Defects of cytoarchitectural proteins can cause left ventricular noncompaction (LVNC), which is often associated with conduction system diseases. We have previously identified a p.D117N mutation in the LDB3-encoding Z-band Alternatively Spliced PDZ motif gene (ZASP) in a patient with LVNC and conduction disturbances. We sought to investigate a role of p.D117N mutation in the LBD3 NM_001080114.1 isoform (ZASP1-D117N) in the regulation of cardiac sodium channel (Nav1.5) that plays an important role in the cardiac conduction system.
Methods and Results
Effects of ZASP1-wt and ZASP1-D117N on Nav1.5 were studied in HEK-293 cells and neonatal rat cardiomyocytes (NRCMs). Patch-clamp study demonstrated that ZASP1-D117N significantly attenuated INa by 27% in HEK-293 cells and by 32% in NRCMs. In addition, ZASP1-D117N rightward shifted the voltage-dependent activation and inactivation in both systems. In silico simulation using Luo-Rudy phase 1 model demonstrated that altered Nav1.5 function can reduce cardiac conduction velocity by 28% compared to the control. Pull-down assays showed that both wt and ZASP1-D117N can complex with Nav1.5 and telethonin/T-Cap, which required intact PDZ domains. Immunohistochemical staining in NRCMs demonstrates that ZASP1-D117N did not significantly disturb the Z-line structure. Disruption of cytoskeletal networks with ML-7 and cytochalasin D abolished the effects of ZASP1-D117N on the Nav1.5.
ZASP1 can form protein complex with telethonin/T-Cap and Nav1.5. The LVNC-specific ZASP1 mutation can cause loss-of-function of Nav1.5 without significant alteration of the cytoskeletal protein complex. Our study suggests that electrical remodeling can occur in LVNC subject due to a direct effect of mutant ZASP on Nav1.5.
ZASP; sodium channel; cardiac conduction disturbance; left ventricular noncompaction
For many years, the brain has been the primary focus for research on eating behavior. More recently, the discovery of the endocannabinoids (EC) and the endocannabinoid system (ECS), as well as the characterization of its actions on appetite and metabolism, has provided greater insight on the brain and food intake. The purpose of this review is to explain the actions of EC in the brain and other organs as well as their precursor polyunsaturated fatty acids (PUFA) that are converted to these endogenous ligands. The binding of the EC to the cannabinoid receptors in the brain stimulates food intake, and the ECS participates in systemic macronutrient metabolism where the gastrointestinal system, liver, muscle, and adipose are involved. The EC are biosynthesized from two distinct families of dietary PUFA, namely the n-6 and n-3. Based on their biochemistry, these PUFA are well known to exert considerable physiological and health-promoting actions. However, little is known about how these different families of PUFA compete as precursor ligands of cannabinoid receptors to stimulate appetite or perhaps down-regulate the ECS to amend food intake and prevent or control obesity. The goal of this review is to assess the current available research on ECS and food intake, suggest research that may improve the complications associated with obesity and diabetes by dietary PUFA intervention, and further reveal mechanisms to elucidate the relationships between substrate for EC synthesis, ligand actions on receptors, and the physiological consequences of the ECS. Dietary PUFA are lifestyle factors that could potentially curb eating behavior, which may translate to changes in macronutrient metabolism, systemically and in muscle, benefiting health overall.
food intake behavior; endocannabinoids; polyunsaturated fatty acids; appetite; cannabinoid receptors; brain and neuronal function
Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS) in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA) are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25 μM of arachidonate (AA) or docosahexaenoate (DHA), 25 μM of EC [anandamide (AEA), 2-arachidonoylglycerol (2-AG), docosahexaenoylethanolamide (DHEA)], 1 μM of CB1 antagonist NESS0327, and CB2 inverse agonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.
endocannabinoid system; C2C12 myoblasts; cannabinoid receptors; glucose uptake; gene expression; DHEA; polyunsaturated fatty acids
In this study we present data to support the role for Cdk2ap2 in regulating self-renewal of mouse embryonic stem cells (mESCs) under permissive conditions, and cell survival during differentiation of the mESCs into terminally differentiated cell types. To understand the function of Cdk2ap2 during early development, we generated mESCs with homozygous disruption of the endogenous Cdk2ap2 locus (Cdk2ap2tr/tr). The Cdk2ap2tr/tr mESCs, when grown in a complete growth medium containing leukemia inhibitory factor (LIF), showed an early differentiation phenotype characterized by flattened colonies and a distinct intercellular boundary. We also observed downregulation of Nanog and upregulation in markers of mesoderm and endoderm differentiation, including Brachyury (T), Afp, and S100a, when compared to Wt mESCs. Cdk2ap2tr/tr mESCs were able to form embryoid bodies (EBs); however, those EBs were unhealthy and had an increased level of apoptosis. Furthermore, Cdk2ap2tr/tr mESCs were unable to form teratomas in severe combined immunodeficiency (SCID) mice. Cdk2ap2 under normal conditions has a biphasic expression, suggesting regulatory roles in early-versus-late stem cell differentiation. These data begin to add to our understanding of how Cdk2ap2 may be involved in the regulation of self-renewal of stem cells during early embryogenesis.
