Closed-loop anesthesia: foundations and applications in contemporary perioperative medicine.

A closed-loop automatically controls a variable using the principle of feedback. Automation within anesthesia typically aims to improve the stability of a controlled variable and reduce workload associated with simple repetitive tasks. This approach attempts to limit errors due to distractions or fatigue while simultaneously increasing compliance to evidence based perioperative protocols. The ultimate goal is to use these advantages over manual care to improve patient outcome. For more than twenty years, clinical studies in anesthesia have demonstrated the superiority of closed-loop systems compared to manual control for stabilizing a single variable, reducing practitioner workload, and safely administering therapies. This research has focused on various closed-loops that coupled inputs and outputs such as the processed electroencephalogram with propofol, blood pressure with vasopressors, and dynamic predictors of fluid responsiveness with fluid therapy. Recently, multiple simultaneous independent closed-loop systems have been tested in practice and one study has demonstrated a clinical benefit on postoperative cognitive dysfunction. Despite their advantages, these tools still require that a well-trained practitioner maintains situation awareness, understands how closed-loop systems react to each variable, and is ready to retake control if the closed-loop systems fail. In the future, multiple input multiple output closed-loop systems will control anesthetic, fluid and vasopressor titration and may perhaps integrate other key systems, such as the anesthesia machine. Human supervision will nonetheless always be indispensable as situation awareness, communication, and prediction of events remain irreplaceable human factors.

Safety, tolerability, and activity of the active C1s antibody riliprubart in cold agglutinin disease: a phase 1b study.

ABSTRACT: Cold agglutinin disease is a rare autoimmune hemolytic anemia characterized by complement pathway-mediated hemolysis. Riliprubart (SAR445088, BIVV020), a second-generation classical complement inhibitor, is a humanized monoclonal antibody that selectively inhibits only the activated form of C1s. This Phase 1b study evaluated the safety, tolerability, and effect on hemolysis of riliprubart in adult patients with cold agglutinin disease. On day 1, 12 patients received a single IV dose of either 30 mg/kg (n = 6) or 15 mg/kg (n = 6) of riliprubart and were subsequently followed for 15 weeks. Riliprubart was generally well tolerated; there were no treatment-emergent serious adverse events, or treatment-emergent adverse events leading to death or permanent study discontinuation. There were no reports of serious infections, encapsulated bacterial infections including meningococcal infections, hypersensitivity, or thromboembolic events. Rapid improvements in hemoglobin (day 5) and bilirubin (day 1) were observed in both treatment cohorts. Mean hemoglobin levels were maintained at >11.0 g/dL from day 29 and mean levels of bilirubin were normalized by day 29; both responses were maintained throughout the study. Improvements in clinical markers closely correlated with a sustained reduction in the 50% hemolytic complement (CH50) throughout the study. Mean C4 levels, an in vivo marker of treatment activity, increased 1 week after treatment with either dose of riliprubart and were sustained throughout the study. In conclusion, a single IV dose of riliprubart was well tolerated, and led to rapid classical complement inhibition, control of hemolysis, and improvement in anemia, all of which were sustained over 15 weeks. This trial was registered at www.ClinicalTrials.gov as #NCT04269551.

First-in-human study with SAR445088: A novel selective classical complement pathway inhibitor.

SAR445088 is an anti-C1s humanized monoclonal antibody that inhibits activated C1s in the proximal portion of the classical complement system and has the potential to provide clinical benefit in the treatment of complement-mediated diseases. A phase I, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of SAR445088 was conducted in 93 healthy participants to evaluate the safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Single (intravenous [i.v.] and subcutaneous [s.c.]) ascending doses (SAD) and multiple (s.c.) ascending doses (MAD) of SAR445088 were well-tolerated. The PK of SAR445088 was characterized by slow absorption after the s.c. dose and a long half-life (mean terminal half-life [t1/2 ] 8-15 weeks). Two PD assays were used to measure inhibition of the classical complement pathway (CP): Wieslab CP and complement mediated hemolytic capacity (CH50). The estimated half-maximal inhibitory concentration (IC50 ) and 90% inhibitory concentration (IC90 ) for the Wieslab CP assay were 96.4 and 458 µg/ml, respectively, and 16.6 and 57.0 µg/ml, respectively, for the CH50 assay. In summary, SAR445088 was well-tolerated and had favorable PK and PD profiles after SAD (i.v. or s.c.) and MAD (s.c.) in humans. These findings warrant further clinical investigations in patients with classical complement-mediated disorders.

Genetic architecture of natural variation of cardiac performance from flies to humans.

Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome-wide associations studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict transcription factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart-specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.

Epi-MEIF: detecting higher order epistatic interactions for complex traits using mixed effect conditional inference forests.

