Patients within the WHO-subgroup of t-positive acute myeloid leukemia (AML) differ from other AML subgroups as they are characterised by younger age and a grim prognosis. Leukemic transformation can often be attributed to single chromosomal aberrations encoding oncogenes, in the case of t-AML to the fusion protein DEK-CAN . As being a rare disease there is the urgent need for models of t-AML. The only cell line derived from a t-AML patient currently available is FKH1. By using phospho-proteomics on FKH1 cells, we found a strongly activated ABL1 kinase. Further investigation revealed the presence of ETV6-ABL1. This finding renders necessary to determine DEK-CAN- and ETV6-ABL1-related features when using FKH1. This can be done as ETV6-ABL1 activity in FKH1 is responsive to imatinib. Nevertheless, we provided evidence that both SFK and mTOR activation in FKH1 are DEK-CAN-related features as they were activated also in other t and DEK-CAN-positive models. The activation of STAT5 previously shown to be strong in t-AML and activated by DEK-CAN is regulated in FKH1 by both DEK-CAN and ETV6-ABL1. In conclusion, FKH1 cells still represent a model for t-AML and could serve as model for ETV6-ABL1-positive AML if the presence of these leukemia-inducing oncogenes is adequately considered.Taken together, all our results provide clear evidence of novel and specific interdependencies between leukemia-inducing oncogenes and cancer signaling pathways which will influence the design of therapeutic strategies to better address the complexity of cancer signaling.The online version contains supplementary material available at 10.1007/s00277-022-04905-9. In acute myeloid leukemia (AML) the study of specific disease-defining translocations, such as t15;17)-PML-RARα, t-RUNX1-ETO or t-DEK-CAN  has allowed a better understanding of the molecular mechanisms of disease and recognition of the central role of aberrant transcription regulators in the pathogenesis of AML [5;17-PML-Three subtype specific translocations, namely t, t and t, are notable for being associated with a small number of co-existing mutations, mainly in c-KIT or FLT3. In association with these translocations mutations in c-KIT or FLT3 are not essential either for induction or maintenance of the disease  and our AML with the t6;9) is defined as a distinct entity by the WHO classification because of its particular biological and clinical features and unmet clinical needs . In cont;9 is defThe t6;9) represents the only structural chromosomal aberration present at diagnosis and encodes the DEK-CAN fusion protein  represen, 11, itsIt is known that the expression of the leukemogenic nuclear factor DEK-CAN activates the mTOR and STAT5 signaling , 15 strodek-can chimeric transcript and was factor-dependent as it grew only in the presence of 10 ng/ml recombinant human granulocyte colony-stimulating factor (G-CSF), with a doubling time of 54 h. Its capacity of multi-lineage myeloid differentiation with the presence of macrophages, basophils, eosinophils and neutrophils suggested that FKH1 cells originated from a pluripotent myeloid stem cell [For our investigation, we selected FKH1 cells as a model because it is the only t-AML cell line available. The FKH1 cell line was described in 1998 as first permanent cell line with t derived from the peripheral blood of a Japanese patient with t AML. It was transformed from a Philadelphia chromosome (Ph)-negative myeloproliferative neoplasia (MPN). The FKH1 expressed the tem cell . AlthougTo investigate a network of activated signaling pathways in t-AML we first performed phospho-proteomics on FKH1 cells bioinformatically elaborated by the Search Tool for the Retrieval of Interacting Genes (STRING) algorithm in order to evidence signaling pathways activated in t-AML. The findings were then confirmed in other models of DEK-CAN induced leukemia.We used quantitative phospho-proteomics to evidence proteins involved in aberrantly activated signaling pathways in t-positive FKH1 cells in order to generate new hypotheses for further testing via biochemical and cell biological methods. Therefore, we compared FKH1 cells to DEK-CAN-negative U937. The total number of phosphopeptides reported in one run was 5569 . After filtering out the peptides matching to multiple proteins, 5534 phosphopeptides were kept for further analysis corresponding to 3851 unique peptide sequences that carried identical modifications. In this set, serine phosphorylation was detected on 3534 peptides (91.7%), threonine phosphorylation on 492 peptides (12.8%) and tyrosine phosphorylation on 69 peptides (1.8%). For each phosphopeptide, the position of the phosphorylation site(s) in the master protein was determined. The final intensity values were calculated by averaging the intensities across all phosphopeptides that contributed the same phosphorylation sites in the master protein. Of the resulting 3304 phosphoproteins, 1377 were differentially phosphorylated between FKH1 and U937 with a fold change difference of ≥ 1.5 up or down Fig. .Fig. 1ABWe focused on the 40 top upregulated phosphoproteins in FKH1 Fig.  and analConstitutive ABL1 activity plays a crucial role in Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) and AML as well as in Ph-negative MPN, Ph-like ALL and acute Tcell ALL (T-ALL) –22. As cNext, we studied the localisation of ABL1 activity in FKH1 and compared it to that of BCR-ABL1 in K562 cells. U937 cells were used as negative controls. Localisation was assessed by confocal laser scan microscopy (CLSM) using an anti p-ABL1-Y245 antibody (red fluorochrome) and an anti-total ABL1 antibody (green fluorochrome). The integrity of the nucleus was confirmed by DAPI staining. As shown in Fig. To establish activated ABL1 as a survival signal, we investigated the effect of ABL1 kinase inhibition on growth of t-positive cells. Hence, we exposed U937, OCI-AML3, KG1, HL60 together with FKH1 to increasing dosages of imatinib for 5 days. As positive controls, we used BCR-ABL1-positive K562 and Kasumi1 (positive for the c-KIT N822K). Growth arrest was assessed by an XTT cytotoxicity assay. The dosage dependent response of FKH1 cells to imatinib was comparable to that seen in Kasumi1 and K562 cells. All other cells did not respond to dosages of imatinib up to 2 µM Fig. .These data provide evidence for ABL1 kinase activity being indispensable for the growth of t-DEK-CAN-positive FKH1 cells.In order to exclude receptor mediated ABL1 activation, we exposed FKH1 cells to a selection of inhibitors targeting the most important RTKs known in AML . A response in FKH1 cells was seen only when the inhibitor was able to target ABL1 activity Fig. .Fig. 2ABAlthough cytogenetically normal, the development of FKH1 cells in time led to a loss of factor-dependency   and K562 (positive control). ABL1 activity was revealed by (i) its autophosphorylation detected by antibodies against pABL1-Y245 and total-ABL1; and (ii) its substrate phosphorylation of STAT5 detected by pSTAT5-Y694, and-total STAT5. As shown in Fig. To further exclude that DEK-CAN is able to induce ABL1 activity, we studied U937 cells stably expressing DEK-CAN. As shown in Fig. Although ETV6-ABL1-driven neoplasms are well responsive to ATP-competitive TKI such as imatinib, nilotinib and dasatinib, nothing is known to the best of our knowledge about the effects of allosteric inhibition on ETV6-ABL1. Differently to ATP-competitors, allosteric inhibitors inhibit the enzyme activity from outside the active center. The first in class allosteric inhibitor for ABL1-kinases is ABL001 (asciminib). It has been developed starting from GNF2 and 5 . TherefoThe presence of an ETV6-ABL1 activity complicates the use of FKH1 as a model for t-AML and requires confirmation by other models independent of ETV6-ABL1. One characteristic of t-AML is a strong STAT5 activity as revealed by proteomics on over 500 AML-patients . STAT5 aFirst, we studied the capacity of DEK-CAN to phosphorylate STAT5. Therefore, we investigated STAT5 in primary syngeneic AMLs by comparing DEK-CAN-, PML-RARα- and RUNX1-ETO-induced leukemia. STAT5 activation was assessed by its phosphorylation detected by an anti-pSTAT5-Y694 antibody in IFC assays. Here, we show that STAT5 was phosphorylated not only in human t-AML  to a higSTAT5 activation can be triggered also by SFK and an involvement of activated members of SFK family in the regulation of STAT5 was already predicted by our phospho-proteomics and STRING analyses Fig. . DasatinNext, we studied the respective contribution of ABL1 and SFKs to the regulation of STAT5 by a sort of subtractive approach. We reasoned that if both, ETV6-ABL1 and SFKs were involved in the activation of STAT5 in FKH1 cells, the combined inhibition of ABL1 by imatinib and SFK by PP2 would have a stronger effect than the selective ABL1 inhibition alone. As a control we used BCR-ABL1-positive K562. Here we show that in FKH1 cells the combination of imatinib and PP2 had a stronger effect on the pSTAT5 signal than imatinib alone, whereas in K562 PP2 did not add to the effect of imatinib alone Fig. .These findings provide evidence that in FKH1 STAT5 activation depends on both ETV6-ABL1 and SFK activation, whereas in K562 it depends on BCR-ABL1.In order to understand the significance of SFK activation for the phenotype of FKH1 cells, we wanted to answer the question of whether SFK inhibition by PP2 would interfere with the growth of these cells. We exposed FKH1 cells to the SFK inhibitor PP2 and showed that their growth assessed by XTT was inhibited in a dosage dependent manner. Only Kasumi1 cells were more sensitive than FKH1 Fig. .Fig. 4SRTKIs used for the treatment of Ph + leukemia such as dasatinib are dual ABL1/SFK inhibitors that combine in one compound the inhibition of two kinases shown to be indispensable for the growth of FKH1 cells. Thus, we exposed FKH1 cells to increasing concentrations of dasatinib to investigate its effect on the SFKs SRC and LYN, as well as on STAT5. SFK and STAT5 activation was addressed by their autophosphorylation. As controls, we used U937 and HEL cells. HEL are dependent on JAK2-V617F, Thus, we inhibited the JAK2/STAT5 axis in these cells by ruxolitinib as a control. Dasatinib suppressed SFK LYN and SRC as well as the activation of STAT5 Fig. .As nothing is known about the relationship between DEK-CAN and SFK we investigated the effects of dasatinib on the growth of t-positive leukemia in three different models: FKH1 cells, t-positive leukemic blasts and syngeneic DEK-CAN AML cells . Growth As DEK/CAN was not able to activate ABL1 kinase, we attributed the effects of dasatinib mainly to its ability to inhibit SFK members in these models. Therefore, we conclude that SFK activity contributes to the leukemic phenotype induced by DEK-CAN.Another feature in t-AML is the activation of mTOR signaling by DEK-CAN . In ordeTo confirm AKT phosphorylation via mTOR activation (mTORC2) and not via PI3K, we exposed FKH1 cells to three pharmacological inhibitors with distinct inhibitory properties: Torin1, a combined mTORC1/C2 inhibitor, NVP-BEZ235 (BEZ), a dual PI3K and mTORC1/C2 inhibitor, and NVP-BKM120 (BKM), a selective PI3K inhibitor. Only those compounds with mTOR inhibiting activity (Torin1 and BEZ) suppressed the activation of the AKT/S6K cascade in FKH1 cells Fig. .Activation of mTOR signaling is central for cell survival, cell growth and proliferation . To deteThese data clearly showed that mTOR signaling is crucial for the survival of t-AML.Recently, a direct relationship between SFK and mTOR signaling has been described . In factThe functional relationship between LYN and mTOR signaling raised the question whether this could be exploited for a therapeutic approach in order to improve treatment of patients with t-AML. Therefore, we investigated the effects of mTOR- and SFK-inhibition alone and in combination on primary t-AML blasts from two different patients. Once