The cardiac conduction system is an anatomically discrete segment of specialized myocardium that initiates and propagates electrical impulses to coordinate myocardial contraction. To define the molecular composition of the mouse ventricular conduction system we used microdissection and transcriptional profiling by serial analysis of gene expression (SAGE). Conduction-system-specific expression for Id2, a member of the Id gene family of transcriptional repressors, was identified. Analyses of Id2-deficient mice demonstrated structural and functional conduction system abnormalities, including left bundle branch block. A 1.2 kb fragment of the Id2 promoter proved sufficient for cooperative regulation by Nkx2-5 and Tbx5 in vitro and for conduction-system-specific gene expression in vivo. Furthermore, compound haploinsufficiency of Tbx5 and Nkx2-5 or Tbx5 and Id2 prevented embryonic specification of the ventricular conduction system. We conclude that a molecular pathway including Tbx5, Nkx2-5, and Id2 coordinates specification of ventricular myocytes into the ventricular conduction system lineage.

Abnormal myocardial perfusion is present in most patients following primary or rescue PCI in AMI, despite restoration of brisk epicardial coronary flow. In high risk patients achieving TIMI-3 flow after intervention, the myocardial blush score may be used to stratify prognosis into excellent, intermediate and poor survival. Further study is warranted to examine whether adjunctive mechanical or pharmacologic strategies can further improve myocardial perfusion and survival of patients with acute myocardial infarction undergoing intervention.

The genesis of the septal structures of the mammalian heart is central to understanding the ontogeny of congenital heart disease and the evolution of cardiac organogenesis. We found that Hedgehog (Hh) signaling marked a subset of cardiac progenitors specific to the atrial septum and the pulmonary trunk in the mouse. Using genetic inducible fate mapping with Gli1CreERT2, we marked Hh-receiving progenitors in anterior and posterior second heart field splanchnic mesoderm between E8 and E10. In the inflow tract, Hh-receiving progenitors migrated from the posterior second heart field through the dorsal mesocardium to form the atrial septum,including both the primary atrial septum and dorsal mesenchymal protrusion(DMP). In the outflow tract, Hh-receiving progenitors migrated from the anterior second heart field to populate the pulmonary trunk. Abrogation of Hh signaling during atrial septal progenitor specification resulted in atrial and atrioventricular septal defects and hypoplasia of the developing DMP. Hedgehog signaling appeared necessary and sufficient for atrial septal progenitor fate:Hh-receiving cells rendered unresponsive to the Hh ligand migrated into the atrium in normal numbers but populated the atrial free wall rather than the atrial septum. Conversely, constitutive activation of Hh signaling caused inappropriate enlargement of the atrial septum. The close proximity of posterior second heart field cardiac progenitors to pulmonary endoderm suggested a pulmonary source for the Hh ligand. We found that Shh is required in the pulmonary endoderm for atrial septation. Therefore, Hh signaling from distinct pulmonary and pharyngeal endoderm is required for inflow and outflow septation, respectively. These data suggest a model in which respiratory endoderm patterns the morphogenesis of cardiac structural components required for efficient cardiopulmonary circulation.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N-methyladenosine (mA) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several mA readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of mA in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These mA-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Despite Bridging INtegrator 1 ( BIN1 ) being the second most statistically-significant locus associated to Late Onset Alzheimer’s Disease, its role in disease pathogenesis remains to be clarified. As reports suggest a link between BIN1, Tau and extracellular vesicles, we investigated whether BIN1 could affect Tau spreading via exosomes secretion. We observed that BIN1-associated Tau-containing extracellular vesicles purified from cerebrospinal fluid of AD-affected individuals are seeding-competent. We showed that BIN1 over-expression promotes the release of Tau via extracellular vesicles in vitro as well as exacerbation of Tau pathology in vivo in PS19 mice. Genetic deletion of Bin1 from microglia resulted in reduction of Tau secretion via extracellular vesicles in vitro , and in decrease of Tau spreading in vivo in male, but not female, mice, in the context of PS19 background. Interestingly, ablation of Bin1 in microglia of male mice resulted in significant reduction in the expression of heat-shock proteins, previously implicated in Tau proteostasis. These observations suggest that BIN1 could contribute to the progression of AD-related Tau pathology by altering Tau clearance and promoting release of Tau-enriched extracellular vesicles by microglia.

