Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia.

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Systematic identification of biomarker-driven drug combinations to overcome resistance.

The ability to understand and predict variable responses to therapeutic agents may improve outcomes in patients with cancer. We hypothesized that the basal gene-transcription state of cancer cell lines, coupled with cell viability profiles of small molecules, might be leveraged to nominate specific mechanisms of intrinsic resistance and to predict drug combinations that overcome resistance. We analyzed 564,424 sensitivity profiles to identify candidate gene-compound pairs, and validated nine such relationships. We determined the mechanism of a novel relationship, in which expression of the serine hydrolase enzymes monoacylglycerol lipase (MGLL) or carboxylesterase 1 (CES1) confers resistance to the histone lysine demethylase inhibitor GSK-J4 by direct enzymatic modification. Insensitive cell lines could be sensitized to GSK-J4 by inhibition or gene knockout. These analytical and mechanistic studies highlight the potential of integrating gene-expression features with small-molecule response to identify patient populations that are likely to benefit from treatment, to nominate rational candidates for combinations and to provide insights into mechanisms of action.

Molecular Engineering of Stabilized Silicon-Rosindolizine Shortwave Infrared Fluorophores.

Fluorescence-based biological imaging in the shortwave infrared (SWIR, 1000-1700 nm) is an attractive replacement for modern in vivo imaging techniques currently employed in both medical and research settings. Xanthene-based fluorophores containing heterocycle donors have recently emerged as a way to access deep SWIR emitting fluorophores. A concern for xanthene-based SWIR fluorophores though is chemical stability toward ambient nucleophiles due to the high electrophilicity of the cationic fluorophore core. Herein, a series of SWIR emitting silicon-rosindolizine (SiRos) fluorophores with emission maxima >1300 nm (up to 1550 nm) are synthesized. The SiRos fluorophore photophysical properties and chemical stability toward nucleophiles are examined through systematic derivatization of the silicon-core alkyl groups, indolizine donor substitution, and the use of o-tolyl or o-xylyl groups appended to the fluorophore core. The dyes are studied via absorption spectroscopy, steady-state emission spectroscopy, solution-based cyclic voltammetry, time-dependent density functional theory (TD-DFT) computational analysis, X-ray diffraction crystallography, and relative chemical stability over time. Optimal chemical stability is observed via the incorporation of the 2-ethylhexyl silicon substituent and the o-xylyl group to protect the core of the fluorophore.

A multiscale predictive digital twin for neurocardiac modulation.

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high-throughput prediction of novel neuromodulatory therapy. KEY POINTS: A multi-layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate-and-fire neurons generates firing patterns in close agreement with experiment. A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells. We utilized atomic-scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the ß-adrenergic receptor. Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias. The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease.

Rate-dependent effects of state-specific sodium channel blockers in cardiac tissue: Insights from idealized models.

A common side effect of pharmaceutical drugs is an increased propensity for cardiac arrhythmias. Many drugs bind to cardiac ion-channels in a state-specific manner, which alters the ionic conductances in complicated ways, making it difficult to identify the mechanisms underlying pro-arrhythmic drug effects. To better understand the fundamental mechanisms underlying the diverse effects of state-dependent sodium (Na+) channel blockers on cellular excitability, we consider two canonical motifs of drug-ion-channel interactions and compare the effects of Na+ channel blockers on the rate-dependence of peak upstroke velocity, conduction velocity, and vulnerable window size. In the literature, both motifs are referred to as "guarded receptor," but here we distinguish between state-specific binding that does not alter channel gating (referred to here as "guarded receptor") and state-specific binding that blocks certain gating transitions ("gate immobilization"). For each drug binding motif, we consider drugs that bind to the inactivated state and drugs that bind to the non-inactivated state of the Na+ channel. Exploiting the idealized nature of the canonical binding motifs, we identify the fundamental mechanisms underlying the effects on excitability of the various binding interactions. Specifically, we derive the voltage-dependence of the drug binding time constants and the equilibrium fractions of channels bound to drug, and we then derive a formula that incorporates these time constants and equilibrium fractions to elucidate the fundamental mechanisms. In the case of charged drug, we find that drugs that bind to inactivated channels exhibit greater rate-dependence than drugs that bind to non-inactivated channels. For neutral drugs, the effects of guarded receptor interactions are rate-independent, and we describe a novel mechanism for reverse rate-dependence resulting from neutral drug binding to non-inactivated channels via the gate immobilization motif.

