A global dataset of carbon pumping by the world's largest tropical rivers.

The eco-morphodynamic activity of large tropical rivers interacts with riparian vegetation causing implications for the carbon cycle within inland waters. Through a multi-temporal analysis of satellite data spanning the years 2000-2019, we analyzed rivers exceeding 200 m in width across the tropical regions, revealing a Carbon Pump mechanism driving an annual mobilization of 12.45 million tons of organic carbon. The study identifies fluvial eco-morphological signatures as proxies for carbon mobilization, emphasizing the link between river migration and carbon dynamics. To enhance accessibility, our results are encapsulated in a visually compelling WebGIS application, offering a comprehensive understanding of the eco-geomorphological influences on the global carbon cycle within large tropical rivers. Our findings are instrumental in determining the carbon intensity of future hydropower dams, thereby contributing to informed decision-making in the realm of sustainable energy infrastructure. This study elucidates the intricate relationships that govern the nexus of tropical river dynamics, riparian ecosystems, and the global carbon cycle.

Polarizing Macrophage Functional Phenotype to Foster Cardiac Regeneration.

There is an increasing interest in understanding the connection between the immune and cardiovascular systems, which are highly integrated and communicate through finely regulated cross-talking mechanisms. Recent evidence has demonstrated that the immune system does indeed have a key role in the response to cardiac injury and in cardiac regeneration. Among the immune cells, macrophages appear to have a prominent role in this context, with different subtypes described so far that each have a specific influence on cardiac remodeling and repair. Similarly, there are significant differences in how the innate and adaptive immune systems affect the response to cardiac damage. Understanding all these mechanisms may have relevant clinical implications. Several studies have already demonstrated that stem cell-based therapies support myocardial repair. However, the exact role that cardiac macrophages and their modulation may have in this setting is still unclear. The current need to decipher the dual role of immunity in boosting both heart injury and repair is due, at least for a significant part, to unresolved questions related to the complexity of cardiac macrophage phenotypes. The aim of this review is to provide an overview on the role of the immune system, and of macrophages in particular, in the response to cardiac injury and to outline, through the modulation of the immune response, potential novel therapeutic strategies for cardiac regeneration.

Adult Multipotent Cardiac Progenitor-Derived Spheroids: A Reproducible Model of In Vitro Cardiomyocyte Commitment and Specification.

BACKGROUND: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. METHODS: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. RESULTS: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. CONCLUSIONS: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.

Eco-morphodynamic carbon pumping by the largest rivers in the Neotropics.

The eco-morphodynamic activity of large tropical rivers in South and Central America is analyzed to quantify the carbon flux from riparian vegetation to inland waters. We carried out a multi-temporal analysis of satellite data for all the largest rivers in the Neotropics (i.e, width > 200 m) in the period 2000-2019, at 30 m spatial resolution. We developed a quantification of a highly efficient Carbon Pump mechanism. River morphodynamics is shown to drive carbon export from the riparian zone and to promote net primary production by an integrated process through floodplain rejuvenation and colonization. This pumping mechanism alone is shown to account for 8.9 million tons/year of carbon mobilization in these tropical rivers. We identify signatures of the fluvial eco-morphological activity that provide proxies for the carbon mobilization capability associated with river activity. We discuss river migration-carbon mobilization nexus and effects on the carbon intensity of planned hydroelectric dams in the Neotropics. We recommend that future carbon-oriented water policies on these rivers include a similar analysis.

Streptozotocin-Induced Type 1 and 2 Diabetes Mellitus Mouse Models Show Different Functional, Cellular and Molecular Patterns of Diabetic Cardiomyopathy.

The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.

Pharmacological clearance of senescent cells improves cardiac remodeling and function after myocardial infarction in female aged mice.

Cardiovascular diseases (CVD) are predominantly an aging disease. Important sex-specific differences exist and the mechanism(s) by which this sex-by-age interaction influences CVD development and progression remains elusive. Accordingly, it is still unknown whether cell senescence, a main feature of cardiac male aging, is a significant feature also of the female aged mouse heart and whether senolytics, senescence-clearing compounds, promote myocardial repair and regeneration after myocardial infarction (MI) in aged female mice. To this aim, the combination of two senolytics, dasatinib and quercetin (D+Q) or just their vehicle was administered to 22-24 months old C57BL/6 female mice after MI. D+Q improved global left ventricle function and myocardial performance after MI whereby female cardiac aging is characterized by accumulation of cardiac senescent cells that are further increased by MI. Despite their terminal differentiation nature, also cardiomyocytes acquire a senescent phenotype with age in females. D+Q removed senescent cardiac non-myocyte and myocyte cells ameliorating cardiac remodeling and regeneration. Senolytics removed aged dysfunctional cardiac stem/progenitor cells (CSCs), relieving healthy CSCs with normal proliferative and cardiomyogenic differentiation potential. In conclusions, cardiac senescent cells accumulate in the aged female hearts. Removing senescent cells is a key therapeutic target for efficient repair of the aged female heart.

