure. It was found that the delamination would induce significant stress redistribution resulting in brittle and ductile damage of the coating, and, consequently, affecting the formation of plowing.In the automotive coating system, scratch-induced delamination was dominated by the shear strength and fracture energy. Therefore, enhancing these properties of the basecoat/primer interface could improve scratch resistance.In this study, the interlaminar failure and its effect on the scratch damage of an automotive coating system were numerically investigated. Firstly, the scratch-induced delamination of the automotive coating system was effectively simulated using a CZM-based FE model. Then, the correlation between interlaminar failure and plowing damage of the automotive coating was analyzed via the observation of stress evolution. Finally, the effect of interface properties on delamination was investigated numerically. The main conclusions are given as follows:Ultimately, the model developed in this work does not simulate coating damage, hence actual material removal could not be reproduced in the simulation. Intralayer damage to coatings will be considered in future research.


For the crosstalk of these organelles TPCs seem essential. Allergic reactions and anaphylaxis were previously shown to be associated with the endolysosomal two-pore channel TPC1. The release of histamine, controlled by intracellular Ca2+ signals, was increased upon genetic or pharmacologic TPC1 inhibition. Conversely, stimulation of TPC channel activity by one of its endogenous ligands, namely nicotinic adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate P2), were found to trigger the release of Ca2+ from the endolysosomes; thereby improving the effect of TPC1 on regulated mast cell degranulation. In this review we discuss the importance of TPC1 for regulating Ca2+ homeostasis in mast cells and the overall potential of TPC1 as a pharmacological target in anti-inflammatory therapy.Two-pore channels (TPCs) are ligand-gated cation-selective ion channels that are preserved in plant and animal cells. In the latter, TPCs are located in membranes of acidic organelles, such as endosomes, lysosomes, and endolysosomes. Here, we focus on the function of these unique ion channels in mast cells, which are leukocytes that mature from myeloid hematopoietic stem cells. The cytoplasm of these innate immune cells contains a large number of granules that comprise messenger substances, such as histamine and heparin. Mast cells, along with basophil granulocytes, play an essential role in anaphylaxis and allergic reactions by releasing inflammatory mediators. Signaling in mast cells is mainly regulated via the release of Ca TPCs plthe pore ,7,8. Figthe pore .Arabidopsis thaliana by means of X-ray crystallography [Mus musculus and Homo sapiens, respectively, using cryo-electron microscopy. A representative cryo-electron microscopy (EM) derived three-dimensional structure of the source organism Mus musculus in the apo state (inactive) is shown in TPCs are subdivided into TPC1, TPC2, and TPC3. Compared to TPC1 and TPC2, TPC3 was not found in human, primate, or rodent cells . Howeverlography . Only re2+ signals. We further discuss the potential role of TPC1 as a pharmacological target for a wide range of diseases, from anaphylaxis [In this review we focus on the cation channel TPC1 and its function in mast cells, regulating intracellular Caphylaxis ,16.Mast cells and basophils are innate leukocytes that both develop from hematopoietic stem cells. Mast cells can be found in almost all organs and are often located near neurons, blood vessels, and lymphatic vessels to transmit local inflammatory signals . The int3; depicted in 3 then binds to the IP3 receptor (IP3R), which is located in the endoplasmic reticulum (ER) membrane. The ER is one of the most important organelles for the regulation of intracellular Ca2+ and the largest Ca2+ store in the cell [3 to IP3R results in a release of Ca2+ from the ER, which, in turn, triggers a regulated exocytosis of secretory vesicles and the accompanying release of histamine from the cell. It was recently demonstrated that this process is fine-tuned by TPC1 in the endolysosomal membrane [2+-permeable TPC1 can regulate the level of ER-Ca2+ by facilitating the release and uptake (likely indirectly) of Ca2+ from and into the endolysosome, respectively, and thus support the regulated exocytosis of histamine containing vesicles  using trans-Ned-19 [2+ by means of TPC1 in the endolysosome [3 due to repeated allergen exposure, there is an increased release of the previously augmented ER-Ca2+ due to the aforementioned inhibition of TPC1. This, in turn, results in enhanced exocytosis, as shown in Allergen exposure and binding to IgE pre-coupled to FcεR in the plasma membrane of a mast cell activates phospholipase C (PLC); thus leading to the release of inositol 1,4,5-triphosphate lysosome, which subsequently provokes a secretion of perforin and granzyme from the secretory granules into the extracellular space [It is well known that TPCs can be activated by NAADP see , a Ca2+ ng agent ,34,35. Far space ,37. In par space ,39. Therar space ,41. The 2 Pchannels ,13,42,43a2+ flux ,13,42,442 leads to an increase in lysosomal exocytosis, whereas activation by means of NAADP decreases lysosomal exocytosis due to a change in vesicular pH. This has been previously described for NAADP and can probably be attributed to the agonist-specific effect on proton permeability [2 alone P2, is relatively controversial was previously taken up in several articles [2+ and Na+) and that activation is possible via both NAADP and PIP2. It was assumed that this could not always be clearly demonstrated in the past due to inadequate mouse models in which the TPC function was sometimes unintentionally preserved [In this context it is vital to mention a recently published work by Gerndt et al. , who foueability ,46. Howelone see  does notarticles ,48,49,50reserved ,48.+ selective channels and are also activated by PIP2 instead of NAADP [Arabidopsis thaliana) and mammalian HsTPC2 (Homo sapiens). By the implementation of structure-directed mutagenesis, the nonselective AtTPC1 was transformed into a Na+ selective channel similar to HsTPC2. This identified the most important filter residues that are central to the definition of ion selectivity of TPCs. Ultimately, however, there are still some gaps in this context that must be filled with future experiments, especially since PIP2 can regulate TPC1 in addition to NAADP and this more likely leads to a Na+-specific current. The recent discovery of two promising agonists for TPC2, which seem to selectively trigger Na+- or Ca2+-conductance, might help to shed light onto this conundrum [+ currents. The second agonist, termed TPC2-A1-N, in turn triggers an increased Ca2+ signal. Based on these results, it has already been speculated that TPC2-A1-N could be an imitator of NAADP, whereas TPC2-A1-P probably mimics PIP2 [When distinguishing between plant and mammalian TPC channels, the latter have been suggested as potential receptors for NAADP ,34,51. Hof NAADP ,50. A deof NAADP , who peronundrum . The firIP2 . HoweverIP2 ,41. TherThe plant alkaloid tetrandrine was mentioned several times before as an inhibitor for TPC1 and TPC2 ,53. In aIn another study , TPC2 ag2+-activating messenger NAADP, trans Ned-19 physiological questions. Consequently, the potential of TPCs as pharmacological targets for the treatment of diseases, such as anaphylaxis or infectious diseases, has to be investigated further and more comprehensively. Furthermore, one should consider that expression and function of TPCs could be cell type-specific, as this has only recently been discussed [+ (T-helper) cells and CD8+ T- Lymphocytes (CTLs) needs to be investigated in more detail. The precise localization of TPCs in various intracellular vesicles, such as the granules in granulocytes and mast cells as well as in lytic vesicles of Natural Killer (NK) cells or CTLs, is still unclear. In this context, there are many open intriguing questions that need to be answered.While 2D and 3D electron microscopic methods including element analysis and immunocytochemistry could clarify how organelles interact to maintain Caiscussed . In thisiscussed . For exa