SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Insurance denials and diagnostic rates in a pediatric genomic research cohort.

PURPOSE: This study aimed to assess the amount and types of clinical genetic testing denied by insurance and the rate of diagnostic and candidate genetic findings identified through research in patients who faced insurance denials. METHODS: Analysis consisted of review of insurance denials in 801 patients enrolled in a pediatric genomic research repository with either no previous genetic testing or previous negative genetic testing result identified through cross-referencing with insurance prior-authorizations in patient medical records. Patients and denials were also categorized by type of insurance coverage. Diagnostic findings and candidate genetic findings in these groups were determined through review of our internal variant database and patient charts. RESULTS: Of the 801 patients analyzed, 147 had insurance prior-authorization denials on record (18.3%). Exome sequencing and microarray were the most frequently denied genetic tests. Private insurance was significantly more likely to deny testing than public insurance (odds ratio = 2.03 [95% CI = 1.38-2.99] P = .0003). Of the 147 patients with insurance denials, 53.7% had at least 1 diagnostic or candidate finding and 10.9% specifically had a clinically diagnostic finding. Fifty percent of patients with clinically diagnostic results had immediate medical management changes (5.4% of all patients experiencing denials). CONCLUSION: Many patients face a major barrier to genetic testing in the form of lack of insurance coverage. A number of these patients have clinically diagnostic findings with medical management implications that would not have been identified without access to research testing. These findings support re-evaluation of insurance carriers' coverage policies.

Recurrent FOXP4 nonsense variant in two unrelated patients: Association with neurodevelopmental disease and congenital diaphragmatic hernia.

De novo variants in FOXP4 were recently associated with a neurodevelopmental disorder characterized by speech and language delay, growth abnormalities, hypotonia, and variable congenital abnormalities, including congenital diaphragmatic hernia, cervical spine abnormalities, strabismus, cryptorchidism, and ptosis. The variant spectrum in this small cohort was limited to de novo missense except for one frameshift, the inheritance of which was unknown. Variants tested in vitro exhibited reduced repressor transcriptional activity, indicating loss of function is the likely mechanism of disease, but only one frameshift variant was reported. Here, we report four affected individuals from two unrelated families heterozygous for a nonsense variant, c.1893C > G, p.Tyr631*, in FOXP4. The phenotype of the affected children includes developmental delay, feeding difficulties in infancy, and similar facial features. In both cases, the variant was inherited from a parent with mild or even subclinical features. Interestingly, one patient presented with congenital diaphragmatic hernia, as reported in two other FOXP4 patients. This report implicates FOXP4 truncating variants in human disease and highlights the wide phenotypic spectrum and variable expressivity.

Jansen-de Vries syndrome: Expansion of the PPM1D clinical and phenotypic spectrum in 34 families.

Jansen-de Vries syndrome (JdVS) is a neurodevelopmental condition attributed to pathogenic variants in Exons 5 and 6 of PPM1D. As the full phenotypic spectrum and natural history remain to be defined, we describe a large cohort of children and adults with JdVS. This is a retrospective cohort study of 37 individuals from 34 families with disease-causing variants in PPM1D leading to JdVS. Clinical data were provided by treating physicians and/or families. Of the 37 individuals, 27 were male and 10 female, with median age 8.75 years (range 8 months to 62 years). Four families document autosomal dominant transmission, and 32/34 probands were diagnosed via exome sequencing. The facial gestalt, including a broad forehead and broad mouth with a thin and tented upper lip, was most recognizable between 18 and 48 months of age. Common manifestations included global developmental delay (35/36, 97%), hypotonia (25/34, 74%), short stature (14/33, 42%), constipation (22/31, 71%), and cyclic vomiting (6/35, 17%). Distinctive personality traits include a hypersocial affect (21/31, 68%) and moderate-to-severe anxiety (18/28, 64%). In conclusion, JdVS is a clinically recognizable neurodevelopmental syndrome with a characteristic personality and distinctive facial features. The association of pathogenic variants in PPM1D with cyclic vomiting bears not only medical attention but also further pathogenic and mechanistic evaluation.

Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes.

PURPOSE: This study aimed to provide comprehensive diagnostic and candidate analyses in a pediatric rare disease cohort through the Genomic Answers for Kids program. METHODS: Extensive analyses of 960 families with suspected genetic disorders included short-read exome sequencing and short-read genome sequencing (srGS); PacBio HiFi long-read genome sequencing (HiFi-GS); variant calling for single nucleotide variants (SNV), structural variant (SV), and repeat variants; and machine-learning variant prioritization. Structured phenotypes, prioritized variants, and pedigrees were stored in PhenoTips database, with data sharing through controlled access the database of Genotypes and Phenotypes. RESULTS: Diagnostic rates ranged from 11% in patients with prior negative genetic testing to 34.5% in naive patients. Incorporating SVs from genome sequencing added up to 13% of new diagnoses in previously unsolved cases. HiFi-GS yielded increased discovery rate with >4-fold more rare coding SVs compared with srGS. Variants and genes of unknown significance remain the most common finding (58% of nondiagnostic cases). CONCLUSION: Computational prioritization is efficient for diagnostic SNVs. Thorough identification of non-SNVs remains challenging and is partly mitigated using HiFi-GS sequencing. Importantly, community research is supported by sharing real-time data to accelerate gene validation and by providing HiFi variant (SNV/SV) resources from >1000 human alleles to facilitate implementation of new sequencing platforms for rare disease diagnoses.

