Specifically, we demonstrate our approach by developing dynamical systems to compute the solution for the Boolean NAE-K-SAT (K ≥ 4) problem as well as solve the Max-K-Cut of a hypergraph. Our work advances the potential of the physics-inspired ‘toolbox’ for solving COPs.The Ising model provides a natural mapping for many computationally hard combinatorial optimization problems (COPs). Consequently, dynamical system-inspired computing models and hardware platforms that minimize the Ising Hamiltonian, have recently been proposed as a potential candidate for solving COPs, with the promise of significant performance benefit. However, prior work on designing dynamical systems as Ising machines has primarily considered quadratic interactions among the nodes. Dynamical systems and models considering higher order interactions among the Ising spins remain largely unexplored, particularly for applications in computing. Therefore, in this work, we propose Ising spin-based dynamical systems that consider higher order (> 2) interactions among the Ising spins, which subsequently, enables us to develop computational models to  A significant driving force behind the effort to realize a so-called ‘Ising machine’ is that the solution to the Ising model can be mapped to many computationally intractable problems in combinatorial optimization  among others)18. Consequently, this creates the possibility of realizing Ising machine-inspired custom accelerators that can offer the possibility of significant performance benefits. However, dynamical system formulations that have been used to ‘solve’ the Ising model typically consider only pair-wise coupling; examples include, oscillator Ising machines, coherent Ising machines etc. From an application standpoint, while these characteristics capture quadratic interactions, the dynamical systems and their supporting computational models cannot be applied directly to solve problems that require higher order interaction among the spins20. Therefore, the objective of this work is two-fold: (1) define dynamical systems that model higher order (> 2) interactions among the Ising spins; and (2) map the resulting dynamics to relevant computational problems. We consider two examples: computing the solutions for the NAE-K-SAT  problem and the Max-K-Cut of a hypergraph. Our motivation behind selecting these two combinatorial optimization problems was that their objective functions directly map to the solution of the higher order Ising models, and therefore, help illustrate the principle of how dynamical systems for the higher order Ising models can be used in combinatorial optimization. Also, we emphasize here that presently our focus is on defining the Ising machine dynamics that capture the higher order interactions, and not on the physical implementation of the higher order interactions.The minimization of the Ising Hamiltonian using dynamical systems such as coupled electronic21; the NAE-K-SAT problem is considered here since it directly maps to the general form of Eq. . Without loss of generality, we assume that one spin state 18, here we explore this concept for hypergraphs (that entail higher order interactions) by considering the example of solving the Max-K-Cut of a hypergraph.In the prior section, we exploited the binary nature of the Ising spins . We now ‘extend’ the definition of the ‘spin’ in order to facilitate the design of computational models for an even broader spectrum of COPs that would benefit from the use of > 2 states for each node/spin. To facilitate this, we express the possible states of a spin as 27. The Max-K-Cut problem and its comparison with the archetypal MaxCut problem are illustrated in Fig. Computing the Max-K-Cut on a hypergraph is defined as the challenge of partitioning the nodes of a hypergraph into entclass1pt{minima18. Consequently, if the hyperedge satisfies the criterion for the Max-K-Cut i.e., that the nodes that are connected by it belong to at least two sets, the corresponding To develop the objective function for the problem, each hyperedge of the graph can be expressed as As an example, considering a hypergraph where the maximum number of nodes connected by a hyperedge is 3, the objective function for the Max-K-Cut problem can be expressed as:For a hypergraph with hyperedges having more than 3 nodes, the objective function entails the use of higher order interactions among the spins.18. The resulting energy function can be described as,To formulate a dynamical system for minimizing the above objective function, we express We note that n in Eq.  is a Lya18. Furthermore, using Eq. .We now evaluate our proposed model on a representative hypergraph. We consider a hypergraph where each hyperedge has 3 vertices. The corresponding dynamics for this case can then be written as,Figure In this work, we develop computational models for Ising machines that consider higher order interactions (beyond quadratic/pairwise) among Ising spins. Our approach enables the direct formulation of analog computing models for many COPs that entail such interactions without the need for problem decomposition and reduction. Furthermore, using the combination of higher order interactions along with ‘expanding’ the number of ‘spin’ states to greater than 2, we can directly map and solve an even broader class of problems on hypergraphs. This has been summarized in Fig. Supplementary Information.


Introduction: Acquired cold-induced urticaria is a form of physical urticaria that is usually spontaneous. However, reports have shown that bees, wasps, or jellyfish stings can trigger it. We report the first case of cold-induced urticaria following black ant bite-induced anaphylaxis.Case Report: A 41-year-old lady with no chronic illness with a known black ant bite allergy history. Three years ago, she sustained a black ant bite that required an emergency room visit to treat anaphylaxis. A few days later, she developed attacks of generalized hives on exposure to cold air and objects. She was started on desloratadine tablets which controlled her symptoms. The patient was given EpiPen and instructed to avoid black ants’ approach and exposure to cold. She was then followed up in our clinic.Discussions and Conclusions: Acquired cold-induced urticaria is a form of physical chronic inducible urticaria. Physical urticarias account for 25 % of chronic urticarias. The patient can have wheals, angioedema, or both in response to the cold exposure. Symptoms can be mild or severe, limiting the patient’s quality of life. Acquired cold urticaria is idiopathic; however, cases have been reported after different triggers, such as insect stings . The black Samsum ant is a recognized trigger of allergic reactions in Qatar and the Gulf region. In a study done in Qatar, 23.5% of anaphylaxis cases were due to black ant stings. There are no validated or standardized skin tests or immunotherapy for the black Samsum ant, which necessitates physicians to be careful in assessing such patients and focus on taking a detailed history. The limitation of testing and immune therapy makes history the tool for diagnosis, and avoidance is the mainstay of treatment.


Recent studies have revealed that the mode of the fatal stress-induced extracellular release of nuclear ProTα from cortical neurons in primary cultures, astrocytes and C6 glioma cells has two steps: ATP loss-induced nuclear release and the Ca2+-mediated formation of a multiple protein complex and its extracellular release. Under the serum-starving condition, ProTα is diffused from the nucleus throughout the cell due to the ATP loss-induced impairment of importin α–mediated nuclear transport. Subsequent mechanisms are all Ca2+-dependent. They include the formation of a protein complex with ProTα, S100A13, p40 Syt-1 and Annexin A2 (ANXA2); the fusion of the protein complex to the plasma membrane via p40 Syt-1–Stx-1 interaction; and TMEM16F scramblase-mediated ANXA2 flop-out. Subsequently, the protein complex is extracellularly released, leaving ANXA2 on the outer cell surface. The ANXA2 is then flipped in by a force of ATP8A2 activity, and the non-vesicular release of protein complex is repeated. Thus, the ANXA2 flop-out could play key roles in a new type of non-vesicular and non-classical release for DAMPs/alarmins, which is distinct from the modes conducted via gasdermin D or mixed-lineage kinase domain-like pseudokinase pores.Nuclear protein prothymosin α (ProTα) is a unique member of damage-associated molecular patterns (DAMPs)/alarmins. ProTα prevents neuronal necrosis by causing a cell death mode switch in serum-starving or ischemic/reperfusion models in vitro and in vivo. Underlying receptor mechanisms include Toll-like receptor 4 (TLR4) and G Damage-associated molecular patterns (DAMPs)/alarmins are extracellularly released from the cell upon various types of stress and exert inflammatory or inflammation-related actions. Representative DAMPs/alarmins include high mobility group box 1 (HMGB1) protein, heat shock proteins, extracellular cold-inducible RNA-binding protein (eCIRP) and various isoforms of S100 proteins ,3,4. As 2+-binding proteins are involved in molecular processes, such as tethering, docking, priming and fusion, in which Ca2+ sensor synaptotagmin-1, vesicular SNARE synaptobrevin (and homologue) and target SNAREs syntaxin/SNAP-25 (and homologues) play key roles in vesicle docking and fusion to the plasma membrane of nerve endings. Details have been reported elsewhere [The classical mode of secretion mechanisms has been discussed as exocytosis. It is a regulated type of exocytosis of hormones, neurotransmitters and digestive enzymes. In neurons and endocrine cells, a small percentage of secretory vesicles are fused with the plasma membrane upon cell stimuli, whereas the majority remains in a filamentous network of synapsins or actin in the case of neurons and endocrine cells, respectively, in reserve for subsequent stimulation . In theslsewhere .Unlike regulated exocytosis, there is a non-classical or constitutive vesicular exocytosis during the secretion of materials, such as collagen and extracellular matrix proteins (fibroblasts)  or bone Bacterial endotoxin lipopolysaccharides (LPS), when administered to the body at high levels, cause septic shock. LPS is known to stimulate Toll-like receptor 4 and produce proinflammatory cytokines such as IL-1β and TNFα via the activation of NF-κB. LPS is also reported to produce IL-1β and IL-18 via the activation of caspase-1 and release these cytokines through a GSDMD pore in the plasma membrane . On the 5 cells/cm2, LD) conditions (5 cells/cm2 (HD) showed an increase in survival activity; apoptotic features, such as nuclear fragmentation (TEM); and immunocytochemical changes, including the externalization of annexin V and activated caspase-3 [We discovered prothymosin α (ProTα), which inhibits necrotic neuronal death, from a conditioned medium of cortical neurons  and obsenditions A, more tnditions B. Transmaspase-3 . When thaspase-3 . The addaspase-3  and an iaspase-3  and a moaspase-3 .In the serum-free culture of primary neurons, ProTα release into the CM started as early as 1 h after the start of culturing, and it was time-dependent till 12 h . ImmunocThe mechanisms of non-classical and non-vesicular ProTα release induced by serum deprivation in C6 glioma cells could be divided into two stages. The first stage is related to the loss of cellular ATP under the condition of serum deprivation . The add2+-binding protein, interact at a ratio of 1:1; the interaction was Ca2+ concentration-dependent and in the range of 0.1 to 200 μM. The KD value for Strep-tagII-S100A13 in the binding to GST-ProTα was 211.9 and 69.8 nM in the absence and presence of 100 μM Ca2+, respectively. In the study using deletion mutant proteins, C-terminal regions of ProTα (102–111) and S100A13 (88–98) were found to play the most important roles in the interaction. In the fluorescence resonance energy transfer (FRET) analysis using ProTα-EGFP and DsRed2-S100A13, the FRET signal was markedly increased following serum deprivation stress in the presence of amlexanox, which inhibits the release of both proteins [2+. It should be noted that the decrease in extracellular Ca2+ levels by the addition of EGTA also reversed the FRET increase, which is consistent with the previous finding that serum deprivation causes a Ca2+ influx via voltage-dependent N-type Ca2+ channels [2+-concentration, possibly due to the serum-deprivation-stress-induced Ca2+ influx via the voltage-dependent N-type Ca2+ channel. When C6 glioma cells expressing S100A13 (Δ88–98) were used, serum deprivation did not show the extracellular release of ProTα, and the S100A13 mutant was lost [A pull-down assay using CM from serum-deprived C6 glioma cells and anti-ProTα IgG detected two protein bands at 14 and 10 kDa using Coomassie Brilliant Blue staining. The 14 kD protein was identified as ProTα by immunoblotting with anti-ProTα IgG, while the protein at 10 kDa was identified as S100A13 by MALDI-TOF MS/MS analysis . In the proteins . As expechannels . These rwas lost . This fiwas lost ,37,38,39ProTα is in the nuclei of neurons, astrocytes and C6 glioma cells in culture in the absence of serum-free or serum deprivation stress, which causes necrosis and causes the extracellular release of ProTα. However, when C6 glioma cells were given apoptosis-inducing chemicals, such as staurosporin, tunicamycin or etoposide, ProTα was no more localized in the nucleus but distributed throughout the cell . These c2+, we attempted to perform pull-down experiments using StrepTactinTM MicroPrep® resin beads and cytosol fraction from C6 glioma cells expressing Strep-tagII-S100A13. As S100A13 plays a role as a cargo molecule for the non-classical release of many proteins, it was expected that many molecules would be obtained in the pull-down assay. For this reason, we followed a previous study [2+ sensor synaptotagmin-1 (Syt-1). Syt-1 is known to play in the facilitation of SNARE complex formation [2+-dependent manner, a single positive band was detected at 40 kDa, corresponding to p40 Syt-1, which is deficient of the membrane-spanning domain of p65 Syt-1 [6-p40 Syt-1 attached to an anti-His6 IgG-coated sensor chip, Strep-tagII-S100A13 interacts with His6-p40 Syt-1 in a Ca2+ concentration-dependent manner with EC50 (Ca2+) = 83.19 μM. Under the condition of 100 μM Ca2+, the KD of S100A13 vs. His6-p40 Syt-1 was 17.5 μM, which is higher than in the case of the QCM assay, in which the KD of Strep-tagII-S100A13 vs. GST-ProTα was 69.8 nM in the presence of 100 μM Ca2+ [2+ channel and the Ca2+-induced Ca2+ release from an endoplasmic or sarcoplasmic reticulum [2+ concentrations are developed under the condition of serum deprivation stress. Compared with this, the interaction between ProTα and S100A13 may occur by the time of the development of high levels of Ca2+.To examine the molecules that interact with S100A13 in the presence of Caus study , in whicormation . In the 65 Syt-1 . When C6 μM Ca2+ . Consideeticulum , it is pWe used the Duolink™ in situ proximity ligation assay  for the detection, visualization, and quantification of the S100A13 and Syt-1 interaction. In this assay, both proteins in the fixed cell preparation were first labeled with specially designed oligonucleotides conjugated with each antibody/IgG against S100A13 and Syt-1. The preparation was then used for the amplification of the signal by generating a DNA surrogate of the protein using a polymerase. For the purpose of detecting the serum-deprivation-induced intracellular interaction of both proteins, amlexanox is useful to prevent the extracellular release. In the presence of amlexanox, the S100A13–Syt-1 interaction became evident throughout the cell time-dependently at 1.5 and 3 h after the start of serum deprivation. Furthermore, the intracellular injection of Syt-1 IgG also prevented the stress-induced release of ProTα and S100A13 . All the2+ sensor [2+. When neurons are activated and Ca2+ influx occurs, Syt-1 rebinds to the SNAREpin via voltage-gated Ca2+ channels. Thus, these Ca2+-dependent processes may trigger synchronous membrane fusion [Syt-1 (p65 form) is a Ca+ sensor  and prom+ sensor , which i+ sensor , under te fusion .Based on the comparison to exocytotic vesicular release, we speculated the possible binding of p40 Syt-1 to t-SNAREs in the serum-deprivation-induced release of ProTα and S100A13 by the use of different species of Clostridium botulinum neurotoxins (BoNTs), such as type A, B and C1 . Among t2+-mediated cellular processes, such as exocytosis-related membrane traffic, endocytosis and mitotic signaling [Strep-tagII-S100A13, a member of the S100A family in the absence of Ca2+, ANXA2 dimer was detected, while in the presence of 100 μM Ca2+, higher amounts of ANXA2 dimer and β-actin were detected. On the other hand, an immunoblot assay revealed that serum deprivation caused a time-dependent (0.5–3 h) decrease in S100A13 cell contents but an increase in CM contents. However, there was no change in ANXA2 cell contents, suggesting that S100A13 is extracellularly released, while ANXA2 remains in the cell. In the ELISA-based protein binding assay, the interaction between His6-ANXA2 and Strep-tagII-S100A13 bound to the Streptavidin plate was increased in a His6-p40 Syt-1 concentration-dependent manner in the presence of Ca2+.Annexins play roles in Caignaling ,50. Anneignaling ,52 and aignaling . When we2+ in the ELISA-based protein-binding assay [2+ influx via the N-type voltage-dependent Ca2+ channel. The involvement of ANXA2 flop-out in the serum-deprivation-induced release of ProTα, S100A13 and p40 Syt-1 was evidenced by the study using the intracellular delivery of anti-ANXA2 IgG. Interestingly, both S100A13 and Syt-1 signals were localized at the edge of the plasma membrane even in the absence of amlexanox. Similarly, ProTα was detected throughout the cell but was not localized in the nucleus.Under serum deprivation stress, S100A13 is extracellularly released from C6 glioma cells, but ANXA2 remains in the cell in the immunoblot analysis; however, there is a tight interaction between both proteins in the cytosol of C6 glioma cells in the presence of p40 Syt-1 and Cang assay . The tig2+-dependent scrambling of acidic phospholipid PS in the membrane. As the asymmetrical localization of phosphatidylserine (PS) is maintained by P4-type ATPases (flippases) [2+ influx in C6 glioma cells [The mechanisms underlying ANXA2 flop-out are interesting topics to investigate. One of the mechanisms would be related to the Caippases) , the ATPma cells , which mma cells ,56. Furtma cells  and thatma cells . When C6ma cells . The trema cells . These fupper panel, as follows: (1) under the serum-deprivation condition in culture or ischemia–reperfusion stress in vivo, ProTα is diffused from the nucleus throughout the cell due to an ischemic ATP decrease (energy crisis); (2) at the same time, a Ca2+ influx occurs via N-type voltage-dependent Ca2+ channels; (3) S100A13 is then associated with cytosol ProTα, Syt-1 (p65 or p40) and ANXA2 in a Ca2+-dependent manner. The second step is the membrane association and extracellular release of the ProTα/S100A13 complex (lower panel), as follows: (1) the tethering of the S100A13 complex to the membrane with the help of the Ca2+-dependent association between Ca2+ sensor Syt-1 and t-SNARE Stx-1; (2) the amplification of the intracellular concentration of Ca2+ via a Ca2+-induced Ca2+ release (CICR) from the endoplasmic reticulum; (3) the tight binding of ANXA2 to PS on the inner leaflet of the membrane; (4) the PS-ANXA2 flop-out by Ca2+-dependent TMEM16F scramblase activity; (5) the extracellular release of ProTα, S100A13 and p40Syt-1 complexes; (6) the recycling of PS-ANXA2 by flipping in via a force of ATP8A2. This mechanism may be involved in the repeated ProTα/S100A13 complex release.From these findings, the author proposes a new type of non-vesicular release mechanism for ProTα, a member of DAMPs/alarmins upon fatal stress. According to this working hypothesis, the initial step is the formation of the S100A13 complex , as shown in 2+-mediated extracellular flop-out system.Unlike in the case of cultured neurons and astrocytes, ProTα is not localized in the nucleus of cultured microglia. In addition, ProTα release was not caused by serum deprivation stress A. Mechan2+ (extracellular concentration), exogenously administered ProTα may block the tPA binding and subsequent MMP-mediated damage. At initial or weak MCAO, released endogenous ProTα may also inhibit the binding of naturally occurring tPA -induced focal ischemia model, the bilateral common carotid artery occlusion-induced global ischemia model and the retinal ischemia–reperfusion model ,30. The ring tPA .2+ concentrations [2+ and plays a role in vesicle budding, fusion, internalization and membrane repair via lipid segregation [The ANXA2 flop-out-type non-vesicular release of ProTα is a unique mechanism and, it looks distinct from known mechanisms through the membrane pores made of GSDMD or MLKL. Instead of membrane pores, the proposed mechanism includes TMEM16F scramblase, which plays a role in the process of phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane and is activated by very high Catrations . In addiregation . Althougregation ,35,63. Iregation . The rearegation . So far,regation ,67, sugg


A well-calibrated DFT/PCM protocol is employed in studying the factors that control the metal selectivity in biological systems by modeling typical calcium signaling/buffering binding sites and elucidating the thermodynamic outcome of the competition between the “alien” La3+/Ln3+ and “native” Ca2+, and La3+ − Ln3+ within the lanthanide series. The calculations performed reveal that the major determinant of the Ca2+/Ln3+ selectivity in calcium proteins is the net charge of the calcium binding pocket; the more negative the charge, the higher the competitiveness of the trivalent Ln3+ with respect to its Ca2+ contender. Solvent exposure of the binding site also influences the process; buried active centers with net charge of −4 or −3 are characterized by higher Ln3+ over Ca2+ selectivity, whereas it is the opposite for sites with overall charge of −1. Within the series, the competition between La3+ and its fellow lanthanides is determined by the balance between two competing effects: electronic (favoring heavier lanthanides) and solvation .Lanthanides, the 14 4f-block elements plus Lanthanum, have been extensively used to study the structure and biochemical properties of metalloproteins. The characteristics of lanthanides within the lanthanide series are similar, but not identical. The present research offers a systematic investigation of the ability of the entire Ln Note that the metal in the Ca2+ binding site is 7-coordinated, whereas in its La3+ counterpart lanthanum, it is 8-coordinated, at the expense of an additional first-shell water molecule. A positive free energy for Equation (1) implies a Ca2+-selective site, whereas a negative value suggests a Ln3+-selective one.In Equation (1), [Ca2+-signaling/buffering proteins were modeled, and their metal selectivity was examined. These are designated as Site 1, Site 2 and Site 3, whose optimized Ca2+/La3+-loaded structures, following the experimental observations  are typical of parvalbimun (EF site) and calmodulin (EF-I and EF-IV sites). One/two water molecules complement the first coordination shell of the metal cation A. Glutam2+ cation prefers heptacoordination, whereas lanthanum favors higher coordination numbers in biological systems, CN = 8 in particular [2+-O (ligand) distance is 2.43 Å, while the respective