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The coronavirus disease 2019 (COVID-19) pandemic altered education, exams, and residency applications for United States medical students.To determine the specific impact of the pandemic on US medical students and its correlation to their anxiety levels.An 81-question survey was distributed397 medical students from 29 states were analyzed. Approximately half of respondents reported feeling depressed since the pandemic onset. 62% of participants rated 7 or higher out of 10 when asked about anxiety levels. Stressors correlated with higher anxiety scores included "concern about being unable to complete exams or rotations if contracting COVID-19" (RR 1.34; 95%CI: 1.05-1.72,COVID-19 significantly impacted medical students in numerous ways. Medical student education and clinical readiness were reduced, and anxiety levels increased. It is vital that medical students receive support as they become physicians. Further research should be conducted on training medical students in telemedicine to better prepare students in the future for pandemic planning and virtual healthcare.

Mesodermal progenitors in the second heart field (SHF) express Delta-like-ligand 4 (Dll4) that regulates Notch-mediated proliferation. As cells of SHF lineage mature to assume endocardial and myocardial cell fates, we have shown that Dll4 expression is lost, and the subsequent expression of another Notch ligand Jagged1 regulates Notch-mediated maturation events in the developing heart. A subset of SHF progenitors also matures to form the pharyngeal arch artery (PAA) endothelium. Dll4 was originally identified as an arterial endothelial-specific Notch ligand that plays an important role in blood vessel maturation, but its role in aortic arch maturation has not been studied to date secondary to the early lethality observed in Dll4 knockout mice. We show that, unlike in SHF-derived endocardium and myocardium, Dll4 expression persists in SHF-derived arterial endothelial cells. Using SHF-specific conditional deletion of Dll4, we demonstrate that as SHF cells transition from their progenitor state to an endothelial fate, Dll4-mediated Notch signalling switches from providing proliferative to maturation cues. Dll4 expression maintains arterial identity in the PAAs and plays a critical role in the maturation and re-organization of the 4th pharyngeal arch artery, in particular. Haploinsufficiency of Dll4 in SHF leads to highly penetrant aortic arch artery abnormalities, similar to those observed in the clinic, primarily resulting from aberrant reorganization of bilateral 4th pharyngeal arch arteries. Hence, we show that cells of SHF lineage that assume an arterial endothelial fate continue to express Dll4 and the resulting Dll4-mediated Notch signalling transitions from an early proliferative to a later maturation role during aortic arch development.

Introduction: Prenatal alcohol exposure (PAE) leads to congenital heart defects (CHD) with incomplete penetrance and phenotypic heterogeneity. We have demonstrated a gene-environment interaction in this context wherein acute PAE synergizes with second heart field (SHF) haploinsufficiency of Notch signaling, resulting in highly penetrant outflow tract (OFT) defects. Hypothesis: We hypothesize that acute PAE-induced hyperacetylation at histone marker 3 lysine residue 9 (aH3K9) epigenetically inhibits Notch signaling. This potentiates the impact of a pre-existing genetic defect that results in partial loss of Notch signaling leading to loss of viability of SHF progenitors necessary for OFT elongation and alignment. Methods: We studied the effects of acute PAE in vivo in our murine model (two injections of 3g/kg 30% ethanol on E6.5) on acetylation, SHF viability, OFT length, and Notch intracellular domain (NICD) localization. Complimentarily, we evaluated alcohol exposure in vitro using our established thoracic organ culture model. Acetylation was reversed using pan-histone acetyltransferase inhibitor anacardic acid. Results: At E9.5, acute PAE resulted in a significant increase in H3K9 acetylation in SHF associated with restriction of NICD to the cell membrane. This resulted in a 0.28-fold reduction in Notch signaling, as evidenced by reduced Notch reporter expression, and 0.52-fold reduction in SHF cell proliferation (both pIn vitro thoracic organ culture analyses recapitulated increased acetylation, NICD restriction to cell membrane and reduced SHF proliferation by 60% (p Conclusions: Acute PAE induces hyperacetylation in the SHF and inhibits Notch signaling by restricting NICD translocation. Lack of Notch signaling reduces SHF viability and prevents elongation of the developing OFT. This hereto unexplored impact of alcohol on the Notch pathway provides a novel area of investigation regarding gene-environment interaction in alcohol teratogenesis.

