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OBJECTIVES To describe the characteristics of individuals undergoing toxicology testing at delivery for a sole indication of cannabis use and to evaluate the rate of unexpected positive toxicology testing results among this cohort. METHODS This retrospective cohort study included dyads with a maternal history of cannabis use who underwent peripartum toxicology testing between 2016 and 2020 at 5 birthing hospitals in Massachusetts. We collected information on maternal demographic characteristics and toxicology test results and reviewed records of dyads with unexpected positive results to identify additional social risk factors and clinical outcomes. RESULTS Of 60 608 live births reviewed, 1924 dyads underwent toxicology testing, including 614 (31.9%) for a sole indication of cannabis use. Significantly greater percentages of patients in the cannabis cohort were CONCLUSIONS Toxicology testing of patients for a sole indication of cannabis use, without other risk factors, may be of limited utility in elucidating other substance use and may exacerbate existing disparities in perinatal outcomes.

RationaleCongenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood.ObjectivesTo establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing.MethodsWe developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from CDH patients and preterm and term, non-CDH controls were derived and analyzed by bulk RNA-sequencing, ATAC-sequencing, and air-liquidinterface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH.Measurements and Main ResultsTranscriptomic and epigenetic profiling of CDH and non-CDH basal stem cells reveals a disease-specific, proinflammatory signature independent of severity or hernia size. In addition, CDH basal stem cells exhibit defective epithelial differentiationin vitrothat recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, steroid treatment normalizes epithelial differentiation phenotypes of human CDH basal stem cellsin vitroand in nitrofen rat tracheasin vivo.ConclusionsOur findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

BACKGROUND AND OBJECTIVES: When given within 24 hours of birth, the hepatitis B vaccine is up to 90% effective in preventing perinatal infection. The American Academy of Pediatrics now recommends administration within 24 hours for infants with a birth weight >2 kg, but a national benchmark for compliance with this time frame has not been established. We aimed to increase the monthly average of eligible newborns receiving the vaccine on time from 40% to 80% over a 9-month period. METHODS: A series of plan-do-study-act cycles were conducted to improve timeliness of hepatitis B vaccine birth dose administration among newborns in the level 1 nursery at our academic community hospital. Interventions included staff education, nurse-driven consent and vaccine ordering, and earlier initial newborn assessments performed by nursing staff. Our primary outcome was the monthly percentage of newborns receiving the vaccine within 24 hours of birth, and our secondary outcome was the frequency of nonvaccination events. Statistical process control was used to analyze the effectiveness of interventions. RESULTS: Our mean monthly rate of vaccine administration within the 24-hour time frame increased from 40% to 92%. Predischarge vaccination rate improved from a mean of 13 to 61 cases between infants discharged without vaccination. CONCLUSIONS: Nurse-led interventions, including the ability to obtain consent and incorporation of the vaccine into our nurse-activated admission order set, were significant contributors to improvement in the timeliness of hepatitis B vaccine administration. We propose a mean of 90% compliance with the American Academy of Pediatrics recommendations as a benchmark for other institutions.

OBJECTIVES Necrotizing enterocolitis (NEC) is a severe intestinal inflammatory disease and a leading cause of morbidity and mortality in NICUs. Management of NEC is variable because of the lack of evidence-based recommendations. It is widely accepted that standardization of patient care leads to improved outcomes. This quality improvement project aimed to decrease variation in the evaluation and management of NEC in a Level IV NICU. METHODS A multidisciplinary team investigated institutional variation in NEC management and developed a standardized guideline and electronic medical record tools to assist in evaluation and management. Retrospective baseline data were collected for 2 years previously and prospectively for 3.5 years after interventions. Outcomes included the ratio of observed-to-expected days of antibiotics and nil per os (NPO) on the basis of the novel guidelines and the percentage of cases treated with piperacillin/tazobactam. Balancing measures were death, surgery, and antifungal use. RESULTS Over 5.5 years, there were 124 evaluations for NEC. Special cause variation was noted in the observed-to-expected antibiotic and NPO days ratios, decreasing from 1.94 to 1.18 and 1.69 to 1.14, respectively. Piperacillin/tazobactam utilization increased from 30% to 91%. There were no increases in antifungal use, surgery, or death. CONCLUSIONS Variation in evaluation and management of NEC decreased after initiation of a guideline and supporting electronic medical record tools, with fewer antibiotic and NPO days without an increase in morbidity or mortality. A quality improvement approach can benefit patients and decrease variability, even in diseases with limited evidence-based standards.