Understanding the relationship between genetic variations and variations in complex and quantitative phenotypes remains an ongoing challenge. While Genome-wide association studies (GWAS) have become a vital tool for identifying single-locus associations, we lack methods for identifying epistatic interactions. In this article, we propose a novel method for higher-order epistasis detection using mixed effect conditional inference forest (epiMEIF). The proposed method is fitted on a group of single nucleotide polymorphisms (SNPs) potentially associated with the phenotype and the tree structure in the forest facilitates the identification of n-way interactions between the SNPs. Additional testing strategies further improve the robustness of the method. We demonstrate its ability to detect true n-way interactions via extensive simulations in both cross-sectional and longitudinal synthetic datasets. This is further illustrated in an application to reveal epistatic interactions from natural variations of cardiac traits in flies (Drosophila). Overall, the method provides a generalized way to identify higher-order interactions from any GWAS data, thereby greatly improving the detection of the genetic architecture underlying complex phenotypes.

Predicting personalised remifentanil effect site concentration for surgical incision using the nociception level index: A prospective calibration and validation study.

BACKGROUND: Inadequate antinociception can cause haemodynamic instability. The nociception level (NOL) index measures response to noxious stimuli, but its capacity to predict optimal antinociception is unknown. OBJECTIVE: To determine if NOL index change to a tetanic stimulus in cardiac and noncardiac surgery patients could predict the required remifentanil concentration for haemodynamic stability at skin incision. DESIGN: A prospective two-phase cohort study. SETTING: University hospital. PATIENTS: Patients undergoing remifentanil-propofol target controlled infusion (TCI) anaesthesia. INTERVENTIONS: During the calibration phase, investigators evaluated the tetanic stimulus induced NOL index change under standardised TCI remifentanil-propofol anaesthesia during a no-touch period [bispectral index (BIS) between 40 and 60, NOL index under 15]. If the NOL index change was 20 or greater following tetanic stimulation, investigators repeated the tetanus at higher remifentanil concentrations until the response was blunted. Surgeons incised the skin at this remifentanil concentration. The investigators derived a prediction model and in the validation phase calculated, using the NOL response to a single tetanus, the required incision remifentanil concentration for the start of surgery. MAIN OUTCOME: Haemodynamic stability at incision [i.e. maximum heart rate (HR) < 20% increase from baseline, minimum HR (40 bpm) and mean arterial pressure (MAP) ± <20% of baseline]. RESULTS: During the calibration phase, no patient had hypertension. Two patients had a HR increase slightly greater than 20% (25.4 and 26.7%) within the first 2 min of surgery, but neither of these two patients had a HR above 76 bpm. Two patients were slightly hypotensive after incision (MAP 64 and 73 mmHg). During the validation phase, neither tachycardia nor hypotension occurred, but MAP increased to 21.5% above baseline for one patient. CONCLUSION: During a no-touch period in patients under steady-state general anaesthesia [propofol effect site concentration (Ce) required for BIS between 40 and 60], the NOL index response to a tetanic stimulus under remifentanil antinociception can be used to personalise remifentanil Ce for the start of surgery and ensure stable haemodynamics. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03324269.

Effect of dexmedetomidine on Nociception Level Index-guided remifentanil antinociception: A randomised controlled trial.

BACKGROUND: The effect of dexmedetomidine on Nociception Level Index-guided (Medasense, Israel) antinociception to reduce intra-operative opioid requirements has not been previously investigated. OBJECTIVE: We aimed to determine if low-dose dexmedetomidine would reduce remifentanil requirements during Nociception Level Index-guided antinociception without increasing complications associated with dexmedetomidine. DESIGN: Double-blind randomised controlled trial. SETTING: Two university teaching hospitals in Brussels, Belgium. PATIENTS: American Society of Anesthesiologists 1 and 2 patients (n = 58) undergoing maxillofacial or cervicofacial surgery under propofol--remifentanil target-controlled infusion anaesthesia. INTERVENTIONS: A 30 min infusion of dexmedetomidine, or equal volume of 0.9% NaCl, was infused at 1.2 µg kg-1 h-1 immediately preceding induction and then decreased to 0.6 µg kg-1 h-1 until 30 min before ending surgery. Nociception Level Index and frontal electroencephalogram guided the remifentanil and propofol infusions, respectively. MAIN OUTCOMES: The primary outcome was the remifentanil requirement. Other outcomes included the propofol requirement, cardiovascular status and postoperative outcome. RESULTS: Mean ±â€ŠSD remifentanil (3.96 ±â€Š1.95 vs. 4.42 ±â€Š2.04 ng ml-1; P = 0.0024) and propofol (2.78 ±â€Š1.36 vs. 3.06 ±â€Š1.29 µg ml-1; P = 0.0046) TCI effect site concentrations were lower in the dexmedetomidine group at 30 min postincision and remained lower throughout surgery. When remifentanil (0.133 ±â€Š0.085 vs. 0.198 ±â€Š0.086 µg kg-1 min-1; P = 0.0074) and propofol (5.7 ±â€Š2.72 vs. 7.4 ±â€Š2.80 mg kg-1 h-1; P = 0.0228) requirements are represented as infusion rates, this effect became statistically significant at 2 h postincision. CONCLUSION: In ASA 1 and 2 patients receiving Nociception Level Index-guided antinociception, dexmedetomidine decreases intra-operative remifentanil requirements. Combined frontal electroencephalogram and Nociception Level Index monitoring can measure dexmedetomidine's hypnotic and opioid-sparing effects during remifentanil-propofol target-controlled infusion anaesthesia. TRIAL REGISTRATIONS: Clinicaltrials.gov: NCT03912740, EudraCT: 2018-004512-22.