The negative regulation of gene expression by microRNAs as key driver of inducers and repressors of cardiomyocyte differentiation.

Cardiac muscle damage-induced loss of cardiomyocytes (CMs) and dysfunction of the remaining ones leads to heart failure, which nowadays is the number one killer worldwide. Therapies fostering effective cardiac regeneration are the holy grail of cardiovascular research to stop the heart failure epidemic. The main goal of most myocardial regeneration protocols is the generation of new functional CMs through the differentiation of endogenous or exogenous cardiomyogenic cells. Understanding the cellular and molecular basis of cardiomyocyte commitment, specification, differentiation and maturation is needed to devise innovative approaches to replace the CMs lost after injury in the adult heart. The transcriptional regulation of CM differentiation is a highly conserved process that require sequential activation and/or repression of different genetic programs. Therefore, CM differentiation and specification have been depicted as a step-wise specific chemical and mechanical stimuli inducing complete myogenic commitment and cell-cycle exit. Yet, the demonstration that some microRNAs are sufficient to direct ESC differentiation into CMs and that four specific miRNAs reprogram fibroblasts into CMs show that CM differentiation must also involve negative regulatory instructions. Here, we review the mechanisms of CM differentiation during development and from regenerative stem cells with a focus on the involvement of microRNAs in the process, putting in perspective their negative gene regulation as a main modifier of effective CM regeneration in the adult heart.

Myocardial regeneration protocols towards the routine clinical scenario: An unseemly path from bench to bedside.

Heart failure secondary to cardiomyocyte loss and/or dysfunction is the number one killer worldwide. The field of myocardial regeneration with its far-reaching primary goal of cardiac remuscularization and its hard-to-accomplish translation from bench to bedside, has been filled with ups and downs, steps forward and steps backward, controversies galore and, unfortunately, scientific scandals. Despite the present morass in which cardiac remuscularization is stuck in, the search for clinically effective regenerative approaches remains keenly active. Starting with a concise overview of the still highly debated regenerative capacity of the adult mammalian heart, we focus on the main interventions, that have reached or are close to clinical use, critically discussing key findings, successes, and failures. Finally, some promising and innovative approaches for myocardial repair/regeneration still at the pre-clinical stage are discussed to offer a holistic view on the future of myocardial repair/regeneration for the prevention/management of heart failure in the clinical scenario. Funding: This research was funded by Grants from the Ministry of University and Research PRIN2015 2015ZTT5KB_004; PRIN2017NKB2N4_005; PON-AIM - 1829805-2.

Unraveling and Targeting Myocardial Regeneration Deficit in Diabetes.

Cardiomyopathy is a common complication in diabetic patients. Ventricular dysfunction without coronary atherosclerosis and hypertension is driven by hyperglycemia, hyperinsulinemia and impaired insulin signaling. Cardiomyocyte death, hypertrophy, fibrosis, and cell signaling defects underlie cardiomyopathy. Notably, detrimental effects of the diabetic milieu are not limited to cardiomyocytes and vascular cells. The diabetic heart acquires a senescent phenotype and also suffers from altered cellular homeostasis and the insufficient replacement of dying cells. Chronic inflammation, oxidative stress, and metabolic dysregulation damage the population of endogenous cardiac stem cells, which contribute to myocardial cell turnover and repair after injury. Therefore, deficient myocardial repair and the progressive senescence and dysfunction of stem cells in the diabetic heart can represent potential therapeutic targets. While our knowledge of the effects of diabetes on stem cells is growing, several strategies to preserve, activate or restore cardiac stem cell compartments await to be tested in diabetic cardiomyopathy.

Diabetes-Induced Cellular Senescence and Senescence-Associated Secretory Phenotype Impair Cardiac Regeneration and Function Independently of Age.

