Control of Metastable States by Heat Flux in the Hamiltonian Potts Model.

The local equilibrium thermodynamics is a basic assumption of macroscopic descriptions of the out of equilibrium dynamics for Hamiltonian systems. We numerically analyze the Hamiltonian Potts model in two dimensions to study the violation of the assumption for phase coexistence in heat conduction. We observe that the temperature of the interface between ordered and disordered states deviates from the equilibrium transition temperature, indicating that metastable states at equilibrium are stabilized by the influence of a heat flux. We also find that the deviation is described by the formula proposed in an extended framework of the thermodynamics.

Etv2 regulates enhancer chromatin status to initiate Shh expression in the limb bud.

Sonic hedgehog (Shh) is essential for limb development, and the mechanisms that govern the propagation and maintenance of its expression has been well studied; however, the mechanisms that govern the initiation of Shh expression are incomplete. Here we report that ETV2 initiates Shh expression by changing the chromatin status of the developmental limb enhancer, ZRS. Etv2 expression precedes Shh in limb buds, and Etv2 inactivation prevents the opening of limb chromatin, including the ZRS, resulting in an absence of Shh expression. Etv2 overexpression in limb buds causes nucleosomal displacement at the ZRS, ectopic Shh expression, and polydactyly. Areas of nucleosome displacement coincide with ETS binding site clusters. ETV2 also functions as a transcriptional activator of ZRS and is antagonized by ETV4/5 repressors. Known human polydactyl mutations introduce novel ETV2 binding sites in the ZRS, suggesting that ETV2 dosage regulates ZRS activation. These studies identify ETV2 as a pioneer transcription factor (TF) regulating the onset of Shh expression, having both a chromatin regulatory role and a transcriptional activation role.

Stochastic order parameter dynamics for phase coexistence in heat conduction.

We propose a stochastic order parameter model for describing phase coexistence in steady heat conduction near equilibrium. By analyzing the stochastic dynamics with a nonequilibrium adiabatic boundary condition, where total energy is conserved over time, we derive a variational principle that determines thermodynamic properties in nonequilibrium steady states. The resulting variational principle indicates that the temperature of the interface between the ordered region and the disordered region becomes greater (less) than the equilibrium transition temperature in the linear response regime when the thermal conductivity in the ordered region is less (greater) than that in the disordered region. This means that a superheated ordered (supercooled disordered) state appears near the interface, which was predicted by an extended framework of thermodynamics proposed in Nakagawa and Sasa [Liquid-Gas Transitions in Steady Heat Conduction, Phys. Rev. Lett. 119, 260602 (2017)PRLTAO0031-900710.1103/PhysRevLett.119.260602.].

Effective Langevin equations leading to large deviation function of time-averaged velocity for a nonequilibrium Rayleigh piston.

We study fluctuating dynamics of a freely movable piston that separates an infinite cylinder into two regions filled with ideal gas particles at the same pressure but different temperatures. To investigate statistical properties of the time-averaged velocity of the piston in the long-time limit, we perturbatively calculate the large deviation function of the time-averaged velocity. Then, we derive an infinite number of effective Langevin equations yielding the same large deviation function as in the original model. Finally, we provide two possibilities for uniquely determining the form of the effective model.

Complex hereditary spastic paraplegia associated with episodic visual loss caused by ACO2 variants.

Most patients with homozygous or compound heterozygous pathogenic ACO2 variants present with muscular hypotonia features, namely, infantile cerebellar-retinal degeneration. Recently, two studies reported rare familial cases of ACO2 variants presenting as complex hereditary spastic paraplegia (HSP) with broad clinical spectra. Here, we report the case of a 20-year-old Japanese woman with complex HSP caused by compound heterozygous ACO2 variants, revealing a new phenotype of episodic visual loss during febrile illness.

Generation of human endothelium in pig embryos deficient in ETV2.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney1,2. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages3-7. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.

A conserved HH-Gli1-Mycn network regulates heart regeneration from newt to human.