Cardiac output (CO) is a useful measure of myocardial performance. CO monitoring is frequently performed in critically ill adults in order to guide physicians’ treatment strategies. However, standard methods of determining CO in children are not without risk and can be problematic secondary to their invasive nature and other technical problems. COstatus® system (Transonic Systems Inc, NY, USA), which is based on ultrasound dilution technology, works off in situ catheters and uses an innocuous indicator to allow for routine measurements of cardiac output and blood volumes in pediatric patients. The purpose of this study was to validate CO measured by COstatus® with those obtained by the clinical standard technique of pulmonary artery (PAC) thermodilution.
This was a prospective evaluation performed at a single institution. Any child with a structurally normal heart undergoing hemodynamic evaluation in the cardiac catheterization laboratory was included. A prograde right heart catheterization was performed, and CO was first determined by using the PAC thermodilution technique. Thermodilution results were then compared with CO measurements obtained using the COstatus system. The results were analyzed by standard correlation, Bland-Altman, and Crichtley and Critchley analyses.
Twenty-eight patients were evaluated with a median age of 8 yrs and a median weight of 31 kg. The mean thermodilution cardiac index = 3.18 L/min (+/− 1.35 L/min), and the mean COstatus® cardiac index = 3.17 L/min (+/− 1.31 L/min). Standard Pearson correlation tests revealed an excellent correlation coefficient of 0.95 (p<0.0001). Bland-Altman analysis revealed good clinical agreement with a mean difference of −0.004 L/min with a precision of 0.8 L/min/ at 2 SD. A percentage error of 25.4% was noticed in this study which is less than the clinically acceptable limit.
The ultrasound dilution technique of determining CO using the COstatus® system provides a less invasive method than the traditional pulmonary artery thermodilution for accurately determining cardiac output in children. This is the first validation of the COstatus® system in pediatric patients. Further studies are required to establish its accuracy in pediatric patients with cardiac shunts and other hemodynamically unstable conditions.
Objective: To examine tumor control, hearing preservation, and complication rates after frameless fractionated stereotactic radiosurgery (SRS) in patients with vestibular schwannomas (VS).
Methods: Thirty-seven patients treated with fractionated SRS from 2002 to 2011 were retrospectively analyzed. Ninety-five percent were treated with 25 Gy in five fractions, targeting a median tumor volume of 1.03 cc (range 0.14–7.60).
Results: With a median follow-up of 4.25 years (range, 15 months–9 years), no tumors required an additional treatment resulting in 100% tumor control rate. Radiographic control rate was 91% in 32 patients at a median follow-up of 3 years. Of the 14 patients with serviceable hearing and with audiograms, the hearing preservation rate was 78% at a median follow-up of 18 months. Twenty-six patients with serviceable hearing pretreatment, were evaluated by a phone survey with a hearing preservation rate of 73% at a 5 year median follow-up. There were two cases that developed both new increased trigeminal parasthesias and facial spasms but there were no cases of facial weakness. Patient had 96% of good to excellent satisfaction rate with the treatment at a median follow-up of 5 years.
Conclusion: Frameless fractionated SRS treatment of VS results in good rate of tumor control. Hearing preservation rate and rates of cranial nerve toxicity are comparable to what is reported in the literature. Patients choose this modality because of its non-invasive nature and are generally very satisfied with their long term outcome.
