The ability to separate enzymes, nucleic acids, cells, and viruses is an important asset in life sciences. This can be realised by using their spontaneous asymmetric partitioning over two macromolecular aqueous phases in equilibrium with one another. Such phases can already form while mixing two different types of macromolecules in water. We investigate the effect of polydispersity of the macromolecules on the two-phase formation. We study theoretically the phase behavior of a model polydisperse system: an asymmetric binary mixture of hard spheres, of which the smaller component is monodisperse and the larger component is polydisperse. The interactions are modelled in terms of the second virial coefficient and are assumed to be additive hard sphere interactions. The polydisperse component is subdivided into sub-components and has an average size ten times the size of the monodisperse component. We calculate the theoretical liquid–liquid phase separation boundary , the critical point, and the spinodal. We vary the distribution of the polydisperse component in terms of skewness, modality, polydispersity, and number of sub-components. We compare the phase behavior of the polydisperse mixtures with their concomittant monodisperse mixtures. We find that the largest species in the larger (polydisperse) component causes the largest shift in the position of the phase boundary, critical point, and spinodal compared to the binary monodisperse binary mixtures. The polydisperse component also shows fractionation. The smaller species of the polydisperse component favor the phase enriched in the smaller component. This phase also has a higher-volume fraction compared to the monodisperse mixture. The ability to separate enzymes, nucleic acids, cells, and viruses is an important asset which can be realised by using spontaneous asymmetric partitioning over two aqueous macroscopic phases . The forThe formation of separate aqueous phases is usually studied using mixtures of two monodisperse components. However, components in nature are usually not that simple or well-defined. Often, components will exhibit polydispersity in terms of their size, shape, and charge, which is often ignored when studying phase behavior. In order to study the effects of polydispersity theoretically, we chose the following model system: hard spheres. Their separation into two phases is driven by two different physical mechanisms. One mechanism involves only excluded volume interactions, where the minimum distance between the particles is determined by the sum of their respective radii , where oIn most studies on the phase behavior of binary mixtures, the polydispersity of the components is ignored. However, from experiments with for example gelatin and dextran, it is found that polydispersity has an influence on the phase behavior. The polydispersity of both components leads to significant fractionation, especially for dextran .Polydispersity has an effect on the depletion interaction. With increasing polydispersity, the repulsive barrier decreases, leading to an enhanced rate of flocculation of the large colloidal particles . AdditioStudying the phase behavior of polydisperse mixtures is challenging, since a polydisperse component effectively consists of a large number of sub-components, each with a different size and possibly also different shape or charge . Some thIn this study, we aim to gain a better understanding of how size polydispersity influences the liquid–liquid phase behavior in binary mixtures, mainly on the position of the phase boundary, the spinodal, and the critical point. Next, we aim to predict the fractionation of the polydisperse component between the phases. We model the interactions between the different components using the second virial coefficient. We note that the validity of the second-order virial coefficient approximation is limited to dilute systems for hard spheres, but depending on the type of system, as in the aqueous polymeric systems in which we are interested for the above-mentioned practical applications, the validity may increase to the polymer overlap concentration and beyond . This waWe start the theoretical considerations by reviewing the interaction in a simple system of a solute in a solvent . In SectWe start by deriving the equations of state for dilute solutions. Next, we derive the virial expansion for solutions with one solute component. Subsequentl, y we derive the second virial coefficient for mixtures with an arbitrary number of distinguishable components. This gives us all the parameters we need to define the stability boundary, the critical point, and phase boundary of a mixture. The resulting system of equations is solved in Matlab R2017b.V at a temperature T. The total number of particles in the system is then We consider a two-component solution, in which one component is the solvent and the other component is the solute. We define nsemble) .H) of the system, given by:i, i and j, and i.The sum of the kinetic h is Plank’s constant and k is Boltzmann’s constant.The canonical partition function Other thermodynamic variables, such as the Helmholtz free energy, the pressure and the chemical potential can be expressed in terms of this function or its derivatives. The Helmholtz free energy given by :(5)A is given by:i is given by:Therefore, the pressure Z .(18)U(rW) is defined according to: The potential of mean force for dissolved particles (Using Equations  and 19)19), the The Helmholtz free energy Equation  of the sSimilar to the expansion of the universal gas law by a virial expansion for real gasses, we can write a virial expansion for the osmotic pressure, n different particles.With T), and the volume (V) are fixed. The restraint of constant number of particles becomes tedious when accounting for the interaction between the different particles, therefore we will use the grand canonical ensemble to derive the virial coefficients. In the grand canonical ensemble, the temperature (T) and the volume (V) are fixed, as well as the chemical potentials Until now, we have been using the canonical ensemble to describe the system. In the canonical ensemble, the number of particles Ξ–(17)), the cluster integrals can also be written as diagrams :(32)ρ=NVInverting the series we obtain for the activity :(39)z=ρ−This can be substituted in the equation for the pressure (Equation ):(40)βΠ=Comparing this equation to Equation , we see For hard spheres, we define the interaction potentials as for components with a diameter The second virial coefficient then becomes:n the total number of distinguishable compounds, or species, there are two main types of two particle interactions that can occur:interactions between indistinguishable components—i.e., components of the same species: interactions between distinguishable components, i.e., components of different species: When there are more distinct compounds ): x and y can be of any type In which The general equation for the osmotic pressure then becomes (comparable to Equation ):(50)βΠ=For the second virial coefficient, we obtain two types (comparable to Equation (41)): x and y can be of any type In which For additive hard sphere interaction, the interaction potential for particles of different species is given by:With For the second virial coefficient we find (comparable to Equation ): (54)BxA is polydisperse. The concentration of each of the particles in the polydisperse component can be represented by:With:Then:Each of the components in this mixture has a corresponding virial coefficient and cross virial coefficient.a and b, Let us investigate the equations for a simple polydisperse mixture, in which the polydisperse component consists out of two sub-components  . For theWith:The stability matrix becomes:This results in the following determinant for the stability matrix:By increasing the number of sub-components, the number of terms in this determinant increases rapidly. This forms an incentive to try and treat the polydisperse component as if it is effectively one component. A natural and convenient choice for this route is coupled to the experimental determination of virial coefficients using membrane osmometry . Namely,The number averaged virial coefficient of a mixture can be written as:ith particle, ith particle and the jth particle, and ith particle, In which Using this definition, we can map the polydisperse mixture by a For the mixture we considered in Equation , the effThe effective stability matrix for this mixture then becomes:The determinant then becomes:It is clear that Equations  and 71)71) are dIn a binary mixture, the critical point is a stable point which lies on the stability limit   and wherThere are two criteria that have to be used to find critical points. The first one is et(M2)=0 ,37, wherMatrix For a monodisperse binary mixture, this results in the following two matrices for the critical point:In addition:The set of equations that needs be solved for the critical point is:a and b, For the earlier considered polydisperse mixture containing the two sub-components βΠIn components:For a system that separates into two phases, we obtain, using Equations  and 60)60), the This set of equations has This extra set of equations stems from the fact that during phase separation no particles are lost and no new particles are created. The total number of components in the system is therefore given by:V, of the system does not change. The total volume of the system is given by:Additionally, the total volume, The concentrations of each component in each phase are thus given by:The total number of compounds in the system can be found using:This can be rewritten to:With:n equations and one more unknown  distribution of the polydisperse component becomes untraceable.We are calculating the transition from one-phase to two-phase systems and approach this point from the one-phase lower-concentration regime.A and a larger hard sphere B with a size ratio B . We again calculated the phase diagram using both the simplified effective Now that we have a bit of an understanding of how polydispersity influences the critical point and the phase boundary, we increase the number of sub-components of B in B in B for B in B in mixture B in B component at the critical point respectively.From the figure and table, we can conclude that the standard deviation of the polydisperse component plays a big role in moving the critical point and phase boundary. The standard deviation for B in mixtures in B compared to the mixture with the left skewed distribution.The distributions of A, whilst the other is enriched in component B. Even though each phase is enriched in one component, the other component is still present in lower concentrations. We investigated the phase separation of a specific parent mixture  increases with polydipsersity. The bottom phase has a higher volume fraction of component B with higher polydispersity. In general, but most pronounced for the mixture in A), while the larger sub-components favor the bottom phase (the phase enriched in the larger particles B). We like to note that these observations as obtained from the virial approach are in line with previous theoretical work using the UNIQUAC model by Kang and Sandler [The polydispersity in the sizes of component  Sandler  and the  Sandler  and also Sandler ,40,41. R Sandler  also not Sandler  found thB in the parent phase. For these mixtures, the degree of polydispersity is at its lowest for the phase enriched in A.Depending on the type of distribution, phase separation causes the degree of polydispersity to decrease. This is in both phases for all mixtures with a symmetric distribution of We find that the largest species in the polydisperse component causes the largest shift in the position of the phase boundary, critical point, and spinodal compared to the binary monodisperse binary mixtures. Upon phase separation, the polydisperse component fractionates. The smaller species of the polydisperse component favor the phase enriched in the small component, while the larger species remain in the phase enriched in the larger component. The top phase, the phase enriched in the small component, has a larger volume and this volume increases with polydispersity. The virial approach we used yields results in line with those of previous theoretical and experimental work on polydisperse mixtures, and at the same time allows for direct experimental testing using the virial coefficients obtained from membrane osmometry.


Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl− secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.TMEM16A is a Ca When2+  21]. No. No2+ (2ds (LPL) . In the 2, which activated TMEM16A overexpressed in HEK293 cells, but had little effects on endogenous TMEM16A in HT29 cells  TMry small . (iii) Er ETX001 . Cinnama2+ rise in HT29 cells, but showed some inhibitory effects on [Ca2+]i in Cal33 and M1 cells  found endophilin A2 regulated TMEM16A by autophagy-mediated degradation i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate intracellular calcium ([Ca2+]i) was [Ca2+]i =Kd × (R − Rmin)/(Rmax − R) × (Sf2/Sb2), where R is the observed fluorescence ratio. The values Rmax and Rmin (maximum and minimum ratios) and the constant Sf2/Sb2 (fluorescence of free and Ca2+-bound Fura-2 at 380 nm) were calculated using 1 µmol/L ionomycin , 5 µmol/L nigericin , 10 µmol/L monensin , and 5 mmol/L EGTA to equilibrate intracellular and extracellular Ca2+ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2•Ca2+ complex was taken as 224 nmol/L. Control of experiment, imaging acquisition, and data analysis were done with the software package Meta-Fluor .Cells were seeded on glass cover slips and loaded with 2 µM Fura-2/AM and 0.02% pluronic F-127  in Ringer solution .If not indicated otherwise, all chemicals were from Sigma-Aldrich. Data are shown as individual traces or as summaries with mean values ± SEM and number of experiments or cells given in each figure’s legend. For statistical analysis, paired or unpaired Student’s


Beyond the consensus definition of G-quadruplex-forming motifs with tracts of continuous guanines, G-quadruplexes harboring bulges in the G-tetrad core are prevalent in the human genome. Here, we study the incorporation of a duplex hairpin within a bulge of a G-quadruplex. The NMR solution structure of a G-quadruplex containing a duplex bulge was resolved, revealing the structural details of the junction between the duplex bulge and the G-quadruplex. Unexpectedly, instead of an orthogonal connection the duplex stem was observed to stack below the G-quadruplex forming a unique quadruplex–duplex junction. Breaking up of the immediate base pair step at the junction, coupled with a narrowing of the duplex groove within the context of the bulge, led to a progressive transition between the quadruplex and duplex segments. This study revealed that a duplex bulge can be formed at various positions of a G-quadruplex scaffold. In contrast to a non-structured bulge, the stability of a G-quadruplex slightly increases with an increase in the duplex bulge size. A G-quadruplex structure containing a duplex bulge of up to 33 nt in size was shown to form, which was much larger than the previously reported 7-nt bulge. With G-quadruplexes containing duplex bulges representing new structural motifs with potential biological significance, our findings would broaden the definition of potential G-quadruplex-forming sequences. Guanine-rich oligonucleotides have a propensity to adopt non-canonical nucleic acid structures known as G-quadruplexes in the presence of monovalent cations, such as potassium and sodium . The G-qMYC .Unlabeled and site-specific 15N labeling. Spectral assignments were completed by NOESY, COSY, TOCSY and 13C–1H-HSQC experiments. Spectra were processed and analyzed by using the program FELIX .NMR experiments were performed on 600 and 700 MHz Bruker spectrometers at 25°C, unless otherwise specified. DNA samples were dissolved in a buffer containing 30 mM KCl and 20 mM KPi (pH 7). The DNA concentration of NMR samples was typically 0.2–1.0 mM. Resonances for guanine residues were assigned unambiguously using 2–4% site-specific B4-dx2 were calculated using the program XPLOR-NIH  and planarity  restraints were maintained throughout the course of refinement. Out of 100 conformers, 10 best structures with the lowest overall energy were selected. The structures were displayed using the program PyMOL (DeLano Scientific LLC).Structures of the duplex–quadruplex construct PLOR-NIH . HydrogeTm) is defined as the temperature for which there are equal fractions of folded and unfolded species. For each sequence, the average mT value from the folding and unfolding experiments is presented. For clarity, only the folding curves are presented.Circular dichroism (CD) experiments were performed on a JASCO-815 spectropolarimeter. Unless otherwise stated, CD spectra were recorded at 25°C over the range of 220−320 nm using a 1-cm path-length quartz cuvette with a reaction volume of 500 μl. The bandwidth, scan speed and data pitch were set at 2 nm, 200 nm/min and 1 nm, respectively. The oligonucleotide concentration was typically 3–5 μM. For each sample, an average of three scans was taken, the spectrum of the buffer was subtracted, and the data were zero-corrected at 320 nm. For CD melting experiments, cooling and heating were successively performed across the temperature range of 15−95°C. The CD signals were recorded at intervals of 0.5°C and at the wavelength of 263 nm. Two baselines corresponding to the completely folded (low temperature) and completely unfolded (high temperature) states were manually drawn in order to determine the fractions of folded and unfolded species during the melting/folding process. The melting temperature GTGGGTGGGTGGGT  imino proton NMR spectrum of D imino pm Figure , supportm Figure . OverallB4-dx2. Guanine imino and aromatic protons were unambiguously assigned (15N-labeled (4%) samples (B4-dx2 was completed by using through-bond (COSY and TOCSY) and through-space (NOESY) NMR experiments. The imino proton of the isolated guanine G20 was clearly detected at the region of 10.5–12 ppm H8 and (n – 1)H1′/H2′/H2″/H3′/H4′ are clearly observed along the G-columns and two strands of the duplex hairpin . The bases of these loops project outwards from the G-tetrad core.The structure of 2 Figure . The G-tB4-dx2 –G20(H1), T19(CH3)–G24(H1), T19(CH3)–G24(H8) and T19(H6)–G24(H8). To accommodate a duplex hairpin insertion within the context of a quadruplex bulge, the strand separation (defined as the distance between matching pairs of phosphate groups) at the A5-T19 step is narrower (11.3 Å), as compared to the next T6-A18 step (17.3 Å) and a normal Watson-Crick base pair step (∼18 Å) within the duplex stem  are positioned on top of the G-tetrad core; the last thymine T33 is stacked below the bottom G-tetrad. The duplex at the bulge formed between G4 and G20 includes five Watson–Crick base pairs, while the immediate possible base pair step at the quadruplex–duplex junction, A5–T19, is broken up Figure . The dup2 Figure  as compaT Figure  in the rT Figure  is consim Figure . Moreovem Figure . This coB4-dx2 only A5 was projected outward from the overall helix and continuous base stacking between the quadruplex and duplex was observed. Across other quadruplex–duplex hybrid structures, various adaptor base pairs or structural elements have been observed to bridge the two helical domains  ranging from 46 to 50.5°C in the presence of ∼60 mM K+ , indicating that a G-quadruplex with a duplex bulge exhibited significantly higher thermal stability than a G-quadruplex containing a non-structured bulge of the same length.These structures exhibited a high thermal stability, with the melting temperature   . The majed loops . On the pressors . BCL2 coing used . The exiectivity . Pyrroleectivity ,86. HighIn this study, we showed that a duplex stem can be incorporated into a bulge of a G-quadruplex. The NMR solution structure of a G-quadruplex containing a duplex bulge was presented, showing a unique quadruplex–duplex junction with the duplex bulge stacking below the 3′-end G-tetrad. Breaking up of the immediate base pair step at the quadruplex–duplex junction, coupled with a narrowing of the duplex groove within the context of the bulge, led to a progressive transition between the quadruplex and duplex segments. Duplex bulges can occur at various positions of a G-quadruplex scaffold and increasing the length of a duplex bulge does not lead to a decrease in the stability of the overall G-quadruplex core. The formation of a long duplex bulge within G-quadruplexes would expand the current knowledge on predicting G-quadruplex-forming sequences. Potential existence of such structures in the human genome may serve as unique targets for designing ligands for specific G-quadruplex targeting.B4-dx2 have been deposited in the Protein Data Bank (PDB ID: 7CLS).The coordinates for the NMR solution structure of gkaa738_Supplemental_FileClick here for additional data file.


Myocardial remodelling is a common phenomenon in cardiovascular diseases, which threaten human health and the quality of life. Due to the lack of effective early diagnosis and treatment methods, the molecular mechanism of myocardial remodelling should be explored in depth. In this study, we observed the high expression of MBNL1 in cardiac tissue and peripheral blood of an isoproterenol (ISO)‐induced cardiac hypertrophy mouse model. MBNL1 promoted ISO‐induced cardiac hypertrophy and fibrosis by stabilizing Myocardin mRNA in vivo and in vitro. Meanwhile, an increase in MBNL1 may induce the apoptosis of cardiomyocytes treated with ISO via TNF‐α signalling. Interestingly, MBNL1 can be activated by p300 in cardiomyocytes treated with ISO. At last, Myocardin can reverse activate the expression of MBNL1. These results suggest that MBNL1 may be a potential target for the early diagnosis and clinical treatment of myocardial remodelling. As key participants in post‐transcriptional regulation, RBPs control RNA processing at multiple levels, including alternative splicing, RNA stability, RNA localization and translation efficiency.We used bioinformatics tools and found that Myocardin mRNA and the promoter region of MBNL1 contain multiple MBNL1 binding sites and a Myocardin‐binding site, respectively. Therefore, we speculate that there may be an interaction between MBNL1 and Myocardin. This article focuses on the interaction between MBNL1 and Myocardin and their underlying molecular mechanism in regulating myocardial hypertrophy.22.1In this study, all animal‐related experiments were approved by the Institutional Animal Care and Use Committee of the Tianyou Hospital Affiliated to Wuhan University of Science and Technology. And all animal experiments conformed to the ethical standards outlined in the Guide for the Care and Use of Laboratory Animals .2.2Two‐month‐old wild‐type C57BL/6 mice were purchased from the Animal Experimental Centre of Hubei Academy of Preventive Medicine. All mice were fed in SPF level animal laboratory under controlled environmental conditions (12 hours light/dark cycle and room temperature 20 ± 5°C). And all mice were free to obtain standard laboratory food and water. Mice with different treatments were infused with ISO for 2 weeks and saline‐infused mice served as controls. After 14 days, mice were anaesthetised with 50 mg/kg pentobarbital sodium (Sigma) via intraperitoneal injection. No toe pinch reflex confirmed adequate anaesthesia. Then, the mice were killed by cervical dislocation, and hypertrophic analysis was performed.2.3Primary neonatal mouse cardiomyocytes were isolated from the heart of 2‐ to 3‐day‐old C57BL/6 mice with mild enzymatic (0.1% collagenase type II and 0.08% trypsin) digestion and gentle mechanical attrition in sterile environment. After centrifugation, cells were incubated in DMEM/F‐12 (Gibco.) containing 10% foetal bovine serum (Gibco.), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.1 mmol/L bromodeoxyuridine.2.42.The Cos‐7 (CRL‐1651) and HEK 293T/17 (CRL‐11268) cells used in this study were purchased from ATCC. The cells were seeded in High Glucose DMEM (Hyclone) supplemented with 10% FBS (Gibco.), penicillin (100 U/mL) and streptomycin (100 U/mL) at 37°C in humidified air with 5% CO2.5MBNL1 was inserted into a lentivirus vector pCDH‐αMHC. The plasmids pCDH‐αMHC‐MBNL1 or pCDH‐αMHC (as control) were cotransfected with psPAX2 and pMG2.G into 293T cells (approximately 70%‐80% confluency). After 8 hours of transfection, the medium was replaced, and then, supernatant was filtered after 48 hours to obtain active lentivirus.2.6The target shRNAs against mouse MBNL1, Myocardin and p300 were inserted into pαMHC‐clone26. A negative control showing no significant homology to any mouse or human gene was inserted into pαMHC‐clone26. ShRNAs lentivirus were produced by cotransfecting 293T cells with the lentivirus expression plasmid and packaging plasmid. Interference efficiency was measured by realtime PCR and Western blotting.2.7Lentivirus was added to primary mouse cardiomyocyte cultured in low glucose DMEM supplemented with 10% FBS at a ratio of 50 multiplicity of infection (MOI). After overnight incubation, fresh medium was replaced.2.8According to the manufacturer's protocol, the total RNA was extracted by using the Total RNA Extraction Kit (OMEGA). Then, the total RNA extracted was reverse transcribed using M‐MLV Reverse Transcriptase (Promega). Finally, realtime PCR was performed in a StepOne Realtime PCR System (Thermo) with Fast SYBR Green Master Mix (Thermo). The relative expression levels of related genes were normalized to GAPDH. The primers for the realtime PCR analysis are as follows: MBNL1, F: 5′‐CTTGCTCACGACCAGAC‐3′, R: 5′‐ATTCCGCCCATTTATC‐3′; Myocardin, F: 