Epigenetic alterations strongly contribute to the development of various types of cancer, including blood cancers such as acute myeloid leukemia (AML). Unlike genetic mutations, epigenetic changes in cancer are reversible. Therefore, targeted modification of epigenetic regulators, such as histone deacetylases (HDACs), is under intense investigation. Here, we analyzed gene expression for all four classes of HDACs in AML patients compared to healthy controls. We observed significant overexpression of various HDACs, including the relatively unexplored HDAC class IIA members. To investigate the cellular consequences of HDAC inhibition, we treated AML cell lines with the class IIA HDAC inhibitor TMP269 and observed significant effects on the cellular proteome and the growth of AML cells. We further demonstrate that the combination of TMP269 and venetoclax results in enhanced cell apoptosis. Our work provides new data for the HDAC inhibitor TMP269 and suggests that TMP269 might be an alternative compound for polytherapy in AML.Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML. Acute myeloid leukemia (AML) is one of the most common malignant diseases of the hematopoietic system in adults, with a median age at diagnosis of 68 years . AML is Histone deacetylases (HDACs) are enzymes that regulate gene expression by changing the acetylation state of N-terminal lysine residues of core histone proteins and of some non-histone proteins, including oncogenes, tumor suppressor genes, transcription factors, chaperones, and other cell signaling molecules, thereby altering protein stability or biological function ,11. In mIn this study, we describe novel effects of the specific class IIA HDAC inhibitor TMP269  on AML cWe propose that TMP269 mediates its anti-leukemic effects by downregulation of cancer-associated RPs, which synergize with the anti-leukemic/pro-apoptotic effects of venetoclax. Based on our results, TMP269 in combination with venetoclax may provide a novel and potent polytherapy option for AML patients.TMP269, BML-210, and venetoclax were obtained from eubio . MS-275 (entinostat) and bufexamac were purchased from Biozol . Azacitidine was obtained from Selleckchem . All compounds were dissolved in 100% DMSO and stored at −70 °C.2. All cell lines were obtained from the Leibniz-Institute DSMZ GmbH (https://www.dsmz.de accessed on 22 November 2022) with the following accession numbers: MOLM-13 (ACC 554), MOLM-14 (ACC 777), MV4-11 (ACC 102), HL-60 (ACC 3), OCI-AML3 (ACC 852), KG-1a (ACC 421).The human AML cell lines KG-1a, HL-60, OCI-AML3, MOLM-13, MOLM-14, and MV4-11 were cultured in RPMI 1640 medium , Iscove’s Modified Dulbecco’s Medium (IMDM) (MV4-11), or Minimum Essential Medium Eagle (MEM) (OCI-AML3), supplemented with 10%  or 20%  heat-inactivated FBS, 1% L-glutamine, 1% penicillin, and streptomycin. Cells were kept at 37 °C in a humidified incubator with 5% CO® Universal Probe qPCR Master Mix  on the Rotor-Gene 3000 . The amount of mRNA was normalized to the reference gene large ribosomal protein P0 (RPLP0). The relative mRNA expression was determined by calculating the difference between the cycle threshold of the gene of interest and the reference gene (ΔCT). The values are depicted as 2−ΔCT. The following primers (Sigma) were used: HDAC1: forward 5′- CTATCGCCCTCACAAAGCCAATGC -3′, reverse 5′- CAGCACTTGCCACAGAACCACCAG -3′; HDAC2: forward 5′- TGGACCCATCACCCAAGCAGTG -3′, reverse 5′- CCACAGGGAGGATGCCAGAACA -3′; HDAC3: forward 5′- TCGTGCTGGGTGGTGGTGGTTA -3′, reverse 5′- GATGCGGGTGCTGACATCTGGA-3′; HDAC8: forward 5′- ACGGCTCGATGCTGGACATACTTG -3′, reverse 5′- GTTGAGGATTTGTTGGATTCGGTGG -3′; HDAC4: forward 5′- CCCTGCAAATGGATGGCTTGTG -3′, reverse 5′- TGGAGACGGGAGCGGTTCTGTTA -3′; HDAC5: forward 5′- CGAAGTCAAAGGAGCCCACACCA -3′, reverse 5′- CCAGGCAAAGGCAGTTTGTAGGAG -3′; HDAC7: forward 5′- GTAGCAGCACGCCCGCATCA -3′, reverse 5′- AGCAAGGACACTGTCGGCAAGG -3′; HDAC9: forward 5′- GCGGTTGGCATGGATGGATTAGA -3′, reverse 5′- TGCTCAGGGTGGGTGGTGGAA -3′; HDAC6: forward 5′- ACTCATACTCCTGTGCCTGCCTGG -3′, reverse 5′- GCGGTGTTTCTGTTGAGCATAGCG -3′; HDAC10: forward 5′- GCCCTAGAGTCCATCCAGAGTGCC-3′, reverse 5′- GCAACAGCGGTGCGGACAGAG-3′; RPLP0: forward 5’- GGCACCATTGAAATCCTGAGTGATGTG -3’, and reverse 5’- TTGCGGACACCCTCCAGGAAG -3’.Total RNA of cultured cells was extracted using TRI Reagent  according to the manufacturer’s instructions, and reverse-transcribed into complementary DNA (cDNA) with RevertAid H Minus M-MulV reverse transcriptase . RT-PCR was performed with Luna5 cells were seeded in 500 µL medium in a 48-well plate and treated with the desired concentrations of the appropriate HDAC inhibitors for 48 h. After harvesting, cells were manually counted using a Neubauer chamber and proliferation was assayed by flow cytometry on a BD FACS Canto II . Fixable viability dye eFluor780 (eBioscience) was used at 1:2000 to exclude dead cells. Results were analyzed using FlowJo analysis software (BD Biosciences).Cells were labeled with a cell proliferation dye  diluted 1:5000 in PBS . Then, 1 × 10Apoptosis was determined by staining the cells with Annexin V Apoptosis Detection Kit eFluor-450 and 7-AAD (eBioscience) according to the manufacturer’s instructions. For titration of single drugs, MOLM-13 cells were treated with the indicated concentrations of TMP269, azacitidine, or venetoclax for 24 h. For combinatory treatments, MOLM-13 or HL-60 cells were treated with 12.5 µM TMP269, 1 µM azacitidine, 25 nM venetoclax, or a combination thereof (TMP269 + venetoclax or azacitidine + venetoclax) for 24 h. Further, MOLM-13 cells were treated with 5 µM BML-210, 25 nM venetoclax, or a combination thereof (BML-210 + venetoclax) for 24 h. Cell viability and apoptosis were assessed by flow cytometry using a FACS Canto II flow cytometer (BD Biosciences) and the data were analyzed using FlowJo Software (BD Biosciences).v/v) Triton X-100 for 30 min prior to harvesting to achieve maximum LDH release.MOLM-13 cells were treated with different concentrations of HDAC inhibitors for 48 h. Cytotoxicity was assessed by quantification of lactate dehydrogenase (LDH) release into cell supernatant by measuring the decrease in NADH absorbance at 340 nm. As a positive control, cells were lysed with 0.10%  study research program and comprises whole-genome analysis data from 542 AML patients and 74 healthy donors of a total sample size of 2096 from 11 participating centers on three continents [https://geoparse.readthedocs.io/ accessed on 12 December 2022) and the analysis was performed using Python.The public genome dataset GSE13159 from NCBI’s Gene Expression Omnibus (NCBI-GEO) was used to screen all HDAC classes and to compare the ntinents . In all 6 MOLM-13 cells were treated with 12.5 µM TMP269 for 24 h. To analyze the proteome of the differentially treated MOLM-13 cells, label-free quantification (LFQ) was conducted. In short, the cells were lysed followed by proteolysis with trypsin and clean-up of the generated peptides by suspension trapping in an S-trap™ column . Subsequently, the peptides were separated in a reversed-phase high-performance liquid chromatography system coupled to a quadrupole Orbitrap mass spectrometer. Peptide spectra were acquired in data-dependent mode, whereby each scan cycle consisted of a full scan at a resolution setting of 70,000 at m/z 200, followed by 15 higher-energy collisional dissociation scans at 32% normalized collision energy at a resolution setting of 17,500 at m/z 200. The generated peptide spectra were processed utilizing MaxQuant 2.0.1.0 [Homo sapiens (Human) from 3 February 2022 was used applying a 1% false discovery rate and a reversed sequence decoy database. The obtained protein groups were further processed using the Perseus software platform [Three batches of 1 × 10 2.0.1.0  with def 2.0.1.0  includinplatform . More dep-values were below 0.05. Proteins reported by MaxQuant were identified by UniProt accession numbers, and the corresponding gene symbols were derived by annotation mapping provided by the Ensembl database (release 107). Only proteins with a unique mapping were kept in the dataset. Principal component analysis was visualized with the plot_pca function from the DEP R package (v 1.18.0) [p-value was less than 0.05. All analyses were performed in R (v 4.2.0) and Bioconductor (v 3.15). The String database (https://string-db.org/ accessed on 6 September 2022) was used to search for protein