The neonatal intensive care unit (NICU) is a high-acuity, stressful unit for both parents and staff. Up to 50% of mothers and partners experience emotional distress (i.e., depression, anxiety, or posttraumatic stress) during NICU hospitalization and 30-60% continue to experience distress after discharge. Similarly, up to 50% of NICU staff report burnout and emotional distress. Although healthcare providers have developed interdisciplinary guidelines to enhance psychosocial resources for parents and staff, standardized psychosocial services are lacking. The purpose of this short communication is to describe: (1) the need for psychosocial interventions for NICU parents and staff; (2) existent psychosocial programs and their gaps and limitations; and (3) future directions for psychosocial care in NICU settings. We reviewed the current literature and propose a new conceptual model to inform psychosocial interventions for the NICU. We argue that brief, evidence-based, resiliency, and relationship-based programs are needed to enhance parent and staff outcomes and, ultimately, child development and the NICU unit culture.Given the lack of standardized psychosocial care, new interventions for NICU families and staff are needed more than ever. Resiliency, relationship-based interventions that leverage multidisciplinary support may be an innovative way to enhance NICU outcomes and care.• 40-50% of parents in the NICU report elevated emotional distress and 30-50% of staff report burnout. • Psychosocial interventions for parents and staff are needed, yet lacking.• Interventions that focus on resiliency and relationships may improve the culture of the NICU. • New multidisciplinary collaborations and approaches are needed to improve implementation.

Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease.

During the COVID-19 pandemic, thousands of pregnant women have been infected with SARS-CoV-2. The implications of maternal SARS-CoV-2 infection on fetal and childhood well-being are unknown. We aimed to characterize the fetal immune response to maternal SARS-CoV-2 infection. We performed single-cell RNA sequencing and T-cell receptor (TCR) sequencing on cord blood mononuclear cells (CBMC) from newborns of mothers infected with SARS-CoV-2 in the third-trimester (cases) or without SARS-CoV-2 infection. We identified widespread gene expression changes in CBMC from cases, including upregulation of interferon-stimulated genes and Major Histocompatibility Complex genes in CD14 + monocytes; transcriptional changes suggestive of activation of plasmacytoid dendritic cells, and activation and exhaustion of NK cells and CD8 + T-cells. Lastly, we observed fetal TCR repertoire expansion in cases. As none of the infants were infected with SARS-CoV-2, our results suggest that SARS-CoV-2 maternal infection might modulate the fetal immune system in the absence of vertical transmission., BRIEF SUMMARY SARS-CoV-2 infection in the third-trimester of pregnancy without vertical transmission promotes widespread gene expression changes and TCR repertoire clonal expansion in cord blood leukocytes.