5′‐CTCAACCCTTGTCCCA‐3′, R: 5′‐GTCATTTGCTGCTTCACT‐3′; ACTN2, F: 5′‐AAACCCGATGAAAGAGC‐3′, R: 5′‐GCCAGGGGATTGTGC‐3′; ANP, F: 5′‐GGGCTTCTTCCTCGTC‐3′, R: 5′‐TCTCCTCCAGGTGGTCTA‐3′; p300, F: 5′‐TGAGGATACAAAGGAGGCTA‐3′, R: 5′‐CTGACGAAAGGGAAGAGATT‐3′; TNF‐α, F: 5′‐TGTCTACTGAACTTCGGGGT‐3′, R: 5′‐ TTGGTGGTTTGTGAGT GTGA‐3′; GAPDH, F: 5′‐CAGTGCGGTGTCCAAC‐3′, R: 5′‐GACCTCCCCAGT CCAG‐3′.2.9For Western blotting analysis, protein samples were extracted from the cells through protein extraction reagent (Thermo) and the concentration of protein samples was determined by BCA Quantitative Kit (Beyotime). Then, the difference of protein expression was analysed by Western blotting analysis. The antibodies used in this process were as follows: rabbit anti‐Myocardin , rabbit anti‐MBNL1 , mouse anti‐ACTN2 , rabbit anti‐ANP , rabbit anti‐TNF‐α , rabbit anti‐p300  and rabbit anti‐GAPDH . GAPDH expression was used as an internal control.2.105/well, 24‐well plates) were cotransfected with pcDNA3.1 (as control) or Myocardin in combination with pGL3‐MBNL1‐WT or pGL3‐MBNL1‐MUT. The Dual Luciferase Assay Kit (Promega) was used to measure the difference of luciferase activity in each experimental group.The wild type and mutant of MBNL1 promoter were inserted into pGL3 and amplified with primers as follow: F1: GGCGACGCGTGTAGGCTAAGCTAGCCTT GGG, F2: GCGGACGCGTTCACATTCCATCCAGTTGCT, R: TATACTCGAGGC CCCTCCCCCGCGCCA. Cos‐7 cells  for 15 minutes and washed with PBS, the samples were observed by laser scanning confocal microscope (OLYMPUS).2.12In vitro, Myocardin mRNA and TNF‐α mRNA were transcribed by T7 RNA polymerase (Sigma) and the RNA (Roche) was labelled with the Biotin RNA Labeling Mix (Roche). Whole‐cell lysates (1 mg) of primary mouse cardiomyocytes were then incubated with 3 µg of purified biotinylated transcripts for 1 hour at 25°C. Proteins bound to transcripts in pull‐down materials were detected by streptavidin agarose beads (Invitrogen) combined with Western blotting.2.137 primary mouse cardiomyocytes were taken to carry out RIP experiments combined with anti‐IgG or anti‐MBNL1. The RNA in the coprecipitation was then extracted and detected by realtime PCR. The gene‐specific primer used to detect Myocardin is as follows: F: 5′‐CTCAACCCTTGTCCCA‐3′, R: 5′‐GTCATTTGCTGCTTCAC T‐3′.2 × 102.14The excised mouse hearts were fixed with formaldehyde at 4°C for 72 hours and embedded in paraffin. Then, the paraffin‐embedded tissues were cut into 3‐4 µm slices. The sections were then dewaxed with xylene for 15 minutes, rehydrated in gradient alcohols, and stained with haematoxylin‐eosin (H&E) or wheat germ agglutinin (WGA) or Masson trichrome. Five random fields were selected for capture under an inverted microscope .2.15After different lentiviral transductions, apoptotic cells in each well (24‐well plates) or in each section (3‐4 µm slices) were visualized using TUNEL according to the manufacturer's instructions (Promega). The experimental results of TUNEL assay were observed under a confocal microscope (OLYMPUS F3000).2.16g) for 5 minutes. Then, the supernatant was subjected to ELISA assay.Enzyme‐linked immunosorbent assay  was used to detect the secretion of TNF‐α in cell cultures according to the manufacturer's instructions. Briefly, supernatant of primary cardiomyocytes with different treatment was collected and centrifuged  according to the manufacturer's instructions. Briefly, each group to be tested was incubated with 1% formaldehyde to cross‐link DNA‐protein complexes. Cells were then collected, washed three times with ice‐cold PBS and lysed in SDS lysis buffer. After centrifugation, the lysates were ultrasonically treated and the DNA was cut into 200‐1000 bp fragments. We then immunoprecipitate the cross‐linked protein at 4°C overnight using an anti‐Myocardin antibody . IgG acted as the negative control. Finally, DNA from the coprecipitated material was extracted and used as a template for PCR to find the binding site of Myocardin. The primers for PCR analysis are shown below: F: 5′‐CTGCATGAGTCAGTTTTCCA‐3′, R: 5′‐ATT AACT TGTCGGCAGAGAAG‐3′.2.18In this study, we chose TSA (Sigma) as an inhibitor of p300. We treated primary cardiomyocytes with 100 ng/mL TSA for 24 hours. Then, cells were replaced the normal medium and carried out ISO induction. After ISO induction, proteins were collected from cells for Western blotting analysis.2.19Primary cardiomyocytes were transduced with MBNL1 or sh‐MBNL1 lentivirus. Transcription was stopped using Actinomycin D , and RNAs were extracted at various time points  following Actinomycin D treatment. Realtime PCR assay was detected the remaining of Myocardin mRNA and adjusted by GAPDH levels. The primer for the realtime PCR analysis is as follows: Myocardin, F: 5’‐CTCAACCCTTGTCCCA‐3’, R: 5’‐GTCATTTGCTGCTTCACT‐3’. The corrected density was then plotted as a percentage of different time points value against control, with the decay rate constant derived from the slope of the decay curve.2.20GSE129090). Quantitative data are expressed as mean ± SEM. Statistical analysis of differences between two groups was performed by Student's t test. A one‐way analysis of variance followed by Tukey test was performed to compare differences among multiple groups. Statistical analysis was performed with GraphPad Prism 8. P < .05 indicated statistically significant difference.High throughput sequencing data were obtained from the GEO database (33.1GSE129090), we found that MBNL1 was highly expressed myocardial hypertrophy samples , suggesting that Myocardin may be a potential target for MBNL1 Figure . The RNA3.4MBNL1 can regulate myocardial hypertrophy; Myocardin expression was previously confirmed. Here, we explored whether MBNL1 can regulate myocardial hypertrophy via Mycoardin using Myocardin‐knockdown (sh‐Myocardin) mice. First, the loss of Myocardin protein in the hearts of mice was confirmed by using Western blotting Figure . We overPrimary cardiomyocytes were used to verify the results of the above experiments. Phalloidin staining confirmed that MBNL1 could not effectively induce myocardial hypertrophy with Myocardin knockdown Figure . Similar3.5Excessive cardiac hypertrophy or myocardial fibrosis leads to cardiomyocyte apoptosis. In this part, we wanted to identify the effect of MBNL1 on cardiomyocyte apoptosis in ISO‐induced myocardial remodelling. As shown in Figure 3.6We have revealed the function and molecular mechanism of MBNL1 in regulating myocardial remodelling; however, it is unclear how MBNL1 is activated during myocardial remodelling. Therefore, we tried to explore which signalling pathways, related to myocardial remodelling, are involved in MBNL1 activation. First, ISO was used to induce primary cardiomyocytes after treatment with inhibitors of MAPK, JNK, PI‐3K or PKC signalling pathways. As a result, the expression of MBNL1 was inhibited in ISO‐induced primary cardiomyocytes with blocked MAPK or JNK pathways Figure . In othe3.7We analysed the promoter region of MBNL1 and found a Myocardin‐binding site CarG box. Does this mean that Myocardin can also regulate the expression of MBNL1? By using realtime PCR and Western blotting, we confirmed that Myocardin can significantly up‐regulate the expression of MBNL1 when Myocardin is overexpressed in cardiomyocytes; the opposite results were obtained after knocking down Myocardin Figure . Then, w4Hibernating myocardium has great effects on myocardial remodelling, which is caused by the decrease of coronary artery blood flow, leading to the continuous impairment of myocardial and left ventricular function. When the coronary artery blood supply improves or the myocardial oxygen consumption decreases, the cardiac function can be partially or completely restored to normal. Therefore, local microvascular reconstruction and functional recovery have become one of the important ways to reverse hibernating myocardium.In this study, we confirmed that MBNL1 can increase the expression of Myocardin by inhibiting the degradation of Myocardin mRNA, which implicate myocardial hypertrophy and its subsequent pathophysiology. Myocardin is an important transcription factor in cardiovascular system development. The occurrence and development of myocardial hypertrophy are accompanied by the overexpression of Myocardin in cardiomyocytes. Previous studies have also confirmed that the forced overexpression of Myocardin in cardiomyocytes can induce myocardial hypertrophy.Increasing evidence has demonstrated that a large number of factors have important effects on the expression or activity of Myocardin. The transcription factors Nkx2.5, KLF4 and TET2 can regulate the expression of Myocardin at the transcriptional level.In response to environmental changes, the activation of intracellular signalling pathways results in upstream to downstream signalling, resulting in changes in the expression of certain genes, which in turn further lead to physiological or pathological changes such that adaptation to external pressure is achieved. In terms of a single gene, its expression is regulated at multiple levels, including transcription, post‐transcription, translation and apparent modification. Among them, the post‐transcriptional regulation is particularly delicate. During adaptive responses to environmental changes, the post‐transcriptional regulatory mechanism plays an important role. Various factors can be involved in post‐transcriptional regulation processes; these factors include lncRNAs, miRNAs and RBPs (RNA‐binding proteins). RBPs account for 6%‐8% of all proteins encoded and can control the fate of RNAs . Despite the importance of the functions of RBPs, only a very small number of RBPs have been identified, especially in diseases.In recent years, many studies have focused on the involvement of RBPs in cardiac remodelling. The inhibition of RBM20 (RNA‐binding motif protein 20) expression leads to dilated cardiomyopathy (DCM) in adults.+ channel current and SCN5A mRNA abundance.In healthy hearts, fibroblasts play an important role in maintaining normal structure, electrical conduction, mechanical contraction and diastolic function. In main acting cells at pathological state, fibroblasts gather in the heart through various sources and pathways, and transform into myofibroblasts. The secretion of a large number of collagen and bioactive factors implicates myocardial fibrosis and provides a pathological basis for the occurrence and development of the disease. Previous reports have confirmed that MBNL1 can repair myocardial injury by up‐regulating the expression of SRF and promote the occurrence of fibrosis in the case of myocardial injury.Moreover, myocardial remodelling can cause cardiomyocyte apoptosis through the production of TNF‐α in large amounts and lead to heart failure.With the development of antisense oligonucleotide (ASO) technology, the expression or activity of some genes can be controlled. Our results suggest that MBNL1 may be a potential target for the treatment of myocardial remodelling. If ASOs or other small molecular inhibitors are designed for the combination of MBNL1/Myocardin and MBNL1/TNF‐α, it may be possible to inhibit cardiac hypertrophy and prevent the apoptosis of cardiomyocytes.Multiple intracellular signalling pathways, including MAPK, JNK, PKC and PI3K, are important factors in myocardial remodelling.In this study, we explored the relationship between MBNL1 abundance in peripheral blood and cardiac hypertrophy which may provide a new direction for the early diagnosis of myocardial hypertrophy in the future. Myocardial remodelling is the result of multiple factors, including inflammatory factors, macrophages and so on. Among them, macrophage depletion can increase myocardial injury and accelerate the process of myocardial remodelling.Interestingly, we found that Myocardin can reverse activate the transcription of MBNL1. When cardiac hypertrophy occurred, the overexpression of Myocardin also increased MBNL1 protein levels, which further strengthened the stability of Myocardin mRNA; this in turn maintained the increased abundance of Mycoardin and promoted myocardial remodelling.Our study revealed the effect and molecular mechanism of MBNL1 in myocardial remodelling. Specifically, under the effects of hypertrophy stimulators such as ISO, MBNL1 can be activated by MAPK and JNK via p300. MBNL1 can up‐regulate the stability of Myocardin mRNA, resulting in the promotion of myocardial hypertrophy and myocardial fibrosis. Mycoardin can reverse activate the expression of MBNL1 and further accelerate myocardial remodelling. Meanwhile, the sustained activation of MBNL1 can induce cardiomyocytes to secrete TNF‐α and promote the apoptosis of cardiomyocytes Figure . These fThe authors declare that they have no conflict of interest.Yao Xu: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Investigation (lead); Project administration ; Resources (lead); Supervision ; Validation (lead); Visualization ; Writing‐original draft (lead). Chen Liang: Software (supporting); Validation (supporting); Visualization (supporting). Ying Luo: Data curation (supporting); Formal analysis (supporting); Supervision (supporting); Validation (supporting); Writing‐original draft (supporting). Tongcun Zhang: Conceptualization ; Funding acquisition (lead); Project administration ; Supervision .Fig S1Click here for additional data file.