again, we used dasatinib as SFK inhibitor and BEZ as mTOR inhibitor. Both SFK and AKT/mTOR inhibitors alone strongly reduced growth, whereas the combination had only slight if any additive effect on the growth of t-AML blasts from both patients Fig. . The lacTaken together these data identify SFK and mTOR signaling as as closely related but valid only as individual nodes for therapeutic targeting in a network of activated signaling pathways characterising DEK-CAN-positive AML cells.We provide evidence for an interplay of activated signaling pathways in t-AML that are depending on the expression of the DEK-CAN. Our findings show that at least STAT5, mTOR, and SFKs are involved in this interplay of signaling pathways creating a network with nodes that represent targets for molecular therapy approaches , 15.Until now, the main focus regarding aberrant kinase signaling in AML lied on receptor tyrosine kinases (RTKs), e.g. c-KIT or FLT3 and related signaling pathways . This leFKH1 cells have to be carefully evaluated as a model for t-AML because of the presence of ETV6-ABL1. ETV6-ABL1 is the result of the t that in most of the cases is cryptic. Its presence in FKH1 cells is most likely due to the fact they were derived from a t-AML developed from a previous Ph- MPN . The rolSTAT5 activation is highly significant for the survival of t-AMLs and therefore its regulation is critical. Interestingly, the axis JAK2/STAT5 seems to be interrupted as ruxolitinib did not show any effect on activated STAT5 in t-AML -AML and thus to the poor prognosis of t-AML patients. Accumulating evidence suggests that SFKs are activated in AML and contribute to stemness, cell survival and drug resistance –41 whichDoes DEK-CAN need co-factors such as ETV6-ABL1 for the induction and maintenance of AML? A high proportion of t patients harbour FLT3-ITD , which aHere, we present data that will modify current concepts of pathogenesis of AML. Our findings suggest that at least for certain subgroups of AML some of the mutations, such as DEK-CAN or PML-RARα are able by themselves to aberrantly activate signaling pathways essential for the survival and the biology of the respective AML cell. Interestingly, there is no need for these factors to be mutated in order to be constitutively activated. This provides further evidence for the need of analyses such as phospho-proteomics during the development of targeted therapies to understand not only the activation status of the entire signaling structure within a tumor cell but also the effects of an inhibitor on this network and potential positive feed-back effects that might lead to drug resistance.Taken together our here presented data will modify the design of molecular therapy by suggesting the need for a broader approach aimed to include not only one but more signaling pathways, which eventually requires a well-studied combination of inhibitors.www.selleckchem.com). For all compounds 1000 × stock solutions were obtained by dissolving the compounds in dimethyl sulfoxide (DMSO)(Sigma) then further diluted to working concentrations in the corresponding cell culture medium or semi-solid medium prior to use.All inhibitors used in this study were purchased from Selleckchem (2+) [All cell lines  were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany, and were kept in RPMI-1640 medium supplemented with 10% FCS except for FKH1 and Kasumi-1 cells which were maintained in 20% FCS. The U937 clones were created either by M Ruthardt’s laboratory or a kind gift from P.G. Pelicci : MTB45 (empty vector-control), PR9 (expressing PML-RARa), RUNX-1/ETO (expressing HA-RUNX-1/ETO), BCR/ABL or DEK/CAN (expressing HA-DEK/CAN)(10). All these cells expressed their respective transgene under the control of MT-1 promoter inducible by 100 µM ZnSO4 (Zn2+) .2 and thawed for further analysis were obtained from leukemic mice injected with primary murine HSPCs transduced with leukemia-inducing oncogenes  as described elsewhere [Different types of primary syngeneic leukemic cells stored in liquid Nlsewhere , 15. As lsewhere , 15.Primary human AML samples were obtained with informed consent and used in agreement with the Declaration of Helsinki upon the approval of the local ethic committees of the Goethe University Frankfurt . Samples were maintained in X-Vivo10 medium (Lonza) supplemented with 10% FCS (Hyclone/Perbio Science), with the addition of 20 ng/ml hIL-3, 50 ng/ml hSCF, 25 ng/ml hTPO and 50 ng/ml hFLT3-ligand .©, Phospho-Akt Pathway Antibody Sampler Kit©, anti-pSTAT5-Y694, anti-pABL1-Y245 and Y412, anti-pSRC-Y416, anti-SRC, and anti-pLCK-Y505 . Anti-ABL1, anti-STAT5 (Santa Cruz Biotechnology); anti-pLYN-Y396 (Abcam), anti-LYN (BD Biosciences), anti-α-Tubulin (Lab Vision). Blocking and antibody incubation were performed in 5% low-fat dry milk (Carl Roth). Washing was performed in Tris-buffered saline containing 0.1% Tween20 (TBS-T) followed by incubations either with secondary Ab coupled with horseradish-peroxidase for staining with enhanced chemiluminiscence substrate or the IRDye R© 800 and 680 goat anti rabbit or anti mouse ABs (LI-COR Biosciences). Blots were “stripped” using RestoreWestern blot Stripping Buffer© (Perbio Science). Imaging and elaboration were performed with the LI-COR Odyssey Fc system (LI-COR Biosciences).Immunoblot analyses were performed according to widely established protocols. The following kits and antibodies were used: mTor Substrates Antibody Sampler Kit5 cells/sample were fixed with Cytofix buffer (BD Biosciences) according to the manufacturer’s protocol. Permeabilisation was performed with ice-cold 90% methanol for 30 min. Cells were then incubated with the primary antibody pSTAT5-Y694-AlexaFluor 647 (BD Biosciences), pAKT-S473-PE-Vio770, pCRKL-Y207-APC or IgG control for 40 min at RT (Miltenyi). For ABL1 staining, the cells were incubated with the non-labelled pABL1-Y245  followed by secondary goat anti-rabbit IgG AlexaFluor-488 Fab2 . Washing was performed with PBS containing 1% FCS and 0.1% sodium azide. Fluorescence was measured immediately after staining on a FACS Fortessa (BD Biosciences).5 × 10Cells were cultured over-night on poly-D lysine covered chamber slides (Corning), washed with TBS , fixed in 4% paraformaldehyde (AppliChem) for 15 min, and permeabilised with 0.1% Triton-X in TBS. Blocking was performed in 3% (w/v) BSA (Sigma) and 0.1% Tween20 in TBS for 60 min. Cells were incubated with polyclonal rabbit anti–pABL1 (Y245) or monoclonal anti–c-ABL (Santa Cruz) followed by. Alexa Fluor 647-conjugated goat anti-rabbit or anti-mouse AlexaFluor-488 Fab2 Ig antibodies (Life Technologies). Nucleus staining was obtained using DAPI (Life Technologies). The slides were mounted with Moviol (Sigma). Images were acquired by a Leica TCS-SP5 confocal microscope (Leica Microsystems) under identical conditions for pinhole opening, laser power, photomultiplier tension and layer number. Identical parameters were applied for all samples during data elaboration by Imaris (Oxford Science). Final picture elaboration  was performed with open access GIMP-software.Proliferation was assessed by using the XTT proliferation kit according to the manufacturer’s instructions (Roche).For phosphoproteomic analysis the FKH1 and the U937 cells were splitted at a density of 250,000 cells/ml and allowed to rest overnight before lysis.Aliquots of 100 µg of up to 10 samples per experiment were digested with trypsin , labeled with TMT ten plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific) and the labeled samples pooled. This pooled sample was then desalted using a SepPak cartridge (Waters) and subjected to TiO2-based phosphopeptide enrichment according to the manufacturer’s instructions (Pierce). The phospho-enriched sample was evaporated to dryness and then resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific).−1. Solvent A was 0.1% formic acid and solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionised by nano-electrospray ionisation at 2.0 kV using a stainless-steel emitter with an internal diameter of 30 μm and a capillary temperature of 275 °C. All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 2.0 software (Thermo Fisher Scientific) and operated in data-dependent acquisition mode using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120,000, with an automatic gain control (AGC) target of 200,000 and a max injection time of 50 ms. The TopN most intense ions were selected for MS/MS. Precursors were filtered according to charge state (to include charge states 2–7) and with monoisotopic precursor selection. Previously interrogated precursors were excluded using a dynamic window (40 s ± 10 ppm). The MS2 precursors were isolated with a quadrupole mass filter set to a width of 1.2 m/z. ITMS2 spectra were collected with an AGC target of 5000, max injection time of 120 ms and CID collision energy of 35%. For FTMS3 analysis, the Orbitrap was operated at 60 000 resolution with an AGC target of 50,000 and a max injection time of 120 ms. Precursors were fragmented by high energy collision dissociation (HCD) at a normalised collision energy of 55% to ensure maximal TMT reporter ion yield. Synchronous Precursor Selection (SPS) was enabled to include up to 5 MS2 fragment ions in the FTMS3 scan.The TMT-labelled phospho-enriched sample was fractionated using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). In brief, peptides in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific). After washing with 0.5% (vol/vol) acetonitrile 0.1% (vol/vol) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Thermo Fisher Scientific) over a 150-min organic gradient, using 6 gradient segments  with a flow rate of 300 nl minThe raw data files were processed and quantified using Proteome Discoverer software v2.1 (Thermo Scientific) and searched against the Uniprot Human database  using the SEQUEST algorithm. Peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 0.6 Da. Search criteria included oxidation of methionine (+ 15.9949) and phosphorylation of serine, threonine and tyrosine (+ 79.966) as variable modifications and carbamidomethylation of cysteine (+ 57.0214) and the addition of the TMT mass tag (+ 229.163) to peptide N-termini and lysine as fixed modifications. Searches were performed with full tryptic digestion and a maximum of 1 missed cleavage was allowed. The reverse database search option was enabled and all peptide data was filtered to satisfy false discovery rate (FDR) of 5%. All additional bioinformatic elaboration was performed in Rstudio.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031429.www.uniprot.org). STRING acts as a meta-database that examines the potential of both physical and functional protein–protein interactions. The outcome is based on information from numerous sources, including experimental repositories, computational prediction methods and public text collections.For the analysis, the STRING package provided on the uniprot website was used , mIL-6 (20 ng/mL) and mSCF (100 ng/mL) (Stem-Cell Technologies). On day 10 after plating, the number of colonies was determined. Freshly thawed bone marrow cells derived from t-positive AML patients were cultured in semi-solid medium  in the presence of the indicated compounds for 14 days, and the colony number was determined in comparison to the untreated samples.The statistical analysis of the proliferation inhibition results was performed using GraphPad Prism9. The dose–response curves were fitted using the nonlinear regression model and the log(inhibitor) vs normalised response-variable slope function. The comparison between groups was performed using one-way analysis of variance and the non-parametric Kruskal–Wallis test. The multiple comparisons between conditions were corrected by controlling the false-discovery rate using the Benjamin-Hochberg method.Supplementary file1 (PDF 270 KB)Below is the link to the electronic supplementary material.