The mammalian heart has a limited regenerative capacity and typically progresses to heart failure following injury. Here, we defined a hedgehog (HH)-Gli1-Mycn network for cardiomyocyte proliferation and heart regeneration from amphibians to mammals. Using a genome-wide screen, we verified that HH signaling was essential for heart regeneration in the injured newt. Next, pharmacological and genetic loss- and gain-of-function of HH signaling demonstrated the essential requirement for HH signaling in the neonatal, adolescent, and adult mouse heart regeneration, and in the proliferation of hiPSC-derived cardiomyocytes. Fate-mapping and molecular biological studies revealed that HH signaling, via a HH-Gli1-Mycn network, contributed to heart regeneration by inducing proliferation of pre-existing cardiomyocytes and not by de novo cardiomyogenesis. Further, Mycn mRNA transfection experiments recapitulated the effects of HH signaling and promoted adult cardiomyocyte proliferation. These studies defined an evolutionarily conserved function of HH signaling that may serve as a platform for human regenerative therapies.

Unattainability of Carnot efficiency in thermal motors: Coarse graining and entropy production of Feynman-Smoluchowski ratchets.

We revisit and analyze the thermodynamic efficiency of the Feynman-Smoluchowski (FS) ratchet, a classical thought experiment describing an autonomous heat-work converter. Starting from the full kinetics of the FS ratchet and deriving the exact forms of the hidden dissipations resulting from coarse graining, we restate the historical controversy over its thermodynamic efficiency. The existence of hidden entropy productions implies that the standard framework of stochastic thermodynamics applied to the coarse-grained descriptions fails in capturing the dissipative feature of the system. In response to this problem, we explore an extended framework of stochastic thermodynamics to reconstruct the hidden entropy production from the coarse-grained dynamics. The approach serves as a key example of how we can systematically address the problem of thermodynamic efficiency in a multivariable fluctuating nonequilibrium system.

TCM visualizes trajectories and cell populations from single cell data.

Profiling single cell gene expression data over specified time periods are increasingly applied to the study of complex developmental processes. Here, we describe a novel prototype-based dimension reduction method to visualize high throughput temporal expression data for single cell analyses. Our software preserves the global developmental trajectories over a specified time course, and it also identifies subpopulations of cells within each time point demonstrating superior visualization performance over six commonly used methods.

DrImpute: imputing dropout events in single cell RNA sequencing data.

BACKGROUND: The single cell RNA sequencing (scRNA-seq) technique begin a new era by allowing the observation of gene expression at the single cell level. However, there is also a large amount of technical and biological noise. Because of the low number of RNA transcriptomes and the stochastic nature of the gene expression pattern, there is a high chance of missing nonzero entries as zero, which are called dropout events. RESULTS: We develop DrImpute to impute dropout events in scRNA-seq data. We show that DrImpute has significantly better performance on the separation of the dropout zeros from true zeros than existing imputation algorithms. We also demonstrate that DrImpute can significantly improve the performance of existing tools for clustering, visualization and lineage reconstruction of nine published scRNA-seq datasets. CONCLUSIONS: DrImpute can serve as a very useful addition to the currently existing statistical tools for single cell RNA-seq analysis. DrImpute is implemented in R and is available at https://github.com/gongx030/DrImpute .

Etv2-miR-130a-Jarid2 cascade regulates vascular patterning during embryogenesis.