La3+-O (ligand) distance is 2.55 Å. Structurally, the two calcium/lanthanum-loaded binding sites differ mostly in their shape and metal coordination number (CN), as Carticular ,22. The 2+ → La3+ metal exchange (Equation (1)) are also presented in 1 is the major contributor to the free energy of the reaction. As expected, ΔG1 is quite negative , signifying very favorable substitution of the dicationic Ca2+ by the tricationuc La3+ in the gas phase. The solvation, however, strongly attenuates the gas-phase free energy as the desolvation penalty of the incoming La3+ species , and strongly outweigh the free energy gain by the outgoing Ca2+  [3+ substitution. Buried binding sites (ε = 4) are more predisposed to lanthanum attack  than their solvent-exposed counterparts . The trends outlined above for the Ca2+ → La3+ substitution are followed by those for the Ca2+ → Ln3+ exchange as well, where the gas-phase free energies vary between −524.6 and −613.0 kcal mol−1, and the ΔG4/ΔG29 fluctuate between −41.6 and −50.7/−24.0 and −33.3 kcal mol−1, respectively  . Neverthectively .3+/Ln3+ relative free energies, ΔΔGs, were evaluated  as the charge density increases in the same direction  and the highest (least favorable) for Pr3+ . Interestingly, ΔΔGs distribution seems to follow a semi-sinusoidal pattern (3+), followed by a minimum (Dy3+) followed by another (less pronounced) maximum (Er3+). Tracking the changes in the free energies of metal exchange within the lanthanide series is of particular interest. The following reaction was considered:valuated . The gas2+ complex includes two Asp- , one Glu- (bidentately bound), one Asn, a backbone peptide group and a water molecule. The La3+ cation binds to the same set of ligands, with the addition of another water molecule complementing its coordination number to 8. The optimized structures of the Ca2+ and La3+ constructs are presented in This is a typical EF-hand calcium binding site found in calmodulin (EF-II and EF-III), recoverin (EF-II and EF-III) and S100P (EF-II). The hepta-coordinated Ca2+ → La3+ metal exchange are also given in 3+ in Site 2 compared with that in Site 1. This is mainly due to the decreased negative charge density in Site 2 (−3) as compared to that in Site 1 (−4), which affects (unfavorably) the La3+-ligand interactions to a greater extent than the respective Ca2+-ligand interactions.The thermodynamic characteristics of the Ca2+ → Ln3+ exchange in the lanthanide series are summarized in the first half of 2+ competitors in this type of binding site. Furthermore, examining ΔΔG4/29 for the La3+ → Ln3+ substitution, one can conclude that very few of the lanthanides, such as Tb3+, Dy3+ and Lu3+, can successfully compete with La3+ for Site 2 . It has a low negative charge density, comprising one bidentate anionic Glu-, four neutral backbone peptide groups and one or two water molecules, depending on the metal. The structure of the optimized Ca2+ → La3+ substitution decreases further in absolute value as compared with those in Site 1 and Site 2. This weighs heavily on the condensed-phase ΔG4/29, which now are positive, implying calcium binding sites resistant to lanthanum attack.As expected, in view of the low negative charge density of Site 3 , the gas-phase free energy gain upon Ca2+ → Ln3+ exchange in the buried binding sites (ε = 4) remain positive throughout the entire lanthanide series as well . The initial model seven-coordinated Ca2+-bound constructs were subjected to geometry optimization (see below). Consequently, Ca2+ from the optimized structures was replaced by a La3+/Ln3+ cation, followed by a water molecule addition (to complement the lanthanide coordination number to 8), and the resulting constructs were fully optimized. The optimized structures of Ca2+ and La3+ complexes are shown in 3+ complexes does not differ significantly from that of the La3+ counterpart, and thus these are not depicted here. An oxidation state of 3+, typical of the lanthanum and lanthanides, was considered throughout the paper.Model CaDB: 1PAL  .3+ //SDD level of theory to confirm a local minimum on the potential energy surface; no imaginary frequency was detected for any of the structures considered. The frequencies were scaled by an empirical factor of 0.983 [th, comprising zero-point energy and entropies, S. The differences ΔEel, ΔEth and ΔS between the products and reactants in Equation (1) were used to evaluate the metal substitution Gibbs free energy in the gas phase, ΔG1, at T= 298.15 K, according to:Each metal-bound structure was optimized in the gas phase by employing a Gaussian 09 suite of programs . Electroof 0.983  and usedThe basis set superposition error for this type of reactions has been found to be insignificant , and thusolvε, of each metal complex. The outgoing Ca2+ and incoming Ln3+ are considered to be in a bulk aqueous environment (ε = 78) outside the binding pocket. Accordingly, their experimentally evaluated/estimated hydration free energies of −359.7 kcal mol−1  and propanonitrile (ε = 29), respectively. Solvation effects were simulated by performing PCM computations employing the solvation model based on density (SMD) scheme ,53 as imfor Ca2+  and Ln3+, see −1  was emplNote that Equation (4) allows us to separate electronic effects  from other effects such as the solvent accessibility and the effective dielectric constant of the active center, which are controlled by the protein matrix. 2+ in biological systems (to the best of our knowledge). The calculations performed reveal that the major determinant of the Ca2+/Ln3+ selectivity in calcium proteins is the net charge of the calcium binding pocket; the more negative the charge, the higher the competitiveness of the trivalent Ln3+ with respect to its Ca2+ contender  and solvation . The dependency is not straightforward, as ΔΔG4/29 fluctuate between positive and negative values, following semi-sinusoidal-like distribution  are poor competitors to their namesake lanthanum, whereas their counterparts from the middle of the series  are predicted to have greater affinity to the binding pocket than lanthanum. The dielectric properties of the binding cavity only slightly affect the La3+/Ln3+ competition, since ΔΔG4 and ΔΔG29 alternate in relatively narrow limits (Within the series, the competition between Laribution . The calw limits .


Objective: Chronic kidney disease patients have a decreased ability to maintain normal electrolyte concentrations in their blood, which increases the risk for ventricular arrhythmias and sudden cardiac death. Non-invasive monitoring of serum potassium and calcium concentration, [K+] and [Ca2+], can help to prevent arrhythmias in these patients. Electrocardiogram (ECG) markers that significantly correlate with [K+] and [Ca2+] have been proposed, but these relations are highly variable between patients. We hypothesized that inter-individual differences in cell type distribution across the ventricular wall can help to explain this variability.Methods: A population of human heart-torso models were built with different proportions of endocardial, midmyocardial and epicardial cells. Propagation of ventricular electrical activity was described by a reaction-diffusion model, with modified Ten Tusscher-Panfilov dynamics. [K+] and [Ca2+] were varied individually and in combination. Twelve-lead ECGs were simulated and the width, amplitude and morphological variability of T waves and QRS complexes were quantified. Results were compared to measurements from 29 end-stage renal disease (ESRD) patients undergoing hemodialysis (HD).Results: Both simulations and patients data showed that most of the analyzed T wave and QRS complex markers correlated strongly with [K+]  and [Ca2+] (ranging from 0.70 to 0.98). The same sign and similar magnitude of median r was observed in the simulations and the patients. Different cell type distributions in the ventricular wall led to variability in ECG markers that was accentuated at high [K+] and low [Ca2+], in agreement with the larger variability between patients measured at the onset of HD. The simulated ECG variability explained part of the measured inter-patient variability.Conclusion: Changes in ECG markers were similarly related to [K+] and [Ca2+] variations in our models and in the ESRD patients. The high inter-patient ECG variability may be explained by variations in cell type distribution across the ventricular wall, with high sensitivity to variations in the proportion of epicardial cells.Significance: Differences in ventricular wall composition help to explain inter-patient variability in ECG response to [K+] and [Ca2+]. This finding can be used to improve serum electrolyte monitoring in ESRD patients. The cauWe found that the relationships between electrolyte concentrations in the models were similar to those in patients and the variability in the relationships could indeed be explained, partly, by differences in ventricular wall composition.A population of coupled whole-ventricle and torso models was built taking as a basis the computed tomography (CT) data of a patient , as showVm is the transmembrane potential, Cm the membrane capacitance, β the membrane surface-to-volume ratio (the amount of membrane found in a given volume of tissue), Iion the sum of all transmembrane ionic currents, and G the monodomain conductivity tensor GintGext/(Gint + Gext) with Gint and Gext representing the intracellular and extracellular conductivity tensor fields, respectively. The fiber orientations used to compute the conductivity tensors were assigned with a rule-based method , denoted by F is the Faraday constant, R is the gas constant, T is the temperature and Vm is the transmembrane potential (in mV). The updated formulations for f2 and fCass of k = 0 if fCass,inf > fCass and V > − 60 mV, and k = 1 otherwise,Cellular electrophysiology was represented by the human ventricular myocyte model of The initial state for each simulation was pre-calculated from a single cell simulation, one for each cell type: endocardial, midmyocardial (M cells) and epicardial. The values of the model state variables after 1000 paced beats were considered as representative of the cell at steady state.uvw, where C stands for the word “case” and u, v and w denote the thicknesses of the endocardial, midmyocardial, and epicardial layers in tenths of the total wall thickness, respectively   . The popϕext, was computed by solvingVm simulated by the monodomain reaction-diffusion model (1) (ϕext only at a few locations for the computation of the ECG we used a Green’s function of the operator ∇ ⋅ ((Gint + Gext)∇.) for each of these locations to solve this equation efficiently. The ECG leads can be represented by a linear combination of Green’s functions because each recorded ECG lead is a linear combination of ϕext at two or more points. These linear combinations of Green’s functions are termed lead fields  . Since wd fields . Our lead fields . Five-be+], i.e., 5.4 mM, and [Ca2+], i.e., 2 mM. Individual ECG recordings of the same model corresponding to other values of [K+] and/or [Ca2+] were then projected onto the direction of the first PC determined as described above and used for further analysis. We chose to analyze the transformed lead, which represented the main characteristics from the combination of all leads and allowed for an improved morphological characterization.Principal component (PC) analysis was spatially applied to the T waves (or QRS complexes) of the eight independent leads  to enhanA wavelet-based single-lead delineation method was used for QRS detection and wave delineation of each of the 12 leads . Similar+], [Ca2+] and RR values in the patients). T waves  in the first PC were obtained and delineated to compute the below described markers ,• TS/A, representing the ratio between the maximal downward slope  and the amplitude of the T wave (expressed in 1/ms) (• in 1/ms) ,QRSw, representing QRS complex width calculated from QRS complex onset to QRS complex end (expressed in ms), and• QRSa, representing QRS amplitude calculated from minimum to maximum amplitude of QRS complex (expressed in mV).