Course of explosion behaviour of metallic powders - From micron to nanosize.

This work presents an overview about the explosion behaviour of metallic powders from micron to nanosize. Aluminium, magnesium, titanium, iron and zinc were considered and their explosion safety parameters were analysed as a function of their mean primary particle size either determined by BET measurements, particle size distribution. To depict the course of explosion behaviour for these metals, extensive literature review has been performed and additional experimental tests were also performed. Generally, decreasing the particle size in a metallic powder leads to a higher explosion severity. It appears that this statement is true till a critical diameter below which the explosion severity (pmax, dp/dtmax) decreases for all the considered powders. This critical size can be explained by theoretical considerations on the nature of thermal transfer in the flame, namely by analysing the Cassel model. Finally, semi-empirical models were also developed for aluminium to highlight the specific micrometre and nanometre behaviour and the influence of turbulence, particle burning time, diameter and concentration. The influence of these key parameters needs to be further assessed in a future work in order to better understand the mechanisms involved and to extend the scope to other powdered materials.

Transcriptomic analyses reveal rhythmic and CLOCK-driven pathways in human skeletal muscle.

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans.

Interplay between trauma and Pseudomonas entomophila infection in flies: a central role of the JNK pathway and of CrebA.

In mammals, both sterile wounding and infection induce inflammation and activate the innate immune system, and the combination of both challenges may lead to severe health defects, revealing the importance of the balance between the intensity and resolution of the inflammatory response for the organism's fitness. Underlying mechanisms remain however elusive. Using Drosophila, we show that, upon infection with the entomopathogenic bacterium Pseudomonas entomophila (Pe), a sterile wounding induces a reduced resistance and increased host mortality. To identify the molecular mechanisms underlying the susceptibility of wounded flies to bacterial infection, we analyzed the very first steps of the process by comparing the transcriptome landscape of infected (simple hit flies, SH), wounded and infected (double hit flies, DH) and wounded (control) flies. We observed that overexpressed genes in DH flies compared to SH ones are significantly enriched in genes related to stress, including members of the JNK pathway. We demonstrated that the JNK pathway plays a central role in the DH phenotype by manipulating the Jra/dJun activity. Moreover, the CrebA/Creb3-like transcription factor (TF) and its targets were up-regulated in SH flies and we show that CrebA is required for mounting an appropriate immune response. Drosophila thus appears as a relevant model to investigate interactions between trauma and infection and allows to unravel key pathways involved.

Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro.

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.

A Randomized Phase 1 Study to Assess the Safety and Pharmacokinetics of the Subcutaneously Injected Anti-LIGHT Antibody, SAR252067.

LIGHT, a member of the tumor necrosis factor superfamily, is potentially involved in mucosal inflammation associated with inflammatory bowel disease. The safety and pharmacokinetics of the fully human monoclonal anti-LIGHT antibody, SAR252067, was evaluated in healthy volunteers in a phase 1 study as a potential treatment for diseases related to LIGHT-mediated mucosal inflammation. This double-blind, randomized, placebo-controlled, sequential ascending single-dose, single-center, 16-week study randomized 48 subjects to a single subcutaneous dose of SAR252067 (40, 120, 300, 600, 900, or 1200 mg) or placebo. Safety assessments included adverse events (AEs), injection-site reactions, and antidrug antibody (ADA) titer. Pharmacokinetic end points were serum parameters of SAR252067 (Cmax , AUC0-∞ , tmax , t1/2z ). Serum-soluble LIGHT concentrations were also determined. Safety analyses included all 48 participants; pharmacokinetic analyses included 36 subjects who received SAR252067. No serious AEs were reported, and no dose-effect relationship was apparent. Injection-site reactions were minimal. ADAs were not clinically relevant. SAR252067 exposure increased in a near-dose-proportional manner, median tmax ranged from 5.0 to 8.5 days, and t1/2z ranged from 18.0 to 27.0 days. Serum-soluble LIGHT significantly increased after SAR252067 administration with the 40-mg dose only. SAR252067 had a good safety profile, was well tolerated in healthy humans, and displayed a predictable pharmacokinetic profile.