AF susceptibility was assessed in male (M) and female (F) mice (4–5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca2+ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca2+ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca2+ transient was faster in males. Males had 54% higher Na+-Ca2+ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca2+ current (ICaL) was recorded with and without BAPTA, a Ca2+ chelator. ICaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca2+-dependent inactivation in males. CaV1.2 expression was similar between sexes. This study reports major sex differences in Ca2+ homeostasis in mouse atria, with larger Ca2+ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca2+ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. The prevalence of AF is approximately 1–2% in the general population and increases to nearly 10% in elderly individuals ,4. AF isTogether with ageing and genetic predispositions, male sex is one of the major risk factors of AF ,10,11,122+ properties. The remodeling of the atria is a critical process involved in the initiation and maintenance of AF  of 150 nM. The voltage protocol consisted of a holding potential of −40 mV with a ramp from +80 to −120 mV over 2 s. Cells were perfused with a solution containing (in mM): 140 NaCl, 10 TEA-Cl, 12 HEPES, 0.5 MgCl2, 2 CaCl2, and 10 glucose (pH adjusted to 7.4 with NaOH). A MPRE8 Multitube Preheater  was used to ensure rapid and local change of perfusion solutions. A baseline current was recorded in the following bath solution (in mM): 140 NaCl, 10 TEA-Cl, 12 HEPES, 0.5 MgCl2, 4 CaCl2, 1 BaCl2, 0.02 Nifedipine, and 0.005 Ryanodine (pH adjusted to 7.4 with NaOH). Then, the current was recorded again after adding 5 mM of nickel chloride (NiCl2) to obtain INCX as the nickel-sensitive current. High concentrations of internal Na+ and external Ca2+ were used to maximize electrophysiological gradients of NCX1 to generate currents of greater amplitude to compensate for the lower cellular capacitance of atrial myocytes compared to ventricular myocytes [r et al. . INCX wamyocytes ,57.CaL.I Protocols used to record ICaL were adapted from Bögeholz et al. [2. Cells were voltage-clamped in whole-cell configuration using borosilicate glass pipettes filled with (in mM): 120 CsCl, 10 TEA-Cl, 10 NaCl, 20 HEPES, 5 MgATP, and 0.05 cAMP (pH adjusted to 7.2 with CsOH). When specified, BAPTA (10 mM) was added to the pipette solution to buffer intracellular Ca2+. From a holding potential of −80 mV, a 50 ms pre-pulse at −50 mV was applied followed by a series of 250 ms voltage steps ranging from −50 to +60 mV in 5–10 mV increments, at a frequency rate of 0.1 Hz. ICaL was defined as the difference between the peak current and that measured at the end of the 250 ms test pulse. Protocols used to study ICaL kinetics are provided in the z et al. . Briefly2+ transients from mouse atrial myocytes were recorded using adaptation of previously published protocols [2+ Tyrode’s solution for 10 min, and then stimulated for an additional 5 min before starting the recording session. Each recording consisted of 10 s of pacing at a frequency of 2 Hz using field stimulations, 10 s without pacing, and 5 s of local perfusion with Ca2+-free Tyrode’s solution containing caffeine (10 mM) via a rapid solution switcher (MPRE8 Multitube Preheater Cell MicroControls). The recording lasted a total of 40 s to allow complete decay of the caffeine-induced transient. Data were acquired at a sampling rate of 100 Hz. Fluo-4 was excited at 489 nm and emission was read at 505 nm. Data were extracted using Zen Black 2.0 software  and was analyzed manually using Clampfit 10.7 software . Carotocols ,58. For 2+ releases were analyzed manually. The inclusion criterion for spontaneous systolic Ca2+ release was a spontaneous increase in fluorescence during the decay phase of a Ca2+ transient during stimulation at 2 Hz. Spontaneous diastolic Ca2+ releases were defined as transient increases in fluorescence, clearly distinguishable from noise level when cells were unstimulated. Each spontaneous event was confirmed by video visualization using Zen Black 2.0 software .Spontaneous systolic and diastolic CaHypoxanthine Guanine PhosphoribosylTransferase 1 (Hprt1). The relative expression was calculated using the 2-∆∆Ct method. Murine gene-specific primers used for qPCR reactions are presented in Total RNA extraction and qPCR were conducted using previously published protocols . Total RSLC8A1 and CACNA1C in atrial samples from 5 men and 6 women with no history of cardiac diseases. Gene expression was normalized to the housekeeping gene HPRT1. Human gene-specific primers used for qPCR reactions are presented in The protocol used to isolate total RNA from mouse atria was also used on human samples. Atrial biopsies were collected from explanted hearts after transplantation and provided by the human tissue bank of the Montreal Heart Institute (project #2007-43). We quantified the mRNA expression of V1.2, proteins were transferred overnight at RT at low voltage (10 V) onto a polyvinylidene difluoride (PVDF) membrane. The membrane was fixed for 30 min at RT in the following buffer: 70% methanol, 29% acetic acid, and 1% glycerol. The membrane was then blocked in Tris-buffered saline Tween-20 solution (TBST) containing 5% non-fat dry milk for 4 h, at RT, and then incubated overnight at 4 °C with primary antibody . For NCX1, the electrophoretic separation was followed by a 90-min transfer at 350 mA onto PVDF membrane at 4 °C. The membrane was blocked for 90 min with TBST containing 5% BSA at RT and then incubated overnight at 4 °C with mouse anti-NCX1 antibody . The lower sections of both CaV1.2 and NCX1 membranes were cut, and independently blocked with TBST/5% non-fat dry milk and incubated with anti-GAPDH . Following three 5-min washes, all membranes were incubated in secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse) in TBST/5% non-fat dry milk for 90 min at RT. After 3 additional TBST washes, bands of interest were detected using enhanced chemiluminescence reagents  and visualized in film-free chemiluminescence using ChemiDoc apparatus . Densitometry analysis was performed using Bio-Rad Image Lab 6.1. The expressions of CaV1.2 and NCX1 were normalized to their respective loading controls, GAPDH.Protocols used for the isolation of the total protein fractions and Western blot analysis were adapted from our previously published papers ,59. Brie2+ transient experiments, n indicates the number of cells and N represents the number of mice. The compared groups were tested for equality of variance. Unpaired two-tailed Student’s t-tests were used for numerical data, whereas the chi-squared test was used for categorical data such as EPS data and spontaneous Ca2+ releases. Statistical analysis was performed using GraphPad Prism 9.4 software . p-values inferior to 0.05 were considered statistically significant.Results are presented as mean ± standard error of the mean (SEM) unless specified otherwise. For patch-clamp and Ca2+ handling regulation associated with higher NCX1 expression and function and more frequent spontaneous Ca2+ releases in males. Together, these results suggest that males are more prone to developing AF due to increased triggered activity. Equally important, these sex differences were observed independently of risk factors such as aging and comorbidities.This study showed that, as in humans, male mice have a greater vulnerability to AF. Moreover, we identified important sex differences in Ca


Doxorubicin-induced cardiomyopathy (DICM) is one of the complications that can limit treatment for a significant number of cancer patients. In animal models, the administration of statins can prevent the development of DICM. Therefore, the use of statins with anthracyclines potentially could enable cancer patients to complete their chemotherapy without added cardiotoxicity. The precise mechanism mediating the cardioprotection is not well understood. The purpose of this study is to determine the molecular mechanism by which rosuvastatin confers cardioprotection in a mouse model of DICM.Rosuvastatin was intraperitoneally administered into adult male mice at 100 μg/kg daily for 7 days, followed by a single intraperitoneal doxorubicin injection at 10 mg/kg. Animals continued to receive rosuvastatin daily for an additional 14 days. Cardiac function was assessed by echocardiography. Optical calcium mapping was performed on retrograde Langendorff perfused isolated hearts. Ventricular tissue samples were analyzed by immunofluorescence microscopy, Western blotting, and quantitative polymerase chain reaction.P < 0.001) and re-expression of the fetal gene program. However, we found no evidence of maladaptive cardiac hypertrophy or adverse ventricular remodeling in mice exposed to this dose of doxorubicin. In contrast, rosuvastatin-doxorubicin-treated mice maintained their cardiac function . Mechanistically, the effect of rosuvastatin was associated with activation of Akt and phosphorylation of phospholamban with preserved sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (SERCA2)-mediated Ca2+ reuptake. These effects occurred independently of perturbations in ryanodine receptor 2 function.Exposure to doxorubicin resulted in significantly reduced fractional shortening (27.4% ± 1.11% vs 40% ± 5.8% in controls; Rosuvastatin counteracts the cardiotoxic effects of doxorubicin by directly targeting sarcoplasmic calcium cycling. Perturbations in Ca2+ homeostasis are important key events in the failing heart.2+-calmodulin-dependent protein kinase 2 (CaMKII). PKA and CaMKII phosphorylate multiple targets that are important in Ca2+ handling, particularly phospholamban (PLN).2+ transporting 2 (SERCA2) to enhance Ca2+-reuptake into the SR.Cardiac performance relies on the balance between Ca2+ handling by Dox has been observed in animal models of DICM.2) to disrupt Ca2+ transfer between the SR and the cytoplasmic compartments.,2+ handling and the signaling transduction pathways involved in Ca2+ regulation. Herein, we present data that demonstrate that the protective effect of pretreatment with statins is mediated through the maintenance of the Akt/SERCA2 axis for calcium handling, despite exposure to Dox. This effect happens independently of RyR2.Dysregulation of Caad libitum) for 6 hours and given 1 U/kg insulin , i.p. Hearts were isolated 5 or 10 minutes post-insulin administration. Detailed experimental procedures, including echocardiography, confocal immunofluorescence microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Western blotting, densitometry, antibodies, oligonucleotides, and real-time reverse transcriptase quantitative polymerase chain reaction, were performed as described in the All animal usage was in accordance with our institutional animal care guidelines . Male C57BL/6J mice  aged 10-12 weeks (22-26 g) were treated with a single dose of Dox . In a subgroup of mice, rosuvastatin  was given 1 week before Dox at 10 μg/kg per day (10R) or 100 μg/kg per day (100R) i.p. for a further 2 weeks, followed by sacrifice. To study short-term signaling events, mice were starved of food  was added slowly over 5 minutes to the circulating perfusion solution for the optical recording of myocardial calcium dynamics. To minimize motion artifacts, a mechanical uncoupler, blebbistatin  was then added slowly over 5 minutes to the perfusion solution. We proceeded with optical mapping at different pacing rates after an additional 3-5 minutes. Dye fluorescence was stimulated using a xenon light source  and a 530-nm green filter  with the emission light bandpass filtered at 585/40 nm. A high-speed CMOS camera  was used to record dye fluorescence at a sampling rate of 500 frames/second. Image size was 16 mm × 16 mm with a 100 × 100-pixel resolution.Mice were deeply anesthetized with 2%-3% isoflurane, and heparin was injected i.p. Through a mid-transthoracic incision, hearts were extracted. Isolated murine hearts were placed into ice-cold modified Krebs-Henseleit solution containing the following: NaCl, 118 mM; KCl, 4.7 mM; CaCl2+ signals to assess the following calcium transient dynamics.Optical mapping of ventricular epicardial calcium signals was performed using a paced-paused protocol with a Grass S88X stimulator to pace the Langendorff-perfused heart for 30 seconds at 11-13.5 Hz. Fluorescence was recorded at the end of the pacing maneuver. Optical signals recorded during the final 2 seconds of pacing and the first 2 seconds of spontaneous rhythm were analyzed. From an area on the optical maps representing the ventricle, 3 pixels were selected for a feature analysis of Ca2+ amplitude alternans ratio was calculated as y/x, where x is the amplitude of higher and y is the amplitude of smaller Ca2+ transient amplitude. This measure is unitless, and the absence of alternans is indicated by a ratio of 1. The Ca2+ amplitude alternans ratio is an indicator of RyR2 function.The Ca2+ elevation, which is an indicator of RyR2 dysfunction2+ amplitude