• Time- and amplitude-based T wave and QRS complex markers were computed from the last T wave and QRS complex of each simulated ECG projected onto the first PC. The analyzed T wave and QRS complex markers as shown in +] (3 mM) and maximum [Ca2+] (3.2 mM). We considered the reference for the simulated T wave (or QRS complex) in the same way as considered for the ESRD patients so as to perform the comparison on a similar basis. In the patients, the reference was computed at the end of the HD session when [K+] was minimum, closer to 3 mM, and [Ca2+] was maximum, closer to 3.2 mM, as this is the time when the patient was discharged from hospital with restored serum ion levels, thus being an acceptable reference for ambulatory monitoring (Morphology-based T wave and QRS complex markers were computed using the time-warping methodology described previously . For eacnitoring .fs(ts) and the reference simulated T wave (or QRS complex) as fr(tr). fr and fs, with their respective time domains, tr and ts. Let γ(tr) be the warping function that relates tr and ts, such that fs(γ(tr)) denotes the warping of fs(ts) using γ(tr). A dynamic programming algorithm was used to obtain the function γ*(tr) that optimally warps fr(tr) into fs(ts). This function is shown in fs(γ*(tr)), is shown in fr(tr). The simulated T wave (or QRS complex) at a particular electrolyte level was expressed as fs(ts) and fr(tr) as computed in previous studies  and in vector tr, thus not accounting for the sign of such differences. The amplitude marker da,T was computed from the area contained between fr(tr) and fs(γ*(tr)) normalized by the L2-norm of fr(tr), thus quantifying amplitude differences after time warping the two T waves ,• • • • da,Q, representing amplitude variations in QRS morphology (expressed as a %),• • • The set of all morphology-based T wave and QRS complex markers analyzed in this study included+], [Ca2+] and their combination with T wave and QRS complex characteristics in each model of the population, simulations were conducted under varying values of the electrolytes. The range of simulated [K+] values included the default level in the Ten Tusscher-Panfilov model, i.e., [K+] = 5.4 mM, as well as other levels below and above it: [K+] ∈ {3, 4, 5.4, 6.2} mM. In the case of [Ca2+], the range of simulated values included the default level of 2 mM and values around it: [Ca2+] ∈ {1.4, 2, 2.6, 3.2} mM. The combinations of [K+] and [Ca2+] included: , , , and  mM. The simulated ranges are similar to those observed in patients during HD , the percentage of change Y and its sensitivity SY;c;a1,a2 to changes in the proportion of cells was computed as follows . The values of a1 and a2 were taken as the minimum and maximum proportions of cells in each layer, respectively: 10% and 50% for endocardial and midmyocardial cells, and 20% and 60% for epicardial cells.For each T wave and QRS complex marker at each given concentration of , [Ca2+] and their combinations are shown in da,T, +] and [Ca2+]. Large differences between models in the population could be observed for all analyzed T wave markers. +] and [Ca2+] and their combinations, with high variability between models for all markers.T wave markers  and [Ca2+] . Inter-individual variability in the correlation coefficients associated with Tw and TS/A was high in both simulations and patients. For all other T wave morphology markers, the variability between models only partly reproduced the variability between patients. r with [K+] and [Ca2+]. As can be seen from the table, all the analyzed morphology-based T wave markers correlated strongly with [K+] and [Ca2+] in simulations and patients, with part of the inter-patient variability being reproduced by the models.To assess the extent to which our population of models could reproduce the inter-patient variability in T wave markers at concomitantly varying electrolyte levels, a correlation analysis was performed. +] and [Ca2+] are presented in The results of the sensitivity analysis performed to investigate how different proportions of endocardial, midmyocardial and epicardial cells may explain individual T wave responses when varying both [K+] and [Ca2+] in the same direction and with similar averaged magnitude as in ECGs from ESRD patients undergoing HD. In both simulations and patients, high inter-individual ECG variability was observed, which was accentuated at high [K+] and low [Ca2+]. Differences in cell type distribution, particularly in the proportion of epicardial cells, may explain inter-patient variability in T wave and QRS complex response to electrolyte variations within this model.In our whole-heart and torso simulations, T wave and QRS complex duration, amplitude and morphology changed with  (1.4–3.2 mM) and their combinations.We measured commonly used QRS complex and T wave markers describing characteristics related to their duration and amplitude, including T wave and QRS complex widths  . We eval2+] values to assess the impact on the ECG markers. In the patients, total serum calcium concentration ([Ca2+]) was measured, which has higher values than the corresponding ionized calcium concentration. The values of [Ca2+] in our analyzed ESRD patients were in the ranges reported in other studies  from the start to the end of the HD session were not statistically significant  and [Ca2+] that were in line with our observations in ESRD patients during and after HD . In the case of QRS complex markers, QRSa, +] and [Ca2+] in the simulations and the patients (absolute median correlation coefficient ranging from 0.71 to 0.98). These results support the use of our in silico population of models to describe the average response of T wave and QRS complex to variations in electrolyte levels like those seen in ESRD patients during and after HD.For confirmation of the ability of our in silico models could reproduce the general trends of electrolyte-induced changes in ECG markers observed in ESRD patients, we investigated how it may explain the high inter-patient variability in such changes. In agreement with the diversity of patterns of QRS complex and T wave changes in response to [K+] and [Ca2+] variations measured in the patients, we found different ventricular models presenting largely distinct behaviors that covered some of the patterns described in the patients. Models with thin epicardial layers were mainly related with a non-monotonic behavior of the evaluated ECG markers similar to that presented by some ESRD patients. This was particularly clear for the T wave morphology marker +] and [Ca2+] variations.After assessing how our population of patients . For thepatients . Also, tl layers . Therefo+] and [Ca2+], we computed QRS and T wave marker sensitivities to variations in the thickness of the endocardial, midmyocardial and epicardial cell layers. The highest sensitivity values were observed for T wave morphology markers da,Q and +] and low levels of [Ca2+], which may explain the remarkably large inter-patient variability observed at the beginning of the HD, when [K+] was enhanced and [Ca2+] was reduced. In particular, +] = 6.2 mM and [Ca2+] = 1.4 mM, sensitivity values above 110% were found, which corresponded to a coefficient of determination of 0.93 for the relationship between +] and [Ca2+] values.To quantify how different ventricular wall compositions may explain individual ECG responses at a range of  from the ventricular electrophysiology and a torso model. No changes with [Ca2+] were found in QRS duration, while the R wave amplitude and energy diminished with decreasing [Ca2+] (QRSa and 2+] (+] (2+] and high [K+].Several previous studies have characterized ECG features in relation to electrolyte concentrations. g [Ca2+] , which wg [Ca2+]  and incra2+] (+] , which i+] (Other studies have simulated human ventricular electrophysiology in a one-dimensional transmural model and have derived pseudo-ECGs at varying [K+] . IncreasInter-patient variability in the relation between electrolytes and ECG markers has been reported by several authors . We are 2+] relationship as described in several in vitro and in vivo studies (+] values larger than 6 and 8 mM, respectively (2+] reported experimentally. Another model recently proposed by 2+] relationship but requires further testing in 3D tissues. The Bartolucci et al. model could be a good option for future studies, considering its ability to reproduce a physiological APD-[Ca2+] relationship.Our simulations were performed with the most realistic available techniques to model the electrophysiology of the heart and the resulting ECGs. The electrophysiology was represented by a widely-used and detailed model of the human ventricular myocyte  that is  studies . This mo studies  and the  studies , but theectively . SubsequICaL formulation. In any case, the findings of our study remain to be confirmed. If confirmed, this could imply that accurate serum ion concentration estimation in individual patients would require a priori knowledge of the individual patient’s cell type distribution. Methods could be developed to infer such distribution from the ECG and blood data acquired from the patient during one or several prior HD sessions. ECG-based estimators of serum ion concentrations could be subsequently derived as a function of the patient’s cell type distribution.We found that variations in wall composition may explain inter-patient variability in the ECG response to electrolyte changes. While we have shown that variability in the thickness of endocardial, midmyocardial and epicardial layers may be a cause, we cannot exclude additional contributing factors. On top of including other types of ventricular heterogeneities, as discussed above, future studies could also investigate electrophysiological differences across the ventricular wall, e.g., by incorporating transmural differences in the in silico population included 7 whole-ventricle models with different cell type distributions across the ventricular wall, which we used to simulate variations in [K+] from 3 to 6.2 mM and in [Ca2+] from 1.4 to 3.2 mM. Future studies could extend the methods proposed here to add larger numbers of coupled ventricle-torso models and simulate a wider range of [K+] and [Ca2+].Our 2+], which has been reported to be possibly involved in observed alterations in the ECG (2+] variations because serum [Mg2+] levels were not measured in our patients.The effects of other electrolyte concentrations such as magnesium,  and [Ca2+]. Differences in the proportion of ventricular cell types, particularly of epicardial cells, may explain the inter-subject variability in such responses. These findings can pave a path to design better tools for non-invasive serum electrolyte monitoring and prediction of arrhythmic events in the patients.Our