networks and physical protein interactions.The identified protein groups were filtered to remove decoy hits as well as proteins that were only identified by site. Next, the LFQ intensities were log2-transformed and normalized by subtraction of the median. Protein abundances yielded by MaxQuant are shown as LFQ intensities, thus removing protein groups flagged as potential contaminants or as a hit to the reverse database . Further 1.18.0)  on varia 1.18.0) ; enrichmWestern blots were prepared as previously described . Cell lyp-values < 0.05 were considered significant .Statistical analyses were performed with GraphPad Prism 9 software. Comparisons of multiple groups were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. For comparison of two groups, the Mann–Whitney U test was used for non-parametric data. Data from at least three experiments are shown as mean ± standard deviation (SD) or as indicated in the respective figure legend. HDAC1 and HDAC2  and of SIRT3, SIRT5, SIRT6, and SIRT7 (class III) were significantly reduced and HDAC11 (class IV) was significantly increased in AML patients compared to healthy controls  but also HDAC4, HDAC5, and HDAC9 (class IIA HDACs), and HDAC10 (class IIB HDAC) are highly expressed in MOLM-13, MOLM-14, as well as in MV4-11 cells. All of these cell types are representative of the M5 FAB group, which is known to be the most aggressive AML subtype [Having shown that several class I and class IIA/IIB HDAC genes are overexpressed in AML patient samples, we sequentially aimed to investigate whether enhanced class I and class II HDAC gene expression can also be observed in different human AML cell lines. Thus, we monitored the class I and class II HDAC mRNA expression in six AML cell lines derived from patients of different FAB subtypes (M0–M5) E. While  subtype . Therefo subtype .Taken together, these first results identify the increased HDAC gene expression of specific class I and class II HDACs in primary AML patient samples, an expression profile which is also retained in AML patient-derived cell lines.Different HDAC inhibitors have been investigated in AML . One of To better understand the molecular consequences of TMP269 administration, we performed differential proteomic analysis of MOLM-13 cells treated with 12.5 µM TMP269 or DMSO  for 24 h. The concentration of 12.5 µM TMP269 was chosen based on recently published data highlighting that >10 µM TMP269 produces strong inhibition of class IIA HDAC enzyme activity in a cell-free system  and antip-value < 0.05 and no limitations on expression fold change).Our proteomic analysis generated a MaxQuant output file containing an initial 3647 protein groups. After removing those flagged as potential contaminants or as hits to the reverse database, as well as protein groups with missing abundance measurements, and filtering for valid UniProt accession numbers, 2025 protein groups remained for differential abundance analysis. The further removal of 49 protein groups matching more than a single gene and quantified proteins associated to multiple Uniprot IDs gave us a final set of 1976 proteins/genes for downstream analysis. This set contained 20 upregulated and 24 downregulated proteins/genes  upregulated protein was mediator of cell motility 1 (MEMO1) and the most significantly downregulated protein was 60S ribosomal protein L6 (RPL6).Principal component analysis shows a clear separation of the replicates for the TMP269 or the DMSO treatment groups A. Forty-Subsequently, we focused on proteins significantly downregulated by TMP269 treatment and performed gene ontology (GO) enrichment analysis for the biological process (BP), cellular component (CC), and molecular function (MF). Our analysis revealed highly enriched GO terms for ribonucleoprotein complex biogenesis and cytoplasmic translation C, cytosoAIP, NUDC, RPL35A, RPL37A, RPL6, RPS21, RPS25, and SMARCB1) that are overexpressed at the transcriptional level in AML patients but downregulated at the protein level upon TMP269 treatment in MOLM-13 cells  that are upregulated upon TMP269 treatment in MOLM-13 cells but are expressed at lower transcriptional levels in AML patients  and class II  HDAC gene expression in AML patients compared to healthy controls and also confirmed the expression of these genes in various AML cell lines, representative of different AML subtypes.Our work complements the existing data on dysregulated HDAC gene expression in AML patients. We screened the publicly available database from the MILE study for gene expression levels of all classes of HDAC genes in AML patients and compared them to healthy controls. We found specifically increased class I  and RPL6 (most significant). LYAR is a cell growth-regulating protein and part of the 60S ribosomal subunit that is described to control protein translation . In coloRPL6 gene is overexpressed in AML patients compared to healthy controls. Furthermore, the downregulation of RPL6 protein levels induced by TMP269 treatment is associated with increased AML cell apoptosis when the cells are additionally treated with venetoclax.RPL6 is the most significantly downregulated protein in our proteomic analysis. RPL6 is a component of the large ribosomal subunit (60S) . RibosomOur String analysis revealed that RPL6 directly interacts with other RPL proteins such as RPL35A, RPL37A, RPS21, and RPS25, which are also significantly downregulated by TMP269 treatment and upregulated at the gene expression level in AML patients.RPS25 gene was demonstrated to be overexpressed in human leukemia cells which exhibit chemotherapy resistance to adriamycin [RPS25 has recently been shown to play an essential role in the cap-independent initiation of translation, which suggests that RPS25 has an important general function in regulating protein translation . The RPSriamycin . Furtherriamycin  and cellriamycin . HoweverRPL35A knockdown inhibited cell proliferation and migration, promoted apoptosis, and suppressed tumor growth [Another candidate to be investigated based on our proteome analysis is RPL35A, which was also downregulated upon TMP269 treatment but is upregulated at the transcriptional level in AML patients. In gastric cancer, r growth . Furtherr growth . This inAlthough a significant proportion of AML patients can now be cured, studies on newer treatment options such as the BCL-2 inhibitor venetoclax revealed that patients can become resistant to this drug, resulting in lower survival rates . Finding


PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. However, little is known as to whether PC2 contributes to the primary cilium structure. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild-type LLC-PK1 renal epithelial cells. Confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody to identify primary cilia and measure their length. Although primary cilia length measurements did not follow a Normal distribution, the data were normalized by Box-Cox transformation rendering statistical differences under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 μM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length compared to their respective scrambled RNA transfected cells. The data indicate that conditions that regulate PC2 function or gene expression modify the length of primary cilia in renal epithelial cells. Blocking of PC2 mitigates the effects of elevated external Ca2+ concentration on primary cilia length. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.Polycystin-2  is a Ca Shh) and Wnt, which play essential roles in the embryonic phase  and polycystin-2 , respectively, whose localization to primary cilia was an essential clue in directly linking primary cilia to renal cysts    and in Ppithelia  and the se model . In consdel , primary cilium length is maintained by PC2-mediated Ca2+ entry. In a high external Ca2+ concentration (6.2 mM), the length of the primary cilium was reduced, which was consistent with an increased intraciliary Ca2+ and the depolymerization of axonemal microtubules. Either PC2 inhibition by Li+  were cultured as previously described  in Dulbe studies .Confluent cell monolayers exposed overnight to the various experimental conditions were rinsed twice with phosphate-buffered saline (PBS) and fixed for 10 min in a freshly prepared solution of paraformaldehyde (4%) and sucrose (2%). Cells were then washed three times with PBS, blocked for 30 min with BSA (1%) in PBS, and incubated for 60 min with an anti-acetylated-α-tubulin antibody (Santa Cruz Biotechnology) to identify primary cilia . Anti-mo2, LiCl, amiloride, and EGTA, were obtained from Sigma-Aldrich  and diluted at their final concentrations indicated.Unless otherwise stated, chemical reagents, including CaCl2+ solution was prepared as follows: the Ca2+ chelating agent EGTA (ethylene-bis(oxyethylenenitrilo) tetraacetic acid, 100 mM) was dissolved in NaOH and titrated with HCl to reach pH ∼7.0, and used at a 1 mM final concentration. The final Ca2+ concentration was calculated by:QK is the dissociation constant of the Ca2+-chelator complex, [Q] is the concentration of the free chelating agent, and [CaQ] is the concentration of Ca2+ bound to Q. The nominal Ca2+-free solution was estimated to have a Ca2+ concentration of 0.32 nM. Whenever indicated, CaCl2 was added from a 500 mM stock solution to reach either 1.2 mM  or 6.2 mM (high) free Ca2+. The solutions were labeled as 0 Ca, 1.2 Ca, and 6.2 Ca, respectively.The nominal free-CaPKD2 gene expression in cultured LLC-PK1 cells was conducted using the small interfering RNA technique  UUCUCCGAACGUGUCACGU, as scrambled control. siRNA transfection was conducted as follows . Tubes were added either scrambled  or antisense  RNA with Optimum medium (100 μl) in the absence or presence of Lipofectamine (2 μl). Briefly, the tubes were incubated for 5 min at room temperature and then mixed with either Irss or siPKD2 for another 20 min . Incubation was conducted by medium change with a mixture of fresh medium  and 200 μl of the transfection mixture. The total transfection time was three overnights (72 h). Silencing efficiency was confirmed by the Western blot technique, 42 ± 7.91%, n = 4 reduction , as described elsewhere . Brieflyn > 140 measurements/experiment under each condition, were collected from three to four repeats (N experiments). Under all tested experimental conditions, primary cilia length measurements followed a non-Normal distribution (2+). Thus, data were first analyzed by a non-parametric one-way ANOVA test performed by Kruskal–Wallis ranges to assess differences within and among different conditions. Individual experiments showed similar general tendencies among experimental conditions, although some did not support statistical significance. In such cases, the Mann-Whitney U test further tested individual observed differences. To improve and enhance the statistical analysis, experimental data were also processed by Box-Cox transformation , and high Ca2+ (6.2 mM) conditions.Experimental data with ribution –3, showiormation  to aid iormation . Versionribution –3. The Nribution –3. Follo2+ to evaluate the effect of external Ca2+ on the length of primary cilia  , the incubation procedure also produced a dramatic change in cell morphology, suggesting that the phenomenon could occur by mechanisms other than those observed with high Ca2+.Confluent monolayers of wild-type LLC-PK1 cells were exposed overnight to solutions containing either 1.2 mM  or 6.2 mM (high) Cary cilia . Cells wry cilia , and Supry cilia . Data diry cilia , Left, ary cilia , Right. ctively) . Thus, c2+ and 200 μM amiloride , which increases ciliary length in neurons and other cells , such that comparisons of PC2-silenced cells (P1ss) were always made with respect to the Irss control condition and the not with untreated cells. In normal Ca2+  depolymerizing agent . While there is no apparent explanation for this phenomenon, it is possible that cells exposed for several hours to a Ca2+-free environment would suffer several changes, including major rearrangements in the cytoskeleton, and loss of adhesiveness to the substrate, leading to the retraction of the primary cilia. Under normal external Ca2+ conditions (1.2 mM), the primary cilium length is maintained by PC2-mediated Ca2+ entry. In a high external Ca2+ (6.2 mM) concentration, the increased intraciliary Ca2+ reduced the length of the primary cilium. PC2 inhibition by treatment with either Li+ or amiloride or a reduction of its expression (siRNA) would instead contribute to reducing Ca2+ entry to the primary cilium, also decreasing the rate of MT depolymerization, rendering longer primary cilia. The present study suggests that PC2 mediated Ca2+ transport is a key mechanism to the control of primary cilium length in renal epithelial cells.The present study provides evidence that a reduction of PC2 function (amiloride and lithium) or expression (silencing) regulate the length of the primary cilia in renal epithelial cells, in a Cat manner . It is kng agent . Thus, tPKD tissues. The disease is largely associated with mutations in the PKD1 gene, as compared to PKD2, such that essential aspects to the regulation of the PC2 channel may be implicated and not observed in normal tissue. A recent study by Zhou’s group , a lengthening of the primary cilium has been observed in various cultured cells, including neurons and fibroblasts  have a considerable effect on PC2 function (+ could be transported to the ciliary compartment, accumulating and exerting a still unknown structural effect on the ciliary length even at low concentrations. Another possibility is to consider the potential impact of the Li+ interaction with PC2 on Ca2+ transport. In any case, Li+ blockade of PC2 in organelles such as the primary cilium may help explain the impaired sensory function and the therapeutic effects of Li+.Treatment with Lidiseases . Althougroblasts . The priroblasts , and we ated PC2 . Even lofunction , suggestPKD2 gene expression was silenced in the LLC-PK1 cells to prove the functional role of PC2 in regulating the length of the primary cilium. In the presence of normal Ca2+, PKD2-silenced cells showed a longer primary cilium than their respective controls. This is consistent with the fact that the absence of PC2 would have a similar effect to that of PC2 inhibitors, giving rise to longer primary cilia and suggesting that PC2 is necessary for the regulatory Ca2+ entry into the organelle. Interestingly, in the presence of high external Ca2+, PC2-silencing induced a reversal such that the values were similar to the control cells incubated in normal Ca2+. Although there is no explanation for this phenomenon, it may be related to PC2 silencing, which only represented approximately 50% of the inhibited gene product. It is possible that the remaining gene product could compensate and maintain a sustainable Ca2+ influx to the primary cilium. In the absence of PC2 Ca2+ transport could also be potentiated by other Ca2+-permeable channels in the primary cilium, including TRPC1 and TRPV4 (2+ permeability properties, although information in this regard is yet unavailable. PC2 silencing could affect these interactions, thus interfering with the mechano-transduction of the Ca2+ signals associated with primary cilia.nd TRPV4 . PC2 intnd TRPV4  and may 2+ and cAMP signaling regulate PC2 channel activity (2+ and cAMP signals interact with each other leading to changes in the primary cilium length. We also observed how cAMP signals regulate the length of the primary cilium in confluent wild-type LLC-PK1 renal epithelial cells (2+ produced an increase in the length of the primary cilium. This is apparently paradoxical because cAMP signaling and PKA activation both stimulate PC2 channel function. Thus, PC2 may not be the only Ca2+-controlling pathway. Other transport mechanisms may also participate in the regulation of the Ca2+ concentration in the primary cilium, controlling its length. Interestingly, however, exposure of the cells to vasopressin, which increases cAMP in the primary cilia of LLC-PK1 cells, mimicked the effect of 8-Br-cAMP in normal but not in high Ca2+. Thus, the study determined a complicated crosstalk between the cAMP and Ca2+ signals to modulate the length of the primary cilium, in a phenomenon that implicates the presence of PC2. 2+ and cAMP increase the length of embryonic kidney cells. Although the primary cilium could be considered a compartment largely independent of the rest of the cell, both second messenger signals affect the entire cytoplasm and not solely the primary cilium. Expected changes in the concentrations of Ca2+ and cAMP may be associated with the activation of cytoplasmic reactions including Ca2+-dependent phosphodiesterases and adenylyl cyclases that also regulate ciliary length.Caactivity . Howeveral cells . Treatme2+ entry, render ciliary elongation. In contrast, high external Ca2+ leads to a reduction in its length (2+ influx in the onset of ciliopathies and the formation of signals leading to renal cysts, more specifically, in ADPKD.In conclusion, our results indicate that a functional PC2 controls the length of the primary cilium in renal epithelial cells, such that pathways that lead to its inhibition, including gene suppression and possibly a consequent decrease in ciliary Cas length . These r