Rate-dependent effects of lidocaine on cardiac dynamics: Development and analysis of a low-dimensional drug-channel interaction model.

State-dependent sodium channel blockers are often prescribed to treat cardiac arrhythmias, but many sodium channel blockers are known to have pro-arrhythmic side effects. While the anti and proarrhythmic potential of a sodium channel blocker is thought to depend on the characteristics of its rate-dependent block, the mechanisms linking these two attributes are unclear. Furthermore, how specific properties of rate-dependent block arise from the binding kinetics of a particular drug is poorly understood. Here, we examine the rate-dependent effects of the sodium channel blocker lidocaine by constructing and analyzing a novel drug-channel interaction model. First, we identify the predominant mode of lidocaine binding in a 24 variable Markov model for lidocaine-sodium channel interaction by Moreno et al. Specifically, we find that (1) the vast majority of lidocaine bound to sodium channels is in the neutral form, i.e., the binding of charged lidocaine to sodium channels is negligible, and (2) neutral lidocaine binds almost exclusively to inactivated channels and, upon binding, immobilizes channels in the inactivated state. We then develop a novel 3-variable lidocaine-sodium channel interaction model that incorporates only the predominant mode of drug binding. Our low-dimensional model replicates an extensive amount of the voltage-clamp data used to parameterize the Moreno et al. model. Furthermore, the effects of lidocaine on action potential upstroke velocity and conduction velocity in our model are similar to those predicted by the Moreno et al. model. By exploiting the low-dimensionality of our model, we derive an algebraic expression for level of rate-dependent block as a function of pacing frequency, restitution properties, diastolic and plateau potentials, and drug binding rate constants. Our model predicts that the level of rate-dependent block is sensitive to alterations in restitution properties and increases in diastolic potential, but it is insensitive to variations in the shape of the action potential waveform and lidocaine binding rates.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Mechanistic insights into cancer cell killing through interaction of phosphodiesterase 3A and schlafen family member 12.

Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.

Casting a wider net: Immunosurveillance by nonclassical MHC molecules.

Most studies of T lymphocytes focus on recognition of classical major histocompatibility complex (MHC) class I or II molecules presenting oligopeptides, yet there are numerous variations and exceptions of biological significance based on recognition of a wide variety of nonclassical MHC molecules. These include αß and γδ T cells that recognize different class Ib molecules (CD1, MR-1, HLA-E, G, F, et al.) that are nearly monomorphic within a given species. Collectively, these T cells can be considered "unconventional," in part because they recognize lipids, metabolites, and modified peptides. Unlike classical MHC-specific cells, unconventional T cells generally exhibit limited T-cell antigen receptor (TCR) repertoires and often produce innate immune cell-like rapid effector responses. Exploiting this system in new generation vaccines for human immunodeficiency virus (HIV), tuberculosis (TB), other infectious agents, and cancer was the focus of a recent workshop, "Immune Surveillance by Non-classical MHC Molecules: Improving Diversity for Antigens," sponsored by the National Institute of Allergy and Infectious Diseases. Here, we summarize salient points presented regarding the basic immunobiology of unconventional T cells, recent advances in methodologies to measure unconventional T-cell activity in diseases, and approaches to harness their considerable clinical potential.

Dynamics of adrenergic signaling in cardiac myocytes and implications for pharmacological treatment.