during pacing and expressed as an arbitrary unit (AU).Diastolic Ca2+ transient durations (CaTD) were measured from 0% to 50% of repolarization (CaTD50) and from 0% to 80% of repolarization (CaTD80), and were expressed in msec, as described previously.As an indicator of cytosolic calcium extrusion,P values of < 0.05 to be significant. When groups passed the normality test, data evaluation between 2 groups was performed by an unpaired Student t-test. The statistical significance of 3 groups was calculated using analysis of variance (ANOVA) and the Tukey-Kramer multiple comparison post-test. Additional 2-group ANOVAs were performed between all 2-group combinations when 3-group ANOVA was found to be significant. P values were derived from 2-way ANOVA performed for global comparison between groups.All statistical analyses were performed employing GraphPad InStat (version 3.1) and GraphPad Prism . All data are reported as means ± standard error of the mean (SEM). We considered 2-tailed P < 0.001; Several murine models of DICM have been independently reported, with differences in dose and treatment regimens of Dox administered (5-20 mg/kg). When C57BL/6 mice were treated with 20 mg/kg Dox i.p., they developed a significant reduction in fractional shortening (FS) and they succumbed within 1 week (data not shown). In contrast, mice treated with a single injection of 10 mg/kg Dox, a dosage with a relevant LD50,With the lower FS, no evidence was seen of hypertrophic growth or fibrosis in mice treated with Dox or statin-Dox, in comparison to the control group , E-I. HoIntercalation of Dox into DNA results in double-strand breaks that activate the DNA damage response (DDR). This effect can be evaluated by measuring the phosphorylation status of histone variant γH2AX. We found that Dox treatment led to phosphorylation of Serine at position 139 of γH2AX in cardiomyocytes within 3 days, which resolved by 14 days . This ob,,2+ cycling, ex vivo. We observed a significant decrease in the calcium amplitude alternans ratios in Dox-treated hearts compared to controls (P = 00.017). Furthermore, spontaneous calcium elevations were significantly increased in Dox-treated animals (P = 0.007). Together, these results revealed a Dox-mediated defect in calcium release via the RyR230 that is not prevented by rosuvastatin pretreatment.Given that DICM has been associated with disrupted calcium handling ,26,28,3550) and 80% (CaTD80) of maximum were measured. Dox induced a significant prolongation of CaTD50 (P = 0.004) and CaTD80 (P = 0.129), as compared to control (P = 0.001) and CaTD80 (P = 0.002) were significantly lower in the statin-Dox-treated group compared to the Dox-treated group /Akt/mammalian target of rapamycin (mTor) signaling involved in regulating Ca2+ uptake.PLN is a downstream target of Akt and may be directly phosphorylated by Akt independently of PKA and CaMKIIδ activation through SR translocation of Akt leading to SR Ca2 and SERCA2 pathways. Our key finding is that statin pretreatment was able to preserve the Akt/PLN/SERCA2 signaling axis, independent of RyR2, to maintain normal cardiac function. Although DICM has been studied widely, many basic molecular and morphologic questions remain unanswered. In our model, despite activation of the DDR following doxorubicin treatment, cardiomyocyte apoptosis was not observed post-Dox treatment. We concluded that impaired heart function in our model was not driven by cardiomyocyte loss, as reported in other studies, in which even a very low degree of cardiomyocyte apoptosis was sufficient to cause a dilated cardiomyopathy.,,Our model of DICM features a sustained decrease in cardiac function with activation of the DDR in cardiomyocytes, and disruptive Akt/PLN signaling leading to impaired calcium handling through RyR,,Given how well statins are broadly tolerated and the growing need to determine the best therapies in the treatment of DICM, interest in the use of statins in treating DICM has been increasing. Although earlier animal studies using statins showed protection against DICM,2 and SERCA2, which are both vital for normal heart function.,2 and SERCA2 in DICM has been previously shown, although which plays a more principal role remains unclear.,2 and SERCA2 are present post-Dox treatment. However, statin pretreatment helps to protect against SERCA2 abnormalities to preserve calcium handling, independent of RyR2 as illustrated in The dynamic release and reuptake of calcium from the SR stores are dependent upon RyR,2+ flux is not beneficial.2+ reuptake, despite the persistence of cardiac alternans and suppressed RyR2 protein expression, which could have a long-term impact on heart function. Our study leaves open the possibility that statins could be used in combination with a therapy that specifically targets RyR2 to fully restore normal Ca2+ handling in the heart. One limitation of our study is that SR calcium load and L-type calcium current, which may play an important role in DICM, were not assessed. In addition, only male mice were assessed. Future work will address the potential for sex-specific differences.Canonical inactivation of PLN suppression on SERCA2 is mediated by step-wise phosphorylation of PLN by PKA at Ser16 followed by phosphorylation at Thr17 by CaMKIIδ.Although other molecular targets have been identified, effective therapies to prevent DICM have yet to be identified. Thus, DICM, once itdevelops, is widely accepted to be irreversible or only partially reversible in the majority of patients.,,,Intact heart calcium transient analysis may provide limited mechanistic insight. Single-cell patch clamping will be ideal for exploring the precise mechanism; this analysis is beyond the scope of this study. However, studies have demonstrated that calcium transient analysis in intact heart could be a surrogate of intracellular calcium dynamics.


Rabbits are being increasingly used as companion animals, and in research; thus, the need for proper veterinary care for rabbits has increased. Surgical access is more challenging in rabbits under inhalation anesthesia compared to other animals, such as dogs and cats. Rabbits have a very narrow and deep oral cavity, large incisors, and a large tongue. Moreover, their temporomandibular joint has limited mobility, making it more difficult to approach the larynx. Various methods have been proposed to overcome this difficulty. The video laryngoscope was introduced in 1999 and is useful when airway intubation is unsuccessful using a conventional laryngoscope. We postulated that a video laryngoscope with a modified size 1 Macintosh blade  would facilitate the intubation of New Zealand White rabbits. Sixteen specific-pathogen-free male New Zealand White rabbits weighing 3.45–4.70 kg were studied. All rabbits were intubated using the video laryngoscope. Typically, a 3.0 mm endotracheal tube was used for rabbits weighing < 4 kg, while a 3.5 mm tube was used in those weighing > 4 kg. During surgery, anesthesia was well maintained, and there were no major abnormalities in the animals’ conditions. No rabbit developed breathing difficulties or anorexia after recovering from anesthesia. We established an intubation method using a video laryngoscope with a modified blade and stylet in the supine  position and successfully applied it in 16 rabbits. It is useful for training novices and for treating rabbits in veterinary hospitals with few staff members and animal research facilities where there are insufficient human resources.The online version contains supplementary material available at 10.1186/s42826-022-00130-7. Rabbits are popular companion animals worldwide, and are also important for research studies , 2. The Video laryngoscopes (VLs) have been widely used since their development in 1999 for patients in whom conventional laryngoscope (CL) intubation is difficult. Simple, easy-to-use VLs are now available at a comparatively low cost , and canWe postulated that a VL with a modified size 1 Macintosh blade  would facilitate the intubation of New Zealand White rabbits.Sixteen specific pathogen-free male New Zealand White rabbits weighing 3.45–4.70 kg were used . The animals were housed individually in stainless steel cages that could be auto-washed and auto-watered and maintained on a 12 h light–dark cycle (lights off at 8 pm). The room temperature was 18–26 °C, and humidity was 50 ± 10%. The food was Lab Rabbit Chow; antibiotic-free/gamma sterilized diet  and tap water were offered ad libitum. A mineral block , wood block , hay, and fresh organic vegetables were provided for environmental enrichment.The VL intubation technique was developed in six rabbits during a rabbit practical training workshop held by the Laboratory Animal Research Center (LARC), Institute of Biomedical Industry and Department of Laboratory Animal (DOLA), College of Medicine, The Catholic University of Korea. Then it was used to intubate 10 rabbits scheduled for cardiovascular, orthopedic, and gastrointestinal surgery (3 rabbits each) and plastic surgery (1 rabbit).All surgical interventions and pre- and post-surgical animal care were conducted in accordance with the Animal Protection Act, Laboratory Animal Act, and Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine, Catholic University of Korea Songeui Campus .The IACUC and Department of Laboratory Animals of at the Catholic University of Korea (Songeui Campus) were accredited as a Korea Excellent Laboratory Animal Facility (No. 10) by the Korea Ministry of Food and Drug Safety in 2017, and acquired full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care International in 2018.Figure Shiley Hi-Contour Oral/Nasal endotracheal (ET) tubes  with inner diameters (IDs) of 3.0, 3.5, and 4.0 mm and respective outer diameters (ODs) of 4.3, 4.9, and 5.6 mm were used without being cut . A 10Fr stylet  was inserted inside the 3.5 and 4.0 mm ID ET tubes and a 10Fr stylet with the rubber covering stripped from the distal 60% of the stylet using long-nose pliers was used for the 2.5- and 3.0 mm ID ET tubes. Then the stylet was bent approximately 90° at the junction with the ET tube connector and the part inside the ET was bent into a gentle curve  or hydrogen peroxide .The ET tube and stylet were selected according to bodyweight, but smaller sizes were used if it was difficult to pass the tube through the vocal fold during intubation Fig.  8]. The. The8]. 2) was detected on a monitor  intubated six of the rabbits using a VL in a practical techniques workshop about handling, drug administration, blood collection, anesthesia and pre-, peri-, and postoperative care. Subsequently, three LAVs at LARC applied the method and equipment Fig.  to 10 ra2, PE´CO2, and body temperature were recorded every 10–15 min during surgery  was injected using a 1 mL syringe with a 26G needle. After 10–15 min, xylazine  was administered using a 1 mL syringe. Once the animal had been sedated, it was preoxygenated using a face mask, and the marginal ear vein was cannulated using a 24G intravenous (IV) catheter . Then a Luer-Lok adapter  was attached to the IV catheter and flushed with 0.9% normal saline . Next, ketoprofen  or tramadol  was injected as analgesia depending on the surgery. Cefazolin  was given as an antibiotic. Ketamine  or tiletamine and zolazepam  were administered to induce anesthesia. Inhalation isoflurane  or sevoflurane  was used as the inhalation anesthetic in 10 rabbits undergoing various surgeries. Eye ointment  was applied after inducing anesthesia to prevent the cornea from drying. Between drug injection (1–2 mL IV) and fluid therapy (5–10 mg kg–1 h–1 IV) during surgery, 0.9% normal saline was administered slowly [Blood was analyzed before surgery. Local anesthetic cream  was applied to the marginal ear vein of each rabbit 15–20 min before cannulation. Then glycopyrrolate , body temperature, and noninvasive blood pressure (NIBP) were monitored during the surgery in 10 rabbits; an electrocardiogram (ECG) was also performed [The PE´COerformed , 12.t-tests used for the statistical analysis in this study. A p-value < 0.033 was considered significant.GraphPad Prism  was used to perform the one-sample Various complications have been reported during or after anesthesia for rabbit intubation, such as damage to or bleeding of the larynx, laryngeal spasm , 5. MostOnce mastered, a VL can be used for intubation without assistance because the intubation is performed in the supine rather than the sternal position. This is useful for treating rabbits in veterinary hospitals with few staff members or animal research facilities where there are insufficient human resources.To adapt a VL for rabbit intubation, the blade was cut  to an appropriate size for the rabbit mouth under the guidance of a biomedical engineer  described here will be commercialized. We also hope to produce additional modified products for veterinary medicine and research.Additional file 1: Video 1 Intubation with the video laryngoscope.Additional file 2: Video 2 Intubation with the video laryngoscope.