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network.

Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. Here, as a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, we apply an integrated approach to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, we examined H3K4me3 and H3K27ac chromatin landscapes to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. Our results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which we show is essential for differentiated heart function.

Qualitative Dynamical Modelling Can Formally Explain Mesoderm Specification and Predict Novel Developmental Phenotypes.

Given the complexity of developmental networks, it is often difficult to predict the effect of genetic perturbations, even within coding genes. Regulatory factors generally have pleiotropic effects, exhibit partially redundant roles, and regulate highly interconnected pathways with ample cross-talk. Here, we delineate a logical model encompassing 48 components and 82 regulatory interactions involved in mesoderm specification during Drosophila development, thereby providing a formal integration of all available genetic information from the literature. The four main tissues derived from mesoderm correspond to alternative stable states. We demonstrate that the model can predict known mutant phenotypes and use it to systematically predict the effects of over 300 new, often non-intuitive, loss- and gain-of-function mutations, and combinations thereof. We further validated several novel predictions experimentally, thereby demonstrating the robustness of model. Logical modelling can thus contribute to formally explain and predict regulatory outcomes underlying cell fate decisions.

[Cardiac pathologies and aging: lessons from a tiny heart]. / Pathologies et vieillissement cardiaque - Les leçons d'un tout petit cœur.

The high level of conservation of the cardiogenic gene regulatory network as well as of the cellular and physiological characteristics of the cardiomyocytes between fly and human, makes the small heart of this invertebrate the simplest and most flexible genetic system to dissect the fundamental molecular mechanisms that are brought into play during the development, the establishment and the maintenance of the cardiac function. The recent improvements in techniques of measurements of cardiac function made it possible to validate Drosophila as a model of cardiomyopathies and arrhythmias of genetic and metabolic origin or dependent of ageing. The heart of the fly thus represents a model of choice to identify genes and their interactions implicated in cardiac pathologies.

LedPred: an R/bioconductor package to predict regulatory sequences using support vector machines.

UNLABELLED: Supervised classification based on support vector machines (SVMs) has successfully been used for the prediction of cis-regulatory modules (CRMs). However, no integrated tool using such heterogeneous data as position-specific scoring matrices, ChIP-seq data or conservation scores is currently available. Here, we present LedPred, a flexible SVM workflow that predicts new regulatory sequences based on the annotation of known CRMs, which are associated to a large variety of feature types. LedPred is provided as an R/Bioconductor package connected to an online server to avoid installation of non-R software. Due to the heterogeneous CRM feature integration, LedPred excels at the prediction of regulatory sequences in Drosophila and mouse datasets compared with similar SVM-based software. AVAILABILITY AND IMPLEMENTATION: LedPred is available on GitHub: https://github.com/aitgon/LedPred and Bioconductor: http://bioconductor.org/packages/release/bioc/html/LedPred.html under the MIT license. CONTACT: aitor.gonzalez@univ-amu.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion.

OBJECTIVE: Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. METHODS: We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. RESULTS: Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. CONCLUSIONS: Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

Tissue specific RNA isolation in Drosophila embryos: a strategy to analyze context dependent transcriptome landscapes using FACS.

The Hox family of transcription factors defines cell identity along the A/P axis of animal body plan by modulating expression of distinct sets of target genes in a tissue specific manner. Identifying such tissue specific target genes is indispensable if one wants to understand how Hox proteins mediate their context dependent function. Genome wide analysis of transcriptional activity in different tissues and contexts regarding Hox genes activity could help in reaching this goal. Such experiments rely on the possibility to selectively purify the cells of interest from developing embryos and to perform a transcriptomic analysis on such purified cell populations. By combining expression of a fluorescent protein and fluorescent activating cell sorting (FACS) technique, it is possible to obtain highly purified specific cell populations. In this chapter we describe the experimental procedure we have established in Drosophila-starting from a genetically marked small cell population (cardiomyocytes, 104 cells)-to dissociate the embryos in order to turn it into a suspension of individual cells, sort cells according to the expression of the introduced genetic marker and purify the total RNA content of the sorted cells. This can be used to analyze the transcriptome landscape of rare cell populations in wild type and mutant contexts. This technique has shown to be useful in the case of cardiac cells but is virtually applicable to any cell type and mutant backgrounds, provided that specific genetic markers are available.