BACKGROUND: Prenatal alcohol exposure (PAE) is associated with an increased incidence of congenital heart defects (CHD), in particular outflow tract (OFT) defects. However, the variability in the incidence of CHD following PAE has not been fully explored. We hypothesize that a concomitant, relevant genetic defect would potentiate the adverse effect of PAE and partially explain the variability of PAE-induced CHD incidence. METHODS: The OFT is formed by the second heart field (SHF). Our PAE model consisted of two intraperitoneal injections (3 g/kg, separated by 6 hr) of 30% ethanol on E6.5 during SHF specification. The impact of genetic defects was studied by SHF-specific loss of Delta-like ligand 4 (Dll4), fibroblast growth factor 8 (Fgf8) and Islet1. RESULTS: Acute PAE alone significantly increased CHD incidence (4% vs. 26%, p = .015) with a particular increase in OFT alignment defects, viz., double outlet right ventricle (0 vs. 9%, p = .02). In embryos with a SHF genetic defect, acute PAE significantly increased CHD incidence (14 vs. 63%, p < .001), including double outlet right ventricle (6 vs. 50%, p < .001) compared to controls. PAE (p = .01) and heterozygous loss of Dll4 (p = .04) were found to independently contribute to CHD incidence, while neither Islet1 nor Fgf8 defects were found to be significant. CONCLUSIONS: Our model recapitulates the increased incidence of OFT alignment defects seen in the clinic due to PAE. The presence of a concomitant SHF genetic mutation increases the incidence of PAE-related OFT defects. An apparent synergistic interaction between PAE and the loss of DLL4-mediated Notch signaling in OFT alignment requires further analysis.

Heterozygous loss-of-function mutation in Delta-like ligand-4 (Dll4) is an important cause of Adams-Oliver syndrome (AOS). Cardiac defects, in particular outflow tract (OFT) alignment defects, are observed in about one-fourth of patients with this syndrome. The mechanism underlying this genotype-phenotype correlation has not yet been established. Dll4-mediated Notch signaling is known to play a crucial role in second heart field (SHF) progenitor cell proliferation. We hypothesized that the depletion of the SHF progenitor pool of cells due to partial loss of Dll4 is responsible for the OFT alignment defects seen in AOS. To demonstrate this, we studied Dll4 expression by murine SHF progenitor cells around E9.5, a crucial time-point in SHF biology. We used SHF-specific (Islet1-Cre) conditional knockout of Dll4 to bypass the early embryonic lethality seen in global Dll4 heterozygotes. Dll4-mediated Notch signaling is critically required for SHF proliferation such that Dll4 knockout results in a 33% reduction in proliferation and a fourfold increase in apoptosis in SHF cells, leading to a 56% decline in the size of the SHF progenitor pool. A reduction in SHF cells available for incorporation into the developing heart leads to underdevelopment of the SHF-derived right ventricle and OFT. Similar to the clinical syndrome, 32% of SHF-specific Dll4 heterozygotes demonstrate foreshortened and misaligned OFT, resulting in a double outlet right ventricle. Our murine model provides a molecular mechanism to explain the cardiac defects observed in AOS and establishes a novel clinical role for Dll4-mediated Notch signaling in SHF progenitor biology.

Introduction: Wnt5a is a known regulator of planar cell polarity (PCP) signaling in second heart field (SHF) progenitor cells. It is generally believed that Wnt5a ligands are secreted by the SHF; however, SHF-specific conditional deletion of Wnt5a does not recapitulate outflow tract (OFT) defects observed in global Wnt5a mutants. Hypothesis: Given the proximity and interaction between neural crest cells (NCC) and cranial SHF that contributes to the developing OFT, we hypothesize that NCC may serve as an additional source of Wnt5a for SHF progenitors. Methods: Wnt5a was conditionally deleted in the neural crest using transgenic Wnt1-cre mice. Embryos were harvested from control and mutant litter mates and immunofluoresence, in situ hybridization, and hematoxylin-eosin stains were performed on histologic sections using standard techniques. India ink injections were performed to evaluate pharyngeal arch artery and outflow tract morphology in whole mount embryos. Results: Wnt1-cre driven conditional deletion of Wnt5a in NCC did not impact NCC survival or migration into the developing OFT. However, SHF cells in mutant E10.5 embryos showed altered PCP signaling with reduced phalloidin and laminin expression. The resulting loss in polarized directional movement of the SHF led to reduced incorporation into and elongation of the developing OFT. The shortened OFT was mal-aligned resulting in fully penetrant double outlet right ventricle (DORV) at E14.5. In addition, maturation of pharyngeal arch arteries was also impaired such that all mutants express pharyngeal arch artery defects, including aortic arch abnormalities and aberrant right subclavian artery. In contrast, there was no observed effect on PCP in the more caudal SHF cells, and none of the mutant embryos had inflow tract or venous pole defects. Conclusions: Our results demonstrate that NCC are a novel source of Wnt5a. NCC-derived Wnt5a is critically required to regulate PCP signals in the most cranial SHF regulating the development of the OFT and pharyngeal arch arteries.