Exome sequencing in multiplex families with left-sided cardiac defects has high yield for disease gene discovery.

Congenital heart disease (CHD) is a common group of birth defects with a strong genetic contribution to their etiology, but historically the diagnostic yield from exome studies of isolated CHD has been low. Pleiotropy, variable expressivity, and the difficulty of accurately phenotyping newborns contribute to this problem. We hypothesized that performing exome sequencing on selected individuals in families with multiple members affected by left-sided CHD, then filtering variants by population frequency, in silico predictive algorithms, and phenotypic annotations from publicly available databases would increase this yield and generate a list of candidate disease-causing variants that would show a high validation rate. In eight of the nineteen families in our study (42%), we established a well-known gene/phenotype link for a candidate variant or performed confirmation of a candidate variant's effect on protein function, including variants in genes not previously described or firmly established as disease genes in the body of CHD literature: BMP10, CASZ1, ROCK1 and SMYD1. Two plausible variants in different genes were found to segregate in the same family in two instances suggesting oligogenic inheritance. These results highlight the need for functional validation and demonstrate that in the era of next-generation sequencing, multiplex families with isolated CHD can still bring high yield to the discovery of novel disease genes.

Retinoic acid signaling restricts the size of the first heart field within the anterior lateral plate mesoderm.

Retinoic acid (RA) signaling is required to restrict heart size through limiting the posterior boundary of the vertebrate cardiac progenitor field within the anterior lateral plate mesoderm (ALPM). However, we still do not fully understand how different cardiac progenitor populations that contribute to the developing heart, including earlier-differentiating first heart field (FHF), later-differentiating second heart field (SHF), and neural crest-derived progenitors, are each affected in RA-deficient embryos. Here, we quantified the number of cardiac progenitors and differentiating cardiomyocytes (CMs) in RA-deficient zebrafish embryos. While Nkx2.5+ cells were increased overall in the nascent hearts of RA-deficient embryos, unexpectedly, we found that the major effect within this population was a significant expansion in the number of differentiating FHF CMs. In contrast to the expansion of the FHF, there was a progressive decrease in SHF progenitors at the arterial pole as the heart tube elongated. Temporal differentiation assays and immunostaining in RA-deficient embryos showed that the outflow tracts (OFTs) of the hearts were significantly smaller, containing fewer differentiated SHF-derived ventricular CMs and a complete absence of SHF-derived smooth muscle at later stages. At the venous pole of the heart, pacemaker cells of the sinoatrial node also failed to differentiate in RA-deficient embryos. Interestingly, genetic lineage tracing showed that the number of neural-crest derived CMs was not altered within the enlarged hearts of RA-deficient zebrafish embryos. Altogether, our data show that the enlarged hearts in RA-deficient zebrafish embryos are comprised of an expansion in earlier differentiating FHF-derived CMs coupled with a progressive depletion of the SHF, suggesting RA signaling determines the relative ratios of earlier- and later-differentiation cardiac progenitors within an expanded cardiac progenitor pool.

Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors.

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.

Patterning of vertebrate cardiac progenitor fields by retinoic acid signaling.

The influence of retinoic acid (RA) signaling on vertebrate development has a well-studied history. Cumulatively, we now understand that RA signaling has a conserved requirement early in development restricting cardiac progenitors within the anterior lateral plate mesoderm of vertebrate embryos. Moreover, genetic and pharmacological manipulations of RA signaling in vertebrate models have shown that proper heart development is achieved through the deployment of positive and negative feedback mechanisms, which maintain appropriate RA levels. In this brief review, we present a chronological overview of key work that has led to a current model of the critical role for early RA signaling in limiting the generation of cardiac progenitors within vertebrate embryos. Furthermore, we integrate the previous work in mice and our recent findings using zebrafish, which together show that RA signaling has remarkably conserved influences on the later-differentiating progenitor populations at the arterial and venous poles. We discuss how recognizing the significant conservation of RA signaling on the differentiation of these progenitor populations offers new perspectives and may impact future work dedicated to examining vertebrate heart development.

Nr2f1a balances atrial chamber and atrioventricular canal size via BMP signaling-independent and -dependent mechanisms.