Although mentoring is critical for career advancement, underrepresented minority (URM) faculty often lack access to mentoring opportunities. We sought to evaluate the impact of peer mentoring on career development success of URM early career faculty in the National Heart Lung and Blood Institute-sponsored, Programs to Increase Diversity Among Individuals Engaged in Health-Related Research-Functional and Translational Genomics of Blood Disorders (PRIDE-FTG). The outcome of peer mentoring was evaluated using the Mentoring Competency Assessment (MCA), a brief open-ended qualitative survey, and a semi-structured exit interview. Surveys were completed at baseline (Time 1), 6 months, and at the end of PRIDE-FTG participation (Time 2). The following results were obtained. Between Time 1 and Time 2, mentees’ self-assessment scores increased for the MCA (p < 0.01) with significant increases in effective communication (p < 0.001), aligning expectations (p < 0.05), assessing understanding (p < 0.01), and addressing diversity (p < 0.002). Mentees rated their peer mentors higher in the MCA with significant differences noted for promoting development (p < 0.027). These data suggest that PRIDE-FTG peer mentoring approaches successfully improved MCA competencies among URM junior faculty participants with faculty ranking peer mentors higher than themselves. Among URM faculty, peer mentoring initiatives should be investigated as a key strategy to support early career scholar development. For underrepresented minority (URM) faculty pursuing academic careers, enhanced mentoring opportunities in research training are lacking . URM resA national initiative that has succeeded in providing consistent mentoring support for URM faculty is the National Heart Lung and Blood Institute (NHLBI)-sponsored Programs to Increase Diversity Among Individuals Engaged in Health-Related Research (PRIDE) ,6,7. ForTo enhance research mentoring strategies in hematology, an important innovation of the PRIDE-FTG program is the central role of peer mentoring, which has been demonstrated to increase productivity and the efficiency and focus of research projects . The PRIThe present study was a mixed-methods evaluation study that was approved by the Institutional Review Board (IRB) of Augusta University.In the USA, nine unique PRIDE Programs are funded along with a central coordination core. These mentored-research programs address the difficulties experienced by URM junior investigators in establishing independent research projects and achieving higher academic ranks. The PRIDE-FTG at Augusta University, established in 2011, aims to enhance basic and clinical/translational research skills in hematology using functional and translational genomics techniques. Matriculation into the PRIDE-FTG program involves a two-stage application process including a competitive review. The program consists of two in-person Summer Institutes (SI), each lasting 10–14 days, followed by the receipt of a certificate of completion from NHLBI. Each SI trains 8–10 mentees who are assigned a primary research mentor, institutional mentor, and peer mentor as described below. Both SIs are comprised of didactic lectures, hands-on lab practical, grant-writing workshops, and an opportunity for mentees to compete for Small Research Project pilot funding to support future extramural grant applications.During SI1, mentees were given a 2-hour didactic interactive lecture on peer mentoring. Immediately afterwards, they participated in a speed-mentoring event, intended to mimic speed dating, where mentees asked questions of other mentees to identify compatible peer mentors. Mentees were given sample ice-breaker questions to facilitate discussions. Prior to the conclusion of SI1, peer mentor groups were formed, ranging from 2 to 4 peers per group, dependent on cohort size and preferences. Participants could tailor the format, frequency, and goals of the peer mentoring group and were required to submit monthly narrative reports of their groups’ interactions. Peer mentoring groups continued for the 1-year duration of the PRIDE-FTG program, and some peer mentoring groups continued after the conclusion of the program. We were interested in peer mentoring self-assessment of competency over the course of the PRIDE program as well comparisons of peer mentoring competency in self-versus others. Past research has identified discrepancies in mentoring competency assessment with difference when one is rating self or others .The PRIDE-FTG peer mentoring program was established in 2015. Forty-four mentees from Cohorts 4 through 8 (2015–2020) were included in this study. At the time of their entry into PRIDE-FTG, mentees were 93% female, 89% Black, and 86% assistant professors.The peer mentoring aspect of the PRIDE-FTG program was evaluated using four assessments. The first two were the Mentoring Competency Assessment (MCA) , which wAt the start of PRIDE-FTG SI1, mentees were consented by a member of the evaluation team. All but the last cohort included in this sample were consented in person and were given an informed consent to sign and return to PRIDE-FTG staff and a copy was given to them to keep. Due to the COVID-19 pandemic, which led to a virtual SI1 in 2020, the last cohort included in this sample was consented via Zoom . These participants were emailed copies of their informed consent and instructed to sign and return them as a scanned document or photo image.After informed consent was obtained, mentees were emailed a link to the first survey on Qualtrics , a cloud-based surveying program. The first survey, noted as Time 1, consisted of the MCA Self-Assessment and was 5–10 minutes long. Six months into the PRIDE-FTG program, mentees were emailed a second survey that contained a qualitative question requesting suggestions for any improvements to the PRIDE-FTG peer mentoring program. The final survey administration, noted as Time 2 and given at the end of the PRIDE-FTG program participation, consisted of the MCA Self-Evaluation and the MCA Peer Mentor evaluation, lasting approximately 10–20 minutes. Also, at the conclusion of SI2, mentees completed a 30–45-minute interview with a member of the evaluation team. Three of the five cohorts included in this sample were interviewed in person. Recordings were obtained using a digital voice recorder and later transcribed by a member of the evaluation team. Both the audio recordings and the transcriptions were uploaded to Box , a Health Insurance Portability and Accountability Act-compliant cloud-based storage program. Due to the COVID-19 pandemic, the latter two cohorts completed their exit interviews virtually via Microsoft Teams , through which interviews were recorded and transcriptions automatically generated. Transcriptions were downloaded from Microsoft Teams and cleaned for analysis. Both the recording and transcription were uploaded to a secure cloud-based storage server.Quantitative survey responses were downloaded from Qualtrics and imported into IBM SPSS Statistics Software , version 28. Frequencies were used to describe demographic information of the five cohorts. Analyses for quantitative responses included paired samples t-tests for the MCA Self-Assessment at Time 1 and 2 and for comparisons between the MCA Self-Assessment at Time 2 and the MCA Assessment of a Peer Mentor “Between Time 1 and 2, participants’ self-assessment scores increased for both the total MCA and for each of the six competencies. Statistically significant increases were demonstrated for the entire MCA (p < 0.01), as well as with four of the six competencies .“Also increased between Time 1 and 2 was aligning expectations (p < 0.05). An aligning expectations characteristic is the consideration of mentor–mentee differences, which was underscored by one participant who noted:We also read each other’s grants as well. That’s has been a great learning point for me to be able to read other people’s grants” (Mentee 31).“Also, significantly higher at Time 2 was assessing understanding (p < 0.001). A component of this competency, assessing mentees’ knowledge and skills, was exemplified by the following response:Every time I got together with my peer groups, we were able to sort of let it out here, down at a whole different level, in terms of how things were affecting us. You know it was such a heavy time to be honest with you, I never ever want to talk about it again… but it was enough interactions that allowed me to actually let go and sort of start talking about that with a different group of people from what my normal environment would have exposed me to” (Mentee 40).“Lastly, between Time 1 and 2, self-assessment scores significantly increased for addressing diversity (p < 0.01). As an example of an element of the addressing diversity subscale, one participant mentioned the benefits of working with a diverse group of peer mentees, particularly in the wake of the killings of Ahmaud Arbery, George Floyd, and Breonna Taylor:I think I've gained confidence overall in my career advancement in my career trajectory, and… [my] peer group has also made my goal seem more attainable to me” (Mentee 31).“When comparing the Time 2 self-assessment with the peer mentor assessment, PRIDE-FTG mentees rated their peer mentors higher in every competency and the MCA as a whole, when compared to themselves , aligning expectations (p < 0.05), assessing understanding (p < 0.01), and addressing diversity (p < 0.002). However, when comparing themselves to their peer mentors, the most significant differences were noted for promoting development (p < 0.027).et al. attributed long-term (6-year) peer mentor success to the balance of similarities and differences among a group of implementation scientists in similar career phases [The findings of the peer mentors’ report of effective communication, aligning expectations, assessing understanding, and addressing diversity as critical self-assessment competencies is not surprising. Other studies point to the importance of effective communication and diversity in developing peer mentoring relationships among junior faculty members –18. Membr phases . Likewisr phases .The development of peer mentoring relationships has also been observed to promote health care careers, increase access to mentorship, and encourage meaningful mentoring relationships between URM high school and medical students . SeveralWe also found that the informal use of technology such as GroupMe and cell phone text messaging proved to be invaluable resources for effective communication. As a result, several long-term supportive friendships and exchange of ideas for research collaboration have arisen. However, we also acknowledge limitations in our study. Some mentees were noncompliant with program evaluations which contributed to limited sample size for data analysis. We are continuing the peer mentoring programs for future mentees, which will increase sample size and extend the follow-up period to 2 years. Larger cohorts to validate our innovative peer mentoring strategy is critical to further evaluate the program’s efficacy. Nonetheless, our findings and those of other investigators support the critical need for expansion of innovative high-impact junior faculty peer mentoring initiatives similar to the PRIDE-FTG Program and others that achieve a greater degree of communication, support, and collaboration than traditional dyadic mentor-protégé pair relationships ,24. AlthIn conclusion, our innovative peer mentoring program fostered community among URM junior faculty mentees. Two major themes emerged from our data analysis. Mentees experienced an increase in peer mentoring skills over the course of the PRIDE-FTG program. Mentees tended to rate themselves lower than their peers at Time 2, which may be attributable to early career individuals’ experiences of imposter syndrome, the inner experiences of self-doubt, or overestimation of peers’ competence due to relationships. Given the challenges of URM faculty retention at academic institutions, it would be important to address mentees’ underestimation of competency and skills, which might contribute to attrition at higher faculty rank. Since peer mentoring increases productivity and sustained collaborative research relationships, it is quite plausible that efforts focused on effective communication and assessing understanding and diversity can provide a safe and supportive environment for junior faculty to discuss challenges and successes in professional development. The PRIDE-FTG program will continue the peer mentoring initiatives in support of early-career scholar development among URM populations.