We report that the dominant human missense mutations G303E and G296S in GATA4 , a cardiac-specific transcription factor gene, cause atrioventricular septal defects and valve abnormalities by disrupting a signaling cascade involved in endocardial cushion development. These GATA4 missense mutations, but not a mutation causing secundum atrial septal defects (S52F), demonstrated impaired protein interactions with SMAD4, a transcription factor required for canonical bone morphogenetic protein/transforming growth factor-β ( BMP / TGF-β ) signaling. Gata4 and Smad4 genetically interact in vivo: atrioventricular septal defects result from endothelial-specific Gata4 and Smad4 compound haploinsufficiency. Endothelial-specific knockout of Smad4 caused an absence of valve-forming activity: Smad4 -deficient endocardium was associated with acellular endocardial cushions, absent epithelial-to-mesenchymal transformation, reduced endocardial proliferation, and loss of Id2 expression in valve-forming regions. We show that Gata4 and Smad4 cooperatively activated the Id2 promoter, that human GATA4 mutations abrogated this activity, and that Id2 deficiency in mice could cause atrioventricular septal defects. We suggest that one determinant of the phenotypic spectrum caused by human GATA4 mutations is differential effects on GATA4/SMAD4 interactions required for endocardial cushion development.

Objectives: The objective was to determine if use of ultrasound (US) by emergency physicians (EPs) to localize spinal landmarks improves the performance of lumbar puncture (LP).Methods: This was a prospective, randomized, controlled study conducted in a county teaching hospital. Subjects, adults 18 years of age or older who were to receive LPs for routine clinical care in the emergency department (ED), were randomized either to undergo US localization of the puncture site or to have the puncture site determined by palpation of spinal landmarks. Primary outcomes were the number of needle insertion attempts and success of the procedure. Secondary outcomes were pain associated with the procedure, time to perform the procedure, number of traumatic taps, and patient satisfaction with the procedure.Results: One-hundred patients were enrolled in the study, with 50 in each study group. There were no significant differences between the two groups in terms of age, sex, body mass index (BMI), indication for LP, or ease of palpation of landmarks. For both primary outcomes and secondary outcomes there were no significant differences between those undergoing US localization and those with palpation alone.

Atrioventricular septal defects (AVSDs) are a common severe form of congenital heart disease (CHD). In this study we identified deleterious non-synonymous mutations in two cilia genes, Dnah11 and Mks1, in independent N-ethyl-N-nitrosourea-induced mouse mutant lines with heritable recessive AVSDs by whole-exome sequencing. Cilia are required for left/right body axis determination and second heart field (SHF) Hedgehog (Hh) signaling, and we find that cilia mutations affect these requirements differentially. Dnah11 did not disrupt SHF Hh signaling and caused AVSDs only concurrently with heterotaxy, a left/right axis abnormality. In contrast, Mks1 disrupted SHF Hh signaling and caused AVSDs without heterotaxy. We performed unbiased whole-genome SHF transcriptional profiling and found that cilia motility genes were not expressed in the SHF whereas cilia structural and signaling genes were highly expressed. SHF cilia gene expression predicted the phenotypic concordance between AVSDs and heterotaxy in mice and humans with cilia gene mutations. A two-step model of cilia action accurately predicted the AVSD/heterotaxyu phenotypic expression pattern caused by cilia gene mutations. We speculate that cilia gene mutations contribute to both syndromic and non-syndromic AVSDs in humans and provide a model that predicts the phenotypic consequences of specific cilia gene mutations.