Foxm1 regulates cardiomyocyte proliferation in adult zebrafish after cardiac injury.

The regenerative capacity of the mammalian heart is poor, with one potential reason being that adult cardiomyocytes cannot proliferate at sufficient levels to replace lost tissue. During development and neonatal stages, cardiomyocytes can successfully divide under injury conditions; however, as these cells mature their ability to proliferate is lost. Therefore, understanding the regulatory programs that can induce post-mitotic cardiomyocytes into a proliferative state is essential to enhance cardiac regeneration. Here, we report that the forkhead transcription factor Foxm1 is required for cardiomyocyte proliferation after injury through transcriptional regulation of cell cycle genes. Transcriptomic analysis of injured zebrafish hearts revealed that foxm1 expression is increased in border zone cardiomyocytes. Decreased cardiomyocyte proliferation and expression of cell cycle genes in foxm1 mutant hearts was observed, suggesting it is required for cell cycle checkpoints. Subsequent analysis of a candidate Foxm1 target gene, cenpf, revealed that this microtubule and kinetochore binding protein is also required for cardiac regeneration. Moreover, cenpf mutants show increased cardiomyocyte binucleation. Thus, foxm1 and cenpf are required for cardiomyocytes to complete mitosis during zebrafish cardiac regeneration.

The FXYD1 protein plays a protective role against pulmonary hypertension and arterial remodeling via redox and inflammatory mechanisms.

Pulmonary hypertension (PH) consists of a heterogenous group of diseases that culminate in increased pulmonary arterial pressure and right ventricular (RV) dysfunction. We sought to investigate the role of FXYD1, a small membrane protein that modulates Na+-K+-ATPase function, in the pathophysiology of PH. We mined online transcriptome databases to assess FXYD1 expression in PH. We characterized the effects of FXYD1 knockout (KO) in mice on right and left ventricular (RV and LV) function using echocardiography and measured invasive hemodynamic measurements under normal conditions and after treatment with bleomycin sulfate or chronic hypoxia to induce PH. Using immunohistochemistry, immunoblotting, and functional assays, we examined the effects of FXYD1 KO on pulmonary microvasculature and RV and LV structure and assessed signaling via endothelial nitric oxide synthase (eNOS) and inflammatory pathways. FXYD1 lung expression tended to be lower in samples from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with controls, supporting a potential pathophysiological role. FXYD1 KO mice displayed characteristics of PH including significant increases in pulmonary arterial pressure, increased muscularization of small pulmonary arterioles, and impaired RV systolic function, in addition to LV systolic dysfunction. However, when PH was stimulated with standard models of lung injury-induced PH, there was no exacerbation of disease in FXYD1 KO mice. Both the lungs and left ventricles exhibited elevated nitrosative stress and inflammatory milieu. The absence of FXYD1 in mice results in LV inflammation and cardiopulmonary redox signaling changes that predispose to pathophysiological features of PH, suggesting FXYD1 may be protective.NEW & NOTEWORTHY This is the first study to show that deficiency of the FXYD1 protein is associated with pulmonary hypertension. FXYD1 expression is lower in the lungs of people with idiopathic pulmonary artery hypertension. FXYD1 deficiency results in both left and right ventricular functional impairment. Finally, FXYD1 may endogenously protect the heart from oxidative and inflammatory injury.

Direct Activation of Nucleobases with Small Molecules for the Conditional Control of Antisense Function.

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule-induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido-caged nucleobases that are site-specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson-Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule-induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes.

Engineering Small Molecule Switches of Protein Function in Zebrafish Embryos.

Precise temporally regulated protein function directs the highly complex processes that make up embryo development. The zebrafish embryo is an excellent model organism to study development, and conditional control over enzymatic activity is desirable to target chemical intervention to specific developmental events and to investigate biological mechanisms. Surprisingly few, generally applicable small molecule switches of protein function exist in zebrafish. Genetic code expansion allows for site-specific incorporation of unnatural amino acids into proteins that contain caging groups that are removed through addition of small molecule triggers such as phosphines or tetrazines. This broadly applicable control of protein function was applied to activate several enzymes, including a GTPase and a protease, with temporal precision in zebrafish embryos. Simple addition of the small molecule to the media produces robust and tunable protein activation, which was used to gain insight into the development of a congenital heart defect from a RASopathy mutant of NRAS and to control DNA and protein cleavage events catalyzed by a viral recombinase and a viral protease, respectively.