These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves . Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10–50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin’s C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft.Dysferlin-null A/J myofibers generate abnormal Ca Studiesysferlin , and thaysferlin . The subysferlin , has morysferlin , where tysferlin .2+ signaling in healthy muscle, and that the dystrophic phenotypes that arise in its absence may be due in part to changes in the regulation of Ca2+, which are believed to be pathogenic in many diseases of skeletal muscle . These phenotypes were completely reversed by transfection of fibers to express dysferlin or by treatment of the fibers before and during hypoosmotic shock with drugs that block the L-type Ca2+ channels  and ryanodine receptors (RyR1) that mediate the release of Ca2+  or slower kinetics (EGTA) and thus higher concentrations were needed to generate results similar to BAPTA’s. Finally, we examined myofibers expressing dysferlin containing GCaMP6fu (u-DYSF-ΔC2A). This molecule targets the triad junction like wild type (WT) dysferlin and like WT dysferlin it supports Ca2+ signaling at WT levels. In comparison, GCaMP6fu alone is less potent. Our results suggest that leak of Ca2+ into the triad junction underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers. They further suggest that dysferlin’s ability to bind Ca2+ in the triad junction is sufficient to protect muscle from changes in Ca2+ signaling induced by injury or exercise.Here we test this idea by examining the effects of a permeable form of BAPTA, a chelator with an affinity for CaGCaMP6fu  in placeGuide for the Care and Use of Laboratory Animals . Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.All procedures involving mice complied with the Dysferlin-null (A/J) and control  mice were obtained from the Jackson Laboratory  or the National Cancer Institute, Frederick, MD (A/JCr) or bred at the University of Maryland, Baltimore (C57Bl/6). Mice were anesthetized with 2.5–4.5% isoflurane vaporized in oxygen and euthanized by cervical dislocation. Mice were 12–16 weeks of age at the time their tissues were studied.www.jain-foundation.org). The constructs carrying Venus-dysferlin and Venus-dysferlin missing the C2A domain (residues 1–107) have been reported (u sequence (AddGene), we deleted the Venus moiety from Venus-dysferlin missing the C2A domain with NheI and KpnI and replaced it with GCaMP6fu using the same restriction sites. This placed the GCaMP6fu sequence in frame with the rest of the dysferlin ORF.mVenus-dysferlin    was provided by The Jain Foundation  with a Zeiss Duo Laser Scanning Confocal System , equipped with a C-Apochromat 40×/1.20 W Korr objective. Fluorescence excitation and emission were at 488 and >505 nm, respectively, with the laser intensity attenuated to 1%. methods , as desc methods . Venus-d2, 0.3 mM NaH2PO4, 5 mM HEPES, 5.5 mM glucose, 1.8 mM CaCl2.Mice were anesthetized and FDB muscles from both feet were harvested. A 2 week period was allowed after electroporation. Single myofibers were prepared in DMEM with 2% (wt/vol) BSA, 1 μl/ml gentamicin, and 2 mg/ml type II collagenase  for 2 h at 37°C. Myofibers were kept for 12–14 h at 37°C and plated on 96-well plates coated with laminin  1 h before experimentation. Fibers were then washed in normal Tyrode’s solution, pH 7.4, containing 140 mM NaCl, 5 mM KCl, 0.5 mM MgClIsolated FDB fibers were loaded for 45 min at 37°C with 4.4 µM Rhod-2AM in the presence of 0.25% Pluronic F-127 (both from ThermoFisher), diluted in culture medium, then washed with Tyrode’s solution. When BAPTA-AM, Fluo-4AM or EGTA-AM was used, it was included in the same solution at concentrations from 0 to 250 nM.u were excited by light at 488 nm  and fluorescence was measured at wavelengths of >515 nm. Trains of voltage pulses transients were induced by field stimulation (1 Hz for 10 s) every 1 min for 5 min, as reported  and background fluorescence (Fo), normalized to Fo. is reported.Line-scan images were taken in the middle of myofibers at 1.9 ms per line at maximal aperture. ImageJ 1.31v  averaged the profiles for every pixel over time and took the maximal value for each of 10 voltage pulses to calculate the mean maximal value of the Ca2+ transients in electroporated myofibers are higher than those in controls  was induced as described . In brie2 (to yield [Ca2+]free = 100 nM), 1.1 MgCl2 (to yield [Mg2+]free = 1 mM), pH 7.4. The concentrations were calculated with WEBMAXC STANDARD. Line-scan images through the solution were taken at 1.9 ms per line at maximal aperture. Averaged data from 5 experiments were used to build the calibration curve. We used identical conditions to scan fibers preloaded with 10 nM Fluo-4AM.Calibration was done in the glass-bottom chambers used for culturing myofibers. Solutions containing known concentrations of Fluo-4 were added in the solution (mM): 10 HEPES, 264.2 KCl, 5 EGTA, 2.7 CaClImmunoblotting for RyR used antibodies specific for RyR1 (ThermoFisher/Invitrogen), RyR2 (ProteinTech) and RyR3 , diluted 1:500. Gels were 3–8% Tris-Acetate with Tris-acetate-SDS running buffer (NuPAGE/Invitrogen). Secondary antibodies were HRP conjugates , used at 1:10,000. Blots were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) and imaged with a BioRad ChemiDoc MP instrument.t test and Χ2 analysis. A value of p < 0.05 was considered statistically significant.Quantitative data are shown as mean ± SE. Statistical significance was determined with Student’s BAPTA-AM and EGTA-AM were from MilliporeSigma. Fluo-4AM was from Invitrogen. Unless specified otherwise, all other chemicals were from Sigma-Aldrich.2+ transients with field stimulation and recorded the increase in Rhod-2 fluorescence, which tracks [Ca2+]i . DMSO, the vehicle, had no effect in the absence of the chelator . These results show that low concentrations of BAPTA-AM enhance the amplitude of Ca2+ transients in A/J muscle fibers, restoring them to control levels.We first examined the effects of incubating dysferlin-null A/J myofibers with increasing concentrations of BAPTA-AM in the presence of 4.4 µM Rhod-2AM. We elicited Ca [Ca2+]i . Figures by ∼15% , 2A, to 2+ transients of dysferlin-null A/J myofibers after a brief osmotic shock injury (OSI). As above, we recorded the transients via Rhod-2 fluorescence in response to electrical stimulation. As we reported earlier . The results indicate that Fluo-4 reached concentrations in the sarcoplasm of ∼60 nM, or about 6 times higher than its concentration in the bath  are ∼160 and ∼370 nM, respectively, they both have relatively high “on” and “off” rates for Ca2+ and thus are able to chelate Ca2+ rapidly. The affinity of EGTA for Ca2+ is close to that of BAPTA, but its “on” and “off” rates for binding Ca2+ are ∼100-fold slower than BAPTA’s. We used EGTA-AM to test the idea that rapid, transient changes in the concentration of Ca2+, rather than changes in the equilibrium concentration alone, play a role in the abnormal Ca2+ signaling that we observe in A/J myofibers before and after OSI. Our results . These results were far less consistent as a function of concentration than those of GCaMP6fu-DYSF-ΔC2A  and RyR1s , would be expected if the triad junctional regions of dysferlin-null muscle fibers experienced a small Ca2+ leak in the resting state that increased with injury. Although the leak in the resting state does not result in a detectable increase in [Ca2+]i  and if we estimate [Ca2+]I after hypoosmotic shock in the absence of BAPTA is ∼120 nM, then BAPTA would be expected to reduce the resting [Ca2+]I after shock to ∼100 nM. A concentration of 120 nM is in the same range reported for the ability of Ca2+ to activate isolated RyR1, as well as RyR2 (2+]I from 120 nM to <100 nM (2+ waves in injured dysferlin-null myofibers.In contrast to Fluo-4AM, which carries a negative