Listeria monocytogenes (L. monocytogenes) infection. Altogether, our findings proposed the beneficial consequences of MSP on rabbits’ growth, gut health, and immunity. After post-experimental infection of rabbits with L. monocytogenes, administration of MSP during the whole rearing period greatly reduced the detrimental impact of infection and consequently renovated efficient rabbits’ production.Weaning is a crucial period associated with great stress and susceptibility to infection, implying adverse impacts on farmed rabbits’ production. Recently, probiotics have been provided as direct microbial feed supplements, which are considered the ideal antibiotic substitutes during pathogenic infections with an emphasis on promoting rabbits’ growth and modulating their immune functions. Therefore, our experiment was carried out to explore the efficacy of multi-strain probiotics (MSP) on rabbits’ growth, molecular aspects, such as nutrients transporters, cytokines, and intestinal integrity, and effectiveness against Listeria monocytogenes (L. monocytogenes) were evaluated. In the feeding trial, 220 rabbits were fed a control diet or diet supplemented with three MSP graded levels. At 60 days of age, rabbits were experimentally infected with L. monocytogenes and the positive control, enrofloxacin, prophylactic MSP (MSPP), and prophylactic and therapeutic MSP (MSPTT) groups were included. During the growing period, MSP at the level of 1 × 108 CFU/kg diet (MSPIII) promoted the rabbits’ growth, upregulated the nutrient transporters and tight-junction-related genes, and modified cytokines expression. Supplementing MSPTT for L. monocytogenes experimentally-infected rabbits restored the impaired growth and intestinal barriers, reduced clinical signs of severity and mortalities, and attenuated the excessive inflammatory reactions. Notably, enrofloxacin decreased L. monocytogenes and beneficial microbial loads; unlike MSPTT, which decreased pathogenic bacterial loads and sustained the beneficial ones. Histopathological changes were greatly reduced in MSPTT, confirming its promising role in restricting L. monocytogenes translocation to different organs. Therefore, our results suggest the use of MSPTT as an alternative to antibiotics, thereby conferring protection for rabbits against L. monocytogenes infection.Multi-strain probiotics (MSP) are considered innovative antibiotics’ substitutes supporting superior gut health and immunity of farmed rabbits. The promising roles of MSP on performance, intestinal immunity, integrity and transporters, and resistance against  Rabbit breeding is becoming important due to the rapid growth rate, superior productive ability, and easily digestible and healthy meat of rabbits . WeaningListeria monocytogenes (L. monocytogenes) is one of the most devious pathogens associated with listeriosis, and is uniquely difficult to monitor [L. monocytogenes, and their meat is a potential source of listerial foodborne pathogens [L. monocytogenes against eradication is due to its capacity to adapt and persist in a variety of environments, its intracellular site, and the weak intracellular diffusion of some antibiotics, which makes it a serious ongoing concern in animal production [L. monocytogenes arises from its ability for invasion, adhesion, and translocation across the intestinal barrier throughout the gastrointestinal phase of infection [L. monocytogenes at the gastrointestinal stage of infection is the best way to restrict pathogen spread to the deeper tissues and avoid consequent lethality. Despite the fact that antibiotics are the treatment of choice for listeriosis, they can disrupt the gut microbiota and impair host defense immunity [Lactobacillus or Bacillus species are commonly used in rabbits’ feed [L. monocytogenes infection over long-term use is very limited. Moreover, the precise MSP mechanism of actions that assess their immune modulation and promising roles in preventing L. monocytogenes induced intestinal barrier dysfunction need further investigation. Keeping the above-mentioned facts in view, the present work was undertaken to investigate, for the first time, the effects of MSP—in comparison with enrofloxacin—on the growth performance and expression of nutrient transporters, cytokines, and tight-junction-protein-related genes and their roles in fighting L. monocytogenes experimental infection in rabbits. monitor . Previouathogens ,12. The oduction ,14. Addinfection . Therefoimmunity . Besidesimmunity ,18,19,20immunity ,22, suchimmunity ,27,28,29ts’ feed . HoweverLactobacillus acidophilus NBIMCC 8242, Bacillus subtilis DSM 17,299 and Enterococcus faecium NBIMCC 8270, were mixed at equal ratios (1:1:1) before being added into the formulated feed. The probiotic strains were grown separately in de Man, Rogosa, and Sharpe broth  at 37 °C for 48 h. After incubation, the cells were centrifuged and then spray-dried before being added to the rabbit feed.Three probiotic bacterial strains, including L. monocytogenes experimental infection in the clinical trial.A total of 220 weaned New Zealand white male rabbits, aged approximately 30 days and having an 802.8 g average body weight, were obtained from commercial rabbit farms. To conduct the experiment, feeding, experimental infection, and clinical trials were formed. A four-week feeding trial was carried out to examine the effect of supplementing the basal diet with three graded levels of the three probiotic bacterial strains at equal ratios and to choose the appropriate and effective dose of the mixed probiotic strains to be used as a prophylactic and/or a novel therapeutic tool for Lactobacillus acidophilus, Bacillus subtilis, and Enterococcus faecium at graded levels—1 × 106 (MSPI), 1 × 107 (MSPII), and 1 × 108 (MSPIII) colony forming units (CFU)/kg diet, respectively. All rabbits were kept in cages, fed pelleted diets, and provided free access to water and feed throughout the experimental rearing period. The diets were formulated in accordance with the nutrient recommendations for rabbits [For this trial, 220 rabbits were randomly allocated into four equal experimental groups; each had 55 rabbits arranged in 5 replicates (11 rabbits each). The rabbits were offered the basal diet (control or untreated group) or fed experimental diets supplemented with a mixture of equal doses of  rabbits . The ingL. monocytogenes. The experimental infection trial was conducted according to the protocol of an earlier study [L. monocytogenes strain previously recovered from clinically-diseased rabbits. The used strain was revitalized in Listeria enrichment broth  at 37 °C for 18 h under microaerophilic conditions. Subsequently, 0.1 mL of the enrichment broth was inoculated for 24 h at 37 °C onto a Listeria-selective agar base supplemented with Listeria-selective supplement . The typical colonies of L. monocytogenes were confirmed by Gram’s stain and biochemical tests including lactose, sucrose and xylose fermentation, catalase, oxidase and urease according to FDA bacteriological analytical manual. The strain was further confirmed via umbrella shaped motility, hemolysis onto sheep blood agar , CAMP test and molecular identification methods. The inoculum suspension was subsequently prepared to obtain a concentration of 107 CFU/mL [L. monocytogenes inoculum. Establishment of L. monocytogenes infection was verified by observing the characteristic clinical symptoms and post-mortem lesions of the sacrificed rabbits as well as bacteriological re-isolation and identification of the used strain. These trials were conducted on two experimental groups comprised of the control  and MSP  groups. All rabbits were checked to be free from detectable er study  using th7 CFU/mL . At 60 dn = 50/group) were further subdivided into 2 subgroups (n = 25/subgroup and each subgroup contained 5 replicates). Regarding the MSP group, the rabbits in the first subgroup received the MSP prophylactically for 30 days during the previous feeding trial (from 30 to 60 days of age) without further MSP supplementation after L. monocytogenes experimental infection (MSPP); meanwhile, those in the second subgroup were offered the same MSP for an additional 30 days after L. monocytogenes experimental infection . Concerning the control group, the rabbits in the first subgroup were kept infected only and did not receive any treatment, but those in the second subgroup were treated with commercial enrofloxacin hydrochloride (ENR) at a dose of 40 mg/kg for 7 consecutive days, as was recommended by the producer [L. monocytogenes infection.In the clinical trial, the above-mentioned experimental groups . The feed intake (FI) and body weight gain (BWG) were verified and then the feed conversion ratio (FCR) was estimated as following: amount of consumed feed (g)/BWG (g) ,34. AddiL. monocytogenes experimental infection) days of age, 5 experimental rabbits were randomly selected and sacrificed by cervical dislocation. The slaughtering and dissection of rabbits were performed according to the recommendations of the World Rabbit Science Association [®  for subsequent mRNA expression assays. At 7 and 14 days post-experimental infection, L. monocytogenes counts were enumerated in the cecal contents. Finally, samples of liver, brain, and spleen tissues were harvested and fixed in 10% neutral buffered formalin for histopathological examination at 14 days post-infection according to [At 60  and 90 . The extracted DNA quality and concentration were assessed by a Thermo Scientific NanoDrop 2000 spectrophotometer . Ultimately, the purified DNA samples were stored at −80 °C for posterior quantitative PCR analysis. Real-time PCR (RT-PCR) assays were performed to calculate the populations of some intestinal microbial species, including total bacteria, and GAPDH), as a calibrator. All the investigated target genes and the sequences of their appropriate primers are characterized in −ΔΔCt method [The extracted total RNA from frozen jejunum and cecum tissues was done via QIAamp RNeasy Mini kit  in compliance with the manufacturer’s instructions. RNA concentration and purity were assessed through a Nano Drop 2000 spectrophotometer . The expression analysis was determined by one-step qRT-PCR assays using 2× QuantiTect SYBR Green RT-PCR Kit . The mRNA expression levels of genes encoding nutrient transporters including sodium–glucose co-transporter-1 (SGLT-1), glucose transporter-2 (GLUT-2), cationic amino acid transporter-1 (CAT-1), and fatty acid-binding protein-2 (FABP-2); tight junction proteins (TJP) comprising claudins-1 (CLDN-1), junctional adhesion molecule-2 (JAM-2), occludin, and mucin-2 (MUC-2); and cytokines including interleukin-6 (IL-6), IL-8, IL-1β, IL-10, transforming growth factor-beta-1 (TGF-β1), and tumor necrosis factor-alpha (TNF-α) were determined at 60 and 90 days of age. Moreover, the mRNA expression level of gene encoding interferon gamma (IFNγ) was detected at 90 days of age only. All qRT-PCR reactions were run, in triplicate, on a Stratagene Mx3005P real-time thermal cycler . The transcript levels of the genes of interest were normalized to the expression of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase  of paraffin-embedded tissues were stained with eosin and hematoxylin and examined under the light microscope equipped with a computerized digital camera ,41,42. Sp < 0.05.The results were analyzed by the GLM procedure of SPSS and a subsequent Tukey’s post-hoc test was used to estimate the statistically significant differences among the treatment groups. Homogeneity and normality among our groups were evaluated through Levene’s and Shapiro–Wilk’s tests, correspondingly. The achieved data were conveyed as standard error of mean (SEM). For all tests, the significance was determined at p < 0.05) increased with increasing levels of MSP. Moreover, supplementation with MSP at a level of 1 × 108 CFU/kg displayed the most significant (p < 0.05) improvement in rabbits’ FCR (L. monocytogenes significantly (p < 0.05) decreased BWG and impaired FCR. Interestingly, this impaired growth rate and FCR were improved in the probiotics- or enrofloxacin-treated groups. Meanwhile, rabbits receiving MSP (prophylactic and therapeutic) had the highest significant (p < 0.05) BWG and improved FCR. The experimentally-infected