Diabetes mellitus (DM) affects the biology of multipotent cardiac stem/progenitor cells (CSCs) and adult myocardial regeneration. We assessed the hypothesis that senescence and senescence-associated secretory phenotype (SASP) are main mechanisms of cardiac degenerative defect in DM. Accordingly, we tested whether ablation of senescent CSCs would rescue the cardiac regenerative/reparative defect imposed by DM. We obtained cardiac tissue from nonaged (50- to 64-year-old) patients with type 2 diabetes mellitus (T2DM) and without DM (NDM) and postinfarct cardiomyopathy undergoing cardiac surgery. A higher reactive oxygen species production in T2DM was associated with an increased number of senescent/dysfunctional T2DM-human CSCs (hCSCs) with reduced proliferation, clonogenesis/spherogenesis, and myogenic differentiation versus NDM-hCSCs in vitro. T2DM-hCSCs showed a defined pathologic SASP. A combination of two senolytics, dasatinib (D) and quercetin (Q), cleared senescent T2DM-hCSCs in vitro, restoring their expansion and myogenic differentiation capacities. In a T2DM model in young mice, diabetic status per se (independently of ischemia and age) caused CSC senescence coupled with myocardial pathologic remodeling and cardiac dysfunction. D + Q treatment efficiently eliminated senescent cells, rescuing CSC function, which resulted in functional myocardial repair/regeneration, improving cardiac function in murine DM. In conclusion, DM hampers CSC biology, inhibiting CSCs' regenerative potential through the induction of cellular senescence and SASP independently from aging. Senolytics clear senescence, abrogating the SASP and restoring a fully proliferative/differentiation-competent hCSC pool in T2DM with normalization of cardiac function.

From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish.

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.

In vitro CSC-derived cardiomyocytes exhibit the typical microRNA-mRNA blueprint of endogenous cardiomyocytes.

miRNAs modulate cardiomyocyte specification by targeting mRNAs of cell cycle regulators and acting in cardiac muscle lineage gene regulatory loops. It is unknown if or to-what-extent these miRNA/mRNA networks are operative during cardiomyocyte differentiation of adult cardiac stem/progenitor cells (CSCs). Clonally-derived mouse CSCs differentiated into contracting cardiomyocytes in vitro (iCMs). Comparison of "CSCs vs. iCMs" mRNome and microRNome showed a balanced up-regulation of CM-related mRNAs together with a down-regulation of cell cycle and DNA replication mRNAs. The down-regulation of cell cycle genes and the up-regulation of the mature myofilament genes in iCMs reached intermediate levels between those of fetal and neonatal cardiomyocytes. Cardiomyo-miRs were up-regulated in iCMs. The specific networks of miRNA/mRNAs operative in iCMs closely resembled those of adult CMs (aCMs). miR-1 and miR-499 enhanced myogenic commitment toward terminal differentiation of iCMs. In conclusions, CSC specification/differentiation into contracting iCMs follows known cardiomyo-MiR-dependent developmental cardiomyocyte differentiation trajectories and iCMs transcriptome/miRNome resembles that of CMs.

Physical Exercise and Cardiac Repair: The Potential Role of Nitric Oxide in Boosting Stem Cell Regenerative Biology.

Over the years strong evidence has been accumulated showing that aerobic physical exercise exerts beneficial effects on the prevention and reduction of cardiovascular risk. Exercise in healthy subjects fosters physiological remodeling of the adult heart. Concurrently, physical training can significantly slow-down or even reverse the maladaptive pathologic cardiac remodeling in cardiac diseases, improving heart function. The underlying cellular and molecular mechanisms of the beneficial effects of physical exercise on the heart are still a subject of intensive study. Aerobic activity increases cardiovascular nitric oxide (NO) released mainly through nitric oxidase synthase 3 activity, promoting endothelium-dependent vasodilation, reducing vascular resistance, and lowering blood pressure. On the reverse, an imbalance between increasing free radical production and decreased NO generation characterizes pathologic remodeling, which has been termed the "nitroso-redox imbalance". Besides these classical evidence on the role of NO in cardiac physiology and pathology, accumulating data show that NO regulate different aspects of stem cell biology, including survival, proliferation, migration, differentiation, and secretion of pro-regenerative factors. Concurrently, it has been shown that physical exercise generates physiological remodeling while antagonizes pathologic remodeling also by fostering cardiac regeneration, including new cardiomyocyte formation. This review is therefore focused on the possible link between physical exercise, NO, and stem cell biology in the cardiac regenerative/reparative response to physiological or pathological load. Cellular and molecular mechanisms that generate an exercise-induced cardioprotective phenotype are discussed in regards with myocardial repair and regeneration. Aerobic training can benefit cells implicated in cardiovascular homeostasis and response to damage by NO-mediated pathways that protect stem cells in the hostile environment, enhance their activation and differentiation and, in turn, translate to more efficient myocardial tissue regeneration. Moreover, stem cell preconditioning by and/or local potentiation of NO signaling can be envisioned as promising approaches to improve the post-transplantation stem cell survival and the efficacy of cardiac stem cell therapy.

