have formed a joint venture with shared ownership and governance of Baylor Genetics (BG), which performs clinical microarray analysis and clinical exome sequencing. JRL serves on the scientific advisory board of BG. JRL has stock ownership in 23andMe, is a paid consultant for Regeneron Pharmaceuticals, and is a coinventor on multiple US and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting (

Natural killer (NK) cells are innate immune cells that control viral infection and tumorigenic cell growth through targeted cell lysis and cytokine secretion. Human NK cells are classically defined as CD56 + CD3 − in peripheral blood. CD56 is neural cell adhesion molecule (NCAM1), and despite its ubiquitous expression on human NK cells, the role of CD56 in human NK cell cytotoxic function has not been fully explored. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis, in part through interactions with focal adhesion kinase (FAK). Here we describe the generation and use of CD56-deficient human NK cell lines to define a novel requirement for CD56 in target cell lysis. Namely, we demonstrate that deletion of CD56 on the NK92 cell line led to impaired cytotoxic function against multiple susceptible target cell lines. Deletion of CD56 in a second NK cell line, YTS cells, led to a less severe cytotoxicity defect but impairment in cytokine secretion. Confocal microscopy of wild-type and CD56-KO NK92 cells conjugated to susceptible targets revealed that CD56-KO cells failed to polarize during immunological synapse (IS) formation and had severely impaired exocytosis of lytic granules at the IS. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 was decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and IS formation and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 were rescued by the reintroduction of NCAM140 (CD56), into NK92 CD56-KO cells. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells. cytotoxicity | natural killer cell | NCAM | CD56 Correspondence: em3375@cumc.columbia.edu 2 | bioRχiv Mace et al. | CD56 and human NK cell cytotoxicity

Human natural killer (NK) cells are defined as CD56+CD3−. Despite its ubiquitous expression on human NK cells the role of CD56 (NCAM) in human NK cell cytotoxic function has not been defined. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis through interactions with focal adhesion kinase (FAK). Here we demonstrate that deletion of CD56 on the NK92 cell line leads to impaired cytotoxic function. CD56-knockout (KO) cells fail to polarize during immunological synapse (IS) formation and have severely impaired exocytosis of lytic granules. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 is decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 are rescued by the reintroduction of CD56. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells.

BACKGROUND:The COVID-19 pandemic drastically impacted medical student experiences. Little is known about the impact of the pandemic on student well-being and protective factors for burnout. OBJECTIVE: Assess US medical student burnout, stress, and loneliness during the initial phase of the pandemic, compare results to pre-pandemic data, and identify risk factors for distress and protective factors to inform support interventions. DESIGN: Cross-sectional survey of medical students conducted between May and July 2020. PARTICIPANTS: 3826 students from 22 medical schools. MAIN MEASURES: Burnout (MBI-HSS), stress (PSS-10), loneliness (UCLA scale), and student experiences. Compared burnout and stress to pre-pandemic studies (2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020). KEY RESULTS: Of 12,389 students, 3826 responded (31%). Compared to pre-pandemic studies, burnout was lower (50% vs. 52%, P = 0.03) while mean stress was higher (18.9 vs. 16.0, P < 0.001). Half (1609/3247) reported high (≥ 6/9) loneliness scores. Significant differences were found in burnout and stress by class year (P = 0.002 and P < 0.001) and race (P = 0.004 and P < 0.001), with the highest levels in second-and third-year students and Black, Asian, or other racial minority students. Students experiencing financial strain or racism had higher burnout and stress (P < 0.001 for all). Respondents with COVID-19 diagnoses in themselves or family members had higher stress (P < 0.001). Nearly half (1756/3569) volunteered during the pandemic, with volunteers reporting lower burnout [48% (782/1639) vs. 52% (853/1656), P = 0.03]. CONCLUSIONS: While stress was higher compared to pre-pandemic data, burnout was significantly lower. Higher burnout and stress among Black, Asian, and other racial minority students and those who experienced financial strain, racism, or COVID-19 diagnoses likely reflect underlying racial and socioeconomic inequalities exacerbated by the pandemic and concurrent national racial injustice events. Volunteer engagement may be protective against burnout. Schools should proactively support vulnerable students during periods of stress.

Background: Cigarette smoking is an important risk factor for cardiac diseases. In the current study, we sought to assess the effect of electronic cigarette extract (ECE) and conventional cigarette smoke extract (CSE) on cardiomyocytes. Methods: iPSCs-derived cardiomyocytes were used in the study to evaluate cellular toxicities. Cells were exposed to either ECE or CSE for two consecutive days as an acute exposure or every other day for 14 days. Concentration of nicotine in both ECE and CSE were measured by Mass-Spectrometry and Q-Exactive-HF was used to identify other ingredients in both extracts. Fluorescent microscopy was used to measure the oxidative stress after ECE and CSE exposure. Motility and beat frequency of cardiomyocytes were determined using the Sisson-Ammons Video Analysis system. Heart failure target panel genes of exposed cardiomyocytes were compared to control unexposed cells. Results: Despite nicotine concentration in CSE being six-fold higher than ECE (50 μg in CSE and 8 μg in ECE), ECE had similar toxic effect on cardiomyocytes. Both CSE and ECE generate significant cellular reactive oxygen species. The Sisson-Ammons Video Analysis (SAVA) analysis showed significant changes in myocyte function with both CSE and ECE slowing beating and increasing cell death. Chronic exposure of both ECE and CSE significantly decreased cardiomyocytes viability long term at all doses. Target panel gene expression profiles of both ECE and CSE exposed cardiomyocytes were different from controls with distinct pattern of genes that involved cell proliferation, inflammation, and apoptosis.