Photopharmacology is a booming research area requiring a new generation of agents possessing simultaneous functions of photoswitching and pharmacophore. It is important that any practical implementation of photopharmacology ideally requires spatial control of the medicinal treatment zone. Thus, advances in the study of substances meeting all the listed requirements will lead to breakthrough research in the coming years. In this study, CQDs@phosphonate nanohybrids are presented for the first time and combine biocompatible and nontoxic luminescent carbon quantum dots (CQDs) with photoactive phosphonate enabling inhibition of butyrylcholinesterase (BChE), which is a prognostic marker of numerous diseases. The conjunction of these components in hybrids maintains photoswitching and provides enhancement of BChE inhibition. After laser irradiation with a wavelength of 266 nm, CQDs@phosphonate hybrids demonstrate a drastic increase of butyrylcholinesterase inhibition from 38% up to almost 100% and a simultaneous luminescence decrease. All the listed hybrid properties are demonstrated not only for in vitro experiments but also for complex biological samples, i.e., chicken breast. Thus, the most important achievement is the demonstration of hybrids characterized by a remarkable combination of all-in-one properties important for photopharmacology: (i) bioactivity toward butyrylcholinesterase inhibition, (ii) strong change of inhibition degree as a result of laser irradiation, luminescence as an indicator of (iii) bioactivity state, and of (iv) spatial localization on the surface of a sample. A photoswitch is a compound changing structural geometry or conformation as well as chemical properties under optical radiation. As a photoswitch, one primarily uses azobenzenes, quinoline derivatives, diarylethenes, diazocins, or aryl azoindazoles malonate. Diethyl 2-[(4-chlorinephenyl)amino]malonate was prepared from diethyl 2-bromomalonate and the 4-chloroaniline via the procedure adopted from . Chloroaorted in  and trie−1 (Sigma-Aldrich), bovine serum albumin (BSA) (Sigma-Aldrich), butyrylthiocholine chloride (Sigma-Aldrich); HEPES buffer solution  (Sigma-Aldrich); KMnO4, Mn(Ac)2·4H2O, K2CO3  were used for measurements of biological activity.Butyrylcholinesterase from horse blood plasma (EC 3.1.1.8), activity 264 U mgThe structure and composition of CQDs were studied by transmission electron microscopy (TEM)  using the annular dark-field imaging method of sample mapping of scanning electron microscopy (HAADF STEM)  on a Tecnai Osiris microscope  at an accelerating voltage of 200 kV.The element composition of CQDs was studied using X-ray photoelectron spectroscopy (XPS) with AxisUltraDLD  and LAS-3000  spectrometers. Photoelectrons were excited by X-ray radiation from an aluminum anode (AlKα = 1486.6 eV) at a tube voltage of 12 kV and an emission current of 20 mA. The photoelectron peaks were calibrated using the C 1 s carbon and N 1 s nitrogen lines with binding energies of 285 and 400 eV, respectively.Measurements of the sizes/zeta potential of CQDs in suspensions were performed using the Malvern Zetasizer NanoZS instrument .1H, 13C, 31P spectroscopy recorded with a Bruker Advance 400 spectrometer  , the residual signals of the solvent (CDCl3) were used as an internal reference.The structure of the product was confirmed by the data of The optical density spectra were recorded using a Shimadzu UV-3600 spectrophotometer  in the wavelength range of 200–500 nm with a resolution of 1 nm (scanning speed—medium).−2 mg/mL at room temperature. Excitation intensities used for steady-state and kinetic photoluminescence measurements were very low and did not affect the samples.The photoluminescence spectra of the samples were obtained using a Shimadzu RF-6000 fluorimeter . Additional processing of the spectra consisted in smoothing over 10 points using a moving average. The steady-state photoluminescence spectra were measured using a modular spectrofluorometer Fluorolog-3  with a continuous wave Xe lamp as an excitation source. Luminescence kinetics measurements were carried out using LED340 and LED390 . Lifetimes were calculated using the TCSPC technique. All measurements were performed with samples with a CQD concentration of 6.6 × 10–1.The FTIR spectrum of CQD was recorded using a Bruker Invenio R FTIR spectrometer  using an attenuated total reflection (ATR) accessory with a diamond crystal. The resulting spectrum was obtained by averaging 20 scans with a spectral resolution of 4 cm−1 with a resolution of 4 cm−1. In the obtained spectra, the Blackman–Harris apodization function was used and the Mertz phase correction was applied. Due to the low concentration of the investigated substances, the subtraction of the contribution of CQD in isopropanol  using an ATR accessory with a diamond crystal. To obtain the FTIR spectra, the spectrometer configuration with a XT-KBr beam splitter and liquid nitrogen cooled mercury cadmium telluride (MCT-A) detector was used. For the solutions under investigation, the spectra were obtained in the region 650–4000 cmanol see  was perfThe activity of butyrylcholinesterase (BChE) is the initial rate of biocatalytic hydrolysis of butyrylthiocholine, which is determined by the accumulation of thiocholine using a thiol-sensitive sensor . The response to thiocholine formed during the enzymatic hydrolysis of butyrylthiocholine was recorded in the product accumulation mode .2-modified planar electrodes [The BChE activity before and after inhibition was measured using an IPC-micro neurotoxin amperometric analyzer with integrated MnOectrodes ,36.–3:10–7 M), dissolved in the HEPES buffer containing BSA (1 mg/mL), pH 7.5, was placed in a microtube with a buffer solution. Incubation with the substrate was carried out for 10 min . The control (blanc) analysis—the preliminary measurements of BChE activity without inhibitor—was performed to normalize the enzyme activity before each inhibition measurement. In accordance with the literature data, the measurement inaccuracy of an IPC-micro neurotoxin analysis does not exceed 10% [To determine the inhibiting ability of the test substance, the stock solution of the BChE (1 mg/mL), and the substrate butyrylthiocholine chloride (0.5 M) were initially prepared. Then, an aliquot of the inhibitor (PhAM)   with a defocused laser beam (d = 10 mm) in a 1 cm thick quartz cell. The laser irradiation time was 30 min in all these experiments.The experiments with biological objects were carried out on a specially developed experimental setup. A diode laser was used as a source of excitation of CQD to initiate photoluminescence of the hybrids . The laser radiation was focused by a lens onto the surface of a sample placed on a glass slide. The reflected signal (elastic scattering + luminescence) was collected at the entrance slit of a monochromator . An Omega 420ALP edge filter (SKU: W3726) was used to reduce the contribution of scattered light. The obtained photoluminescence spectra were recorded using a PMT . As a model experiment with a complex biological sample, a cut-out piece of chicken breast (across muscle fibers) was used. CQD and Hybrid4 CQD@PhAM were injected with a syringe into the chicken breast to a depth of ca. 1 mm. Part of the substance was poured onto the surface of the chicken breast; then the surface was carefully blotted with fat-free napkins.31P NMR chemical shift of the phosphonate was observed in a strong field at −8.52 ppm. The 31P, 1H, and 13C NMR spectra and their description for the PhAM compound are presented in The phosphorylated arylaminomalonate compound a with a −1  and ethylenediamine (1.08 mL) were used as precursors (precursors ratio 1:1). The prepared mixture of precursors was placed in a Teflon container, which, in turn, was placed in a steel autoclave. Next, the sample was kept in a muffle furnace for 2 h at a temperature of 190 °C. To remove the larger fraction, the resulting highly concentrated aqueous suspension of CQDs was passed through a membrane filter with a pore diameter of 0.22 μm.According to the TEM results, the synthesized CQDs are carbon nanoparticles with an average size of 7.0 ± 0.5 nm a. One ca−1 (corresponding to stretching ν(C-O), bending δ(C-H) vibrations), 1385 cm−1 (corresponding to δ(C-H), ν(C-O) vibrations), 1487 cm−1 (corresponding to ν(N=O), ν(O-O), δ(N-H) vibrations), 1550 cm−1 (corresponding to δ(N-H), ν(C-N), ν(C=C) vibrations) and 1647 cm−1 (corresponding to ν(C-N), ν(C=O), and δ(O-H) vibrations). In the high-frequency region of the spectrum (2700–3800 cm−1), bands attributed to the stretching vibrations of ν(C-H), ν(N-H), and ν(O-H) are observed [According to the XPS studies , synthes−2 M. Hybrid1 CQD-PhAM was obtained by stirring the sample Mix with a magnetic stirrer for 1 h. Hybrid4 CQD-PhAM was obtained as a result of 4 h of stirring. Then the obtained samples were studied with absorption, luminescence, and FTIR spectroscopies as well as IPC-micro neurotoxin amperometric analysis.Phosphonate and carbon quantum dots hybrids (CQD@PhAM) were prepared by steering the components at ambient conditions for different times. To study the effect of the conjunction of the components on the final photo pharmacological properties, we prepared several samples based on phosphonate (PhAM) and carbon quantum dots (CQD)—(1) Mix CQD-PhAM, (2) Hybrid1 CQD@PhAM and (3) Hybrid4 CQD@Ph. Mix CQD-PhAM was prepared as a result of adding PhAM powder to the CQD suspension in isopropanol without stirring: the concentration of CQD suspension in isopropanol was 1 mg/mL and the concentration of phosphonate was 10One can see that the spectra of Mix, Hybrid1, and Hybrid4 are characterized by absorption bands at 240 and 290 nm, typical for phosphorylated 2-arylaminomalonate compound  (1620–1700 cm−1), as well as in the region of stretching hydrogen vibrations in O-H and N-H bonds (3100–3600 cm−1) was observed [−1 (significant contribution from stretching vibrations in the P-O and C-O bonds in PO3 and COO groups as well as stretching vibrations of C-O bonds in P-O-C and C-O-C parts) [FTIR absorbance spectroscopy is a sensitive technique allowing one to observe structural changes, which can take place during hybrid formation. To provide the observed changes in the PhAM component more clearly, the FTIR spectrum of CQD in isopropanol was recorded  so that observed . In addiC parts) ,10,15,42In such a way, absorption, luminescence, and IR spectroscopy reveal the conjunction of the components CQD and phosphorylated arylaminomalonate, and the formation of a hybrid. Then biological activity towards BChE inhibition was measured for Mix, Hybrid 1, and Hybrid 4, and for reference samples—PhAM and CQD. It was found that the CQD isopropanol solution does not have any biological activity towards BChE; PhAM showed BChE inhibition of 25%. The mix sample shows no effect of the CQD on the biological activity of PhAM within measurement error . Hybrid1Summarizing the results of PhAM, CQD, Mix, Hybrid1, and Hybrid4 samples investigation with comprehensive techniques, one can conclude that the process of the conjunction of PhAM and CQD by means of prolonged stirring results in the formation of hybrids possessing chemical interaction of the components via polar groups of PhAM. The properties of the resulting hybrids differ from that of the individual components. This is manifested by changes in the FTIR spectra, by a decrease in the luminescence intensity, and by an increase of BChE inhibition for Hybrid1 and Hybrid4 in comparison with PhAM, CQD, and Mix samples. In such a way, we demonstrated for the first time the formation of hybrids from bioactive organophosphorus compounds and luminescent CQD.However, the chosen organophosphorus compound is characterized by its ability to change the conformation after illumination with resonant laser irradiation . Our earex = 266 nm, power 120 mW) was chosen for irradiation of CQD@PhAM hybrids. In accordance with the absorption spectra presented in The effect of laser irradiation on the photoluminescence properties of Hybrids CQD@PhAM is shown in av, was calculated for a fair comparison with samples before irradiation: I1 and I2 are the amplitudes of the exponential terms with decay constants (lifetimes) τ1 and τ2 [Important information about hybrid formation and the effect of laser irradiation can be extracted from luminescence kinetics measurements. 