Determination of appropriate chamber size is critical for normal vertebrate heart development. Although Nr2f transcription factors promote atrial maintenance and differentiation, how they determine atrial size remains unclear. Here, we demonstrate that zebrafish Nr2f1a is expressed in differentiating atrial cardiomyocytes. Zebrafish nr2f1a mutants have smaller atria due to a specific reduction in atrial cardiomyocyte (AC) number, suggesting it has similar requirements to Nr2f2 in mammals. Furthermore, the smaller atria in nr2f1a mutants are derived from distinct mechanisms that perturb AC differentiation at the chamber poles. At the venous pole, Nr2f1a enhances the rate of AC differentiation. Nr2f1a also establishes the atrial-atrioventricular canal (AVC) border through promoting the differentiation of mature ACs. Without Nr2f1a, AVC markers are expanded into the atrium, resulting in enlarged endocardial cushions (ECs). Inhibition of Bmp signaling can restore EC development, but not AC number, suggesting that Nr2f1a concomitantly coordinates atrial and AVC size through both Bmp-dependent and independent mechanisms. These findings provide insight into conserved functions of Nr2f proteins and the etiology of atrioventricular septal defects (AVSDs) associated with NR2F2 mutations in humans.

Nicotine inhalant via E-cigarette facilitates sensorimotor function recovery by upregulating neuronal BDNF-TrkB signalling in traumatic brain injury.

BACKGROUND AND PURPOSE: Traumatic brain injury (TBI) causes lifelong physical and psychological dysfunction in affected individuals. The current study investigated the effects of chronic nicotine exposure via E-cigarettes (E-cig) (vaping) on TBI-associated behavioural and biochemical changes. EXPERIMENTAL APPROACH: Adult C57/BL6J male mice were subjected to controlled cortical impact (CCI) followed by daily exposure to E-cig vapour for 6 weeks. Sensorimotor functions, locomotion, and sociability were subsequently evaluated by nesting, open field, and social approach tests, respectively. Immunoblots were conducted to examine the expression of mature brain-derived neurotrophic factor (mBDNF) and associated downstream proteins (p-Erk, p-Akt). Histological analyses were performed to evaluate neuronal survival and neuroinflammation. KEY RESULTS: Post-injury chronic nicotine exposure significantly improved nesting performance in CCI mice. Histological analysis revealed increased survival of cortical neurons in the perilesion cortex with chronic nicotine exposure. Immunoblots revealed that chronic nicotine exposure significantly up-regulated mBDNF, p-Erk and p-Akt expression in the perilesion cortex of CCI mice. Immunofluorescence microscopy indicated that elevated mBDNF and p-Akt expression were mainly localized within cortical neurons. Immunolabelling of Iba1 demonstrated that chronic nicotine exposure attenuated microglia-mediated neuroinflammation. CONCLUSIONS AND IMPLICATIONS: Post-injury chronic nicotine exposure via vaping facilitates recovery of sensorimotor function by upregulating neuroprotective mBDNF/TrkB/Akt/Erk signalling. These findings suggest potential neuroprotective properties of nicotine despite its highly addictive nature. Thus, understanding the multifaceted effects of chronic nicotine exposure on TBI-associated symptoms is crucial for paving the way for informed and properly managed therapeutic interventions.

Benzyne-Promoted, 1,2-cis-Selective O-Glycosylation with Benzylchalcogenoglycoside Donors.

Here, we show that the reaction of benzylchalcogenoglycosides with benzyne in the presence of alcohols results in highly 1,2-cis-selective O-glycosylation in a solvent-dependent manner. Thioglycosides, selenoglycosides, and alcohols with a range of nucleophilicities lead to a productive reaction, and unusual protecting groups, auxiliary groups, and additives are avoided.

Parental perception of facilitators and barriers to health among young children with down syndrome: a qualitative study.

Background: Despite high rates of obesity and weight-related conditions observed in children with Down syndrome, little is known about how to prevent these conditions. Purpose: The purpose of this study was to identify parent-perceived facilitators and barriers to health for toddlers (12-36 months old) with Down syndrome. Materials and methods: We conducted in-depth, semi-structured interviews with the mothers of 25 toddlers with Down syndrome. All interviews were conducted using Zoom Video Technology, audio recorded and transcribed before being coded in NVivo software using a structured protocol. Thematic analysis was used to identify themes in perceived facilitators and barriers to health at the level of the child, family, and community. Data were triangulated using reflective journaling, video review of child meals, and member-checking techniques. Results: We identified unique themes for facilitators (on the move and sound sleep) and barriers (co-occurring conditions and eating behaviors) at the level of the child. At the level of the family and community, overarching themes that were viewed as either a facilitator or barrier, depending on the context, were identified (role models matter, time is critical, the importance of place, and social support). Conclusion: These themes can help clinicians and researchers tailor their health promotion interventions to meet the unique needs of children with Down syndrome by using strength-based approaches and providing families with the tools to overcome barriers.

Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice.

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).