charge, BAPTA-AM is uncharged, suggesting that it should permeate the muscle cell membrane more efficiently. Nevertheless, if we assume that BAPTA accumulates like Fluo-4, and not considering the contribution of out-of-focus fluorescence to our calibration, intracellular concentrations of BAPTA should be ∼60 nM after myofibers are loaded with 10 nM BAPTA-AM. If the affinity of BAPTA for Ca as RyR2 . CICR in as RyR2  at conce <100 nM . Our res2+ transient in uninjured A/J fibers. The reduced amplitude of the Ca2+ transient that we observe in A/J myofibers before injury or treatment with BAPTA is likely due to the activation of RyR1 by sarcoplasmic Ca2+ and the consequent small depletion of the luminal Ca2+ stores in the terminal cisternae of the sarcoplasmic reticulum, both of which would be inhibited by chelation of sarcoplasmic Ca2+ by BAPTA. Notably, murine muscle expresses undetectable levels of the two other major forms of the RyR, RyR2 and RyR3 (2+ release is mediated by RyR1. As noted above, RyR1 has been linked to CICR-associated pathogenesis in other diseases of muscle (Our results further indicate that BAPTA has a significant effect on the Caand RyR3 , consistf muscle . Our resAs CICR mediated by the RyR1 in skeletal muscle is thought to be suppressed when the channels are well ordered in LTCC-RyR1 couplons at triad junctions, the absence of dysferlin may well lead to a “couplonopathy” , in whic2+ with BAPTA, but also by placing a mutant form of dysferlin with an enhanced ability to bind Ca2+ in the cleft of the triad junction itself. These experiments took advantage of our earlier observation that dysferlin lacking its N-terminal C2 domain, C2A, trafficked normally to the transverse tubules but did not support normal Ca2+ signaling (2+ with affinities in the micromolar range {2+ waves, it does not restore the amplitude of the transient to control levels and it fails to preserve the amplitude after OSI. We therefore used DYSF-ΔC2A as a backbone to target a Ca2+ binding protein moiety specifically to the junctional cleft by replacing C2A with GCaMP6fu, a calmodulin-based fluorescent Ca2+ indicator with rapid binding kinetics and high affinity for Ca2+ (0.89 μM at room temperature (u concentrated at triad junctions via Dysf-ΔC2A both enhances the amplitude of the Ca2+ transient to control levels in uninjured fibers and protects the transient from decreasing following OSI. GCaMP6fu expressed as a soluble protein in the sarcoplasm is inactive in the former and trends to lower activity in the latter. It also fails to suppress Ca2+ waves. This strongly suggests that chelating Ca2+ within the triad junction of dysferlin-null skeletal muscle is sufficient to suppress abnormal Ca2+ signaling associated with CICR. Considered together with our other results, it further suggests that CICR is triggered primarily by the local elevation of Ca2+ in the junctional cleft and not by more widespread changes in [Ca2+]i.Our evidence suggests that the couplon can be stabilized not only by uniformly chelating sarcoplasmic Caignaling . Notablyperature . We findu-DYSF-ΔC2A and WT dysferlin indicate that a key role of the C2A domain in native dysferlin in the triad junction may be to bind Ca2+ rapidly and with high affinity, consistent with earlier reports of the activity of the isolated C2A domain (2+ signaling following injury in wild type muscle. This activity may help to explain the fact that healthy muscle but not dysferlinopathic muscle recovers quickly from exercise-induced injuries, avoiding necrotic fiber death (The similarities in the effects of GCaMP6fA domain , and thaer death .2+ waves (u in the junctional cleft is sufficient to suppress waves. Further studies with additional constructs that also target the triad junction may be informative in this regard. Such studies may also permit us to use GCaMP-based methods to measure the concentrations of Ca2+ in the junctional cleft in healthy and dysferlin-null muscle, before and after injury.Because DYSF-ΔC2A largely suppresses Ca2+ waves , we do n


FLT3 mutated patients compared to nonmutated patients. Fostamatinib, entospletinib, TAK-659, and RO9021 induced significant apoptosis in primary AML cells, although the proapoptotic effects of the SYK inhibitors were less pronounced than the antiproliferative effects. Finally, most of the SYK inhibitors caused a significant decrease in the release of cytokines and chemokines from primary AML cells, indicating a potent inhibitory effect on the release of these leukemic signaling molecules. We concluded that the SYK inhibitors had antileukemic effects in AML, although larger studies are strongly needed to identify which patient subsets will benefit most from such a treatment.Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal prognosis. The cytoplasmic spleen tyrosine kinase (SYK) is highly expressed by hematopoietic cells and has emerged as a potential therapeutic target. In this study, we evaluated the in vitro antileukemic effects of five SYK inhibitors, fostamatinib, entospletinib, cerdulatinib, TAK-659, and RO9021, in a consecutive AML patient cohort. All inhibitors demonstrated a concentration-dependent antiproliferative effect, although there was considerable heterogeneity among patients. For fostamatinib and TAK-659, the antiproliferative effects were significantly higher in  Acute myeloid leukemia (AML), the most common type of adult acute leukemia, is characterized by rapid cellular proliferation, an aggressive clinical course, and a high mortality rate . DespiteFLT3 gene -thymidine incorporation assay. Detectable proliferation, defined as >1000 counts per minute (cpm), was found in all untreated patient samples. Cerdulatinib (0.01–10 µM) and TAK-659 (0.005–5 µM) showed a strong antiproliferative effect in all seven patient samples. A more divergent antiproliferative effect was demonstrated after inhibition by fostamatinib (0.01–5 µM), entospletinib (0.01–150 µM), and RO9021 (0.01–10 µM) in the same seven patient samples. Overall, all five SYK inhibitors demonstrated a concentration-dependent antiproliferative effect in all seven patient samples, and the results are presented in We tested the ability of primary AML cells to survive and still proliferate seven days after treatment with the five SYK inhibitors: fostamatinib, entospletinib, cerdulatinib, TAK-659, and RO9021. For the initial dose–response curve, we tested seven concentrations with a broad range on a cohort of seven randomly selected AML patients . These c3H]-thymidine incorporation of >1000 cpm was found for 59 (87%) of the 68 patient samples. As seen in We next studied the antiproliferative effect of these five SYK inhibitors in a large cohort of 68 consecutive AML patients for two selected drug concentrations. We aimed to use concentrations resulting in a reduced AML cell proliferation of approximately 30% and 70% compared to the untreated controls. Therefore, based on the results of the initial dose–response study A, the fop-values in Overall, treatment with the SYK inhibitors resulted in a significant antiproliferative effect compared to the untreated controls  by assessing the percent proliferation compared to the controls (set to 100%) using the -thymidine  per well was added. After an additional 20–22 h, cultures were harvested, and nuclear incorporation was measured using the [3H]-thymidine incorporation assay. Detectable proliferation was defined as >1000 cpm, and proliferation was estimated by comparing percent proliferation in treated cultures to treatment-free control cultures.In treated cultures, 100 μL of indicated concentrations of each of the five SYK inhibitors was added in StemSpan SFEM6 cells/mL/well in 24-well plates with 500 μL of StemSpan SFEMTM medium supplemented with G-CSF, SCF, and FLT3-L. In treated cultures, 500 μL of indicated concentrations of SYK inhibitors per well were added. 