Acute myeloid leukemia (AML) in children remains a challenging disease to cure with suboptimal outcomes particularly when compared to the more common lymphoid leukemias. Recent advances in the genetic characterization of AML have enhanced understanding of individualized patient risk, which has also led to the development of new therapeutic strategies. Here, we review key cytogenetic and molecular features of pediatric AML and how new therapies are being used to improve outcomes.WT1, CBFA2T3-GLIS2, and KAT6A fusions, DEK-NUP214 and NUP98 fusions, and specific KMT2A rearrangements, which are associated with poor outcomes. However, outcomes are starting to improve with the addition of therapies such as gemtuzumab ozogamicin and FLT3 inhibitors, initially developed in adult AML.Recent studies have revealed an increasing number of mutations, including The combination of advanced risk stratification and ongoing improvements and innovations in treatment strategy will undoubtedly lead to better outcomes for children with AML. Pediatric acute myeloid leukemia (AML) is a heterogeneous disease with generally poor outcomes compared to childhood lymphoid leukemia. Decades of coordinated efforts through cooperative group trials have improved our understanding of the unique biology underlying pediatric AML and patient outcomes. However, relapse remains frequent with limited available treatment options. As AML is more common and heavily researched in older adults, the field of pediatric AML has advanced in the understanding of AML biology and novel therapeutic strategies by translation of adult studies into the realm of pediatrics. However, there are substantial differences in the mutational landscape in pediatric versus adult AML and tolerance to treatment regimens, thus requiring dedicated risk stratification and treatment considerations in children.Compared to adults, children with AML have superior outcomes due to fewer adverse genetic mutations and the ability to tolerate the high-intensity chemotherapy currently necessary for cure. While complete remission (CR) rates are high in pediatric AML at approximately 90%, event-free survival (EFS) and overall survival (OS) remain suboptimal at 45% and 65%, respectively, at 3 years, and nearly half of children will relapse , 2. EvenCEBPA and NPM1 mutations may be cured with chemotherapy alone [KMT2A, located on chromosome 11q23, were also included in initial WHO classifications, but the prognostic significance was unclear until recently [Treatment decisions, namely the need for SCT, in pediatric AML are primarily driven by genetic risk classification which is quickly evolving. The World Health Organization (WHO) began defining recurrent genetic groups in myeloid malignancies in 2001 due to high incidence of specific mutations in AML, unique underlying biology, and comparable outcomes amongst patients with these lesions . Howeverpy alone –6. In copy alone , 8. Rearrecently , 10••. AAlong with improving our understanding of the biology of AML, identification of recurrent mutations has provided an opportunity to develop new treatment approaches. Targeted therapeutics, epigenetic modifiers, immune-based strategies, and novel metabolic pathway inhibition have all been utilized recently with varying degrees of success in adult AML. These strategies are now being employed in children with the aim of improving cure rates and providing more options in the relapse setting. Here, we discuss the current progress in understanding the unique mutational landscape of pediatric AML, implications on prognosis, and the use of new treatment strategies to optimize therapy and outcomes in children with AML.NPM1 mutations, and CEBPA mutations, each of which carries EFS and OS rates of approximately 65–70% and 80%, respectively [NPM1 and CEBPA are more recent additions to the favorable prognostic group and are similarly associated with an improved prognosis in both children and adults [CEBPA encodes a transcription factor that regulates expression of myeloid-specific genes with recurrent mutations occurring in two functional domains in approximately 4% of pediatric AML [NPM1 is a primarily nucleolar protein which plays a role in regulating p53 function and other cellular processes and, when mutated in AML, aberrantly localizes to the cytoplasm [NPM1 mutations are more common with increasing age but do occur in about 5–10% of children compared to 30% of adults [NPM1 mutations, particularly in cytogenetically normal AML or when they occur in the context of HR mutations where the presence of mutated NPM1 may improve outcomes compared to the HR genetic feature alone [Risk stratification in pediatric AML has previously been based on the presence of a handful of genetic features and responses to therapy. Those with favorable genetics include t8;21), inv16), , inv(16)ectively , 11. t is a recurrently mutated gene in AML, occurring in 20–25% of children and 30% of adults [FLT3 mutations lead to constitutive activation of the tyrosine kinase domain (TKD) causing uninhibited growth and are characterized as either TKD or internal tandem duplication (ITD) mutations with FLT3-ITD being significantly more common in children  has been refined over the last decade. Previous trials demonstrated that an AR > 0.4 conferred a poorer prognosis compared to an AR < 0.4, and so FLT3-targeted therapy was included only for the high AR group [FLT3-ITD AR > 0.1 may benefit from FLT3 inhibitor therapy and SCT in first remission with this lower threshold being used in upcoming trials.FMS-like tyrosine kinase 3  inhibition to limit cell growth. Mutations in Ras pathway genes are also found to associate with rearrangements in KMT2A, though this relationship appears to be most common in infant acute lymphoblastic leukemia [Mutations in the Ras pathway are the most common mutations occurring up to 40% of pediatric AML patients, primarily in either AS genes •. Similasubtypes . For exagorithms . Importaleukemia .KIT gene occur in 12% of pediatric AML patients and typically lead to unregulated function of the KIT protein [KIT mutations in pediatric AML as a whole, others have shown an improved OS associated with KIT mutations [KIT mutations in the favorable-risk CBF AML group, as KIT mutations occur in 24–36% of this population with a slight predominance for exon 17 mutations [KIT mutations, such mutations within the t subgroup of AML increase the risk of relapse and lead to an inferior OS of only 50% at 4 years [Mutations in the tyrosine kinase receptor  protein •, 24. Th protein . Though utations •, 21. Thutations , 26. Whi 4 years , 26.KMT2A (formerly MLL) regulates gene expression via methylation of histone residues and epigenetic modifications. It is located on chromosome 11q23 and is frequently involved in gene rearrangements in AML with a multitude of fusion partners. KMT2A rearrangements were included in the original WHO AML classification, but the prognostic significance in adults was unclear. KMT2A rearrangements are more common in children than adults with a particularly high occurrence in infant AML, and the fusion partner profile is also unique to each age group [KMT2A rearrangements are clearly associated with inferior outcomes with an EFS of 44% and an OS of 56% along with higher rates of early death and relapse, the true prognostic value is highly dependent on the fusion partner gene  is the most common KMT2A rearrangement in children, yet over 100 different fusion partners have been identified [MLLT10, the fusion partner in t, also pairs with other genes in novel fusion events [KMT2A rearrangements such as t and t are now considered HR fusions due to their associated high relapse risk [KMT2A fusions, not all rearrangements have well-defined outcomes, but careful consideration should be given to the fusion partner when considering the treatment approach for such patients.Lysine methyltransferase 2a encoded by ge group , 27••. Wentified , 16. Of ng from t;11 report showed a superior EFS in those with t that also harbored FLT3-ITD, likely due to the fact that all FLT3-ITD-positive patients received SCT in 1st CR [DEK-NUP214 AML [AML with t, a translocation leading to with AML –33. DEK ytoplasm , 32. Thin 1st CR , 32. In P214 AML , 32.KAT6A, a histone acetyltransferase, with a partner gene, most commonly CREBBP on 16p13. In adults, this fusion is often found in younger patients with unique clinical characteristics such as leukemia cutis, disseminated intravascular coagulation, and hemophagocytosis [KAT6A fusions, current reports demonstrate a 5-year EFS and an OS of 55–60% in those who undergo therapy with curative intent and often undergo SCT in first remission [KAT6A fusions, where spontaneous remission often occurs, though 50% will subsequently have return of their disease [Translocations involving 8p11 are a newly described entity in AML, which lead to fusion of ocytosis . In chilocytosis . Amongstemission . Relapse disease .WT1) gene is commonly mutated across different malignancies and regulates quiescence in hematopoietic progenitors as well as differentiation of myeloid cells [WT1 occur in approximately 10–15% of childhood AML and are more likely to be clonal than in adults [WT1 mutations, both alleles are affected either by mutation or by deletion [WT1 mutations frequently co-occur with aberrations such as FLT3-ITD and NUP98 fusions [WT1 mutations are independent poor prognostic factors with a 5-year OS of 35% and EFS of 22% in children [WT1 mutations [WT1 mutation and FLT3-ITD confers an even poorer prognosis with an OS of 21% and EFS of 15–30% and a significantly higher rate of relapse, though these outcomes do not reflect the most recent clinical trials [Wilms’ tumor 1 , was originally described in infant AML and associated with an extremely poor prognosis with a 3-year EFS and OS of 0% [MNX1-ETV6 has been identified outside of infancy where it frequently co-occurs with trisomy 19 and has better outcomes than previously reported [ETV6 has also been found to fuse with numerous other genes in pediatric AML and to be associated with inferior survival, leading to the recent inclusion of ETV6 fusions in the HR genetic stratification [ETV6 via chromosome 12p deletion is also associated with a poor prognosis [Fusions with OS of 0% , 38. Recreported . ETV6 hafication ••, 30•. rognosis .CBFA2T3-GLIS2 fusion results from a cryptic translocation in chromosome 16 [CBFA2T3-GLIS2 has a predisposition for African American children which make up nearly 1/3 of patients with the fusion [CBFA2T3-GLIS2 fusion does not co-occur with traditional HR karyotypes [CBFA2T3-GLIS2 had far inferior outcomes with only 50% achieving CR at the end of first induction and 35% having persistent MRD after induction 2. Both EFS and OS are poor at only 20–40% at 5 years regardless of having the AMKL phenotype [osome 16 . It was osome 16 –41. CBFAe fusion . These cryotypes . On retrhenotype , 39. TheNUP98, a nucleoporin gene, has been of recent interest due to its fusion with NSD1 in t in less than 5% of pediatric AML patients [NUP98-NSD1 is not found in combination with other cytogenetic rearrangements, but it frequently co-occurs with FLT3-ITD or WT1 mutations, with one or both genes mutated in 80% of patients [NUP98 fusions in children independently increase the risk of relapse and decrease CR, EFS, and OS [NUP98 fusions combined with either FLT3-ITD or WT1 mutations have an even worse prognosis than NUP98 fusions alone, with a relapse rate over 75% and an OS of 20–40% [NUP98 is the NUP98-KDM5A fusion, identified in 2% of children from recent American and European trials [NUP98-NSD1, the NUP98-KDM5A fusion is more common in young children and most common in AMKL where it was originally described [NUP98-KDM5A fusion rarely co-occurs with WT1 or TK mutations, yet still has a very poor prognosis. The 5-year EFS and OS of this group are 16–39% and 33–34%, respectively [.patients •, 21. NUpatients , 42. NUP, and OS •, 21, 42f 20–40% . A seconn trials . In contescribed , 43. Theectively , 43. Impectively .RPN1- and RUNX1-MECOM fusions, NPM1-MLF1, and FUS-ERG [Additional rare gene fusions and mutations exist which are associated with inferior survival. However, the small number of children in which these mutations occur has limited the ability to study such genetics in detail, including accurate survival statistics •, 44. Th FUS-ERG , 30•. MaWhile outcomes in pediatric AML have improved over the decades, these advances have been primarily due to early intensification of therapy, early use of SCT in HR patients, and improvements in supportive care. Current regimens still rely heavily on high-dose chemotherapy including cytarabine and anthracyclines in order to induce remissions. These agents carry a serious risk of toxicity including infection and cardiac dysfunction. Despite high doses of cytotoxic chemotherapy, relapse remains a frequent problem in pediatric AML. The newer treatment approaches discussed below have only recently been introduced in pediatric clinical trials Table . These aFLT3-ITD mutations, KMT2A rearrangements, single-nucleotide polymorphisms in ABCB1 and CD33, and high CD33 expression, but these are not currently factored into treatment decisions [Targeted therapy which exploits cell surface markers unique to cancer cells has been a significant therapeutic advance in cancer treatment. In drug-antibody conjugates, antibody targeting can deliver a drug, such as the small molecule calicheamicin, directly to cells of interest. Gemtuzumab ozogamicin (GO) is one such treatment where calicheamicin is conjugated to an anti-CD33 antibody and has potent anti-tumor effects on CD33-expressing cells, found in a majority of AML cases , 46. Desecisions , 53–55.+ T cells in recognizing and eliminating CD123-expressing cells [CD123, the IL-3 receptor α-chain, has also been a cell surface target of interest in AML owing to its expression in a majority of AML cases, including leukemic stem cells . CD123-tng cells , 58. TheInterest has also recently grown in the use of checkpoint inhibitors in AML to enhance the immune response to cancerous cells. This strategy has been used successfully in adult solid tumors but remains experimental in AML. Currently available checkpoint inhibitors include nivolumab and pembrolizumab, anti-PD-1 antibodies, and ipilimumab, which targets CTLA-4. Early clinical trials in adult AML have shown that checkpoint inhibition, either as monotherapy or in combination with other treatments, is a feasible and safe strategy now being explored in larger studies . NivolumImmunotherapy with chimeric antigen receptor T cells (CAR-T) for AML has lagged significantly behind lymphoid leukemias in development and effectiveness. The heterogeneity of AML has made it challenging to identify adequate leukemia-specific antigens which do not also produce on-target off-tumor effects on normal tissue. CD33 and CD123 are expressed in 90% and 75% of AML, respectively, with less than 5% of cases being negative for both . While CThe prevalence of epigenetic dysregulation in AML development has led to a rising interest in targeting epigenetic modifiers as part of therapy. Hypomethylating agents (HMAs) such as decitabine and azacitidine are DNA methyltransferase (DNMT) inhibitors which alter DNA methylation patterns, leading to increased expression of tumor suppressors and apoptosis . In adulHistone deacetylases (HDACs) contribute to epigenetic regulation via the removal of acetyl groups from histones condensing chromatin and decreasing gene transcription. Inhibition of HDAC activity remodels chromatin in AML cells with attendant decreased expression of DNA repair genes, depletion of CXCR4, cell cycle arrest, and ultimately apoptosis, with these effects being synergistic with standard chemotherapy regimens , 70. TheFLT3-mutated AML has been a specific disease subset of interest due to the targetable nature of tyrosine kinases. FLT3 inhibitors are small molecules which bind to the adenosine triphosphate–binding site on FLT3 leading to competitive inhibition of kinase activity [FLT3 mutation status in younger adults with AML, but also increased toxicity [FLT3-ITD AML, including 1 year of maintenance therapy in the most recent COG trial. Preliminary results demonstrate that addition of sorafenib increases CR and EFS and decreases relapse risk in children with high AR FLT3-ITD, providing rationale for ongoing use of FLT3 inhibitors in children [activity . The druactivity . In additoxicity , 78. Eartoxicity . Sorafenchildren .FLT3 mutation status [Midostaurin, the first FDA-approved FLT3 inhibitor, is a first-generation type I FLT3 inhibitor with off-target effects on KIT and was shown to decrease blast percentages in patients with relapsed/refractory AML when used as a single agent regardless of n status . The larn status . Preclinn status .FLT3-mutated AML after demonstrating a tolerable safety profile with a 20–30% CR rate and significantly prolonged survival when given as monotherapy [FLT3-mutated AML in combination with chemotherapy in both the relapse and upfront settings . Newer FLT3 inhibitors such as quizartinib and crenolanib are currently being investigated as well and appear to have similar efficacy to the approved FLT3 inhibitors [Gilteritinib is a second-generation type I FLT3 inhibitor which also inhibits AXL, a tyrosine kinase which enhances FLT3 activation and serves as a mechanism for FLT3 inhibitor resistance . It was otherapy , 86. Preotherapy . While phibitors .Venetoclax inhibits BCL-2, an anti-apoptotic protein whose overexpression is a mechanism of resistance in AML . In adulA liposomal formulation of traditional chemotherapy agents cytarabine and daunorubicin, termed CPX-351, optimizes pharmacodynamics and synergistic effects via maintenance of the optimal 5:1 molar ratio which extends the half-life and enhances uptake in the bone marrow , 94. ThePneumocystis jirovecii pneumonia (PJP) [Other therapeutic strategies rely on rebranding older medications for new purposes, such as atovaquone, an antibacterial and antimalarial medication which inhibits electron transport and is approved for use in the prevention of ia (PJP) . Recent ia (PJP) . In precia (PJP) . In adulia (PJP) . In chilia (PJP) .The outcomes in pediatric AML, while improved in recent decades, remain suboptimal with high rates of relapse and few options for therapy when initial treatment regimens fail. Genetic risk grouping has gone a long way to target the intensity of therapy to those at the highest risk of relapse and death; however, the heterogeneous nature of AML has left many HR genetic features unidentified until recently.KMT2A fusions, DEK-NUP214 and WT1 mutations, ETV6 rearrangements, and CBFA2T3-GLIS2, MECOM, MLLT10, and NUP98 fusions are now included in various cooperative group risk algorithms that recommend SCT in first CR. The potential for benefit of SCT transplant in first CR for these patients remains to be seen. Additionally, challenges surrounding attainment of an MRD-negative CR using traditional chemotherapy may limit the utility of this approach and continue to highlight the need for additional treatment options for such patients.Through the use of comprehensive mutation testing, collaborative international efforts, and integration of data from adult trials, critical information is now known regarding even rare genetic lesions that alter outcomes. This has primarily led to a growing list of HR genetic features. Specific The treatment of AML is quickly changing in adults, yet the advances in pediatrics are slower to evolve due to a lack of drugs developed specifically for the pediatric population and the challenges of evaluating novel agents for a relatively rare disease. Standard therapies in adult AML, such as GO and FLT3 inhibitors, have only recently been incorporated into pediatric trials, and the optimal way in which to incorporate them into therapy is still under investigation. In addition, newer agents such as HMAs and DNMT inhibitors have been developed primarily for adults who cannot tolerate intensive cytotoxic therapy, and therefore, acceptable outcomes in this population differ greatly from the desired results in children. However, the use of these agents may prove beneficial when incorporated into relapse regimens in children with pediatric AML.The continual expansion of recurrent genetic lesions in AML and their effects on outcomes, along with the growing list of therapeutic options, may ultimately provide an opportunity to individualize regimens in pediatric AML. The addition of GO and FLT3 inhibitors to standard chemotherapy regimens is now becoming a standard of care, but more studies are needed on other new agents to determine their utility in children. Through ongoing collaboration and innovation, treatment strategies will become more tailored to the underlying genetics of the disease with resultant improvements in outcome.