rabbits showed various clinical signs in the form of depression, anorexia and diarrhea, and generalized septicemia in PM examination of freshly dead rabbits. Notably, MSP over the whole rearing period (MSPTT) reduced the severity of both clinical signs and PM lesions. Moreover, the mortality percentages were significantly (p < 0.05) reduced, especially after administration of MSP during the whole rearing period when compared with the positive control group (24 vs. 6%) (The results regarding the growth performance attributes of rabbits are shown in its’ FCR . At the  vs. 6%) .L. monocytogenes experimental infection), MSP supplementation at various levels resulted in a differential increase in the abundance of the total aerobic bacterial (Lactobacillus (Bifidobacterium (Enterobacteriaceae counts (8 CFU/kg remarkably elevated (p < 0.05) the abundance of Lactobacillus and Bifidobacterium counts by 3.45 and 2.51 CFU/g of the ileal contents and 4.65 and 4 CFU/g of the cecal contents, respectively. In contrast, Enterobacteriaceae counts were notably decreased when increasing the levels of MSP in rabbits’ diets. At 90 days of age, rabbits experimentally infected with L. monocytogenes exhibited the highest (p < 0.05) colonization of harmful Enterobacteriaceae and the lowest (p < 0.05) populations of beneficial ones  in the ileum and cecum. Notably, enrofloxacin-treated rabbits exhibited the lowest (p < 0.05) ileal and cecal total bacteria .At 60 days of age , the data indicated that GLUT-2 gene expression levels were significantly (p < 0.05) increased when increasing the MSP levels . Moreover, the transcriptional levels of CAT-1 genes (p < 0.05) upregulation of GLUT-2 genes (p < 0.05) upregulation in SGLT-1  in groups fed MSP at a level of 1 × 108 CFU/kg when compared with the control group . Moreover, CLDN-1 (p < 0.05) post-supplementation with MSP in a dose-dependent manner. At 90 d of age, the maximum expression levels of MUC-2 (JAM-2 (p < 0.05) impaired in rabbits treated with enrofloxacin. Of note, the expression results showed significant (p < 0.05) upregulation of CLDN-1, the relative expression levels of IL-8 (IL-6 (p < 0.05) decreased with increasing the levels of dietary MSP, while TNF-α (IL-1β (p < 0.05) decreased in rabbits fed MSP at levels of 1 × 107 and 1 × 108 CFU/kg unlike the control group. Moreover, IL-10 (TGF-β1 (p < 0.05) upregulated, especially with higher MSP supplementation levels. At 90 d of age, other remarkable findings emerged from the data analyses were the prominant decreases in the expression levels of the IL-8 (IL-6 (TNF-α (IL-1β (p < 0.05) IL-10 (TGF-β1 (p < 0.05) upregulation of the IFNγ gene  IL-10 e and TGF (TGF-β1 f transcrFNγ gene g was detp < 0.05) lower log10 copies of L. monocytogenes populations were found in the cecal contents of rabbits treated with enrofloxacin, followed by those fed MSP during the whole rearing period  (L. monocytogenes counts reached their lowest levels in rabbits either treated with enrofloxacin or those that were supplemented with MSP during the whole rearing period (MSPTT) at 14 days post-infection  . Anothernfection .L. monocytogenes experimentally-infected rabbits (positive control group) showed congestion of the hepatic blood vessels and sinusoids. Moreover, thrombus was formed within blood vessels and neutrophilic infiltrations were seen within sinusoids. The brain showed suppurative meningitis, which is characterized by an increase in the thickness of the meninges due to meningeal congestion and leukocytic infiltrations, mainly neutrophils. The spleen showed shrinkage of some lymphoid follicles and necrotic changes of some lymphoid elements besides dilated splenic sinusoids (L. monocytogenes and treated with enrofloxacin are shown in L. monocytogenes (MSPP) are shown in L. monocytogenes are shown in At 14-days post-infection, the liver of inusoids a. The hiListeria monocytogenes is an opportunistic foodborne pathogen that can infect a wide range of farm animals including rabbits resulting in life-threatening listeriosis [L. monocytogenes ubiquity renders it a dangerous ongoing concern in animal production and food safety because of growth depression and great economic losses [L. monocytogenes disrupts the host’s normal microbiotic balance and leads to the development of antimicrobial-resistant micro-organisms. Thus, an awareness of excessive antibiotics use can encourage the development of alternative safe ways, particularly, on the prophylaxis level.teriosis ,12. The c losses . IndiscrL. monocytogenes infection. Especially in rabbits, mechanisms of how MSP modulate their tight junctions and immunity and counteract listeriosis are not fully understood until now. In the current study, use of MSP at a level of 1 × 108 CFU/kg diet during the growing period contributed to the highest growth performance parameters and good health conditions of rabbits, suggesting the growth-promoting role of selected MSP. In accordance, supplementing rabbits’ diets with B. subtilis improved growth performance, immune organs’ indices, intestinal homeostasis, innate immune response, and disease resistance [Bacillus species could produce extracellular enzymes and some necessary nutrients and provide essential growth factors to boost animal growth [L. acidophilus alone or a combination of B. subtilis and L. acidophilus [L. acidophilus- and B. subtilis-supplemented rabbits [L. monocytogenes experimental infection were compensated in groups supplemented with MSP, suggesting their potential role in attenuating L. monocytogenes infection in rabbits. In the same line, L. monocytogenes infection impaired the growth performance parameters of rabbits; however, L. acidophilus administration recovered the final BWG of rabbits [Among these safe alternatives, we claimed that MSP could block the pathogen attachment or invasion of epithelial cells and consequently improve the rabbits’ growth performance and confer protection against sistance . Moreovesistance ,45,46 fol growth . Similardophilus . These f rabbits ,49. Inte rabbits . In this rabbits ,52. Addi rabbits , ability rabbits , improve rabbits .SGLT-1 and GLUT-2 engage in the intestinal absorption of monosaccharides and their concentrations, and, consequently, control monosaccharide uptake in the small intestine. SGLT-1 is specified in the apical membrane and it mediates glucose uptake from the intestinal lumen through the brush-border membrane into the intestinal enterocytes [GLUT-2 is the primary fructose and glucose transporter in the basolateral membrane [. FABP-2 is implicated in the trafficking of intracellular free fatty acids and eventually enhances intestinal nutrient absorption [CAT-1 can transport cationic amino acids  from enterocytes to the vascular supply or vice versa. In this respect, probiotic strains have been previously shown to improve nutrient absorption [SGLT-1, GLUT-2, CAT1, and FABP-2 genes associated with nutrient transportation even after L. monocytogenes experimental infection. Similarly, the relative gene expression of glucose and protein transporters showed a significant (p < 0.05) upregulation after feeding on multi-strain probiotics in broiler chickens [The digestion end products of dietary ingredients in the small intestine are principally absorbed through nutrient transporters that play a crucial role in nutrient absorption and feed utilization ,56. The erocytes ,58, whilmembrane ,60,61. Fsorption . Moreovesorption ; howeverchickens .8 CFU/kg diet shifted the ileal and cecal microbiome composition of growing rabbits at 60 days of age towards the beneficial bacteria when compared to the control group. In contrast, the abundance of Enterobacteriaceae opportunistic pathogens was decreased after MSP supplementation, especially at higher doses. In accordance, dietary probiotics increased the numbers of beneficial bacterial organisms in the rabbits GIT than the pathogenic ones [L. acidophilus led to higher concentrations of cecal acetic acids and total volatile fatty acids and decreased intestinal coliform colonization [B. subtilis/L. acidophilus-supplemented diets showed greater intestinal lactobacilli counts than those fed diets supplemented with B. subtilis alone [L. monocytogenes and treatment with enrofloxacin, all counted bacterial populations were decreased, while early and continuous MSP supplementation after infection enhanced beneficial bacterial communities and decreased the colonization of harmful ones in the cecum and ileum, which are in agreement with previous studies [The intestinal microbiota could greatly affect the host’s gut health via modulating intestinal pH, transporter gene expression, and mucosal immunity . Probiotnic ones . Moreovenic ones ,68. The nic ones . Moreovenic ones . Additionization ,71. Moreis alone . This cois alone . On the is alone . Notably studies ,75. Here studies ,77. Mean studies .Lactobacillus species triggered the immune system by boosting the expression of anti-inflammatory cytokine genes, IFN-γ and IL-10 [TNF-α, IL-6, and IL-8 [IL-10 production, which blunts the excessive inflammatory response [L. monocytogenes could upregulate TNF-α and IL-6 genes, thereby leading to an increase in the permeability of the intestinal epithelium [L.monocytogenes [IL-1β is a key pro-inflammatory cytokine that stimulates its own expression and the expression of other pro-inflammatory cytokines and chemokines, which in turn recruit inflammatory responses and trigger the induction of antimicrobial cells [TNF-α, IL-6, IL-8 and IL-1β) genes were noted in rabbits fed a basal diet and experimentally infected with L. monocytogenes compared with those experimentally infected and treated with either enrofloxacin or probiotics. Similarly, probiotics could blunt intestinal inflammation [IFNγ, its initial production is a crucial step for producing an immune response and regulating L. monocytogenes infection [IL-10 has a predominantly antagonistic impact on inflammation, besides its crucial role in depressing the inflammatory and immune responses [IFNγ, which promoted effective L. monocytogenes clearance and upregulated IL-10 and TGFβ genes that suppress excessive inflammation and maintain intestinal immune homeostasis. The robust inflammatory response in the L. monocytogenes experimentally-infected group was effectively counteracted by MSP administration over the whole rearing period (prophylactic and therapeutic), thereby indicating their strong anti-inflammatory properties. In previous studies, higher transcript levels of IFN-γ genes were detected after B. subtilis pre-treatment, even after Citrobacter rodentium infection [Lactobacillus species triggered pathogen clearance and suppressed intestinal inflammation via stimulating the anti-inflammatory cytokines (IL-10 and TGFβ) production [IL-2, IL-8, L-1β, and IL-6 transcript levels [Cytokines are known to have a key regulatory impact in monitoring the intestinal inflammatory response. Probiotic bacteria exert immunostimulatory and immunoregulatory activities, and thus they can be extensively applied in the treatment of numerous diseases ,80. The nd IL-10 ,83, or band IL-8 ,85. Addiresponse . On the response . In thisithelium . Stimulaytogenes . IL-1β ial cells . As antiammation  through ammation . Regardinfection . Moreoveesponses . In the nfection ,94. In aoduction ,96. On toduction . In a seoduction . Moreovet levels ,99.ZO-1, CLDN-1, and occludin, could lead to barrier integrity impairment. In this context, probiotics have a diverse mechanism of action to enhance the function of the intestinal barrier and sustain homeostasis, and thus it may counteract the weaning stress. Herein, higher expression levels of ZO-1, CLDN-1, occludin, MUC-2, and JAM-2 in the intestine of rabbits described that MSP, especially at higher doses, enhanced TJPs. In accordance, probiotics could enhance gut barrier function by increasing the expression of TJP genes [Lactobacillus species, such as L. plantarum, abated barrier disruption via upregulation of TJPs [L. monocytogenes infection. However, after supplementing MSP during the whole rearing period (prophylactic and therapeutic), these reductions were abrogated. This could be attributed to the beneficial roles of MSP in fortifying the intestinal epithelial resistance to pathogens by sustaining