500 μL StemSpan SFEMTM medium was added to treatment-free control cultures. After 48 h, the AML cells were washed twice with cold PBS. The cell pellets were resuspended in binding buffer and double stained with annexin V and PI in the dark for 15 min. Cell viability, apoptosis, and necrosis were analyzed with Pacific BlueTM Annexin V Apoptosis Detection Kit with PI , staining according to the manufacturer’s protocol. Unstained and single-stained control cells were included in each experiment. Measurements were performed using flow cytometry FACS Verse (BD FACSVerse 8 color flow) and the FlowJo 10.0.7 software  [AML cells were seeded at 1 × 10OR, USA) . A total6 cells/mL in 1 mL/well) were seeded in 24-well plates with StemSpan SFEMTM medium supplemented with the cytokines G-CSF, SCF, and FLT3-L and treated with one of the five SYK inhibitors. StemSpan SFEMTM medium supplemented with cytokines G-CSF, SCF, and FLT3-L was treatment-free control. Supernatants were harvested after 48 h and stored at −80 °C until analyzed. Human Magnetic Luminex Assays  were used to analyze all supernatant samples according to the manufacturer’s protocol. Cytokine release was estimated as a ratio to untreated control for each cytokine and SYK inhibitor.AML cells  [Graphs and figures were made using GraphPad Prism v. 5.02 . For statistical analysis, Fisher’s exact test was performed by using Stata 17 Software   and WilcNJ, USA) .To conclude, our present results suggested that disrupting SYK signaling can alter the growth, viability, and mediator release of primary AML cells. Thus, SYK inhibitors can directly affect the leukemic cells and may also potentially, indirectly affect leukemogenesis. In the current study, we investigated five SYK inhibitors with different pharmacodynamic and pharmacokinetic properties. The specificity of the different inhibitors for SYK and other tyrosine kinases varies somewhat, and it is therefore not surprising that they had different effects in different AML patient samples. All the inhibitors, however, demonstrated significant concentration-dependent antiproliferative effects in our study. This study supports further investigation of the utilization of SYK inhibition in AML, and further preclinical, translational, and clinical studies should be performed to capture the features and benefits of SYK inhibition in AML.


The nutritional composition of the diets for growing rabbits should be well-balanced, both to meet the requirements of the high-producing hybrids currently used and to avoid nutrient losses. The improvement of the feed conversion ratio represents a key point in increasing rabbit farmers’ profitability because of the high feeding costs. This study evaluated the growth performance, digestive efficiency and meat quality of growing rabbits belonging to the most diffused commercial crossbred rabbit breeds, Grimaud and Hyla, feeding them diets with different energy and protein levels. The two genotypes showed similar growth performance, but Grimaud rabbits achieved higher slaughter yield than Hyla rabbits. The administration of high-energy diets improved feed conversion and increased nutrient digestibility. Conversely, low dietary protein contents reduced nitrogen excretion without negative effects on growth, feed efficiency and carcass traits of fast-growing rabbits.p ≤ 0.01) compared to G rabbits, while exhibiting lower slaughter yield and lower carcass fatness (p ≤ 0.01). As DE level increased, feed intake decreased and feed conversion improved (p < 0.001), while caecal volatile fatty acid concentration increased and caecal pH and N-ammonia decreased (p ≤ 0.01). The reduction in the CP level did not affect performance and carcass traits. No significant interaction was detected between genotype and diet. In conclusion, G rabbits may have an advantage over H rabbits because of the higher slaughter yield. As for the nutritional characteristics of diets for high-producing crossbred rabbits, high energy levels are confirmed to enhance feed efficiency, whereas dietary CP can be reduced to 144 g/kg without negative effects on performance and carcass traits.The present study evaluated the effect of digestible energy (DE) and crude protein (CP) levels on growth, digestive efficiency, slaughter yield and meat quality in growing rabbits of two genotypes . A total of 384 crossbred rabbits (192 G and 192 H), half males and half females, were divided into eight experimental groups according to a bi-factorial arrangement  and reared in individual cages. From 33 to 64 days of age, rabbits were fed four diets obtained by combining two DE levels  and two CP levels . Then, all rabbits received the same fattening diet  until slaughtering (77 days of age). Growth performance did not differ between genotypes for the entire trial , but H rabbits showed a faster growth during the post-weaning period ( During the last two decades, rabbit meat consumption is progressively declining in the European Union (EU) due to several issues, including high production costs, consumers’ concerns for animal welfare (cage-farming systems), unsuitable presentation of the end product (whole carcasses with head), increased popularity of rabbits as pet animals and criticism about the environmental sustainability of rabbit farms . MoreoveGenetic selection, feeding and nutritional strategies aimed at optimising growth rate and feed efficiency are the main tools to reduce feed conversion ratio (FCR) and enhance farmer profitability ,7. In faAs for protein requirements in growing rabbits, they are age dependent, being higher in the first post-weaning phase as a consequence of the rapid tissue accretion, gut development, and the need for maintaining intestinal mucosa functionality . HoweverMoreover, both growth performance and carcass composition could be more or less affected by changes in DE and CP levels depending on the genotype, with fast-growing rabbits especially affected, and on the animals’ resilience in the presence of dietary nutritional changes.Therefore, the present study aimed at evaluating the effects of the levels of dietary DE and CP on growth performance, diet digestibility, caecal fermentation activity and carcass and meat quality traits in fast-growing rabbits of the two commercial crossbred lines of French origin most commonly reared in France and in Italy.The trial was realised at the experimental farm “L. Toniolo” of the University of Padova, in a brick shed and under a natural photoperiod during the months of April and May. Extraction fans and an automatic heating system were used to control air circulation and relative humidity and to maintain the temperature within 17–24 °C.A total of 384 crossbred rabbits: 192 Grimaud  and 192 Hyla , with half being females and half males, were selected on two commercial farms (one farm per genotype). On both farms the rabbit does were synchronised and inseminated on the same day, 18 days post-partum, and housed in multifunctional cages. Rabbits were selected from healthy litters (standardised at 9 kits per doe) of multiparous does (3 to 4 kindling) and moved the same day to the experimental facilities at 33 days of age. Rabbits were individually identified using ear marks, housed in individual cages (285 mm × 410 mm × 285 mm) and randomly allocated to eight experimental groups , homogeneous by average live weight and standard deviation, according to a bi-factorial arrangement with two genotypes (G: Grimaud vs. H: Hyla) and four experimental post-weaning diets obtained from the combination of two DE levels  and two CP levels .The ingredients and chemical compositions of the experimental diets are listed in The post weaning diets were administered from 33 to 64 d of age. The increase in DE from LE diets to the HE diets was realised by increasing the inclusion rates of barley (+155 g/kg on average), soybean oil (+10 g/kg on average), soybean meal (+60 g/kg on average) and dried beet pulp (+95 g/kg on average) at the expense of dehydrated alfalfa meal (−234 g/kg on average), wheat bran (−50 g/kg on average) and sunflower meal (−35 g/kg on average). This way, the HE diets were characterised by higher contents of lipids (+7 g/kg on average) and starch (+44 g/kg on average) and lower contents of neutral detergent fibre , acid detergent fibre  and lignin (sa) (−18 g/kg on average) with respect to LE diets. The higher CP level of HP diets compared to LP ones was related to the partial or total substitution of the low-CP dehydrated alfalfa meal (14.3 g CP/100 g) with the high-CP dehydrated alfalfa meal (16.5 g CP/100 g), besides a higher inclusion rate of soybean and/or sunflower meals.Due to the presence of enteric diseases in the commercial farms where the rabbits were born, all post-weaning diets (fed from 33 to 64 days of age) were supplemented with an antibiotic , besides the standard coccidiostat supplementation .From 65 days of age until slaughtering (77 days of age), a