1 and τ2 . The ana−1 (v(C=O) in COO groups), 1500 cm−1 (δ(NCH) and δ(CCH) in the benzene ring), 1288 cm−1 (v(P=O) in the PO3 group and v(C-O) inside the COO groups), 1028 and 860 cm−1 (v(C-O), δ(OCO) in and near COO groups and ρ(CH3)). At the same time, there is an increase in absorption for the peak at 1051 cm−1  in P-O-C). In addition, a new peak corresponding to v(C=O) appears at about 1705 cm−1, which differs from v(C=O) in PhAM and CQD. Noteworthy, C=O bond is highly polar and, therefore, sensitive to its environment and hydrogen bond formation. Hybrid4 displays more pronounced changes in comparison to Hybrid1. Thus, it can be assumed that during irradiation, changes occur as a result of PhAM-CQD interaction through the polar groups COO and PO3, and also through hydrogen bond formation.To study structural changes in the CQD@PhAM hybrids after laser irradiation, which significantly affects absorption and luminescence properties, FTIR spectroscopy was applied. Similar to FTIR spectroscopy provided earlier, the spectrum of CQD in isopropanol was subtracted from the spectra under study. When comparing the spectra of Hybrid1 and Hybrid4 before and after irradiation , a numbeChecking the effect of laser irradiation on the bioactivity of hybrids, we have found that BChE inhibition is increased more than twofold and reached 65% (versus 29%) in the case of Hybrid1 and almost 100% (versus 38%) in the case of Hybrid4 . If we cAs subsequent experiments showed , the samThe control of the location of the drugs in the organs and tissues of the body is a fundamental factor in the treatment of a wide list of localized diseases  or diseases requiring site-specific drug delivery . The solution to this problem can be implemented using luminescent substances for drug mapping. However, the visualizing of luminescent objects in biological tissues can be difficult due to autofluorescence—the intrinsic property of cells. Thus, a study of the possibility of detecting a luminescent marker against the background of autofluorescence is an essential test for biomedical applicability.ex = 405 nm) chicken breast sample without injection, and after CQD, Hybrid4, and Hybrid4_LI injection (from top to bottom). One can see clear visualization of CQD and Hybrid4 against the background of chicken breast autofluorescence (as seen without injection): CQD and Hybrid4 photoluminescence stands out not only in intensity but also in color. Hybrid4_LI photoluminescence also stands out against the background of biotissue autofluorescence; however, a strong decrease in the intensity as compared to CQD and Hybrid4 luminescence and the transformation of turquoise color to blue, similar to the color of autofluorescence, is clearly visible.As a biological object in our experiments, a cross-section cut out of a chicken breast (across muscle fibers) was used. CQD and Hybrid4 CQD@PhAM were injected with a syringe into the chicken breast to a depth of ca. 1 mm. The visual observation is confirmed by emission spectra of chicken breast, non-irradiated Hybrid4 and laser-irradiated Hybrid4_LI on the chicken breast a. EmissiIt can be concluded that the change of the luminescence decay resulting from the fitting procedure can be used as a reliable indicator to determine the “non-illuminated” or “illuminated” hybrid state even in such a complex environment as biological tissue. Thus, for in vitro experiments, the observed features of non-irradiated and laser-irradiated CQD@PhAM hybrids (luminescence and kinetic decay) are preserved on the biological test sample. Moreover, in spite of unfavorable conditions—autoluminescence of the biosample in a close spectral region, the visualization of the localization of CQD@PhAM hybrids on the chicken breast is readily observed. Furthermore, the hybrid state “non-irradiated” or “irradiated” is readily distinguished by observation.In such a way, the series of experiments on laser irradiation of Hybrids results in considerable changes in critical properties—luminescence and bioactivity. Furthermore, the photoactivation ability typical for phosphonates is preserved for Hybrids, which allowed for obtaining compounds with highly different values of BChE inhibition before and after laser irradiation. An extremely important observation is the strong difference in luminescence kinetics behavior for non-irradiated and laser-irradiated Hybrids. It is revealed in a change of the decay curve shape and in significantly different lifetimes. It should be noted that the luminescence features are observed not only for in vitro experiments with Hybrids solutions but also for biosamples injected with Hybrids. In such a way, luminescence kinetics can be considered as an indicator of the state of biological activity of CQD@PhAM Hybrids—mono-exponential decay testifies to non-irradiated hybrid with moderate bioactivity, biexponential decay testifies to hybrid with “switched on” bioactivity. Thus, the obtained CQD@PhAM hybrids are unique multifunctional materials  combininIn summary, we demonstrated for the first time the synthesis and characterization of organic-inorganic hybrids based on luminescent carbon quantum dots and a photoactive phosphorylated 2-arylaminomalonate compound. The synthesis of the hybrids was obtained in the course of stirring components at ambient conditions. The formation of hybrids was confirmed by complementary techniques—absorption, luminescence, FTIR spectroscopies, and bioactivity investigation—checking of butyrylcholinesterase inhibition. It was found that CQD@PhAM Hybrids are characterized by red-shifted bands and lower luminescence intensity in comparison to CQD, while BChE inhibition of Hybrids is higher than that for the phosphorylated 2-arylaminomalonate compound. FTIR data analysis revealed the formation of Hybrids by means of phosphonates adsorption on CQDs surface via polar groups. All the obtained results confirm the active interaction of PhAM with CQDs. The stability of the CQD@PhAM hybrids was confirmed by biological tests on butyrylcholinesterase inhibition after 90 days of storage at ambient conditions.Further experiments were aimed at studying the Hybrid properties after exposure to laser irradiation with a wavelength of 266 nm (corresponding to the intrinsic absorption band of the phosphonate compound). It was interesting to discover that not only the luminescent properties and the bioactivity of the CQD@PhAM Hybrids were retained after laser irradiation but also the steep increase of BChE inhibition up to almost 100% and the simultaneous luminescence intensity decrease. In such a way, laser irradiation of Hybrids results in a considerable change of critical properties—luminescence and bioactivity. Furthermore, the photoactivation ability typical for phosphonates is preserved for Hybrids that allow obtaining compounds with highly different values of BChE inhibition in states before and after laser irradiation. A highly relevant observation is the strong difference in the luminescence kinetics behavior for non-irradiated and laser-irradiated Hybrids, which is revealed in a change in the shape of the decay curve and in significantly different lifetimes. In such a way, the luminescence kinetics can be considered as an indicator of the state of biological activity of the CQD@PhAM Hybrids—mono-exponential decay testifying to non-irradiated hybrid with moderate bioactivity, and biexponential decay testifying to hybrid with “switched on” bioactivity. These features were observed both for in vitro experiments and for a biological test sample—chicken breast. We have demonstrated that the CQD@PhAM hybrids luminescence can be registered against the autoluminescence background of biological tissues. Thus, the obtained CQD@PhAM hybrids are unique multifunctional materials providing all-in-one important functions for photopharmacology: (i) bioactivity toward BChE inhibition, (ii) ability to change the degree of inhibition as a result of laser irradiation, (iii) luminescence kinetics as an indicator of biological activity state and (iv) luminescence as a visualization of Hybrid spatial localization on the surface of a sample.


Moreover, activated JNK2 enhances diastolic SR Ca2+ leak in 24 h and 48 h alcohol-exposed HL-1 atrial myocytes as well as HEK-RyR2 cells (inducible expression of human RyR2) with the overexpression of tGFP-tagged active JNK2-tGFP or inactive JNK2dn-tGFP. Meanwhile, the SR Ca2+ load and systolic Ca2+ transient amplitude were both increased in ventricular myocytes, along with the preserved cardiac function in 2 Mo alcohol-exposed mice. Moreover, the role of activated JNK2 in SR Ca2+ overload and enhanced transient amplitude was also confirmed in long-term alcohol-exposed HL-1 atrial myocytes. In conclusion, our findings suggest that long-term alcohol-activated JNK2 is a key driver in preserved cardiac function, but at the expense of enhanced cardiac arrhythmogenicity. Modulating JNK2 activity could be a novel anti-arrhythmia therapeutic strategy.Long-term alcohol consumption leads to cardiac arrhythmias including atrial fibrillation (AF), the most common alcohol-related arrhythmia. While AF significantly increases morbidity and mortality in patients, it takes years for an alcoholic individual undergoing an adaptive status with normal cardiac function to reach alcoholic cardiomyopathy. The underlying mechanism remains unclear to date. In this study, we assessed the functional role of JNK2 in long-term alcohol-evoked atrial arrhythmogenicity but preserved cardiac function. Wild-type (WT) mice and cardiac-specific JNK2dn mice (with an overexpression of inactive dominant negative (dn) JNK2) were treated with alcohol . Confocal Ca Long-term alcohol consumption leads to cardiac arrhythmias, including both atrial and ventricular arrythmias, with atrial fibrillation (AF) being the most common alcohol-related arrhythmia; however, it can take years for an alcoholic individual undergoing adaptive cardiac changes, with normal function, to reach the point of alcoholic cardiomyopathy ,2,3,4. TIn the United States and South America, more than half of all women, and 60% to 80% of all men consume alcohol, while at least 50% of adults are active consumers of alcohol in Europe ,8.2+ homeostasis, which could underly enhanced arrhythmogenicity [2+ activities in intact mouse atria, using high-resolution confocal Ca2+ imaging in Langendorff-perfused intact mouse hearts . We have previously reported that cardiac diastolic sarcoplasmic reticulum (SR) Ca2+-handling dysfunction is a key abnormal ectopic trigger that initiates arrhythmias, and that this Ca2+ mishandling even causes cardiac pathological remodeling [2+ handling was measured in cardiac myocytes freshly isolated from 2 Mo alcohol-exposed mice, and in a heterologous cellular model of HER-RyR2 cells with an inducible expression of human RyR2 channels and other SR Ca2+ handling proteins, as previously characterized [2+-triggered arrhythmic activities, but also contributes to the maintenance of normal cardiac function. Modulating JNK2 activity could be a potential novel therapeutic strategy for alcoholic-related AF.It is known that an excessive alcohol intake activates the stress-response kinase JNK, leading to organ injury ,12,13,14genicity ,16. Our genicity ,18,19,20modeling . The functerized . Our finAll animal studies followed the Guide for the Care and Use of Laboratory Animals , and were approved by the Institutional Animal Care and Use Committees of The Ohio State University, Rush University Medical Center, and Loyola University at Chicago. The terminal experiments were performed within 24 h following the conclusion of the alcohol course treatments. Wild-type (WT) C57B/6j male mice  and cardiac-specific JNK2dn transgenic (Tg) mice with an overexpression of inactive JNK2 dominant-negative protein ,20,23 we2+ leak, the HL-1 myocytes were treated with 50 mM ethanol for 24 or 48 h with and without the presence of 170 nM JNK2 inhibitor JNK2I , as previously described [A cultured HL-1 atrial myocyte line was used for our studies, as previously described ,19,20,21escribed ,19,20,212+ load-handling proteins, as previously described [2+ imaging. In brief, stable doxycycline-inducible HEK-293 cells with heterologous WT-RyR2 (RyR2-HEK-293) were cultured on coverslips, and RyR2 expression was induced with doxycycline (2 μg/mL). The plated cells were then transfected with either JNK2-tGFP (active human JNK2) or JNK2dn-tGFP (inactive human JNK2) mammalian expression vectors in 2 mL of DMEM, as previously described [2+ imaging of identifiable tGFP-positive cells  was conducted.In addition, we used a well-characterized heterologous HEK cell line with doxycycline-inducible expression of human RyR2 and other SR Caescribed ,25. HEK-escribed . FollowiTM Kinase assay kit . The reaction was performed using a kinase reaction buffer system , in the presence of 20 µM ATP for 45 min, as previously described [The JNK2-isoform-specific activity was measured in 2 Mo alcohol-exposed mouse hearts versus sham controls from immunoprecipitating (IP) JNK2 protein, using anti-JNK2-specific antibodies , followed by measurement of the production of ADP in the kinase reaction with a JNK-specific enzyme substrate c-Jun, using an ADP-Gloescribed ,21,26.2+ imaging studies, the mice were anesthetized using 2–4% isoflurane delivered with 100% oxygen, and were subsequently euthanized in a humane manner. The freshly harvested intact mouse hearts were cannulated, and subjected to Langendorff perfusion with oxygenated Ca2+-free Tyrode’s solution  at room temperature, to eliminate blood in the coronary system. Subsequently, each heart was loaded with the Ca2+-sensitive dye Rhod2-AM   dissolved in DMSO) for 30 min. Following the dye staining, the intact heart was transferred to oxygenated 1.8 mM Ca2+ Tyrode’s perfusion at 37 °C. The imaging of Ca2+ transients in the intact mouse atria was performed using a confocal microscope . The Rhod-2 dye was excited using an argon laser at 560 nm, and the emitted light was collected at a wavelength of 590 nm. The diastolic threshold was established as the minimum voltage required to achieve 1:1 atrial capture. Confocal line scans at 2 µs/pixel were conducted during sinus rhythm and after pacing, at frequencies of 8 Hz, 10 Hz, and 20 Hz.For the obtention of the whole mouse hearts for the confocal Ca2+ transient signals were normalized to the background level of fluorescence (ΔF/F0), where F0 represents the resting fluorescence during diastole in the same cardiac myocyte. The Ca2+ transient decay constant τ was calculated as the time required for the Ca2+ transient peak to decay to approximately 63.2% of its systolic peak value. Ca2+ waves were identified as abnormal Ca2+ release events from the RyR2 that were unsynchronized with normal sinus rhythm Ca2+ transients. The frequency of Ca2+ waves was quantified in terms of waves per millimeter per second. The intracellular Ca2+ transient decay constant τ and Ca2+ wave frequency were analyzed using a well-established customized MATLAB-based algorithm.The Ca2+ imaging studies. Prior to the imaging, the cells were loaded with Fluo-4 AM dye  for 15 min at 34.5 °C, followed immediately by confocal Ca2+ imaging studies . During imaging, the myocytes were continuously perfused with normal Hanks’ salt solution at 35 °C, and physiological pH . The Fluo-4 AM dye was excited with an argon laser at a wavelength of 488 nm, and the emitted light was collected at a wavelength of 515 nm.Cultured HL-1 mouse atrial myocytes, freshly isolated mouse ventricular myocytes, and transfected HEK-RyR2 cells were used for cellular confocal Ca2+ leak, the myocytes were superfused with 0 Na+/0 Ca2+ Tyrode’s solution  for 10 min. A pre-tetracaine confocal recording was taken, after which the cells were superfused with tetracaine , and a post-tetracaine recording was taken. The Ca2+ leak was calculated as the intracellular Ca2+ fluorescence decline following the superfusion of the cells with tetracaine. The rapid decline in fluorescence signal after the tetracaine exposure represents the diastolic SR Ca2+ that “leaked” through the RyR2 channel, given that tetracaine rapidly lowers the RyR2 open probability with blocked NCX activity via the 0 Na+/0 Ca2+ Tyrode’s solution. The fluorescence readings were defined as the collected steady-state fluorescence readings at the beginning of the recording (F0). The Ca2+ transient signals were reported as ΔF/F0, which is the change in fluorescence normalized to the baseline (F0) readings.To measure the SR Ca2+ load, the myocytes were, instead, superfused with 1.8 mM Ca2+ Tyrode’s solution at room temperature , and a baseline recording was taken. Then, the monocyte layer was superfused with caffeine , to obtain a post-caffeine image. The Ca2+ transient amplitude was defined as the peak values of the Ca2+ transients against the diastolic background (F0). The SR Ca2+ leak, load, and transient amplitude were all analyzed using a well-established customized MATLAB -based algorithm.To measure the SR Cat-test. When a heterogeneity of variance was observed, a nonparametric Mann–Whitney U test or nonparametric one-way ANOVA was performed. A p-value of <0.05 was considered significant.All data are presented as mean ± SEM. The differences between multiple groups or any two groups were evaluated using one-way ANOVA with the post-hoc Tukey test or Student’s We first assessed the cardiac JNK2 activity in 2-month (2 Mo) alcohol-exposed mouse hearts by immunoprecipitating (IP) the JNK2 proteins with a JNK2-isoform-specific antibody, followed by JNK2 enzyme activity measurement (reflected in the ADP production), using the ADP-Glo kinase assay kit , as we previously described ,26. In t2+ triggers are critical in generating abnormal ectopic activities and enhanced arrhythmogenesis in heart failure and AF [2+-triggered activities, high-resolution confocal Ca2+ imaging was conducted on Langendorff-perfused intact mouse hearts. We firstly recorded line-scan confocal Ca2+ transient images of intact atria  during their intrinsic sinus rhythm. 2+ transient decay constant τ was prolonged in 2 Mo alcohol-exposed WT mice compared to sham WT mice (55.9 ± 2.74 ms vs. 30.03 ± 6.72 ms in WT sham), reflecting the elevated level of diastolic intracellular Ca2+, and the increased risk of generating arrhythmic activities. Next, hearts were electrically paced, and then the pacing was stopped, to permit the inherent sinus rhythm to resume. The summarized data in 2+ waves compared to sham controls following 8 Hz (10.20 ± 2.07 waves/(mm × s) vs. 1.99 ± 1.33 waves/(mm × s) in the sham), 10 Hz (15.38 ± 2.94 waves/(mm × s) vs. 4.84 ± 1.99 waves/(mm × s) in the WT sham), and 20 Hz (24.05 ± 5.98 waves/(mm × s) vs. 7.59 ± 2.72 waves/(mm × s)) electrical pacing. Thus, long-term alcohol exposure enhances Ca2+-triggered atrial arrhythmogenicity.We and others have previously shown that abnormal Cae and AF ,27,28,292+ waves following 8 Hz (10.20 ± 2.07 waves/(mm × s) in 2 Mo Alc-WT vs. 2.71 ± 1.03 waves/(mm × s) in JNK2dn-Alc), 10 Hz (15.38 ± 2.94 waves/(mm × s) in WT Alc vs. 3.11 ± 0.96 waves/(mm × s) in JNK2dn-Alc), and 20 Hz (24.05 ± 5.98 waves/(mm × s) in WT-Alc vs. 7.34 ± 1.53 waves/(mm × s) in JNK2dn-Alc) burst pacing (2+-triggered arrhythmic activities in intact atria.JNK activity is known to be reflected by the phosphorylation status of the amino acid residues of Threonine 183 and Tyrosine 185 on the “TPY motif” (JNK-P) ,31. To st pacing C. Similat pacing B. Taken 2+ mishandling is a crucial factor in Ca2+-triggered arrhythmic events, with enhanced diastolic SR Ca2+ leak leading to increased abnormal Ca2+ waves. Here, we used our well-validated tetracaine-sensitive SR Ca2+ leak measurement under confocal microscopy, as previously described [2+ leak, compared to the sham controls , compared to sham controls. To further assess whether the JNK2 activity is a key factor in its function in SR Ca2+ leak, we transfected HEK-RyR2 cells with a JNK2dn vector, to express inactive JNK2dn proteins. 2+ leak, seen in sham controls, suggesting that JNK2 is responsible for the abnormal diastolic SR Ca2+ leak.To further determine the specific action of activated JNK2 in diastolic SR Caescribed ,25. We t2+ load is essential to supplying enough systolic Ca2+ released from the SR to generate the normal systolic Ca2+ transients required for normal contractions of the cardiac muscles. In the myocyte relaxation phase, the SR Ca2+ release almost completely shuts off (∼99%), and most of the remaining cytosolic Ca2+ is pumped back into the SR by the Ca2+ pump (SERCA2) [2+ load via enhanced SERCA2 pump activity suggest that JNK2 could play a key role in maintaining the normal cardiac output as an adaptive mechanism in response to binge alcohol exposure [2+ load in long-term alcohol-exposed HL-1 myocytes. We found a significantly increased SR Ca2+ load by measuring the peak of the 10 mM caffeine-induced Ca2+ transients in both 24 h and 48 h alcohol-exposed HL-1 atrial myocytes, compared to sham controls  . A reduc(SERCA2) ,33. Howe(SERCA2) ,21 of JNexposure ,21. Here2+ load in freshly isolated ventricular myocytes from 2 Mo alcohol-exposed mice. The summarized cellular confocal Ca2+ data reveal that there is significantly elevated SR Ca2+ load  alcohol-driven JNK activation seems to be common, and contributes to alcohol-related cell death and tissue injury [2+ leak and triggered arrhythmias. Further, this long-term alcohol-activated JNK2 is also essential in maintaining cardiac contractile function, through the JNK2-specific action in augmented SR Ca2+ load for a sufficient Ca2+ transient amplitude, in order to maintain a normal cardiac output.Our findings for the JNK2-specific actions in [Cae injury ,13,29,45e injury ,46. To de injury ,47,48. O2+ handling is known to play a key role in cardiac adaptive and maladaptive remodeling during the development of heart failure [2+-triggered SR Ca2+ release produces a large intracellular Ca2+ transient that drives cell contraction, while Ca2+ release stops during the diastolic phase, and excess cytosolic Ca2+ ions are removed from the cytosol, either through pumping Ca2+ back to SR (via the SERCA2 function), or extruding Ca2+ from the cell [2+ leak will reduce the SR Ca2+ load and, consequently, reduce the systolic fractional SR Ca2+ release for a given L-type voltage-gated Ca2+ current (ICa) trigger, consequently reducing the systolic Ca2+ transient amplitude, as commonly seen in the failing heart [2+ waves can be triggered by an excessive diastolic NCX-mediated influx from elevated intracellular Ca2+, which may produce abnormal triggered activities , and initiate arrhythmias [2+ events and AF susceptibility via an enhanced diastolic SR Ca2+ leak, while this JNK2-driven SR Ca2+ leak was associated with an elevated SR Ca2+ load in both aged and binge-alcohol-exposed hearts [2+ leak depletes the SR load that occurs in heart failure myocytes [2+ transient amplitude in both the atria and ventricles are unaffected by JNK2 activation induced by aging or binge alcohol exposure [2+ load in order to maintain a normal Ca2+ transient amplitude, despite a greater diastolic leak concordantly existing in long-term alcohol-exposed myocytes. In other words, long-term alcohol-activated JNK2, as a stress-induced regulator, maintains a high level of SR Ca2+ load in order to preserve cardiac function but, on the other hand, causes an enhanced arrhythmogenicity, due to diastolic SR Ca2+ leak. Thus, the functional outcome of the long-term activation of JNK2 could explain how an individual with a long history of excessive alcohol consumption has a significantly increased risk of cardiac arrhythmias, and yet their cardiac function remains normal for years.Dysfunctional SR Ca failure ,45,49,50ng heart ,29,45. Phythmias ,45. Our d hearts ,20. Thismyocytes ,45. Howeexposure ,20,21. I2+-triggered ectopic triggers, to promote cardiac arrhythmia onset. While modulating JNK2 activity could be a promising approach to stopping Ca2+-triggered activity, and improving myocardial conduction and, consequently, eliminating AF in chronic alcohol users, further investigation is clearly warranted, to understand whether the inhibition of JNK2 would also allow the maintenance of abnormal cardiac function in long-term alcohol-exposed hearts. Exploring new drugs targeting JNK2 activity as a novel anti-arrhythmia therapeutic strategy for patients with an issue of long-term alcohol intake could be a future research direction.It is known that abnormal activities in cells in the pulmonary veins have been shown to underlie the initiation of AF; the maintenance of AF is believed to be due to an arrhythmogenic substrate characterized by abnormal impulse propagation. This substrate includes structural  and molecular  remodeling ,52. We r