Chemically Acylated tRNAs are Functional in Zebrafish Embryos.

Genetic code expansion has pushed protein chemistry past the canonical 22 amino acids. The key enzymes that make this possible are engineered aminoacyl tRNA synthetases. However, as the number of genetically encoded amino acids has increased over the years, obvious limits in the type and size of novel side chains that can be accommodated by the synthetase enzyme become apparent. Here, we show that chemically acylating tRNAs allow for robust, site-specific incorporation of unnatural amino acids into proteins in zebrafish embryos, an important model organism for human health and development. We apply this approach to incorporate a unique photocaged histidine analogue for which synthetase engineering efforts have failed. Additionally, we demonstrate optical control over different enzymes in live embryos by installing photocaged histidine into their active sites.

Procedural simplification of left atrial appendage occlusion using the VersaCross connect system: First in-human experience.

BACKGROUND: Left atrial appendage occlusion (LAAO) has emerged over the last two decades as an efficient and safe alternative to oral anticoagulation for stroke prevention. However, LAAO remains challenging due to the variety of anatomies and the multiple steps required to complete the procedure. AIMS: We report the first series of in-human experience of the new all-in-one VersaCross Connect system designed to access the left atrium in conjunction with the delivery sheath for deployment of the WATCHMAN FLX device. METHODS: We prospectively included the first nine consecutive cases of LAAO using the new VersaCross Connect system for WATCHMAN FLX device implantation at the Montreal Heart Institute and Vancouver General Hospital and collected procedural duration (defined as time from femoral access to closure) and time from transseptal puncture to device delivery. RESULTS: VersaCross Connect system use for WATCHMAN FLX implantation was successful in all patients. No procedural complication was reported. Mean procedural time was 31 ± 6.3 min with a fluoroscopy time of 6.7 ± 4.9 min. The mean delay between the transseptal puncture and device implantation was 12.2 ± 1.9 min. CONCLUSIONS: We showed that the VersaCross Connect system was safe and successfully used in all first nine cases. This new system helped improve the efficiency of the procedure.

How workplace-based assessments guide learning in postgraduate education: A scoping review.

INTRODUCTION: Competency-based medical education (CBME) led to the widespread adoption of workplace-based assessment (WBA) with the promise of achieving assessment for learning. Despite this, studies have illustrated tensions between the summative and formative role of WBA which undermine learning goals. Models of workplace-based learning (WBL) provide insight, however, these models excluded WBA. This scoping review synthesizes the primary literature addressing the role of WBA to guide learning in postgraduate medical education, with the goal of identifying gaps to address in future studies. METHODS: The search was applied to OVID Medline, Web of Science, ERIC and CINAHL databases, articles up to September 2020 were included. Titles and abstracts were screened by two reviewers, followed by a full text review. Two members independently extracted and analysed quantitative and qualitative data using a descriptive-analytic technique rooted in Billett's four premises of WBL. Themes were synthesized and discussed until consensus. RESULTS: All 33 papers focused on the perception of learning through WBA. The majority applied qualitative methodology (70%), and 12 studies (36%) made explicit reference to theory. Aligning with Billett's first premise, results reinforce that learning always occurs in the workplace. WBA helped guide learning goals and enhanced feedback frequency and specificity. Billett's remaining premises provided an important lens to understand how tensions that existed in WBL have been exacerbated with frequent WBA. As individuals engage in both work and WBA, they are slowly transforming the workplace. Culture and context frame individual experiences and the perceived authenticity of WBA. Finally, individuals will have different goals, and learn different things, from the same experience. CONCLUSION: Analysing WBA literature through the lens of WBL theory allows us to reframe previously described tensions. We propose that future studies attend to learning theory, and demonstrate alignment with philosophical position, to advance our understanding of assessment-for-learning in the workplace.

Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts.

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2 × 106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

Optical Control of MicroRNA Function in Zebrafish Embryos.

MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate hundreds of transcripts, which can make it difficult to study their role in the timing and location of specific developmental processes using conventional morpholino oligonucleotide (MO) knockdown or genetic deletion approaches. We demonstrate that light-activated circular morpholino oligonucleotides (cMOs) can be applied to the conditional control of microRNA function. We targeted miR-430 in zebrafish embryos to study its role in the development of the embryo body and the heart. Using 405 nm irradiation, precise spatial and temporal control over miR-430 function was demonstrated, offering insight into the cell populations and developmental timepoints involved in each process.

Optogenetic Protein Cleavage in Zebrafish Embryos.

A wide array of optogenetic tools are available that allow for precise spatiotemporal control over cellular processes. These tools are particularly important to zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and light-triggered apoptosis.

Incidentally identified left ventricular apical aneurysm in a patient with Fabry disease.

Fabry disease is a rare X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme, galactosidase A, that can result in a progressive increase in the left ventricle (LV) wall thickness from glycosphingolipid deposition leading to myocardial fibrosis, conduction abnormalities, arrhythmias, and heart failure. We present a case of a patient with advanced Fabry cardiomyopathy, in whom a small LV apical aneurysm was incidentally discovered on abdominal imaging, which could have easily evaded detection on standard transthoracic echocardiography. The LV apex should be thoroughly interrogated in patients with Fabry cardiomyopathy, as the finding of LV aneurysm could have important management implications with respect to the prevention of stroke and sudden cardiac death.

Zebrafish heart regenerates after chemoptogenetic cardiomyocyte depletion.

BACKGROUND: Zebrafish can regenerate adult cardiac tissue following injuries from ventricular apex amputation, cryoinjury, and cardiomyocyte genetic ablation. Here, we characterize cardiac regeneration from cardiomyocyte chemoptogenetic ablation caused by localized near-infrared excited photosensitizer-mediated reactive oxygen species (ROS) generation. RESULTS: Exposure of transgenic adult zebrafish, Tg(myl7:fapdl5-cerulean), to di-iodinated derivative of the cell- permeable Malachite Green ester fluorogen (MG-2I) and whole-body illumination with 660 nm light resulted in cytotoxic damage to about 30% of cardiac tissue. After chemoptogenetic cardiomyocyte ablation, heart function was compromised, and macrophage infiltration was detected, but epicardial and endocardial activation response was much muted when compared to ventricular amputation. The spared cardiomyocytes underwent proliferation and restored the heart structure and function in 45-60 days after ablation. CONCLUSIONS: This cardiomyocyte ablation system did not appear to activate the epicardium and endocardium as is noted in other cardiac injury models. This approach represents a useful model to study specifically cardiomyocyte injury, proliferation and regeneration in the absence of whole organ activation. Moreover, this system can be adapted to ablate distinct cell populations in any organ system to study their function in regeneration.

Small Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger Reduction.

Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small molecule-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger reduction. This is enabled by a highly flexible linker design that offers opportunities for the installation of chemically activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small molecule-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.

Dusp6 attenuates Ras/MAPK signaling to limit zebrafish heart regeneration.

Zebrafish regenerate cardiac tissue through proliferation of pre-existing cardiomyocytes and neovascularization. Secreted growth factors such as FGFs, IGF, PDGFs and Neuregulin play essential roles in stimulating cardiomyocyte proliferation. These factors activate the Ras/MAPK pathway, which is tightly controlled by the feedback attenuator Dual specificity phosphatase 6 (Dusp6), an ERK phosphatase. Here, we show that suppressing Dusp6 function enhances cardiac regeneration. Inactivation of Dusp6 by small molecules or by gene inactivation increased cardiomyocyte proliferation, coronary angiogenesis, and reduced fibrosis after ventricular resection. Inhibition of Erbb or PDGF receptor signaling suppressed cardiac regeneration in wild-type zebrafish, but had a milder effect on regeneration in dusp6 mutants. Moreover, in rat primary cardiomyocytes, NRG1-stimulated proliferation can be enhanced upon chemical inhibition of Dusp6 with BCI. Our results suggest that Dusp6 attenuates Ras/MAPK signaling during regeneration and that suppressing Dusp6 can enhance cardiac repair.

Impact of targeted pulmonary arterial hypertension therapy in patients with combined post- and precapillary pulmonary hypertension.