Aβ25–35, however, induced a Ca2+ diminution in Aβ-preconditioned astrocytes as a result of the potentiation of the plasma membrane Ca2+ ATPase (PMCA). The PMCA and CX protein expression was observed with immunostaining in the brain tissue of hAPPJ20 AD mouse model. We also observed both Ca2+-independent and Ca2+-dependent glutamate release upon astrocytic Aβ exposure, with the former mediated by CX hemichannel and the latter by both anion channels and lysosome exocytosis. Our results suggest that Aβ peptide causes state-dependent responses in astrocytes, in association with a multiphasic release of signaling molecules. This study therefore helps to understand astrocyte engagement in AD-related amyloidopathy.Excessive amounts of amyloid β (Aβ) peptide have been suggested to dysregulate synaptic transmission in Alzheimer’s disease (AD). As a major type of glial cell in the mammalian brain, astrocytes regulate neuronal function and undergo activity alterations upon Aβ exposure. Yet the mechanistic steps underlying astrocytic responses to Aβ peptide remain to be elucidated. Here by fluorescence imaging of signaling pathways, we dissected astrocytic responses to Aβ25–35 peptide, a neurotoxic Aβ fragment present in AD patients. In native health astrocytes, Aβ25–35 evoked CaThe online version contains supplementary material available at 10.1186/s40478-021-01146-1. Toxic Aβ peptides are implicated in the development of cognitive deficits of AD . In the 2+ signaling [2+ activity has been shown to be upregulated by Aβ peptides [2+ hyperactivity was found to compromise neuronal survival [2+-dependent protein phosphatase calcineurin in astrocytes ameliorates synaptic function in AD mouse model [Astrocytes are the major glial cell-type in the mammalian brain . Albeit ignaling , which iignaling , 91, theignaling , the perignaling  as well ignaling . Astrocypeptides , 85, andpeptides , 47. Aβ-se model . In addise model , 90, 98.2+ signals in primary astrocytes derived from mouse cortex, which involved the activation of metabotropic P2Y receptor and the opening of CX hemichannel. In contrast, Aβ25–35 caused a Ca2+ diminution in Aβ-preconditioned astrocytes that involved the potentiated Ca2+ extrusion via PMCA and the activation of cAMP signal. We further observed both Ca2+-independent and -dependent glutamate release in astrocytes upon Aβ25–35 exposure, which relied respectively on CX hemichannel, anion channels and lysosome exocytosis. These results show a state-dependent adaptation in astrocyte responses to neurotoxic Aβ peptide, and suggest molecular targets to control astrocyte functions in AD amyloidopathy.Here, we imaged astrocytic signaling cascades in response to Aβ25–35, a neurotoxic Aβ fragment found in AD patients , 46, 57.2 atmosphere in Dulbecco's Modified Eagle Medium  supplemented with 5% fetal bovine serum , penicillin , and streptomycin . Recordings were made during the following week at room temperature  in the standard extracellular saline containing (in mM): 140 NaCl, 5.5 KCl, 1.8 CaCl2, 1 MgCl2, 20 glucose, 10 HEPES . The hAPPJ20 AD mouse model was obtained from The Jackson Lab (No: 34836-JAX), expressing mutated human amyloid precursor protein (hAPP) monogene comprising the Swedish (K670N/M671L) and the Indiana (V717F) mutations [Our laboratory follows the European Union and institutional guidelines for the care and use of experimental animals (Council directive 86/609EEC). The care of experimental animals was also in conformity with the French National Charter on the ethics of animal experimentation. Primary astrocytes were cultured from the neocortex of P0-1 NMRI mice of either sex adapted from the published protocol , 56. Astutations , 60. Theutations , 60. Bre2+-indicator dyes Oregon Green BAPTA-1 AM (OGB-1 AM) or Xrhod-1 AM (Invitrogen) were loaded into astrocytes by incubating them in dye-containing extracellular solutions . To label lysosomes, astrocytes were incubated in 6.7 µM FM4-64 (Invitrogen) for 30 min. To fluorescently label ATP accumulation compartments in live cells, astrocytes were incubated in 50 µM MANT-ATP (Invitrogen) for 1 h. Prior to live cell imaging, cells were thoroughly washed during at least 30 min, and they were continuously perfused by dye-free solution (~ 0.5 ml/min) during imaging. BAPTA AM  was used to chelate astrocyte intracellular Ca2+. Plasmids encoding fluorescent sensors were obtained from Addgene unless otherwise indicated. Lipofectamine 2000 (Invitrogen) was used for transfecting cDNA plasmids into astrocytes following the standard protocol provided by the supplier. Cells were used ~ 24 h after transfection. Suramin, MPEP, thapsigargin and Gap26 were purchased from Tocris, 2-APB from Ascent Scientific, forskolin from Abcam, Aβ25–35 from Bachem, and all other compounds from Sigma-Aldrich. Dual-channel local perfusion system was used to switch smoothly between control solution and specific pharmacological manipulations, and controlled by electric valves operated by a TTL trigger box commanded by MetaMorph (Molecular Devices). Immunostaining for mouse brain slices was performed following the standard protocol as previously described [The chemical Caescribed . As for +/Kr+ multi-line laser (CVI Melles Griot) with an acousto-optical tunable filter (AA.Opto). Laser beam was directed onto the glass/water interface at a super-critical angle, thereby enabling the total reflection of the excitation beam and the generation of evanescent field on the side of astrocyte substrate. The penetration depth (1/e2-intensity decay) of the evanescent field was estimated of the order of 200 nm [Total internal reflection fluorescence (TIRF) imaging was performed on a custom-made inverted microscope via the through-the-objective configuration  . The 488f 200 nm , therebyBackground was estimated from the autofluorescence signal in non-labeled cells of the same preparation, and then subtracted from the fluorescent images. The contour of the footprint of single astrocytes was delineated with the ImageJ plugin Cell Outliner or with MetaMorph segmentation tool, from which the mean fluorescence was measured over time to generate the time courses of specific signals. The FRET ratio of the cAMP sensor GFP(nd)-EPAC1(dDEP)-mCherry and the donor/acceptor bleed-through control was obtained as previously reported . During p < 0.05, and **p < 0.01.All data are expressed as mean ± standard deviation (SD), and the t-test was used for assessing the significance. Comparison of non-normally distributed data was also validated using their median ± absolute deviation and the non-parametric tests (Kolmogorov–Smirnov or Mann–Whitney U-test). All statistical operations were performed with Matlab (The MathWorks), with n.s., denoting non significant, *2+ transients in primary astrocytes cultured from mouse cortex. With the cytosolic Ca2+ indicator OGB-1 AM, we observed an oscillatory Ca2+ increase upon the local application of submicromolar Aβ25–35  and longer-lasting Ca2+ signals following a temporal delay  to image near-membrane Cak-GCaMP3   store [2+ rise. Compared to control condition , which caused a prominent discharge of Ca2+ from ER store  receptor, we examined the effect of its blocker 2-Aminoethoxydiphenyl borate  and we also observed an inhibition impact  store , 31, 85.2+ release from the internal store is recruited by the activation of metabotropic receptors. Astrocytes express a variety of receptors, among which metabotropic glutamate receptor 5 (mGluR5) and purinergic P2 receptors respond with Ca2+ rises to glutamate and ATP, respectively [p = 0.6; Fig. 2+ signals were fully abolished by the combination of P2 receptor antagonists PPADS (100 µM) and suramin  to induce Ca2+ influx [2+ influx through astrocytic SOCs [2+ upon, we used a standard protocol to image SOC-mediated Ca2+ influx [2+-free solution, and then Ca2+ added back to generate SOC-mediated Ca2+ influx  in response to the subsequent acute Aβ25–35 challenge, and over time started diminishing the basal Ca2+ level . After a 2-h preconditioning, most astrocytes responded to Aβ25–35 with a diminution in basal Ca2+ level , were also inhibited by an acute application of Aβ25–35  channel regulates basal Ca2+ level in astrocytes [2+ entry. Yet blocking SOC and TRPA1 channels by the wide-spectrum blocker gadolinium  [2+ diminution might be due to the potentiation of Ca2+ extrusion. We noted that chronic pre-conditioning of astrocytes with low Aβ25–35 gradually elevated intracellular Ca2+ level  that utilizes the Na+ gradient to export intracellular Ca2+ and the plasma membrane Ca2+ ATPase (PMCA) driven by ATP hydrolysis [+-free external solution showed no effect on Aβ-caused Ca2+ diminution . This manipulation proportionally altered Aβ-induced Ca2+ decline  [2+ diminution induced by Aβ25–35  , 81 faildrolysis , 26, 73.m Mg-ATP , 92, we  (50 µM) , 82, lar2+ so as to prevent its overload [2+ level independent of its elevation, suggesting other signaling pathways than Ca2+ had been recruited. Another second messenger cAMP is also known to potentiate PMCA activity [2+ level (peak dF/F0 = −7.5 ± 3.1%) in primary cultured astrocytes, which was counteracted by the PMCA blocker La3+ ‐mCherry [2+ extrusion.We next examined the signaling link between Aβ25–35 and PMCA potentiation. PMCA is activated upon intracellular rise of Caoverload , 62. In activity , 40. Ind‐mCherry , 87, ast2+ export with H+ influx [2+ dynamics during Aβ application. As the fluorescence of GFP protein can be quenched by H+ [2+ sensor protein GECO-R [2+. We observed a concomitant decrease in GFP fluorescence (dF/F0 = −4.0 ± 3.7%) upon Aβ25–35-induced Ca2+ diminution  to Aβ25–35, we observed stepwise H+ influx recorded by GFP quenching , a selective blocker of connexin43 hemichannel [2+ elevation and glutamate release as compared to control and to the inactive scrambled peptide of Gap26 . As expected, this treatment reduced the glutamate release during the phase prior to Caced Fig. a, corrobde Gap26 0 µM, a sichannel , also in2+-sensitive [2+-dependent glutamate release. One way to clarify this issue would be to block CX hemichannels, which however is known to interfere with Aβ-induced Ca2+ signal , a compound permeabilizing lysosomes by osmotic swelling [2+-dependent glutamate release.Another pathway for glutamate release could be Caocytosis , 50, 101ocytosis , 75. Theocytosis  was obse AM Fig. a1. Folloing Fig. a2, a3. Wing Fig. b. CD63-ping Fig. b. A simi2+ homeostasis in association with a multiphasic release of signaling transmitters have been observed in primary cortical astrocytes. As illustrated in Additional file 2+ concentration is maintained at physiological low level (~ 100 nM) in astrocyes in basal conditions [2+ elevation to cause further glutamate release. In basal conditions, Aβ25–35 plays an excitatory role in upregulating astrocyte Ca2+ signals. In Aβ-preconditioned astrocytes  receptor, has been approved for the treatment of moderate to severe AD [Our results suggest that Aβ-evoked Cad manner  (but seed manner ). In APPd manner . In addid manner . The relvesicles . Althougvesicles , lysosomvesicles , 50, 84.vesicles . Hence, vesicles , 98. Memevere AD .It has been noted that globally ablating pathologically altered astrocytes in AD mouse model worsens the disease . Thus, uAdditional file 1. Figure S1–S6 and Table S1–S2.


Direct MF bouton Ca2+ imaging revealed slow Ca2+ decay kinetics of action potential (AP) burst‐evoked Ca2+ transients. Finally, we observed that asynchronous release was preferentially mediated by Ca2+ influx through P/Q‐type voltage‐gated Ca2+ channels, while the contribution of N‐type VGCCs was limited. Overall, our results uncover the determinants of long‐lasting asynchronous release from MF terminals and suggest that asynchronous release could influence CA3 pyramidal cell firing up to seconds following termination of granule cell bursting.Action potentials trigger two modes of neurotransmitter release, with a fast synchronous component and a temporally delayed asynchronous release. Asynchronous release contributes to information transfer at synapses, including at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse where it controls the timing of postsynaptic CA3 pyramidal neuron firing. Here, we identified and characterized the main determinants of asynchronous release at the MF–CA3 synapse. We found that asynchronous release at MF–CA3 synapses can last on the order of seconds following repetitive MF stimulation. Elevating the stimulation frequency or the external Ca 2+ dynamics are the major determinants of this type of release rate. Asynchronous release was preferentially mediated by P/Q‐type voltage‐gated Ca2+ channels, while the contribution of N‐type VGCCs was limited.Asynchronous release at MF‐CA3 synapses can last on the order of seconds and presynaptic Ca Synchronous and asynchronous release both depend on a common presynaptic Ca2+ influx, but with different dependency on the Ca2+ concentration  axons to hippocampal CA3 pyramidal cells. Granule cells can fire action potentials in bursts  from a granule cell can trigger postsynaptic CA3 spiking  contribute to asynchronous glutamate release from MF remains unexplored.In giant MF terminals, action potential‐evoked Ca2+ dynamics to tune the amount of asynchronous release at MF terminals. Furthermore, we show that Ca2+ influx through P/Q‐type but not N‐type VGCCs significantly contributes to asynchronous release at this synapse.Here, we report that asynchronous release is long lasting at the MF–CA3 synapse. The frequency of presynaptic APs is the major factor that determines presynaptic Ca22.1Asynchronous release occurs following presynaptic firing and can last on the order of hundreds of milliseconds following stimulus termination. However, the properties of asynchronous release at MF–CA3 pyramidal cells remain generally undefined. We first aimed to investigate the dynamics of asynchronous release at MF–CA3 synapses.2+ concentration to 6 mM while voltage‐clamp recordings were performed from CA3 pyramidal cells. About 10 stimuli were delivered at 20 Hz, following which asynchronous EPSCs were automatically detected 10 ms following the last stimulus in the train . This increase in EPSC frequency lasted on the order of seconds  while the external Cad Figure . We obsen Figure . Therefo2.42+ and the stimulation frequency. To understand the presynaptic Ca2+ dynamics gating asynchronous release, we next quantified the temporal dynamics of single action potential‐ and burst‐evoked Ca2+ elevations in MF boutons.Asynchronous release strongly depends on presynaptic Ca2+ dynamics recordings were obtained using random access two‐photon imaging, allowing recordings with both high spatial and temporal resolutions   with a fast rise time  and slow decay kinetics  a finding consistent with presynaptic terminals of large volume , in line with our previous results  Figure . Under t+ Figure . Followi2+ transients using the single‐compartment model (methods) in order to be able to model AP‐evoked presynaptic Ca2+ dynamics at different stimulation patterns under dye‐free conditions. We observed that the decay time of burst‐evoked presynaptic Ca2+ transients was slow, with Ca2+ removal rate constant in the range of 0.2–0.4 ms‐1  used by us in . We assumed the presence of three major endogenous Ca2+ buffers  and computed Ca2+ traces for the four different stimulation scenarios  or ω‐Conotoxin GVIA   or CTx   Figure . We obse) Figure . Applicad Figure . Synchros Figure . We nexts Figure . The effs Figure , CTx hads Figure . Thus, t2.72+ removal from MF boutons gates long‐lasting asynchronous release. We next performed experiments to explore how manipulating the intracellular Ca2+ buffering dynamics would alter asynchronous release.Our results implicate that slow Ca2+ chelator EGTA–AM is expected to reduce the initial frequency of asynchronous release, but should make asynchronous release longer as it lengthens Ca2+ decay from the bouton  of vesicles remains unknown.Previous reports indicated that distinct Ca2+ transients are sustained during trains, we cannot exclude that a combination of factors acting in opposite direction could maintain a sustained Ca2+ transients’ amplitude. For example, we cannot exclude the possibility that the Ca2+ flux through P/Q‐ and N‐type VGCCs may be modulated during trains by activity‐ or Ca2+‐dependent modulation of channels. If present, such modulation of channels would complicate our interpretations for the proposed roles of P/Q‐ and N‐type VGCCs in asynchronous release.Another factor could complicate our interpretation. While we find that Ca2+ influx at this synapse : NaCl 87, NaHCO3 25, KCl 2.5, NaH2PO4 1.25, MgCl2 7, CaCl2 0.5, glucose 25, and sucrose 75 . The brain was hemisected and dissected on a petri dish while immersed in the cutting solution and was oriented to preserve the MF axons  for 30 min, at which time the incubator was turned off and the slices were allowed to recover for an additional 30 min.All experiments involving the use of animals were performed in accordance with Université Laval's guidelines for animal welfare. C57BL/6 mice (P17–P30) of either sex were anesthetized with isoflurane. Following decapitation, the brain was rapidly extracted and immersed in ice‐cold cutting solution saturated with 95% O4.22+, the Mg2+ concentration was adjusted to 2.5 mM. The Mg2+ concentration was reduced to 0 mM for experiments performed in 6 mM Ca2+. Whole‐cell recordings were performed at 32°C. CA3 pyramidal cells were visually identified using a 40×  water‐immersion objective. Microelectrodes were pulled from borosilicate filaments and filled with an intracellular solution containing (in mM): K‐gluconate 120, KCl 20, HEPES 10, MgCl2 2, Mg2ATP 2, NaGTP 0.3, phosphocreatine 7, EGTA 0.6 . CA3 pyramidal cells were voltage‐clamped at −70 mV. The MF were stimulated by positioning an electrode connected to a stimulus isolator  in stratum lucidum. The stimulating electrode was moved along stratum lucidum until facilitating EPSCs with rapid kinetics were identified.Acute slices were transferred to a recording chamber mounted on an upright microscope. Slices were held under a U‐shaped harp and perfused with oxygenated ACSF (described above) at a speed of 2–3 ml/min. For experiments performed in 1.2 mM Ca4.3V2.1 and CaV2.2 were used to block and investigate the role of Ca2+ flux through P/Q‐ and N‐type VGCCs in asynchronous release. P/Q‐type VGCCs were blocked with 200 nM ω‐Agatoxin IVA  and N‐type VGCCs were blocked with 200 nM ω‐Conotoxin GVIA . In all electrophysiology and Ca2+ imaging experiments, the brain slices were perfused for 10 min with the toxins to allow full diffusion and complete blockade of VGCCs. Following recordings, MF identity was confirmed by application of DCG‐IV .Selective toxins for Ca4.42+ imaging was performed on a custom‐built random‐access (RAMP) two‐photon microscope operated in LabView   was directed to the back aperture of the water‐immersion objective  by a pair of acousto‐optic deflectors . Emitted photons were collected through an oil condenser (NA = 1.4) and short‐pass filtered at 720 nm . Photons were separated in two channels based on their wavelength by a 580 nm dichroic mirror  and band‐pass filtered at 500–560 nm in the green channel  or 595–665 nm for the red channel. Photons were detected by external GaAsP photomultiplier tubes in both channels . The data were recorded with a custom‐written software in LabView and recorded on a personal computer. For Ca2+ imaging experiments, the intracellular solution was supplemented with the morphological indicator Alexa‐594 (40 µM) and the calcium indicator Fluo‐5F (385 µM) or Fluo‐4FF (375 µM). Whole‐cell recordings were obtained from visually identified granule cells using a 25X Leica objective (NA = 0.95). The morphological indicator and the calcium‐sensitive dye were left to passively diffuse in the granule cell axon for 1 hr before starting recordings. Granule cells with cut axons axon were discarded. For neurons with an intact axon, it was followed to the CA3 region where giant MF boutons could be identified 22σ2, where tiAP denotes the times of peaks of Ca2+ currents during each action potential. To take into account our experimental conditions [Ca2+]ext= 2.5 mM . We assumed that a MF terminal contains three endogenous buffers . The complete set of model parameters and Ca2+ binding reactions, including concentrations and binding properties of the endogenous buffers can be found in  profiles from the single‐compartment Ca2+ dynamics model in the stochastic simulations −1, β = 5,800 s−1, b = 0.25, χ = 2.94 (μM s)−1, δ = 130 s−1, γ1 = 0.417 × 10‐3 s−1, γ2 = 6,000 s−1, γ3 = 6,000 s−1. The repriming rate was constrained using the experimental release rate values at the start of asynchronous release . Peak amplitude measurements and analysis of decay kinetics were performed in Igor Pro 6.3. Individual trials were first averaged in space for all points recorded in the bouton and averaged across trials. Amplitude of Ca4.8t test. Differences were deemed significant if p < .05.Spontaneous and asynchronous EPSCs were automatically detected using the Clampfit software Event Detection module. Asynchronous release was measured in a 500 ms window starting 10 ms after the last stimulus termination. The average frequency of spontaneous EPSCs, measured before the stimulation train, was subtracted from the frequency of spontaneous EPSCs following the train to obtain the asynchronous component of release. Peak synchronous EPSC amplitudes were measured from averaged trials in Clampfit. Statistical analysis was performed with paired and unpaired Student's S. Chamberland, Y. Timofeeva, K. Volynski, and K. Tóth designed the study. S. Chamberland, A. Evstratova, and K. Tóth performed experiments, Y. Timofeeva, C. A. Norman, and K. Volynski performed computational modeling, S. Chamberland, Y. Timofeeva, K. Volynski, and K. Tóth analyzed the data, S. Chamberland, Y. Timofeeva, K. Volynski, and K. Tóth wrote the paper.


Nod1 or Rip2 in mice could prevent cardiac Ca2+ mishandling induced by sixth nephrectomy (Nx), a model of CKD. We examined intracellular Ca2+ dynamics in cardiomyocytes from Wild-type (Wt), Nod1−/− and −/−Rip2 sham-operated or nephrectomized mice. Compared with Wt cardiomyocytes, Wt-Nx cells showed an impairment in the properties and kinetics of the intracellular Ca2+ transients, a reduction in both cell shortening and sarcoplasmic reticulum Ca2+ load, together with an increase in diastolic Ca2+ leak. Cardiomyocytes from Nod1−/−-Nx and Rip2−/−-Nx mice showed a significant amelioration in Ca2+ mishandling without modifying the kidney impairment induced by Nx. In conclusion, Nod1 and Rip2 deficiency prevents the intracellular Ca2+ mishandling induced by experimental CKD, unveiling new innate immune targets for the development of innovative therapeutic strategies to reduce cardiac complications in patients with CKD.Risk of cardiovascular disease (CVD) increases considerably as renal function declines in chronic kidney disease (CKD). Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) has emerged as a novel innate immune receptor involved in both CVD and CKD. Following activation, NOD1 undergoes a conformational change that allows the activation of the receptor-interacting serine/threonine protein kinase 2 (RIP2), promoting an inflammatory response. We evaluated whether the genetic deficiency of  Chronic kidney disease (CKD) is a complex pathology characterized by a reduced glomerular filtration rate, increased urinary albumin excretion and kidney damage . Recent 2+ levels, acting as a key mediator of excitation-contraction (EC)-coupling. The initial depolarization event of the action potential activates L-type Ca2+ channels (LTCCs) of the sarcolemma, firing an inward voltage-dependent Ca2+ current type l (ICaL) from the extracellular medium. Ca2+ entry triggers a large release of Ca2+ from the sarcoplasmic reticulum (SR) by ryanodine receptor (RyR2) channels, resulting in an increased intracellular Ca2+ concentration ([Ca2+]i) that prompts cell contraction. For relaxation to occur, [Ca2+]i must return to diastolic levels and this occurs mainly by two mechanisms: (i) Ca2+ re-uptake by the Sarco/Endoplasmic Ca2+ pump (SERCA) 2a and (ii) Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX). During diastole, RyR2 channels are mostly closed; however, there is always a low but finite probability that a RyR2 channel will spontaneously open, mediating Ca2+ flux into the cytosol—known as Ca2+ sparks. Ca2+ spark frequency is normally low during diastole but, in some pathological conditions, abnormally large or frequent sparks can activate the RyR2 channels at neighboring release sites, generating SR Ca2+ waves that favor Ca2+ extrusion by NCX, and providing a substrate to initiate a life-threatening arrhythmia. Dysregulation of any of these Ca2+ handling mechanisms is commonly associated with the development of cardiac dysfunction [Cardiac muscle cell contraction is tightly regulated by the change in intracellular Cafunction , but wheThere is a growing body of evidence suggesting that inflammation induced by innate immune system activation can contribute to cardiac dysfunction ,12. Inde2+ mishandling [Nod1/2 or Rip2 deletion, was protective against acute kidney ischemia/reperfusion injury, suggesting that NODs respond to endogenous ligands after injury [Nod1/2 deletion had no impact on the chronic renal damage induced by ureteral obstruction [Several groups, including ours, have analyzed the role of NOD1 in CVD ,33,34,35handling . NOD1 exhandling ,36. Suppr injury . By conttruction .2+ mishandling induced by experimental CKD.The potential role of NOD1 in the cardiovascular complications caused by specific renal disease is unknown. Accordingly, the main goal of the present study was to investigate whether the NOD1-dependent pathway was implicated in cardiac dysfunction and CaWt) mice, accompanied by a reduction in kidney weight (KW). Notably, the KW in Nx mice considers the remaining one-third of the left kidney after the surgery, which is hypertrophied in both Wt and Nod1−/− mice , we measured the density of ICaL using patch-clamp technique in the whole cell configuration in cardiomyocytes isolated from Wt-sham and Wt-Nx mice. CaL density in both experimental groups, indicating that the Nx did no induce any change in the density of Ca2+ entering through LTCCs.Given the link between CKD and the prevalence of cardiac and systolic dysfunction ,3 and thhandling , we evalmyocytes A–D. By cam cells B. These 2+ transients between the different groups were related to changes in the cardiomyocyte SR Ca2+ load by measuring caffeine-evoked Ca2+ transients. 2+ transients in each group. The amplitude of caffeine-evoked Ca2+ transients was significantly lower in Wt-Nx cells than in Wt-sham cardiomyocytes , such as Ca2+ waves and spontaneous Ca2+ transients, in ventricular quiescent cardiomyocytes. 2+ wave (upper panel) and spontaneous Ca2+ transient release (lower panel) from cells isolated from Wt-Nx mice. Results showed that the occurrence of SCR was almost 3-fold higher in Wt-Nx cardiomyocytes than in Wt-sham cells  in a Wt-sham cell and pro-arrhythmogenic Ca2+ transients and waves (lower panel) in a Wt-Nx cell are shown in 2+ events was significantly higher in Wt-Nx cardiomyocytes than in Wt-sham cells, being this pro-arrhythmogenic Ca2+ release more than 2-fold higher in Wt-Nx  the decrease in the Ca2+ transient amplitude; (ii) the increase in their decay time constant; and (iii) the depressed cell contraction. Thus, the loss of NOD1 prevents the decline in systolic Ca2+ release induced by the Nx surgery. This improvement in systolic Ca2+ release can be related to the levels of SR Ca2+ load. Indeed, the reduction in the SR Ca2+ load observed in Wt-Nx cardiomyocytes was also prevented by the loss of NOD1. Thus, both the maintenance of the SR Ca2+ load and the improvement in the systolic Ca2+ release can explain the better cardiac parameters exhibited by Nod1−/−-Nx cells, compared with Wt-Nx counterparts.In contrast to what occurs in 2+ load can result from an increase in the Ca2+ leak during diastole. In this regard, Wt-Nx cardiomyocytes showed an increase in diastolic Ca2+ leak represented by a higher frequency of Ca2+ sparks, Ca2+ waves and spontaneous Ca2+ transients, as compared with Wt-sham cells. Supporting these results, we previously showed that RyR2 channel activity is increased in hearts from Wt-Nx mice [2+ leak observed in Wt-Nx mice is potentially a good substrate for the induction of cardiac arrhythmias since the released Ca2+ diffuses to neighboring RyR2 clusters inducing SCR and triggering cardiac arrhythmias. The genetic deletion of NOD1 reduces the occurrence of Ca2+ sparks, Ca2+ waves and spontaneous Ca2+ transients after Nx surgery, similar to those found in sham-operated Wt and Nod1−/− mice. Thus, NOD1 deficiency prevents the abnormal diastolic Ca2+ leak induced by the Nx, along with a reduction of pro-arrhythmogenic Ca2+ events. A likely explanation for this is that NOD1 deficiency rescues the SR Ca2+ content and improves cell contractility, recovering the impaired cardiac outcome observed in Wt-Nx mice. These features also bear a resemblance to those found in HF, since SCR is ameliorated in Nod1−/−-sham mice with experimental HF and the deletion of NOD1 prevents RyR2 hyperactivity [Depressed SR Ca-Nx mice . The incactivity .2+ cycling-dysregulation evident in our mouse model of CKD, we also determined whether the deficiency of RIP2 plays a role in the regulation of Ca2+ dynamics. RIP2 is the adapter kinase that mediates the majority of NOD1 actions. Accordingly, RIP2-deficient cells are hyporesponsive to signaling through NOD proteins and show severely reduced NFκB activation [2+ mishandling, as cardiomyocytes from Rip2−/−-Nx mice exhibit improved Ca2+ transients amplitude, kinetic rates and cell contractility compared with their Wt-Nx counterparts. This improvement in systolic Ca2+ release can be due to the rescue of the SR Ca2+ content found in the absence of RIP2. Moreover, we also demonstrate that RIP2 deficiency prevents the increased diastolic Ca2+ release observed in Wt-Nx mice, reducing the Ca2+ spark frequency and the occurrence of pro-arrhythmogenic events. These results are in line with those from the analysis of Nod1−/− mice. It would be of great interest to determine whether NOD1/RIP2 antagonists can also prevent the Ca2+ mishandling linked to CKD in future studies.To comprehensively study the pathway involved in Cativation . Althougtivation . We demo2+ mishandling induced by experimental CKD. Our findings suggest that the NOD1 proinflammatory pathway could be targeted for the development of new therapies to reduce the risk of cardiovascular complications in patients with CKD.In conclusion, we establish, for the first time, to our knowledge, that the genetic deletion of two different components of the NOD1 signaling pathway prevents CaNod1−/− and −/−Rip2 mice on a C57BL/6J  background were used. Mice were bred and housed under specific pathogen-free conditions in the Experimental Animal Centre of Instituto de Investigación Hospital Universitario la PAZ, IdiPAZ. Mice were maintained at controlled temperature (23–25 °C) on a 12-hr light/dark cycle with ad libitum access to water and a standard diet. Wild-type (Wt) C57BL/6J mice  were employed as controls. Nod1−/− and −/−Rip2 were kindly provided by Dr. Gabriel Nuñez . Gene expression analysis of Nod1 and Rip2 confirmed the absence of signal in Nod1- and Rip2-deficient mice (not shown). Macroscopic cardiac and kidney parameters were analyzed in all experimental groups as before [The study was conducted following recommendations of the Spanish Animal Care and Use Committee, according to the guidelines for ethical care of experimental animals of the European Union (2010/63/EU), and was approved by the General Direction of Agriculture and the Environment at the Environment Council of Madrid (PROEX: 053/16 and 272.5/20). Male s before ,49.Blood plasma samples were used to analyze the levels of phosphate , blood urea nitrogen (BUN) and urea  and fibroblast growth factor-23 (FGF-23)  following the manufacturers’ instructions.v/v, isoflurane/oxygen) anesthesia and preoperative analgesia  in a two-stage approach, as described [®, 4-0), which was subsequently removed. After a recovery period of one week, the entire right kidney was removed, following ligation of the renal blood vessels and cauterization of the ureter. The peritoneum and skin were then sutured, and the animals were returned to their cages. In control mice, sham surgeries involved midline incision, exposure of both kidneys, but no removal of tissue. The same timings were used as for Nx surgery. Blood plasma was employed for biochemical assays and isolated ventricular cardiomyocytes for Ca2+ recordings.Six-week-old male were randomly assigned to either five-sixth nephrectomy (Nx) or sham surgery under isoflurane   and CaCl2 (0.1 mmol/L). The heart was then removed from the Langendorff apparatus and the ventricles were cut out, finely minced into small pieces and mechanically dissociated in the enzymatic solution (standard Tyrode’s solution containing 0.1 mmol/L CaCl2). The cardiomyocyte cell suspension was filtered through a nylon mesh (250 µm), pelleted by centrifugation for 3 min at 300 rpm and suspended in Tyrode’s solution containing 0.5 mmol/L CaCl2. Cells were centrifuged as before and suspended in a storage solution containing 1 mmol/L CaCl2. Tyrode’s solution comprised (in mmol/L): 130 NaCl, 5.4 KCl, 0.5 MgCl2, 25 HEPES, 0.4 NaH2PO4, 22 glucose; pH = 7.4 adjusted with NaOH. Cardiomyocytes were immediately used for calcium imaging analyses and patch-clamp experiments.Six weeks after the second surgery, ventricular cardiomyocytes were isolated using standard enzymatic digestion . Briefly2+ transients, cells were first loaded with the Ca2+-sensitive probe Fluo-3 , and were then electrically excited at 2 Hz by field stimulation using two parallel platinum electrodes. Fluo-3 was excited at 488 nm and emitted fluorescence was collected at >505 nm. The fluorescence values (F) were normalized by the basal fluorescence (F0) to obtain the fluorescence ratio (F/F0). All Ca2+ images were corrected for the background fluorescence. The decay time constant of Ca2+ transients (Tau) was obtained by fitting the decay trace. Cell contraction was calculated as the difference of cardiomyocyte length between rest and contraction  and expressed as a percentage of shortening of cell length. Spontaneous Ca2+ sparks, and spontaneous Ca2+ transients and waves were acquired once stimulation was stopped. Ca2+ sparks were considered as located and fast increments in Ca2+ fluorescence. Total spark-mediated Ca2+ leak was calculated by multiplying spark frequency × peak × duration × width. SR Ca2+ load was assessed by rapid caffeine (10 mmol/L) application to deplete the SR of Ca2+ stores, after field-stimulation to reach the steady-state. Arrhythmic activity was analyzed as abnormal spontaneous Ca2+ release (SCR) by applying 3 cycles of field electrical stimulation at 2 Hz paced, consisting of 7 electric pulses. Data analysis was performed with homemade routines using IDL 8 software  and Image J 1.50i software (NIH). Images were corrected for background fluorescence. Cardiomyocyte surface area was quantified with the LSM Zeiss Image Browser 4.2 software (Carl Zeiss).Experiments were performed at room temperature (20–23 °C). Images were obtained with a Zeiss LSM 710 Meta confocal microscope , by scanning the cardiomyocytes with an Argon laser every 1 s. Experiments were performed at room temperature (20–23 °C). To record intracellular Cap was reached (p < 0.05) and there was no significant variance in homogeneity, Tukey’s post hoc multicomparison analysis was applied. All statistical analyses were performed with the SPSS 15.0 software  and significance was assumed when p < 0.05.Results are reported as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) or the chi-square test, as appropriate. If a significant level of