Atrial myxomas arise from multipotent cardiac stem cells.

AIMS: Cardiac myxomas usually develop in the atria and consist of an acid-mucopolysaccharide-rich myxoid matrix with polygonal stromal cells scattered throughout. These human benign tumours are a valuable research model because of the rarity of cardiac tumours, their clinical presentation and uncertain origin. Here, we assessed whether multipotent cardiac stem/progenitor cells (CSCs) give rise to atrial myxoma tissue. METHODS AND RESULTS: Twenty-three myxomas were collected and analysed for the presence of multipotent CSCs. We detected myxoma cells positive for c-kit (c-kitpos) but very rare Isl-1 positive cells. Most of the c-kitpos cells were blood lineage-committed CD45pos/CD31pos cells. However, c-kitpos/CD45neg/CD31neg cardiac myxoma cells expressed stemness and cardiac progenitor cell transcription factors. Approximately ≤10% of the c-kitpos/CD45neg/CD31neg myxoma cells also expressed calretinin, a characteristic of myxoma stromal cells. In vitro, the c-kitpos/CD45neg/CD31neg myxoma cells secrete chondroitin-6-sulfate and hyaluronic acid, which are the main components of gelatinous myxoma matrix in vivo. In vitro, c-kitpos/CD45neg/CD31neg myxoma cells have stem cell properties being clonogenic, self-renewing, and sphere forming while exhibiting an abortive cardiac differentiation potential. Myxoma-derived CSCs possess a mRNA and microRNA transcriptome overall similar to normal myocardium-derived c-kitpos/CD45neg/CD31negCSCs , yet showing a relatively small and relevant fraction of dysregulated mRNA/miRNAs (miR-126-3p and miR-335-5p, in particular). Importantly, myxoma-derived CSCs but not normal myocardium-derived CSCs, seed human myxoma tumours in xenograft's in immunodeficient NOD/SCID mice. CONCLUSION: Myxoma-derived c-kitpos/CD45neg/CD31neg CSCs fulfill the criteria expected of atrial myxoma-initiating stem cells. The transcriptome of these cells indicates that they belong to or are derived from the same lineage as the atrial multipotent c-kitpos/CD45neg/CD31neg CSCs. Taken together the data presented here suggest that human myxomas could be the first-described CSC-related human heart disease.

Computational Modeling of a Transcriptional Switch Underlying B-Lymphocyte Lineage Commitment of Hematopoietic Multipotent Cells.

Despite progresses in identifying the cellular mechanisms at the basis of the differentiation of hematopoietic stem/progenitor cells, little is known about the regulatory circuitry at the basis of lineage commitment of hematopoietic multipotent progenitors. To address this issue, we propose a computational approach to give further insights in the comprehension of this genetic mechanism. Differently from T lymphopoiesis, however, there is at present no mathematical model describing lineage restriction of multipotent progenitors to early B-cell precursors. Here, we provide a first model-constructed on the basis of current experimental evidence from literature and of publicly available microarray datasets-of the genetic regulatory network driving the cellular fate determination at the stage of lymphoid lineage commitment, with particular regard to the multipotent-B-cell progenitor transition. By applying multistability analysis methods, we are able to assess the capability of the model to capture the experimentally observed switch-like commitment behavior. These methods allow us to confirm the central role of zinc finger protein 521 (ZNF521) in this process, that we had previously reported, and to identify a novel putative functional interaction for ZNF521, which is essential to realize such characteristic behavior. Moreover, using the devised model, we are able to rigorously analyze the mechanisms underpinning irreversibility of the physiological commitment step and to devise a possible reprogramming strategy, based on the combined modification of the expression of ZNF521 and EBF1.

A control-theoretical approach to the identification of a commitment switch in B lymphopoiesis cell fate determination.

Cellular differentiation is continuously orchestrated by complex networks of transcription factors, signaling molecules and genetic and epigenetic events, a fundamental prerequisite for the design of strategies for reprogramming differentiated cells to immature stem/progenitor cells is a thorough understanding of such complex regulatory machinery. Therefore, mathematical models, along with the associated analysis and control methods, are highly needed in this research field. In the present work, we provide a first model of the genetic regulatory network driving the cellular fate determination at the stage of lymphoid lineage commitment, in particular during lineage restriction of multipotent progenitors to early B-cell committed precursors.

Retroactivity analysis of a chemical reaction network module for the subtraction of molecular fluxes.

The availability of a well-characterised subtraction module is a key step towards the realisation of modular embedded feedback controllers in synthetic biological systems. A well-known problem when dealing with complex biosystems is represented by the retroactivity effect, which can significantly modify the dynamics of interconnected subsystem, with respect to the behaviour they exhibit when disconnected from each other. In this paper, we illustrate a minimal CRN that implements a subtraction operation between two input molecular fluxes. In order to assess its effectiveness as a module of a more complex system, we analyse its retroactivity upon interconnection. More specifically, we connect the subtraction module with an upstream module, which determines the dynamics of the inputs species, and with a downstream transcriptional module, which acts as a load. By comparing the dynamics of the loaded and unloaded subtractor, we show that the retroactivity can be attenuated when the dynamics of the subtractor and of the load system evolve over different timescales. This result, obtained through a singular perturbation analysis, is confirmed by means of numerical simulations.

Validation of a model of the GAL regulatory system via robustness analysis of its bistability characteristics.

BACKGROUND: In Saccharomyces cerevisiæ, structural bistability generates a bimodal expression of the galactose uptake genes (GAL) when exposed to low and high glucose concentrations. This indicates that yeast cells can decide between using either the limited amount of glucose or growing on galactose under changing environmental conditions. A crucial requirement for any plausible mechanistic model of this system is that it reproduces the robustness of the bistable response observed in vivo against inter-individual parametric variability and fluctuating environmental conditions. RESULTS: We show how a control-theoretic analysis of the robustness of a model of the GAL regulatory network may be used to establish the model's plausibility in characterizing the persistent memory of different carbon sources, without the need for extensive simulations. Chemical Reaction Network Theory is used to establish that the proposed network model is compatible with structural bistability. The robustness of each of the two operative conditions against fluctuations of the species concentrations is demonstrated by studying the Domains of Attraction of the corresponding equilibrium points. Finally, we use a global robustness analysis method based on Semi-Definite Programming to evaluate the modification of the bistable steady states induced by multiple parametric variations throughout bounded regions of the parameter space. CONCLUSIONS: Our analysis provides convincing evidence for the robustness, and hence plausibility, of the GAL regulatory network model. The proposed workflow also demonstrates the power of analytical methods from control theory to provide a direct quantitative characterization of the dynamics of multistable biomolecular regulatory systems without recourse to extensive computer simulations.

Structural bistability of the GAL regulatory network and characterization of its domains of attraction.

Bistability is a system-level property, exploited by many biomolecular interaction networks as a key mechanism to accomplish different cellular functions (e.g., differentiation, cell cycle, switch-like response to external stimuli). Bistability has also been experimentally found to occur in the regulatory network of the galactose metabolic pathway in the model organism Saccharomyces cerevisiae. In this yeast, bistability generates a persistent memory of the type of carbon source available in the extracellular medium: under the same experimental conditions, cells previously grown with different nutrients generate different responses and get stably locked into two distinct steady states. The molecular interactions of the GAL regulatory network have been thoroughly dissected through wet-lab experiments; thus, this system provides a formidable benchmark to our ability to characterize and reproduce in silico the behavior of bistable biological systems. To this aim, a number of models have been proposed in the literature; however, we found that they are not able to replicate the persistent memory behavior observed in (Acar et al., 2005 ). The present study proposes a novel model of the GAL regulatory network, which, in addition to reproducing in silico the experimental findings, can be formally analyzed for structural multistability of the network, using chemical reaction network theory (CRNT), and allows the characterization of the domains of attraction (DA). This work provides further insights into the GAL system and proposes an easily generalizable approach to the study of bistability-associated behaviors in biological systems.