BACKGROUND: Combined post- and precapillary pulmonary hypertension (CpcPH) portends poor outcomes in pulmonary hypertension related to left heart disease (PH-LHD). While recent evidence does not support the use of targeted pulmonary arterial hypertension (PAH) therapy in PH-LHD, there is a lack of clinical data on their use in CpcPH. We evaluated the outcomes in patients with CpcPH treated with PAH therapies. METHODS: Retrospectively, 50 patients meeting hemodynamic criteria of CpcPH and started on PAH-targeted drugs were identified. Fifty age- and gender-matched PAH patients were chosen as controls. We evaluated the change in 6-minute walk distance, World Health Organization functional class (FC), tricuspid annular plane systolic excursion, BNP or NT-proBNP, and pulmonary artery systolic pressure at 3, 6, 12, and 24 months of follow-up. RESULTS: After adjusting for age and gender, there was no improvement in World Health Organization FC in CpcPH over 2 years (odds ratio of change to FC I/II 1.01, 95% CI: 0.98-1.04). There was no significant improvement in 6-minute walk distance (ß coefficient 0.21, 95% CI: -0.98 to 1.4), reduction in BNP/NT-proBNP (ß coefficient -12.16, 95% CI: -30.68 to 6.37), increase in tricuspid annular plane systolic excursion (ß coefficient 0.074, 95% CI: 0.010-0.139), or decrease in pulmonary artery systolic pressure (0.996, 95% CI: 0.991-1.011) in CpcPH with therapy. There was higher mortality in CpcPH compared to PAH on treatment (24% vs 4%, P = .003). CONCLUSIONS: There were no improvements in symptoms, exercise capacity, or echocardiographic parameters with PAH-targeted therapy in CpcPH. Further studies into potential treatments benefiting this population are needed.

Closing gigantic left atrial appendage using a LAmbre Closure System: First implant experience in North America.

INTRODUCTION: Despite maturing experience and growing procedural familiarity, there remain challenges in percutaneous left atrial appendage (LAA) closure due to anatomical complexities. METHODS: We report a complex and extremely large LAA that was successfully closed percutaneously using a LAmbre Closure System (Lifetech Scientific Corp.). Cardiac computed tomography angiography demonstrated a gigantic multilobed LAA measuring 48 × 45.3 mm at the level of the ostium that cannot be occluded by the currently approved LAA closure devices in Canada. RESULTS: The manufacturer custom-made a LAmbre 30/50 mm (lobe/disc) device to fit this patient's LAA according to his CTA, which was successfully deployed under fluoroscopy and transesophageal echocardiogram guidance without procedure-related complications. CONCLUSION: The LAmbre device may be considered to close very large LAAs.

A new transseptal solution for enabling left atrial access of large delivery sheaths.

BACKGROUND: Transseptal access for large sheaths may be encumbered by tissue resistance against the sheath-dilator stepped interface. The ExpanSure Large Access Transseptal Dilator (Baylis Medical) is designed as a single introducer and dilation device with a smooth sheath-dilator transition to support transseptal puncture. It may facilitate ease and efficiency of interatrial crossing. METHODS: This study experimentally evaluated the crossing force of ExpanSure relative to a conventional 8.5 F Swartz SL1 transseptal sheath and dilator in a benchtop septum model. Its ability to reduce the subsequent crossing force of a 14 F WATCHMAN delivery sheath was also tested. The clinical use of ExpanSure, including procedure time, was then validated in a series of left atrial appendage closure (LAAC) procedures. RESULTS: In a benchtop septum model (N = 12), less peak force (1.90 ± 0.08 N vs. 2.36 ± 0.09 N; p < .001) and overall work (17.3 ± 1.2 mJ vs. 28.0 ± 1.9 mJ; p < .001) were required to advance ExpanSure relative to a conventional SL1 transseptal sheath and dilator system. Peak force (2.34 ± 0.24 N vs. 2.65 ± 0.21 N; p < .003) and overall work (28.5 ± 3.9 mJ vs. 35.4 ± 2.1 mJ; p < .001) to advance a WATCHMAN sheath were also significantly lower after using ExpanSure than after using a conventional transseptal system. In 19 LAAC procedures, ExpanSure crossed the septum smoothly and integrated readily, which enabled efficient procedure completion (mean total procedure time 37.6 ± 13.5 min), with 100% success and no procedure-related complications. CONCLUSION: Experimental force measurements, combined with early clinical experience using ExpanSure, suggest that the tapered design with smooth transition without dilator-sheath step-up and the larger diameter, both facilitated ease and efficiency of interatrial crossing.