Dense innervation of the heart by the sympathetic nervous system (SNS) allows cardiac output to respond appropriately to the needs of the body under varying conditions, but occasionally the abrupt onset of SNS activity can trigger cardiac arrhythmias. Sympathetic activity leads to the release of norepinephrine (NE) onto cardiomyocytes, activating ß1-adrenergic receptors (ß1-ARs) and leading to the production of the second messenger cyclic AMP (cAMP). Upon sudden activation of ß1-ARs in experiments, intracellular cAMP can transiently rise to a high concentration before converging to a steady state level. Although changes to cellular cAMP concentration are important in modulating the overall cardiovascular response to sympathetic tone, the underlying mechanisms of the cAMP transients and the parameters that control their magnitude are unclear. We reduce a detailed computational model of the ß1-adrenergic signaling cascade to a system of two differential equations by eliminating extraneous variables and applying quasi-steady state approximation. The structure of the reduced model reveals that the large cAMP transients associated with abrupt ß1-AR activation are generated by the interplay of production/degradation of cAMP and desensitization/resensitization of ß1-ARs. The reduced model is used to predict how the dynamics of intracellular cAMP depend on the concentrations of norepinephrine (NE), phosphodiesterases 3 and 4 (PDE3,4), G-protein coupled receptor kinase 2 (GRK2), and ß1-AR, in healthy conditions and a simple model of early stages of heart failure. The key findings of the study are as follows: 1) Applying a reduced model of the dynamics of cardiac sympathetic signaling we show that the concentrations of two variables, cAMP and non-desensitized ß1-AR, capture the overall dynamics of sympathetic signaling; 2) The key factors influencing cAMP production are AC activity and PDE3,4 activity, while those that directly impact ß1-AR phosphorylation are GRK2 and PKA1. Thus, disease states that affect sympathetic control of the heart can be thoroughly assessed by studying AC activity, PDE3,4, GRK2 and PKA activity, as these factors directly impact cAMP production/degradation and ß1-AR (de) phosphorylation and are therefore predicted to comprise the most effective pharmaceutical targets in diseases affecting cardiac ß1-adrenergic signaling.

Synchronization of Electrically Coupled Resonate-and-Fire Neurons.

Electrical coupling between neurons is broadly present across brain areas and is typically assumed to synchronize network activity. However, intrinsic properties of the coupled cells can complicate this simple picture. Many cell types with electrical coupling show a diversity of post-spike subthreshold fluctuations, often linked to subthreshold resonance, which are transmitted through electrical synapses in addition to action potentials. Using the theory of weakly coupled oscillators, we explore the effect of both subthreshold and spike-mediated coupling on synchrony in small networks of electrically coupled resonate-and-fire neurons, a hybrid neuron model with damped subthreshold oscillations and a range of post-spike voltage dynamics. We calculate the phase response curve using an extension of the adjoint method that accounts for the discontinuous post-spike reset rule. We find that both spikes and subthreshold fluctuations can jointly promote synchronization. The subthreshold contribution is strongest when the voltage exhibits a significant post-spike elevation in voltage, or plateau potential. Additionally, we show that the geometry of trajectories approaching the spiking threshold causes a "reset-induced shear" effect that can oppose synchrony in the presence of network asymmetry, despite having no effect on the phase-locking of symmetrically coupled pairs.

Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells.

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics.

High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.

The role of long-range coupling in crayfish swimmeret phase-locking.

During forward swimming, crayfish and other long-tailed crustaceans rhythmically move four pairs of limbs called swimmerets to propel themselves through the water. This behavior is characterized by a particular stroke pattern in which the most posterior limb pair leads the rhythmic cycle and adjacent swimmerets paddle sequentially with a delay of roughly 25% of the period. The neural circuit underlying limb coordination consists of a chain of local modules, each of which controls a pair of limbs. All modules are directly coupled to one another, but the inter-module coupling strengths decrease with the distance of the connection. Prior modeling studies of the swimmeret neural circuit have included only the dominant nearest-neighbor coupling. Here, we investigate the potential modulatory role of long-range connections between modules. Numerical simulations and analytical arguments show that these connections cause decreases in the phase-differences between neighboring modules. Combined with previous results from a computational fluid dynamics model, we posit that this phenomenon might ensure that the resultant limb coordination lies within a range where propulsion is optimal. To further assess the effects of long-range coupling, we modify the model to reflect an experimental preparation where synaptic transmission from a middle module is blocked, and we generate predictions for the phase-locking properties in this system.

Correction to: Influence of Single-Dose Antibiotic Prophylaxis for Early-Onset Pneumonia in High-Risk Intubated Patients.

Due to an error introduced during the production process, J. Dedrick Jordan's name was improperly tagged in the original publication of this article. It is tagged correctly here.

Influence of Single-Dose Antibiotic Prophylaxis for Early-Onset Pneumonia in High-Risk Intubated Patients.

BACKGROUND: Early-onset pneumonia (EOP) after endotracheal intubation is common among critically ill patients with a neurologic injury and is associated with worse clinical outcomes. METHODS: This retrospective cohort study observed outcomes pre- and post-implementation of an EOP prophylaxis protocol which involved the administration of a single dose of ceftriaxone 2 g around the time of intubation. The study included patients ≥ 18 years who were admitted to the University of North Carolina Medical Center (UNCMC) neuroscience intensive care unit (NSICU) between April 1, 2014, and October 26, 2016, and intubated for ≥ 72 h. RESULTS: Among the 172 patients included, use of an EOP prophylaxis protocol resulted in a significant reduction in the rate of microbiologically confirmed EOP compared to those without prophylaxis (7.4 vs 19.8%, p = 0.026). However, EOP prophylaxis did not decrease the combined incidence of microbiologically confirmed or clinically suspected EOP (32.2 vs 37.4%, p = 0.523). No difference in the rate of late-onset pneumonia (34.6 vs 26.4%, p = 0.25) or virulent organism growth (19.8 vs 14.3%, p = 0.416) was observed. No difference was observed in the duration of intubation, duration of intensive care unit (ICU) stay, duration of hospitalization, or ICU antibiotic days within 30 days of intubation. In hospital mortality was found to be higher in those who received EOP prophylaxis compared to those who did not receive prophylaxis (45.7 vs 29.7%, p = 0.04). CONCLUSIONS: The administration of a single antibiotic dose following intubation may reduce the incidence of microbiologically confirmed EOP in patients with neurologic injury who are intubated ≥ 72 h. A prophylaxis strategy does not appear to increase the rate of virulent organism growth or the rate of late-onset pneumonia. However, this practice is not associated with a decrease in days of antibiotic use in the ICU or any clinical outcomes benefit.

A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.

Robust phase-waves in chains of half-center oscillators.

Many neuronal circuits driving coordinated locomotion are composed of chains of half-center oscillators (HCOs) of various lengths. The HCO is a common motif in central pattern generating circuits (CPGs); an HCO consists of two neurons, or two neuronal populations, connected by reciprocal inhibition. To maintain appropriate motor coordination for effective locomotion over a broad range of frequencies, chains of CPGs must produce approximately constant phase-differences in a robust manner. In this article, we study phase-locking in chains of nearest-neighbor coupled HCOs and examine how the circuit architecture can promote phase-constancy, i.e., inter-HCO phase-differences that are frequency-invariant. We use two models with different levels of abstraction: (1) a conductance-based model in which each neuron is modeled by the Morris-Lecar equations (the ML-HCO model); and (2) a coupled phase model in which the state of each HCO is captured by its phase (the phase-HCO model). We show that one of four phase-waves with inter-HCO phase-differences at approximately 0, 25, 50 or 75 % arises robustly as a result of the inter-HCO connection topology, and its robust existence is not affected by the number of HCOs in the chain, the difference in strength between the ascending and descending nearest-neighbor connections, or the number of nearest-neighbor connections. Our results show that the internal anti-phase structure of the HCO and an appropriate inter-HCO connection topology together can provide a mechanism for robust (i.e., frequency-independent) limb coordination in segmented animals, such as the 50 % interlimb phase-differences in the tripod gate of stick insects and cockroaches, and the 25 % interlimb phase-differences in crayfish and other long-tailed crustaceans during forward swimming.