PPP2R1A neurodevelopmental disorder is associated with congenital heart defects.

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that regulates numerous biological processes. PPP2R1A encodes the scaffolding "Aα" subunit of PP2A. To date, nearly 40 patients have been previously reported with 19 different pathogenic PPP2R1A variants, with phenotypes including intellectual disability, developmental delay, epilepsy, infant agenesis/dysgenesis of the corpus callosum, and dysmorphic features. Apart from a single case, severe congenital heart defects (CHD) have not been described. We report four new unrelated individuals with pathogenic heterozygous PPP2R1A variants and CHD and model the crystal structure of several variants to investigate mechanisms of phenotype disparity. Individuals 1 and 2 have a previously described variant (c.548G>A, p.R183Q) and similar phenotypes with severe ventriculomegaly, agenesis/dysgenesis of the corpus callosum, and severe CHD. Individual 3 also has a recurrent variant (c.544C>T, p.R182W) and presented with agenesis of corpus callosum, ventriculomegaly, mild pulmonic stenosis, and small patent foramen ovale. Individual 4 has a novel variant (c.536C>A, p.P179H), ventriculomegaly, and atrial septal defect. To conclude, we propose expansion of the phenotype of PPP2R1A neurodevelopmental disorder to include CHD. Further, the R183Q variant has now been described in three individuals, all with severe neurologic abnormalities, severe CHD, and early death suggesting that this variant may be particularly deleterious.

Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot.

PURPOSE: Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. METHODS: We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. RESULTS: Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). CONCLUSION: Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

Augmented Pulmonary Vasoconstrictor Reactivity after Chronic Hypoxia Requires Src Kinase and Epidermal Growth Factor Receptor Signaling.

Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. We tested the hypothesis that Src-epidermal growth factor receptor (EGFR) signaling mediates enhanced vasoconstrictor sensitivity after chronic hypoxia through NADPH oxidase-derived superoxide generation. Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone after chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the EGFR inhibitor gefitinib. Inhibition of NADPH oxidase, Rac1 (Ras-related C3 botulinum toxin substrate 1), and EGFR abolished pressure-induced pulmonary arterial tone and endothelin-1 (ET-1)-dependent calcium sensitization and vasoconstriction after chronic hypoxia. Consistently, chronic hypoxia augmented ET-1-induced superoxide production through EGFR signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were also activated by ET-1 after chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction in response to ET-1. A role for matrix metalloproteinase 2 to mediate Src-dependent EGFR activation is further supported by our findings. Our studies support a novel role for an Src kinase-EGFR-NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension after chronic hypoxia.

Altered Lipid Domains Facilitate Enhanced Pulmonary Vasoconstriction after Chronic Hypoxia.

Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. Nicotinamide adenine dinucleotide phosphate oxidase and EGFR (epidermal growth factor receptor) signaling contributes to this response. Caveolin-1 regulates the activity of a variety of proteins, including EGFR and nicotinamide adenine dinucleotide phosphate oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction after CH. Although exposure of rats to CH (4 wk, ∼380 mm Hg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content as assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted pulmonary arteries (∼150 µm inner diameter) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH rats than in those obtained from control rats, and increased superoxide production as assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide antennapedia-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction after CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting that cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction after CH.

Intermittent Hypoxia Augments Pulmonary Vasoconstrictor Reactivity through PKCß/Mitochondrial Oxidant Signaling.

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.

PKCß and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats.

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCß and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCß and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCß inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCß-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCß in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCß activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCß and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

Endoplasmic Reticulum Stress Signalling Induces Casein Kinase 1-Dependent Formation of Cytosolic TDP-43 Inclusions in Motor Neuron-Like Cells.

Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation.

MAGEL2-related disorders: A study and case series.

Pathogenic MAGEL2 variants result in the phenotypes of Chitayat-Hall syndrome (CHS), Schaaf-Yang syndrome (SYS) and Prader-Willi syndrome (PWS). We present five patients with mutations in MAGEL2, including the first patient reported with a missense variant, adding to the limited literature. Further, we performed a systematic review of the CHS and SYS literature, assess the overlap between CHS, SYS and PWS, and analyze genotype-phenotype correlations among them. We conclude that there is neither a clinical nor etiological difference between CHS and SYS, and propose that the two syndromes simply be referred to as MAGEL2-related disorders.

Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity after chronic hypoxia.

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.

Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants.