Remodeling of the primitive vasculature is necessary for the formation of a complex branched vascular architecture. However, the factors that modulate these processes are incompletely defined. Previously, we defined the role of microRNAs (miRNAs) in endothelial specification. In the present study, we further examined the Etv2-Cre mediated ablation of DicerL/L and characterized the perturbed vascular patterning in the embryo proper and yolk-sac. We mechanistically defined an important role for miR-130a, an Etv2 downstream target, in the mediation of vascular patterning and angiogenesis in vitro and in vivo. Inducible overexpression of miR-130a resulted in robust induction of vascular sprouts and angiogenesis with increased uptake of acetylated-LDL. Mechanistically, miR-130a directly regulated Jarid2 expression by binding to its 3'-UTR region. Over-expression of Jarid2 in HUVEC cells led to defective tube formation indicating its inhibitory role in angiogenesis. The knockout of miR-130a showed increased levels of Jarid2 in the ES/EB system. In addition, the levels of Jarid2 transcripts were increased in the Etv2-null embryos at E8.5. In the in vivo settings, injection of miR-130a specific morpholinos in zebrafish embryos resulted in perturbed vascular patterning with reduced levels of endothelial transcripts in the miR-130a morphants. Further, co-injection of miR-130a mimics in the miR-130a morphants rescued the vascular defects during embryogenesis. qPCR and in situ hybridization techniques demonstrated increased expression of jarid2a in the miR-130a morphants in vivo. These findings demonstrate a critical role for Etv2-miR-130a-Jarid2 in vascular patterning both in vitro and in vivo.

Etv2 as an essential regulator of mesodermal lineage development.

The 'master regulatory factors' that position at the top of the genetic hierarchy of lineage determination have been a focus of intense interest, and have been investigated in various systems. Etv2/Etsrp71/ER71 is such a factor that is both necessary and sufficient for the development of haematopoietic and endothelial lineages. As such, genetic ablation of Etv2 leads to complete loss of blood and vessels, and overexpression can convert non-endothelial cells to the endothelial lineage. Understanding such master regulatory role of a lineage is not only a fundamental quest in developmental biology, but also holds immense possibilities in regenerative medicine. To harness its activity and utility for therapeutic interventions, it is essential to understand the regulatory mechanisms, molecular function, and networks that surround Etv2. In this review, we provide a comprehensive overview of Etv2 biology focused on mouse and human systems.

Dpath software reveals hierarchical haemato-endothelial lineages of Etv2 progenitors based on single-cell transcriptome analysis.

Developmental, stem cell and cancer biologists are interested in the molecular definition of cellular differentiation. Although single-cell RNA sequencing represents a transformational advance for global gene analyses, novel obstacles have emerged, including the computational management of dropout events, the reconstruction of biological pathways and the isolation of target cell populations. We develop an algorithm named dpath that applies the concept of metagene entropy and allows the ranking of cells based on their differentiation potential. We also develop self-organizing map (SOM) and random walk with restart (RWR) algorithms to separate the progenitors from the differentiated cells and reconstruct the lineage hierarchies in an unbiased manner. We test these algorithms using single cells from Etv2-EYFP transgenic mouse embryos and reveal specific molecular pathways that direct differentiation programmes involving the haemato-endothelial lineages. This software program quantitatively assesses the progenitor and committed states in single-cell RNA-seq data sets in a non-biased manner.

Liquid-Gas Transitions in Steady Heat Conduction.

We study liquid-gas transitions of heat conduction systems in contact with two heat baths under constant pressure in the linear response regime. On the basis of local equilibrium thermodynamics, we propose an equality with a global temperature, which determines the volume near the equilibrium liquid-gas transition. We find that the formation of the liquid-gas interface is accompanied by a discontinuous change in the volume when increasing the mean temperature of the baths. A supercooled gas near the interface is observed as a stable steady state.

Endoglin integrates BMP and Wnt signalling to induce haematopoiesis through JDP2.

Mechanisms of haematopoietic and cardiac patterning remain poorly understood. Here we show that the BMP and Wnt signalling pathways are integrated in an endoglin (Eng)-dependent manner in cardiac and haematopoietic lineage specification. Eng is expressed in early mesoderm and marks both haematopoietic and cardiac progenitors. In the absence of Eng, yolk sacs inappropriately express the cardiac marker, Nkx2.5. Conversely, high levels of Eng in vitro and in vivo increase haematopoiesis and inhibit cardiogenesis. Levels of Eng determine the activation of both BMP and Wnt pathways, which are integrated downstream of Eng by phosphorylation of Smad1 by Gsk3. By interrogating Eng-dependent Wnt-mediated transcriptional changes, we identify Jdp2 as a key Eng-dependent Wnt target, sufficient to establish haematopoietic fate in early mesoderm when BMP and Wnt crosstalk is disturbed. These studies provide mechanistic insight into the integration of BMP and Wnt signalling in the establishment of haematopoietic and cardiac progenitors during embryogenesis.

Numerical determination of entropy associated with excess heat in steady-state thermodynamics.

We numerically determine the global entropy for heat-conducting states, which is connected to the so-called excess heat considered as a basic quantity for steady-state thermodynamics in nonequilibrium. We adopt an efficient method to estimate the global entropy from the bare heat current and find that the obtained entropy agrees with the familiar local equilibrium hypothesis well. Our method possesses a wider applicability than local equilibrium and opens a possibility to compare thermodynamic properties of complex systems in nonequilibrium with those in the local equilibrium. We further investigate the global entropy for heat-conducting states and find that it exhibits both extensive and additive properties; however, the two properties do not degenerate each other differently from those at equilibrium. The separation of the extensivity and additivity makes it difficult to apply powerful thermodynamic methods to the nonequilibrium steady states.

Clinical Diagnosis of Mendelian Disorders Using a Comprehensive Gene-Targeted Panel Test for Next-Generation Sequencing.

BACKGROUND: Genetic diagnoses provide beneficial information to patients and families. However, traditional genetic diagnoses are often difficult even for experienced clinicians and require recognition of characteristic patterns of signs or symptoms to guide targeted genetic testing for the confirmation of diagnoses. Next-generation sequencing (NGS) is a powerful genetic diagnostic tool. However, whole-genome and whole-exome sequencing (WES) are expensive, and the interpretation of results is difficult. Hence, target gene capture sequencing of gene panels has recently been applied to genetic diagnoses. Herein, we demonstrate that targeted sequencing approaches using gene panel testing are highly efficient for the diagnosis of Mendelian disorders. METHODS: NGS using TruSight one gene panel was performed in 17 families and 20 patients, and we developed a bioinformatic pipeline at our institution for detecting mutations. RESULTS: We detected causative mutations in 6 of 17 (35%) families. In particular, 11 (65%) families had syndromic diagnosis and 6 (35%) had no syndromic diagnosis before NGS testing. The number of positive diagnoses was 5 of 11 (45%) in the syndromic group and were 1 of 6 (17%) among patients of the no syndromic diagnosis group. CONCLUSION: Diagnostic yields in the present study were higher than in previous reports of genetic and chromosomal tests and WES. The present comprehensive gene-targeted panel test is a powerful diagnostic tool for Mendelian disorders.

All trans-retinoic acid analogs promote cancer cell apoptosis through non-genomic Crabp1 mediating ERK1/2 phosphorylation.

All trans retinoic acid (atRA) is one of the most potent therapeutic agents, but extensive toxicity caused by nuclear RA receptors (RARs) limits its clinical application in treating cancer. AtRA also exerts non-genomic activities for which the mechanism remains poorly understood. We determine that cellular retinoic acid binding protein 1 (Crabp1) mediates the non-genomic activity of atRA, and identify two compounds as the ligands of Crabp1 to rapidly and RAR-independently activate extracellular signal regulated kinase 1/2 (ERK1/2). Non-canonically activated ERK activates protein phosphatase 2A (PP2A) and lengthens cell cycle duration in embryonic stem cells (ESC). This is abolished in Crabp1-null ESCs. Re-expressing Crabp1 in Crabp1-negative cancer cells also sensitizes their apoptotic induction by atRA. This study reveals a physiological relevance of the non-genomic action of atRA, mediated by Crabp1, in modulating cell cycle progression and apoptosis induction, and provides a new cancer therapeutic strategy whereby compounds specifically targeting Crabp1 can modulate cell cycle and cancer cell apoptosis in a RAR-independent fashion, thereby avoiding atRA's toxicity caused by its genomic effects.