The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.
We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have ?-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not ?-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic ?-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.
Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.
The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.
Our institution is developing a non-invasive Diastolic Timed Vibrator (DTV) to enhance emergency clearance of acute coronary thrombosis. Sonic frequency diastolic vibro-percussion (i.e. 50 Hz, 2 mm amplitude) applied upon the rib-spaces of the left sternal border has shown to improve left ventricular performance and coronary flow in human volunteers. However, therapeutic acoustic penetrability cannot be assumed depending on varying chest size and lung position which attenuates acoustic transmissions. Furthermore, chest locations enabling a direct lung free pathway overlying the base of the heart (wherein the coronaries arise) should be promoted, while locations overlying the left ventricular apex (site of potential thrombus formation) should be avoided. We therefore set out to determine preferred chest wall placement positions for a vibratory interface suitable for treatment of ST Elevation Myocardial Infarction (STEMI). Inter-Costal Space (ICS) positions to the left or right of the sternum were interrogated in 90 adults following routine Echocardiography to ascertain whether the base of the heart could be imaged (hence inferring acoustic transmissibility), and to determine over what part of the heart each transducer position was overlying. The third and fourth ICS proximate the left sternal border provided acoustic transmissibility in 96 and 100% of cases respectively, with only one unwanted occurrence from the fourth ICS where the transducer overlaid the apical third of the left ventricle. Acoustic transmissibility from third and fourth ICS right sternal border was documented in 53 and 85% of cases respectively. A vibration interface in STEMI treatment should allow for contact overlying the left and right third and fourth ICS generally proximate the sternal borders. As vibration transmission to the cardiac apex and/or left atrium cannot be completely avoided, vibration therapy should be contra-indicated in late presenters for antero-septal apical STEMI, and in cases of new onset atrial fibrillation persisting greater than 48 h which have not been adequately anti-coagulated.
A case of myofibrosarcoma of breast is reported. A female patient aged 81 years presented with a mammary mass lesion. Histologically, the tumor consisted of neoplastic spindle cells arranged in fascicles and with variably cellularity and hyalinization. Immunohistochemical studies showed expression of vimentin, smooth-muscle actin, and Bcl-2, but not desmin, S-100, C-kit, or CD34. Proliferative index identified by Ki67 was approximately 30%. Electron microscopy revealed variable amount of rough endoplasmic reticulum, myofilaments, fibronexus junctions, and fibronectin fibrils. The histological, immunohistochemical, and ultrastructural features of this tumor were consistent with myofibrosarcoma. This case will be one of the very few cases of ultrastructurally confirmed mammary myofibrosarcoma reported in the literature and contributes to the recognition of this rare mammary malignant neoplasm. The literature on mammary myofibrosarcoma and its differential diagnosis is also reviewed.
Our institution is in development of a low frequency, non-invasive Diastolic Timed Vibrator (DTV) for use in emergency treatment of ST Elevation Myocardial Infarction (STEMI). It is preferable to avoid vibration emissions during the IsoVolumetric Contraction Period (IVCP) and at least the majority of mechanical systole thereafter, as systolic vibration may cause a negative inotropic effect in the ischemic heart. Furthermore diastolic vibration should preferably include the IsoVolumetric Relaxation Period (IVRP) which has been shown in clinical studies to improve cardiac performance and enhance coronary flow. Electrocardiographic (ECG) monitoring can be used to enable diastolic tracking, however, the timing of the phases of the cardiac cycle in relation to the ECG waveform must first be verified. The objective of this study was therefore to determine timing of onset of mechanical systole and diastole in reference to the QRS-T Complex. One hundred and twenty-three adult echocardiographic studies were assessed for the point of mitral and aortic valve closure in relation to the QRS complex and T wave in a representative population. We found that onset of mechanical systole occurred on and usually shortly after the peak of a first dominant QRS complex deflection, and onset of diastole occurred at the earliest on and most commonly beyond the peak or midpoint of the T wave. A DTV should ideally be able to stop vibrating on or before the peak of the first dominant deflection of a QRS complex, and begin vibrating near the peak of the T wave. Given early detection of ventricular depolarization can occur 10-20 ms prior to R wave peak, it is proposed that a DTV should preferably be able to stop vibrating within 10 ms of a triggered stop command. Onset of vibration during peak of T wave could be approximated by a rate adapted Q-T interval regression equation, and then fine tuned by manual adjustment during therapy.
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 Gli1(CreERT2), 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.
Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.
The developmental mechanisms underlying human congenital heart disease (CHD) are poorly understood. Atrial septal defects (ASDs) can result from haploinsufficiency of cardiogenic transcription factors including TBX5. We demonstrated that Tbx5 is required in the second heart field (SHF) for atrial septation in mice. Conditional Tbx5 haploinsufficiency in the SHF but not the myocardium or endocardium caused ASDs. Tbx5 SHF knockout embryos lacked atrial septum progenitors. We found that Tbx5 mutant SHF progenitors demonstrated cell-cycle progression defects and that Tbx5 regulated cell-cycle progression genes including Cdk6. Activated hedgehog (Hh) signaling rescued ASDs in Tbx5 mutant embryos, placing Tbx5 upstream or parallel to Hh in cardiac progenitors. Tbx5 regulated SHF Gas1 and Osr1 expression, supporting both pathways. These results describe a SHF Tbx5-Hh network required for atrial septation. A paradigm defining molecular requirements in SHF cardiac progenitors for cardiac septum morphogenesis has implications for the ontogeny of CHD.
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