acoustic neuroma; vestibular schwannoma; cyberknife; fractionated; SRS; radiation
The number of bacterial species estimated to exist on Earth has increased dramatically in recent years. This newly recognized species diversity has raised the possibility that bacterial natural product biosynthetic diversity has also been significantly underestimated by previous culture-based studies. Here, we compare 454-pyrosequenced nonribosomal peptide adenylation domain, type I polyketide ketosynthase domain, and type II polyketide ketosynthase alpha gene fragments amplified from cosmid libraries constructed using DNA isolated from three different arid soils. While 16S rRNA gene sequence analysis indicates these cloned metagenomes contain DNA from similar distributions of major bacterial phyla, we found that they contain almost completely distinct collections of secondary metabolite biosynthetic gene sequences. When grouped at 85% identity, only 1.5% of the adenylation domain, 1.2% of the ketosynthase, and 9.3% of the ketosynthase alpha sequence clusters contained sequences from all three metagenomes. Although there is unlikely to be a simple correlation between biosynthetic gene sequence diversity and the diversity of metabolites encoded by the gene clusters in which these genes reside, our analysis further suggests that sequences in one soil metagenome are so distantly related to sequences in another metagenome that they are, in many cases, likely to arise from functionally distinct gene clusters. The marked differences observed among collections of biosynthetic genes found in even ecologically similar environments suggest that prokaryotic natural product biosynthesis diversity is, like bacterial species diversity, potentially much larger than appreciated from culture-based studies.
A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step towards being able to functionally study larger natural product gene clusters from uncultured bacteria.
Evaluate the outcome and prevalence of viral endomyocardial infection after cardiac transplantation.
Viral myocardial infection causes heart failure, but its role after cardiac transplantation is unclear. We hypothesized that viral infection of the cardiac allograft reduces graft survival.
Between 6/1999 and 11/2004, 94 pediatric cardiac transplant patients were screened for the presence of viral genome in serial endomyocardial biopsies (EMBs) using PCR assays. Graft loss, advanced transplant coronary artery disease (TCAD) and acute rejection (AR) were compared in the PCR-positive (PCR+) (n=37) and PCR-negative (PCR−) (n=57) groups, using time dependent Kaplan-Meier and Cox regression analyses. From 11/2002 to 11/2004, intravenous immunoglobulin therapy (IVIG) was administered to patients with PCR+ EMBs. The outcomes of the IVIG-treated, PCR+ patients (n=20) were compared with IVIG-untreated, PCR+ patients (n=17).
Viral genomes were detected in EMBs from 37 (39%) patients; parvovirus B19, adenovirus, & EBV were the most common. The `PCR+ group' (n=37, 25% graft loss at 2.4 years) had decreased graft survival (p<0.001) compared to the `PCR- group' (n=57, 25% graft loss at 8.7 years) and developed advanced TCAD prematurely (p=0.001). The number of AR episodes was similar in both groups. On multivariate analysis, presence of viral genome was an independent risk factor for graft loss (relative risk 4.2, p=0.015). The time to advanced TCAD after becoming PCR+ was longer in the IVIG-treated patients (p=0.03), with a trend towards improved graft survival (p=0.06).
Viral endomyocardial infection is an independent predictor of graft loss in pediatric cardiac transplant recipients. This effect appears to be mediated through premature development of advanced TCAD. IVIG therapy in this subgroup may improve survival and merits further investigation.
Cardiac Transplantation; Virus; Outcome; Graft Vasculopathy; TCAD
Detection of viral genome in rejecting cardiac transplant patients has been reported, with coxsackievirus and adenovirus causing premature graft failure. Recently, parvovirus B19 (PVB19) genome in myocardial samples has been increasingly reported but its role in cardiac pathology and effect on transplant graft survival are unknown. The objectives were to determine if changes in the viruses identified in the myocardium represent an epidemiologic shift in viral myocardial disease and whether PVB19 adversely affects transplant graft survival.
From 9/2002 to 12/2005, 99 children (3 weeks-18 years) with heart transplants had endomyocardial biopsies evaluated for the presence of viral genome utilizing nested PCR. Cellular rejection was assessed by histology of biopsies, while transplant coronary artery disease (TCAD) was diagnosed by coronary angiography or histopathology.
Seven hundred biopsies were evaluated from 99 patients; 121 biopsies had viral genome with 100 (82.6%) positive for PVB19, 24 for Epstein-Barr virus (EBV; 7 positive for PVB19 and EBV), 3 for CMV and 1 for adenovirus. Presence of PVB19 genome did not correlate with rejection score, nor did higher viral copy number. Children with persistent PVB19 infection (>6 months; n=20), had early development of advanced TCAD (p<0.001).
PVB19 is currently the predominant virus detected in heart transplant surveillance biopsies, possibly representing an epidemiologic shift. While cellular rejection does not correlate with the presence or quantity of PVB19 genome in the myocardium, children with chronic PVB19 infection have increased risk for earlier TCAD, supporting the hypothesis that PVB19 negatively affects graft survival.