TJP abundance. A previous study utilizing different probiotic strains described parallel in-vivo and in-vitro results [Salmonella species [L. monocytogenes-induced intestinal permeability through the preservation of the cell junctional architecture of CLDN-1, occludin, and E-cadherin. Additionally, L. plantarum had protective effects on the intestinal barrier by rearranging TJPs  disturbed by Escherichia coli (E. coli) and accordingly ameliorated the barrier function [L. rhamnosus and L. fermentum significantly improved E. coli-disturbed TJP  [IL-6, IL-8, and TNFa). Aside from their important roles in immunity, cytokines were also demonstrated to affect tight junction integrity as pro-inflammatory cytokines could induce tight junction disruption. Commensal bacteria such as Lactobacillus and Bifidobacterium species in intestinal microflora could improve intestinal barrier functions and regulate cytokines secretion [L. plantarum pre-treatment downregulated the pro-inflammatory cytokine genes  and alleviated the reduction in TJP  caused by E. coli [Lactobacillus species were proved to increase the expression of intestinal mucin [L. monocytogenes-induced MUC-2 loss and apoptotic responses [ZO-1, CLDN-1, and occludin genes post-treatment with enrofloxacin.A key function of the intestinal epithelium is to form a natural barrier, which hinders pathogens and toxic constituents from entering the mucosa and coming into contact with the immune system, thereby ensuring intestinal homeostasis ,101. IntJP genes ,107,108. of TJPs ,110. In  results . It has  results . A recen species . Moreove species  describefunction ,114. Ano CLDN-1) . Notablyecretion . Similar E. coli . Further E. coli . Previoual mucin ,118,119.esponses . Althougesponses . This isesponses  verifiedL. monocytogenes, it enters the host via the intestines and then infects the spleen, liver, and immune cells, where they proliferate until attacked by the host’s immune system [L. monocytogenes infection by probiotics. Our results proved that MSP administration during the rearing period greatly reduced the intestinal counts of L. monocytogenes. In accordance, [L. monocytogenes and inhabit the membrane expressed epithelial Hsp60 receptor sites on epithelial cells to competitively eliminate it. There are several mechanisms underlying the effect of probiotic strains on the inhibition of gut translocation of bacteria to other organs [L. monocytogenes [L. acidophilus, L. plantarum, and E. faecium, or their secondary metabolites, prevented the formation of L. monocytogenes biofilm [L. monocytogenes loads were endorsed by recent results in rabbits [L. monocytogenes, significant histological changes were detected in rabbits’ livers, brains, and spleens with a picture of septicemia (positive control group). Similar findings were previously detected in the organs of rabbits infected with L. monocytogenes [L. monocytogenes to other organs, as evidenced by restoring the normal histopathological architecture of rabbits’ livers, brains, and spleens. Similarly, significant improvements were found in the histological pictures of rabbits` tissues after administration of Lactobacillus acidophilus [Regarding the infection with e system . As an ie system . From the system ,123. Regordance,  describer organs . It is p biofilm ,125,126. rabbits . After iytogenes . Adminisdophilus . This coL. monocytogenes attenuated the severity of clinical signs, PM lesions, and pathogen localization or translocation by decreasing its load in the cecum of rabbits, thereby downregulating the pro-inflammatory cytokines and subsiding the excessive inflammatory response. Hence, our results recommend the application of multi-strain probiotics, as an alternative to antibiotics, to offer protection for rabbits against L. monocytogenes infection.Taken together, our findings suggested the favorable outcomes of multi-strain probiotics during the whole rearing period on rabbits’ growth, immune response, and intestinal barriers, as evidenced by modulating the expression of cytokines and tight-junction-protein-related genes. Additionally, administration of MSP for rabbits infected with


Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast  DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. DSB repair failure can cause loss of chromosome fragments and cell death, while their inaccurate repair can result in chromosome rearrangements, genome instability, and ultimately cancer .DNA DSBs can be repaired by multiple pathways with different levels of fidelity. The main mechanisms are non-homologous end joining (NHEJ), which results in a direct ligation of the broken DNA ends , and homThe choice between DSB repair pathways with different outcomes and mutagenic potential is decisive for the maintenance of genomic integrity and it is subjected to complex regulatory mechanisms. The origin and the structural features of the DSBs, as well as the phase of the cell cycle in which they occur, are important determinants of DSB repair pathway decision. DNA end resection commits DSB repair to HR by creating 3′ single-stranded DNA (ssDNA) overhangs necessary for HR, while destroying the substrate for NHEJ repair ,7. DurinThe ratio between NHEJ and HR varies across phylogenetic groups. HR is generally prevalent in organisms with a small genome and with low abundance of repetitive sequences, such as budding yeast. In mammals and plants, NHEJ is the preferred pathway. On average, 75% of the DSBs that occur in mammalian cells are repaired by NHEJ, while HR repairs the remaining 25%. However, HR is always used as the preferred mechanism to repair DSBs that occur during S phase ,13.The response to DSBs should be considered within the context of chromatin, a highly organized structure in which the DNA is wrapped around octamers of four core histones H2A, H2B, H3, and H4 forming the nucleosomes . NucleosChromatin structure around a DSB can be modified by removing entire nucleosomes, thanks to the action of chromatin remodeling factors, and/or by modifying histones, thus increasing chromatin flexibility and accessibility of repair enzymes to the DSB ,14. HistSaccharomyces cerevisiae and in mammalian cells. Considering the evolutionarily distance between these two organisms and the extensive variations of histone marks during the cell cycle and in different cell types in mammals, one would expect that these chromatin modifications could be extremely different in the two species. In contrast, many histone PTMs and histone-modifying enzymes are well conserved, although more intricate and generally redundant pathways have evolved in metazoans compared to yeast. Taking these differences into account, we believe that the acquired knowledge of chromatin landscape and its effects on DSB repair pathway choice in yeast was and still is pilot for identifying proteins and pathways sensitive to specific histone PTMs also in mammals.An extensive work has been carried out to define the role of histones dynamics in DSB response in both the model organism In this review, we will focus on histone PTMs and discuss recent insights into their functions in DSB repair pathway choice, mainly referring to the latest discoveries in yeast. We and others recently reviewed the findings obtained in mammals and the regulatory models which have been proposed ,19,20,21S. cerevisiae), which recognize aberrant DNA structures and phosphorylate a multitude of targets involved in both repair and checkpoint pathways, thus promoting DSB repair and ensuring its completion before the cells progress through the cell cycle ,43,4]. AHR is initiated by resection of both DSB ends to create 3′-ended ssDNA tails in a two-step process that requires the concerted action of helicases and nucleases. The first resection step (short-range resection) requires MRX/MRN and Sae2/CtIP, whose action is particularly important to process DNA ends with protein adducts or “dirty” ends, while it is dispensable for resection of “clean” DSB ends, as those induced by endonucleases . Mre11 pAfter DSB end resection, the most common HR mechanisms require strand pairing and strand invasion, which strictly depend on the RecA/Rad51/Dmc1 family of recombinases that form a nucleoprotein filament on ssDNA. Rad52 together with a set of Rad51 paralogs, replaces RPA with Rad51 in yeast, while in mammals the main loader of RAD51 is BRCA2, along with several RAD51 paralogs ,4. Once For a long time, scientists interested in HR mechanisms have wondered about this “search problem”, thus showing that repair of a DSB is influenced by the proximity between the damaged and the template sequences. This homology search could also be facilitated by DNA damage-stimulated changes in chromosome movement .After the D-loop formation, HR can proceed via the canonical pathway that involves the formation of a double Holliday junction, whose resolution can generate either crossover or non-crossover products b, centerA specialized homology-dependent DSB repair pathway which does not require strand invasion and D-loop formation is the SSA. This mechanism relies on the annealing between two homologous sequences flanking a DSB and leads to the deletion of one of the repeats and of the intervening region c 5]..5].S. cerevisiae) . F. F131]. Histone H1 mediates higher-order chromatin folding in metazoans and it has a well-established role in chromatin organization during DNA repair . RNF20/4HHO1 inactivation was found to increase the resistance to DNA damage of mutants with defective NHEJ, but not of recombination mutants, suggesting that Hho1 may specifically inhibit HR ..ESA1 genNuA4 is recruited to the DSB ends by the MRX complex. Through the acetylation of both histones and non-histone proteins, NuA4 promotes resection and HR, while it inhibits NHEJ . AcetylaThe SAGA complex is another HAT that contributes to NuA4 recruitment to the DSBs and participates in resection stimulation . SAGA acIn mammalian cells, TIP60 depletion causes defects in DSB repair ,153,154.S. cerevisiae, defects in the acetylation of histones that are part of nucleosomes reconstituted during replication are associated with sensitivity to genotoxic agents [In c agents ,157. Celc agents ,163,164.c agents . This cac agents ,166. Sinc agents , its DNAc agents ,169.H3 K56 acetylation promotes cell survival after transient exposure to genotoxic agents that cause DNA damage during replication . When H3In the mammalian system, both reduction and increase of H3 K56 acetylation have been observed after DNA damage. Deacetylation by Sirt6 and Sirt3 HDACs promotes NHEJ by recruiting the chromatin remodeling enzyme SNF2H and 53BP1 to the DSB sites ,171.Chromatin around a DSB undergoes extensive changes that are regulated at multiple levels to allow DSB repair. Histone PTMs play a pivotal role in the modification of chromatin structure and in the subsequent DSB repair, as highlighted by the hypersensitivity to DSB-inducing agents of cells lacking histone modifier enzymes. In mammalian cells, the regulatory network of histone PTMs involved in DSB response is very complex due to redundancy of the modification machinery and to the variability of the modifications in different cell types. From this point of view, the yeast system has the advantage of a simplified histone PTM machinery. Many components and effectors of this machinery, as well as the biological processes controlled by histone PTMs, are conserved between yeast and vertebrates. Therefore, deciphering the chromatin landscape around a DSB and the effects of histone PTMs on DSB repair pathway choice in yeast should help unveil how these systems operate in mammals.Although inadequate DSB repair is one of the main causes of genomic instability and tumorigenesis, it also offers therapeutic opportunities to selectively kill cancer cells. In this context, targeting histone modifications required for DSB repair is expected to increase the hypersensitivity of cancer cells with defects in other key factors involved in the DSB response. A comprehensive view of histone PTMs in the surroundings of DSBs and their effects on DSB repair pathway choice is needed both to elucidate the contribution of histone modifications in controlling DNA metabolism and to predict the possible effects of targeting histone modifiers in therapy.