unique fattening diet (diet F) without antibiotics and coccidiostats was provided to all rabbits . The pelleted diets  were supplemented with synthetic lysine and methionine, vitamins and macro- and micro-minerals to satisfy the nutritional requirements of rabbits during the post-weaning and fattening periods . All aniDuring the trial, the individual live weight and feed intake of the animals were recorded once a week from the arrival of the rabbits until the day before commercial slaughtering. The health of the animals was monitored daily: rabbits were considered ill when showing diarrhoea and/or mucus in the faeces or a live weight loss during a week.The nutrients’ apparent digestibility and the nutritive value of the diets were measured during two in vivo digestibility trials performed according to the European standardised method . The firAt 50 days of age, 48 rabbits , half of them females and half males, representative of the corresponding experimental groups in terms of mean live weight and standard deviation, were slaughtered to allow sampling of their caecal content in the afternoon (between 13:30 and 15:10).3 solution (25% wt/wt) and stored at −20 °C until the chemical analyses.Rabbits were weighed immediately before slaughtering and killed by cervical dislocation. Stomach and gut were removed and weighed. The caecum was removed and weighed, and the pH of the caecal content was immediately measured. The caecal content was then diluted with a 15% HPOAt 76 days of age, the rabbits were weighed without previous fasting to determine the final live weight and to measure growth performance. At 77 days of age, the rabbits on trial were weighed at the experimental farm before loading and after a 4 h fasting period, as required by the slaughterhouse. All healthy rabbits with a live weight exceeding the minimum weight of 2,300 g required by the slaughterhouse  were properly caged and transported to a commercial slaughterhouse by an authorised truck.The number of rabbits discarded by trial due to illness and/or insufficient live weight was used to calculate the total losses rate as follows:Rabbits slaughtered for sampling of the caecal content were not included in the initial number of animals.The travel from the experimental farm to the commercial slaughterhouse lasted about 1 h. Slaughtering took place about 1 h after the animals arrived at the slaughterhouse, where they were individually weighed, stunned by electro-anesthesia, and killed by jugulating. After 2.5 h of chilling, the commercial carcasses were weighed to calculate the individual slaughter yield , and theThe full gastrointestinal tract weight was recorded individually and expressed as a percentage of the slaughter.Moreover, the proportion of other slaughter wastes  was calculated as the difference between slaughter weight, full gastrointestinal tract weight and carcass weight and expressed as a percentage of slaughter weight according to the the following formula:A total of 160 carcasses , representative of the corresponding experimental groups in terms of average rabbit live weight and standard deviation, were selected, transported to the department laboratory and stored at 3–4 °C for 24 h. Then, the reference carcass and the main cuts for meat quality analyses were obtained according to harmonised dissection procedures described by Blasco and Ouhayon .l. lumborum muscle was measured in duplicate using a pH meter  equipped with a specific electrode (cat. 5232). Duplicate measurements of the L*a*b* colour indexes [l. lumborum muscles were dissected and the meat-to-bone ratio of the hind legs was measured [After that, the pH value of the right  indexes  were obtmeasured .l. lumborum muscles were stored at −18 °C in vacuum-sealed plastic bags until determination of thawing and cooking losses and of shear force. In detail, after thawing for 12 h at room temperature, the whole muscles were weighed to measure thawing loss, sealed in plastic bags, and cooked in a water bath for 1 h at 80 °C. After a 1 h cooling period at room temperature, the l. lumborum were weighed to measure cooking losses, and a cut from the middle part was separated (length: 70 mm). On this section, the maximum shear force was measured by means of a TA.HDI dynamometer  with the Allo–Kramer (10 blades) probe .The right The diets and faeces were analysed to determine the contents of dry matter 934.01), ash (967.05), crude protein (2001.11) and starch  using AOAC  methods..01, ash The thawed samples of caecal contents were centrifuged at 9000 rpm for 10 min. Caecal N ammonia was determined on the supernatant with a pH meter  equipped with ammonia specific electrode (mod. 9663 combined with the reference electrode mod. 5044). Volatile fatty acid (VFA) molar contents were measured on the supernatant with gas chromatography  equipped with a flame ionisation detector FID split–splitless injection system programmable oven on a cross bond capillary column DB–FFAP  (Agilent) according to the method described by Osl .The data were analysed by a two–way ANOVA using the PROC GLM of SAS 9.4 software  . The modp-value < 0.05 were accepted as representing statistically significant differences. Least-square means were compared using the Bonferroni test.Overall, differences among means with a During the trial, six rabbits died  showing diarrhoea and/or mucoid enteritis. At the end of the trial, another seven rabbits  were excluded from the trial and the commercial slaughtering due to the low live weight (<2300 g). The mortality rate in the whole trial was 1.8%, and the total losses rate reached 3.9%, without significant differences among groups (data not reported in tables).p < 0.001); thus, it was used as covariate in the analysis of growth performance, feed intake and FCR. The covariate was significant for live weight at 64 and 76 days of age, and for feed intake and FCR in the post-weaning, fattening and whole trial periods. The H rabbits were heavier than the G rabbits at weaning  and at 64 days of age  and feed intake  during the post-weaning period, whereas they grew and ate less during fattening .The initial live weight significantly differed between G and H rabbits (p < 0.001), fattening  and the whole trial . Rabbits fed LE diets during post-weaning period exhibited a higher daily weight gain during fattening compared to rabbits fed HE diets , whereas no significant difference was observed in the post-weaning or in the whole trial periods. The rabbits fed LE diets also showed a worse FCR compared to those fed HE diets from 37 to 64 days of age , whereas an opposite trend was observed from 64 to 76 days of age , when all rabbits received a unique fattening diet. Through the whole trial, the use of HE diets in the post-weaning period improved FCR compared to LE diets .The increase in the DE content reduced feed intake during post-weaning . No significant interaction was observed between genotypes and diets with respect to rabbit growth performance.The dietary protein level did not affect growth, feed intake or FCR of the rabbits. However, in the post-weaning period, a worse FCR was observed in rabbits fed an LE–HP diet (p < 0.001), whereas the digestibility of the ether extract was higher in H than G rabbits  increased, whereas the digestibility of starch decreased , CP (+3.3%), ether extract (+6.0%), gross energy (+19.2%) and all fibre fractions (p < 0.001). The combination between DE and CP levels led to a DP to DE ratio varying from 10.5 g/MJ (HE–LP diet) to 11.3 g/MJ (HE–HP diet) to 11.5 g/MJ (LE–LP diet) to 13.1 g/MJ (LE–HP diet).The CP level did not affect the nutrient apparent digestibility of the diets. However, the digestibility of dry matter and gross energy reached the lowest value when low energy levels were associated with high CP levels (p = 0.045) and ether extract (p < 0.001) was higher in H rabbits compared to G rabbits. As for the residual effect of post-weaning diets, a lower digestibility of ether extract was observed in rabbits previously fed HE diets (p < 0.05).The apparent digestibility of diet F, administered in the fattening period, was slightly affected by the genotype . In detap = 0.034).At 50 days of age, the genotype did not affect full-gut and full-caecum proportions or caecal fermentative activity . Howeverp = 0.006) and N ammonia content  in rabbits fed HE diets compared to those fed LE diets, besides increasing total VFA concentration .The increase in DE content decreased caecal pH  due to a higher chilled carcass weight  and lower incidence of full gastrointestinal tract  and other slaughter wastes  (p = 0.020) and a lower proportion of dissectible fat ; finally, they showed a feed-to-carcass conversion ratio (feed intake/chilled carcass weight) lower than that of H rabbits . Regarding meat quality traits, only the lightness index was higher in H than G rabbits (p = 0.010).As for the effect of genotype on carcass traits, G rabbits achieved a higher carcass yield compared to H rabbits  . Moreovep = 0.012) and the proportion of hind leg  decreased while slaughter wastes increased . The feed-to-carcass conversion ratio increased in LE diets, especially when combined with a high crude protein level .When rabbits were fed diets with increased DE content, in the post weaning period, transport losses  and productive (milk yield) performances of the does ,38,39. HAccording to our results, Hyla rabbits seemed to be more precocious than Grimaud rabbits and able to achieve a suitable slaughter weight earlier (2500–2700 g depending on the market). Indeed, during the post-weaning period, Hyla rabbits grew more than Grimaud rabbits, reaching a higher body weight at 64 days of age. The higher growth rate was strictly correlated to increased feed consumption, as observed by other authors when comparing rabbits from paternal lines (selected for growth rate) with rabbits from maternal lines (selected for litter size) ,43,44. DThe different carcass yield and main cut proportions between genotypes are explained by different allometric coefficients of body tissues and organs. Indeed, depending on the selection program, rabbits of different genotypes could exhibit some differences in the relative growth of body tissues . The incDifferences in the relative growth of body tissues could be responsible for changes in feed efficiency. In this regard, Szendrő et al.  stated tThe use of low-energy diets (rich in insoluble fibre) is generally recommended in growing rabbits during the post-weaning period to reduce morbidity and mortality rates due to digestive diseases ,16,17. OIn this study, a standard feeding program (low-energy diets during post-weaning and high-energy diets during the fattening period) was implemented as a control group in the form of an LE diet (9.2 MJ DE/kg) in the first four weeks after weaning and a high-energy diet (10.7 MJ DE/kg) during the last twelve days of fattening. From the other side, in the HE groups, rabbits were fed high-energy diets throughout the rearing period (10.9 MJ DE/kg during post-weaning and 10.7 MJ DE/kg during fattening). The increase in DE level significantly reduced feed intake and FCR, in agreement with previous studies ,56,57. IBoth in the present trail and in other studies, the use of high-energy diets increased rabbit digestive efficiency ,59 and mThe caecal fermentation traits changed significantly with the increase in DE level as a consequence of the greater availability of highly-fermentable carbohydrates, which promotes a higher production of VFA and reduces pH and ammonia nitrogen levels ,63. In fAs dietary protein level is concerned, a CP content ranging from 150 to 160 g/kg is recommended in the diets of growing rabbits . HoweverWith regard to fermentative activity, a decrease in dietary CP has been associated with decreased total VFA concentration ,66 and iIn the present trial, the association of high CP level with low DE concentration decreased the digestibility of dry matter and gross energy and worsened the FCR and the feed-to-carcass conversion ratio, confirming that excessive DP to DE ratio reduces the efficiency of protein utilisation . The feeAs reported in the current study, several authors observed that once the energy and protein requirements were met, further increases in the dietary DE and CP contents weakly affected the slaughter results and meat quality traits of growing rabbits .In the conditions of the present trial, Grimaud and Hyla rabbits achieved similar growth performance and final live weights, but the former breed showed higher carcass yield and feed-to-carcass conversion ratio.The increase in DE content to 10.9 MJ/kg in the post-weaning diets, once minimal insoluble fibre contents are guaranteed, can improve feed conversion ratio and reduce feeding costs. Moreover, when diets are supplemented with essential amino acids according to the current recommendations, the dietary CP level can be reduced to about 140 g/kg without affecting the growth and feed efficiency of high-producing fattening rabbits.


Gain-of-function mutations of isocitrate dehydrogenases 1/2 (IDH1/2) play crucial roles in the development and progression of acute myeloid leukemia (AML), which provide promising therapeutic targets. Two small molecular inhibitors, ivosidenib and enasidenib have been approved for the treatment of IDH1- and IDH2-mutant AML, respectively. Although these inhibitors benefit patients with AML clinically, drug resistance still occurs and have become a major problem for targeted therapies of IDH-mutant AML. A number of up-to-date studies have demonstrated molecular mechanisms of resistance, providing rationales of novel therapeutic strategies targeting mutant IDH1/2. In this review, we discuss mechanisms of resistance to ivosidenib and enasidenib in patients with AML. Isocitrate dehydrogenases 1/2 (IDH1/2) are metabolic enzymes catalyzing the oxidative decarboxylation of isocitrate to α-KG and reducing NAD(P)+ to NAD(P)H in the tricarboxylic acid (TCA) cycle  and IDH2Mutations to IDH1/2 are important events in several types of cancers, including acute myeloid leukemia (AML), glioma, angio-immunoblastic T-cell lymphoma, chondrosarcoma, intrahepatic cholangiocarcinoma, and so on –11. In AMultiple studies have investigated the impact of IDH1/2 mutations on the prognosis of AML, with inconclusive results. Generally, IDH1 mutations are associated with an inferior outcome and IDH2 mutations are associated with a relatively favorable prognosis in AML , 16–19. The discovery of IDH mutations in cancers promotes the rapid development of targeted inhibitors. Enasidenib AG-221) and ivosidenib AG-120) are inhibitors approved by FDA for the treatment of refractory or relapsed R/R AML with IDH2 or IDH1 mutations  FAO and OxPHOS. OxPHOS, as a master regulator of mitochondrial biogenesis, and biosynthesis or degradation of FA, activation of PGC1α (peroxisome proliferator-activated receptor-γ coactivator-1) was not reversed after the inhibition of mIDH1. Importantly, FAO is a crucial biochemical process for sustaining OxPHOS and mitochondrial function in AML cells. Analysis of transcriptomic data from four clinical trials in 10 resistant patients demonstrated that genes associated with high OxPHOS function are enriched. Collectively, the OxPHOS phenotype was also confirmed as a nongenetic mechanism of IDHi resistance.Accumulating emergence of somatic mutations in cancers promote the development of clonal heterogeneity . AML hasQuek et al. studied the clonal basis of response and acquired resistance to enasidenib treatment . An analIn summary, this review concludes the resistance mechanisms of approved mIDH1/2 inhibitors and the increased understanding of drug-resistance mechanism would promote the development of corresponding strategies. To better investigate resistance mechanisms, development of novel effective approaches for detecting gene mutations is extremely important, such as next generation sequencing (NGS) and single-cell RNA sequencing (scRNA-seq). Future strategies should pay attention to the development of rational combination therapies with mIDH inhibitors or agents which can overcome the resistance to improve the response duration. The emerging biological mechanisms and clinical insights into these issues will provide guidance for rational treatment in the future.(I) Conception and design: YC, CC, and YP; (II) Administrative support: None; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: None; (V) Data analysis and interpretation: None; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.We thank 100 Top Talents Program of Sun Yat-sen University,National Natural Science Foundation of China , and Sanming Project of Medicine in Shenzhen (No. SZSM201911004) for supporting the manuscript preparation and publication.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.