Peroxisomes play diverse and important roles in plants. The functions of peroxisomes are dependent upon their steady state protein composition which in turn reflects the balance of formation and turnover of the organelle. Protein import and turnover of constituent peroxisomal proteins are controlled by the state of cell growth and environment. The evolutionary origin of the peroxisome and the role of the endoplasmic reticulum in peroxisome biogenesis are discussed, as informed by studies of the trafficking of peroxisome membrane proteins. The process of matrix protein import in plants and its similarities and differences with peroxisomes in other organisms is presented and discussed in the context of peroxin distribution across the green plants.

Enhanced NO-dependent pulmonary vasodilation limits increased vasoconstrictor sensitivity in neonatal chronic hypoxia.

Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor Nω-nitro-l-arginine (l-NNA) on baseline pulmonary vascular resistance (PVR) and vasoconstrictor sensitivity to the thromboxane mimetic U-46619 in saline-perfused lungs (in situ) from 2-wk-old control and CH (12-day exposure, 0.5 atm) Sprague-Dawley rats. Basal tone was defined as that reversed by exogenous NO (spermine NONOate). CH neonates displayed elevated right ventricular systolic pressure (in vivo) and right ventricular hypertrophy, indicative of pulmonary hypertension. Perfused lungs from CH rats demonstrated greater baseline PVR, basal tone, and U-46619-mediated vasoconstriction compared with control rats in the absence of l-NNA. l-NNA markedly increased baseline PVR and reactivity to U-46619 in lungs from CH neonates, further augmenting vasoconstrictor sensitivity compared with control lungs. Exposure to CH also enhanced NO-dependent vasodilation to arginine vasopressin, pulmonary expression of NOS III [endothelial NOS (eNOS)], and eNOS phosphorylation at activation residue Ser1177 However, CH did not alter lung nitrotyrosine levels, a posttranslational modification reflecting [Formula: see text] scavenging of NO. We conclude that, in contrast to our hypothesis, enhanced basal tone and agonist-induced vasoconstriction after neonatal CH is limited by increased NO-dependent pulmonary vasodilation resulting from greater eNOS expression and phosphorylation at activation residue Ser1177NEW & NOTEWORTHY This research is the first to demonstrate enhanced nitric oxide-dependent vasodilation that limits increased vasoconstrictor reactivity in neonatal pulmonary hypertension. These results suggest that augmented vasoconstriction in this setting reflects changes in smooth muscle reactivity rather than a reduction in nitric oxide-dependent pulmonary vasodilation.

Inhibition of Diaphanous Formin Signaling In Vivo Impairs Cardiovascular Development and Alters Smooth Muscle Cell Phenotype.

OBJECTIVE: We and others have previously shown that RhoA-dependent stimulation of myocardin-related transcription factor nuclear localization promotes smooth muscle cell (SMC) marker gene expression. The goal of this study was to provide direct in vivo evidence that actin polymerization by the diaphanous-related formins contributes to the regulation of SMC differentiation and phenotype. APPROACH AND RESULTS: Conditional Cre-based genetic approaches were used to overexpress a well-characterized dominant-negative variant of mDia1 (DNmDia) in SMC. DNmDia expression in SM22-expressing cells resulted in embryonic and perinatal lethality in ≈20% of mice because of defects in myocardial development and SMC investment of peripheral vessels. Although most DNmDia(+)/SM22Cre(+) mice exhibited no overt phenotype, the re-expression of SMC differentiation marker gene expression that occurs after carotid artery ligation was delayed, and this effect was accompanied by a significant decrease in myocardin-related transcription factor-A nuclear localization. Interestingly, neointima growth was inhibited by expression of DNmDia in SMC and this was likely because of a defect in directional SMC migration and not to defects in SMC proliferation or survival. Finally, by using the tamoxifen-inducible SM MHC-CreER(T2) line, we showed that SMC-specific induction of DNmDia in adult mice decreased SMC marker gene expression. CONCLUSIONS: Our demonstration that diaphanous-related formin signaling plays a role in heart and vascular development and the maintenance of SMC phenotype provides important new evidence that Rho/actin/myocardin-related transcription factor signaling plays a critical role in cardiovascular function.

A parametrically constrained optimization method for fitting sedimentation velocity experiments.

A method for fitting sedimentation velocity experiments using whole boundary Lamm equation solutions is presented. The method, termed parametrically constrained spectrum analysis (PCSA), provides an optimized approach for simultaneously modeling heterogeneity in size and anisotropy of macromolecular mixtures. The solutions produced by PCSA are particularly useful for modeling polymerizing systems, where a single-valued relationship exists between the molar mass of the growing polymer chain and its corresponding anisotropy. The PCSA uses functional constraints to identify this relationship, and unlike other multidimensional grid methods, assures that only a single molar mass can be associated with a given anisotropy measurement. A description of the PCSA algorithm is presented, as well as several experimental and simulated examples that illustrate its utility and capabilities. The performance advantages of the PCSA method in comparison to other methods are documented. The method has been added to the UltraScan-III software suite, which is available for free download from http://www.ultrascan.uthscsa.edu.

Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription.

We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure.