Jason T. Maynes

A Population-based Study Evaluating the Association between Surgery in Early Life and Child Development at Primary School Entry

Background:It is unclear whether exposure to surgery in early life has long-term adverse effects on child development. The authors aimed to investigate whether surgery in early childhood is associated with adverse effects on child development measured at primary school entry. Methods:The authors conducted a population-based cohort study in Ontario, Canada, by linking provincial health administrative databases to children’s developmental outcomes measured by the Early Development Instrument (EDI). From a cohort of 188,557 children, 28,366 children who underwent surgery before EDI completion (age 5 to 6 yr) were matched to 55,910 unexposed children. The primary outcome was early developmental vulnerability, defined as any domain of the EDI in the lowest tenth percentile of the population. Subgroup analyses were performed based on age at first surgery (less than 2 and greater than or equal to 2 yr) and frequency of surgery. Results:Early developmental vulnerability was increased in the exposed group (7,259/28,366; 25.6%) compared with the unexposed group (13,957/55,910; 25.0%), adjusted odds ratio, 1.05; 95% CI, 1.01 to 1.08. Children aged greater than or equal to 2 yr at the time of first surgery had increased odds of early developmental vulnerability compared with unexposed children (odds ratio, 1.05; 95% CI, 1.01 to 1.10), but children aged less than 2 yr at the time of first exposure were not at increased risk (odds ratio, 1.04; 95% CI, 0.98 to 1.10). There was no increase in odds of early developmental vulnerability with increasing frequency of exposure. Conclusions:Children who undergo surgery before primary school age are at increased risk of early developmental vulnerability, but the magnitude of the difference between exposed and unexposed children is small.

Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function.

Human dilated cardiomyopathy (DCM) manifests as a profound reduction in biventricular cardiac function that typically progresses to death or cardiac transplantation. There is no effective mechanism-based therapy currently available for DCM, in part because the transduction of mechanical load into dynamic changes in cardiac contractility (termed mechanotransduction) remains an incompletely understood process during both normal cardiac function and in disease states. Here we show that the mechanoreceptor protein integrin-linked kinase (ILK) mediates cardiomyocyte force transduction through regulation of the key calcium regulatory protein sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase isoform 2a (SERCA-2a) and phosphorylation of phospholamban (PLN) in the human heart. A non-oncogenic ILK mutation with a synthetic point mutation in the pleckstrin homology-like domain (ILK(R211A)) is shown to enhance global cardiac function through SERCA-2a/PLN. Thus, ILK serves to link mechanoreception to the dynamic modulation of cardiac contractility through a previously undiscovered interaction with the functional SERCA-2a/PLN module that can be exploited to rescue impaired mechanotransduction in DCM.

Molecular mechanisms underlying the interaction of protein phosphatase-1c with ASPP proteins.

The serine/threonine PP-1c (protein phosphatase-1 catalytic subunit) is regulated by association with multiple regulatory subunits. Human ASPPs (apoptosis-stimulating proteins of p53) comprise three family members: ASPP1, ASPP2 and iASPP (inhibitory ASPP), which is uniquely overexpressed in many cancers. While ASPP2 and iASPP are known to bind PP-1c, we now identify novel and distinct molecular interactions that allow all three ASPPs to bind differentially to PP-1c isoforms and p53. iASPP lacks a PP-1c-binding RVXF motif; however, we show it interacts with PP-1c via a RARL sequence with a Kd value of 26 nM. Molecular modelling and mutagenesis of PP-1c-ASPP protein complexes identified two additional modes of interaction. First, two positively charged residues, Lys260 and Arg261 on PP-1c, interact with all ASPP family members. Secondly, the C-terminus of the PP-1c α, β and γ isoforms contain a type-2 SH3 (Src homology 3) poly-proline motif (PxxPxR), which binds directly to the SH3 domains of ASPP1, ASPP2 and iASPP. In PP-1cγ this comprises residues 309-314 (PVTPPR). When the Px(T)PxR motif is deleted or mutated via insertion of a phosphorylation site mimic (T311D), PP-1c fails to bind to all three ASPP proteins. Overall, we provide the first direct evidence for PP-1c binding via its C-terminus to an SH3 protein domain.

Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase

Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.

Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia

BackgroundCommunity-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury.MethodsVT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT.ResultsVT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed.ConclusionsVT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.

DNM1L Variant Alters Baseline Mitochondrial Function and Response to Stress in a Patient with Severe Neurological Dysfunction

Mitochondria play vital roles in brain development and neuronal activity, and mitochondrial dynamics (fission and fusion) maintain organelle function through the removal of damaged components. Dynamin-like protein-1 (DRP-1), encoded by DNM1L, is an evolutionarily conserved GTPase that mediates mitochondrial fission by surrounding the scission site in concentric ring-like structures via self-oligomerization, followed by GTPase-dependant constriction. Here, we describe the clinical characteristics and cellular phenotype of a patient with severe neurological dysfunction, possessing a homozygous DNM1L variant c.305C>T (p.T115M) in the GTPase domain. For comparative analysis, we also describe a previously identified heterozygous variant demonstrating a rapidly fatal neurocognitive phenotype (c.261dup/c.385:386del, p.W88M*9/E129K*6). Using patient-generated fibroblasts, we demonstrated both DNM1L variants undergo adverse alterations to mitochondrial structure and function, including impaired mitochondrial fission, reduced membrane potential, and lower oxidative capacity including an increased cellular level of reactive oxygen species (ROS) and dsDNA breaks. Mutation of DNM1L was also associated with impaired responses to oxidative stress, as treatment with hydrogen peroxide dramatically increased cellular ROS, with minimal exacerbation of already impaired mitochondrial function. Taken together, our observations indicate that homozygous p.T115M variant of DNM1L produces a neurological and neurodevelopmental phenotype, consistent with impaired mitochondrial architecture and function, through a diminished ability to oligomerize, which was most prevalent under oxidative stress.

Evaluating the effects of general anesthesia on sleep in children undergoing elective surgery: an observational case–control study

Study Objectives Previous research has suggested that general anesthetics can disturb postoperative sleep patterns by affecting the sleep-wake cycle. The objective was to identify the effects of general anesthetics on sleep quality and related behavioral changes in children. Methods This was a prospective, observational case-control study with children, aged 18 months to 8 years, undergoing general anesthesia for elective surgery. Participants wore an actigraph for 7 days on three occasions: prior to surgery, the immediate postoperative period, and 3 months after surgery. Data regarding behavior patterns were collected using behavioral assessments at baseline, the first postoperative week, and 3 months following surgery. Results Thirty-one participants (mean age 4.8 ± 2.0 years, 81% male) underwent urologic or otolaryngologic surgery. The median (interquartile range) anesthetic duration was 132.0 (80.0-184.0) min. No significant differences were found in sleep efficiency, total sleep time, wake time after sleep onset, or sleep onset latency between baseline, 7 day postoperative period, and the 3 month follow-up. No significant differences were found in sleep-related behavioral metrics including internalizing and externalizing behaviors, and executive functioning. Data were compared with a control group of 18 participants (mean age 5.3 ± 1.8 years, 61% male). No significant differences were found in sleep patterns and related behavioral metrics between both groups. Conclusions In this study, general anesthesia did not result in disturbed sleep or associated negative behavioral changes in otherwise healthy children undergoing elective surgeries of low complexity. Physicians can advise parents that a childs surgery and associated general anesthetic exposure may not result in significant changes in postoperative sleep patterns.

Inhalational Anesthetics Induce Neuronal Protein Aggregation and Affect ER Trafficking

Anesthetic agents have been implicated in the causation of neurological and cognitive deficits after surgery, the exacerbation of chronic neurodegenerative disease, and were recently reported to promote the onset of the neurologic respiratory disease Congenital Central Hypoventilation Syndrome (CCHS), related to misfolding of the transcription factor Phox2B. To study how anesthetic agents could affect neuronal function through alterations to protein folding, we created neuronal cell models emulating the graded disease severity of CCHS. We found that the gas anesthetic isoflurane and the opiate morphine potentiated aggregation and mislocalization of Phox2B variants, similar to that seen in CCHS, and observed transcript and protein level changes consistent with activation of the endoplasmic reticulum (ER) unfolded protein response. Attenuation of ER stress pathways did not result in a correction of Phox2B misfolding, indicating a primary effect of isoflurane on protein structure. We also observed that isoflurane hindered the folding and activity of proteins that rely heavily on ER function, like the CFTR channel. Our results show how anesthetic drugs can alter protein folding and induce ER stress, indicating a mechanism by which these agents may affect neuronal function after surgery.

Cardiac regenerative capacity is age- and disease-dependent in childhood heart disease

Objective We sought to define the intrinsic stem cell capacity in pediatric heart lesions, and the effects of diagnosis and of age, in order to inform evidence-based use of potential autologous stem cell sources for regenerative medicine therapy. Methods Ventricular explants derived from patients with hypoplastic left heart syndrome (HLHS), tetralogy of Fallot (TF), dilated cardiomyopathy (DCM) and ventricular septal defect (VSD) were analyzed following standard in vitro culture conditions, which yielded cardiospheres (C-spheres), indicative of endogenous stem cell capacity. C-sphere counts generated per 5 mm3 tissue explant and the presence of cardiac progenitor cells were correlated to patient age, diagnosis and echocardiographic function. Results Cardiac explants from patients less than one year of age with TF and DCM robustly generated c-kit- and/or vimentin-positive cardiac mesenchymal cells (CMCs), populating spontaneously forming C-spheres. Beyond one year of age, there was a marked reduction or absence of cardiac explant-derivable cardiac stem cell content in patients with TF, VSD and DCM. Stem cell content in HLHS and DCM strongly correlated to the echocardiographic function in the corresponding ventricular chamber, with better echocardiographic function correlating to a more robust regenerative cellular content. Conclusions We conclude that autologous cardiomyogenic potential in pediatric heart lesions is robust during the first year of life and uniformly declines thereafter. Depletion of stem cell content occurs at an earlier age in HLHS with the onset of ventricular failure in a chamber-specific pattern that correlates directly to ventricular dysfunction. These data suggest that regenerative therapies using autologous cellular sources should be implemented in the neonatal period before the potentially rapid onset of single ventricle failure in HLHS or the evolution of biventricular failure in DCM.

An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis

Aims Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by right ventricular myocardial replacement and life-threatening ventricular arrhythmias. Desmosomal gene mutations are sometimes identified, but clinical and genetic diagnosis remains challenging. Desmosomal skin disorders can be caused by desmosomal gene mutations or autoantibodies. We sought to determine if anti-desmosome antibodies are present in subjects with ARVC. Methods and results We evaluated ARVC subjects and controls for antibodies to cardiac desmosomal cadherin proteins. Desmoglein-2 (DSG2), desmocollin-2, and N-cadherin proteins on western blots were exposed to sera, in primary and validation cohorts of subjects and controls, as well as the naturally occurring Boxer dog model of ARVC. We identified anti-DSG2 antibodies in 12/12 and 25/25 definite ARVC cohorts and 7/8 borderline subjects. Antibody was absent in 11/12, faint in 1/12, and absent in 20/20 of two control cohorts. Anti-DSG2 antibodies were present in 10/10 Boxer dogs with ARVC, and absent in 18/18 without. In humans, the level of anti-DSG2 antibodies correlated with the burden of premature ventricular contractions (r = 0.70), and antibodies caused gap junction dysfunction, a common feature of ARVC, in vitro. Anti-DSG2 antibodies were present in ARVC subjects regardless of whether an underlying mutation was identified, or which mutation was present. A disease-specific DSG2 epitope was identified. Conclusion Anti-DSG2 antibodies are a sensitive and specific biomarker for ARVC. The development of autoimmunity as a result of target-related mutations is unique. Anti-DSG2 antibodies likely explain the cardiac inflammation that is frequently identified in ARVC and may represent a new therapeutic target.