Point-of-care ultrasound in the COVID-19 era: A scoping review.

In the midst of the COVID-19 pandemic, unprecedented pressure has been added to healthcare systems around the globe. Imaging is a crucial component in the management of COVID-19 patients. Point-of-care ultrasound (POCUS) such as hand-carried ultrasound emerges in the COVID-19 era as a tool that can simplify the imaging process of COVID-19 patients, and potentially reduce the strain on healthcare providers and healthcare resources. The preliminary evidence available suggests an increasing role of POCUS in diagnosing, monitoring, and risk-stratifying COVID-19 patients. This scoping review aims to delineate the challenges in imaging COVID-19 patients, discuss the cardiopulmonary complications of COVID-19 and their respective sonographic findings, and summarize the current data and recommendations available. There is currently a critical gap in knowledge in the role of POCUS in the COVID-19 era. Nonetheless, it is crucial to summarize the current preliminary data available in order to help fill this gap in knowledge for future studies.

The novel P2X7 receptor antagonist PKT100 improves cardiac function and survival in pulmonary hypertension by direct targeting of the right ventricle.

In pulmonary hypertension (PH) a proinflammatory milieu drives pulmonary vascular remodeling, maladaptive right ventricular (RV) remodeling, and right-sided heart failure. There is an unmet need for RV-targeted pharmaco-therapies to improve mortality. Targeting of the P2X7 receptor (P2X7R) reduces pulmonary pressures; however, its effects on the RV are presently unknown. We investigated the effect of P2X7 receptor (P2X7R) inhibition on the pulmonary vasculature and RV remodeling using the novel P2X7R antagonist PKT100. C57BL/6 mice were administered intratracheal bleomycin or saline and treated with PKT100 (0.2 mg·kg-1·day-1) or DMSO vehicle. RV was assessed by right heart catheterization and echocardiography, 21 days posttreatment. Cytokines in serum and bronchoalveolar lavage fluid (BALF) were analyzed by ELISA and flow cytometry. Lungs and hearts were analyzed histologically for pulmonary vascular and RV remodeling. Focused-PCR using genes involved in RV remodeling was performed. Right ventricular systolic pressure (RVSP) was elevated in bleomycin-treated mice (30.2 ± 1.1; n = 7) compared with control mice (23.5 ± 1.0; n = 10; P = 0.008). PKT100 treatment did not alter RVSP (32.4 ± 1.8; n = 9), but it substantially improved survival (93% vs. 57% DMSO). There were no differences between DMSO and PKT100 bleomycin mice in pulmonary inflammation or remodeling. However, RV hypertrophy was reduced in PKT100 mice. Bleomycin decreased echocardiographic surrogates of RV systolic performance, which were significantly improved with PKT100. Four genes involved in RV remodeling (RPSA, Rplp0, Add2, and Scn7a) were differentially expressed between DMSO and PKT100-treated groups. The novel P2X7R inhibitor, PKT100, attenuates RV hypertrophy and improves RV contractile function and survival in a mouse model of PH independently of effects on the pulmonary vasculature. PKT100 may improve ventricular response to increased afterload and merits further investigation into the potential role of P2X7R antagonists as direct RV-focused therapies in PH.NEW & NOTEWORTHY This study demonstrates the therapeutic potential for right-sided heart failure of a novel inhibitor of the P2X7 receptor (P2X7R). Inflammatory signaling and right ventricular function were improved in a mouse model of pulmonary fibrosis with secondary pulmonary hypertension when treated with this inhibitor. Importantly, survival was also improved, suggesting that this inhibitor, and other P2X7R antagonists, could be uniquely effective in right ventricle (RV)-targeted therapy in pulmonary hypertension. This addresses a major limitation of current treatment options, where the significant improvements in pulmonary pressures ultimately do not prevent mortality due to RV failure.

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA.

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5'-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.