Implantable cardioverter-defibrillators have aided the prevention of sudden cardiac death in adults. The hope is to provide similar benefits to the pediatric population as the devices become smaller. Herein, we present the case of a 4.9-kg, 5-week-old infant boy who presented with cardiopulmonary arrest. After emergency defibrillation, conventional treatment options included long-term hospitalization for later cardioverter-defibrillator implantation, or installation of an external defibrillator with subsequent home telemetry. On the basis of the infant's body dimensions, we decided that an epicardial implantable cardioverter-defibrillator was feasible and the best option. We performed a median sternotomy and placed a Vitality® implantable cardioverter-defibrillator with a 25-cm defibrillator coil and a 35-cm bipolar ventricular lead. The patient experienced no postoperative morbidity or rhythm disturbances and was discharged from the hospital on postoperative day 5. He was placed on β-blocker therapy and has remained well for 3 years.
Although external devices can be placed in a small patient, we believe that they are too susceptible to lead damage and lead migration, and that the defibrillator thresholds are less reliable. We think that dysrhythmias even in very small children can be treated effectively and safely with use of an epicardial implantable cardioverter-defibrillator. To our knowledge, this 4.9-kg infant is the smallest patient to have undergone a successful implantation of this kind.
Arrhythmias, cardiac/therapy; defibrillators, implantable; heart diseases/therapy; infant; prosthesis implantation/methods; risk assessment/methods; risk factors; treatment outcome
Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.
docosahexaenoic acid; hippocampal development; long-term potentiation; neurite growth; synaptic function; synaptogenesis
The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.
Wolff-Parkinson-White syndrome (WPW) is a bypass reentrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signaling in the development of annulus fibrosus in mice, it has been proposed that BMP signaling through the type 1a receptor and other downstream components may play a role in preexcitation.
Methods and Results
Using the array comparative genomic hybridization (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these, harbored a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy-number variant in the Database of Genomic Variants and has not been identified in 13,321 individuals from other cohort examined by array CGH in our laboratory.
Our findings demonstrate a novel genomic disorder characterized by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.
Wolff-Parkinson-White syndrome; BMP2; 20p12.3; TGF-beta signaling; JAG1
Long-QT syndrome (LQTS) is an inherited disorder associated with sudden cardiac death. The cytoskeletal protein syntrophin-α1 (SNTA1) is known to interact with the cardiac sodium channel (hNav1.5), and we hypothesized that SNTA1 mutations might cause phenotypic LQTS in patients with genotypically normal hNav1.5 by secondarily disturbing sodium channel function.
Methods and Results
Mutational analysis of SNTA1 was performed on 39 LQTS patients (QTc≥480 ms) with previously negative genetic screening for the known LQTS-causing genes. We identified a novel A257G-SNTA1 missense mutation, which affects a highly conserved residue, in 3 unrelated LQTS probands but not in 400 ethnic-matched control alleles. Only 1 of these probands had a preexisting family history of LQTS and sudden death with an additional intronic variant in KCNQ1. Electrophysiological analysis was performed using HEK-293 cells stably expressing hNav1.5 and transiently transfected with either wild-type or mutant SNTA1 and, in neonatal rat cardiomyocytes, transiently transfected with either wild-type or mutant SNTA1. In both HEK-293 cells and neonatal rat cardiomyocytes, increased peak sodium currents were noted along with a 10-mV negative shift of the onset and peak of currents of the current-voltage relationships. In addition, A257G-SNTA1 shifted the steady-state activation (Vh) leftward by 9.4 mV, whereas the voltage-dependent inactivation kinetics and the late sodium currents were similar to wild-type SNTA1.
SNTA1 is a new susceptibility gene for LQTS. A257G-SNTA1 can cause gain-of-function of Nav1.5 similar to the LQT3.
arrhythmia; death; sudden (if surviving; use heart arrest); ion channels; long-QT syndrome
Andersen-Tawil syndrome is an autosomal dominant condition characterized by dysmorphic features, periodic paralysis, and ventricular arrhythmias. Twiddler syndrome is characterized by intentional or inadvertent manipulation of implanted devices in the pacemaker pocket. We describe an unusual case of an 8-year-old girl who had both syndromes.
Andersen syndrome/genetics; cardiac complexes, premature; defibrillators, implantable/adverse effects; electrodes, implanted/adverse effects; foreign-body migration; KCNJ2 protein, human; long-QT syndrome/genetics; mandibulofacial dysostosis; potassium channels, inwardly rectifying/genetics; self-injurious behavior/complications; syncope; tachycardia, ventricular/genetics; Twiddler syndrome
A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step toward being able to functionally study larger natural product gene clusters from uncultured bacteria. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 833–844, 2010.
natural products; eDNA; metagenomics; TAR