The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE‐GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+]cyt signalling in roots.Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion .Extracellular ATP‐induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root cyt increase and transcriptional response in cngc2 roots were significantly impaired.Extracellular ATP‐induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ influx, causing depolarization leading to [Ca2+]cyt increase and damage‐related transcriptional response.CYCLIC NUCLEOTIDE‐GATED CHANNEL2 is required for eATP‐induced epidermal Ca Extracellular ATP‐induced depolarization of PM voltage has been used as a diagnostic of PM Ca2+ channel activity in single epidermal and cortical root cells. Results show an absolute requirement for CNGC2 but not CNGC4 in the epidermis. Patch clamp electrophysiological analysis of eATP‐induced PM Ca2+ influx conductance of epidermal cells confirmed an absolute requirement for CNGC2. Both root eATP‐induced [Ca2+]cyt signature and transcriptional response were impaired by loss of CNGC2 function.Here, two Arabidopsis dorn1‐1, dorn1‐3, p2k2, and p2k1p2k2 mutants were as described previously  insertion line Salk‐066908) was described previously by Chin et al., cngc2‐3 was generated with the CNGC2 coding sequence under the control of its endogenous promoter (Supporting Information Methods dnd1 cngc2 loss‐of‐function mutant constitutively expressing cytosolic (apo)aequorin was described by Qi et al., cngc4‐5  ecotype. Em of root elongation zone cells was measured using a glass microelectrode. A plant was fixed in a plexiglass chamber and immersed in assay solution (10 ml) containing 2 mM calcium chloride ‐N,N,N′,N′‐tetraacetic acid) (EGTA) or with or without 0.5 mM lanthanum chloride (LaCl3)), 0.1 mM potassium chloride (KCl), 1 mM MES–Tris (pH 6.0) for at least 30 min before impalement. Microelectrode construction, recording circuitry, and impalement are described in Methods Em (> 6 min), eATP  or ATP disodium salt (Na2ATP); Sigma) was added to the chamber . In controls, magnesium sulphate (MgSO4) or sodium sulphate (Na2SO4) was added.Plasma membrane potential et al. aequorin were used for luminescence‐based quantification of [Ca2+]cyt. Roots were placed individually into a 96‐well plate (one root per well) and incubated overnight at room temperature in darkness with 10 µM coelenterazine in 100 µl of buffer: 2 mM CaCl2, 0.1 mM KCl, 1 mM MES–Tris (pH 5.6). CaCl2 was included to maintain a similar level to that of the growth medium. Samples were washed with coelenterazine‐free buffer and left to recover for at least 20 min in darkness. A FLUOstar Optima plate reader  was used to record luminescence as described in Matthus et al. . UBQ10 and TUB4 acted as internal controls. Primers are listed in Table Total RNA was extracted from roots (frozen in liquid nitrogen) using the RNAeasy Plant Mini Kit (Qiagen) and subjected to DNase I treatment . Complementary DNA (cDNA) was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real‐time (qRT)‐PCR was performed in a Rotor‐Gene 3000 thermocycler with the Rotor‐Gene™ SYBRt‐test or Tukey's honestly significant difference was used for parametric data comparison, whereas the Mann–Whitney U test was used to compare the nonparametric data.Data normality was first analysed with the Shapiro–Wilk test in R. Student's Em of a single Col‐0 root elongation zone epidermal cell , and Em recovered fully after 14.7 ± 2.2 min  abolished the response to 300 µM eATP  and the complemented cngc2‐3,CNGC2::CNGC2 mutant   Fig. , but maxl‐0 Fig. . This maant Fig. e. To verant Fig. a–c was aent Fig. . These rdorn1‐3 loss‐of‐function mutant, the dorn1‐1 kinase mutant, and the p2k2 mutant all retained a small but significant depolarization of Em when challenged with 300 µM eATP  also sustained a small but significant depolarization of Em when challenged with 300 µM eATP, but this was not significantly different to that caused by the Na2SO4 control . Under control conditions, the p2k1p2k2 mutant had a significantly more negative Em  than its paired Col‐0 wild‐type (−129.9 ± 4.6 (n = 5); P = 0.005), and this may help explain why sodium ions (Na+) caused a depolarization in this mutant but not in Col‐0. Overall, the results suggest that the two receptors working together are sufficient to initiate the eATP‐induced depolarization of Em and that CNGC2 is an absolute requirement in this cell type.Elongation zone epidermal cells of the ATP Fig. . CNGC2 tion Fig. e. The doet al., et al., cngc4‐5 loss‐of‐function mutant . For Col‐0, 300 µM eATP activated whole‐cell inward current upon membrane hyperpolarization, but not outward current upon membrane depolarization , far from the equilibrium potentials of potassium ions  and chloride ions (−28 mV) and indicating Ca2+ permeability. Extracellular ATP‐activated inward current was significantly inhibited by 100 µM gadolinium ions (Gd3+), a plant Ca2+ channel blocker that is effective against CNGC2  the eATP‐activated current would deliver Ca2+ to the cytosol, which would both elevate [Ca2+]cyt and initiate depolarization. It can be inferred that some eATP‐activated Ca2+ influx should have occurred in membrane voltage trials at the less negative Em caused by EGTA  failed to respond to 300 µM eATP  blocked inward and outward currents in the presence of eATP, but these currents were not investigated further aequorin as a bioluminescent [Ca2+]cyt reporter. The typical monophasic [Ca2+]cyt increase (‘touch response’) after sodium chloride (NaCl) addition (control for mechanostimulation and cation effect of Na2ATP) was observed in individual roots of Col‐0 and dnd1. The amplitude of the touch peak and total [Ca2+]cyt mobilized did not differ significantly between genotypes  in both Col‐0 and dnd1 roots  was observed in previous studies on Arabidopsis roots and seedlings using aequorin  or after ATP (ATP added) was observed between Col‐0 and these two mutants . This indicates a CNGC4‐dependent pathway in the cortex. The residual depolarization in the cngc4‐5 implies involvement of other CNGCs (but not CNGC2) or other transport systems , WRKY DNA‐BINDING PROTEIN 40 (WRKY40), CALCIUM‐DEPENDENT PROTEIN KINASE 28 (CPK28), and the cysteine protease METACASPASE 7 (MC7)  significantly upregulated expression of all four genes in Col‐0, with no significant difference between Col‐0 and cngc2‐3,CNGC2::CNGC2  compared with cngc2‐3,CNGC2::CNGC2  in the root epidermis, but in either event it is the obligate component of the depolarization response given CNGC4's redundancy  did not depolarize dnd1 elongation zone epidermis.Fig. S4 Single receptor mutants supported a small but significant extracellular ATP (eATP)‐induced depolarization of elongation zone epidermal Em.Fig. S5 The p2k1p2k2 double receptor mutant lacked the extracellular ATP (eATP)‐induced depolarization of elongation zone epidermal Em.Fig. S6cngc4‐5 supported a significant extracellular ATP (eATP)‐induced depolarization of elongation zone epidermal Em.Fig. S7 Extracellular ATP (eATP) did not activate inward currents in dorn1‐3 root elongation zone epidermal protoplasts.Fig. S8 Cyclic Nucleotide‐Gated Channel2 (CNGC2) contributed to the extracellular ATP (eATP)‐induced [Ca2+]cyt increase in roots.Fig. S9 Cyclic Nucleotide‐Gated Channel2 (CNGC2) is not required for extracellular ATP (eATP)‐induced depolarization of primary root elongation zone cortical plasma membrane potential but CNGC4 is involved.Methods S1 Genotyping cngc insertional and complemented mutants.Methods S2 Growth conditions.Methods S3 Membrane voltage measurement.Methods S4 Patch clamp recordings.Methods S5 Quantitative real‐time PCR analysis of gene expression.Table S1 Primers used for genotyping transfer DNA mutant lines and quantitative real‐time PCR.Table S2 Mean ± SE membrane voltage Em measurements.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries  should be directed to the Click here for additional data file.