Background COVID-19, the disease caused by the highly infectious and transmissible coronavirus SARS-CoV-2, has quickly become a morbid global pandemic. Although the impact of SARS-CoV-2 infection in children is less clinically apparent, collecting high-quality biospecimens from infants, children, and adolescents in a standardized manner during the COVID-19 pandemic is essential to establish a biologic understanding of the disease in the pediatric population. This biorepository enables pediatric centers world-wide to collect samples uniformly to drive forward our understanding of COVID-19 by addressing specific pediatric and neonatal COVID-19-related questions. Methods A COVID-19 biospecimen collection study was implemented with strategic enrollment guidelines to include patients seen in urgent care clinics and hospital settings, neonates born to SARS-CoV-2 infected mothers, and asymptomatic children. The methodology described here, details the importance of establishing collaborations between the clinical and research teams to harmonize protocols for patient recruitment and sample collection, processing and storage. It also details modifications required for biobanking during a surge of the COVID-19 pandemic. Results Considerations and challenges facing enrollment of neonatal and pediatric cohorts are described. A roadmap is laid out for successful collection, processing, storage and database management of multiple pediatric samples such as blood, nasopharyngeal and oropharyngeal swabs, sputum, saliva, tracheal aspirates, stool, and urine. Using this methodology, we enrolled 327 participants, who provided a total of 972 biospecimens. Conclusions Pediatric biospecimens will be key in answering questions relating to viral transmission by children, differences between pediatric and adult viral susceptibility and immune responses, the impact of maternal SARS-CoV-2 infection on fetal development, and factors driving the Multisystem Inflammatory Syndrome in Children. The specimens in this biorepository will allow necessary comparative studies between children and adults, help determine the accuracy of current pediatric viral testing techniques, in addition to, understanding neonatal exposure to SARS-CoV-2 infection and disease abnormalities. The successful establishment of a pediatric biorepository is critical to provide insight into disease pathogenesis, and subsequently, develop future treatment and vaccination strategies.

Background Collection of biospecimens is a critical first step to understanding the impact of COVID-19 on pregnant women and newborns - vulnerable populations that are challenging to enroll and at risk of exclusion from research. We describe the establishment of a COVID-19 perinatal biorepository, the unique challenges imposed by the COVID-19 pandemic, and strategies used to overcome them. Methods A transdisciplinary approach was developed to maximize the enrollment of pregnant women and their newborns into a COVID-19 prospective cohort and tissue biorepository, established on March 19, 2020 at Massachusetts General Hospital (MGH). The first SARS-CoV-2 positive pregnant woman was enrolled on April 2, and enrollment was expanded to SARS-CoV-2 negative controls on April 20. A unified enrollment strategy with a single consent process for pregnant women and newborns was implemented on May 4. SARS-CoV-2 status was determined by viral detection on RT-PCR of a nasopharyngeal swab. Wide-ranging and pregnancy-specific samples were collected from maternal participants during pregnancy and postpartum. Newborn samples were collected during the initial hospitalization. Results Between April 2 and June 9, 100 women and 78 newborns were enrolled in the MGH COVID-19 biorepository. The rate of dyad enrollment and number of samples collected per woman significantly increased after changes to enrollment strategy (from 5 to over 8 dyads/week, P P P = 0.0007). The highest sample yield was for placenta (96%), umbilical cord blood (93%), urine (99%), and maternal blood (91%). The lowest-yield sample types were maternal stool (30%) and breastmilk (22%). Of the 61 delivered women who also enrolled their newborns, fewer women agreed to neonatal blood compared to cord blood (39 vs 58, P Conclusions Establishing a COVID-19 perinatal biorepository required patient advocacy, transdisciplinary collaboration and creative solutions to unique challenges. This biorepository is unique in its comprehensive sample collection and the inclusion of a control population. It serves as an important resource for research into the impact of COVID-19 on pregnant women and newborns and provides lessons for future biorepository efforts.

Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture.We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates.Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium.Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease.Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.

Objectives As schools plan for re-opening, understanding the potential role children play in the coronavirus infectious disease 2019 (COVID-19) pandemic and the factors that drive severe illness in children is critical. Study design Children ages 0-22 years with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presenting to urgent care clinics or being hospitalized for confirmed/suspected SARS-CoV-2 infection or multisystem inflammatory syndrome in children (MIS-C) at Massachusetts General Hospital were offered enrollment in the Massachusetts General Hospital Pediatric COVID-19 Biorepository. Enrolled children provided nasopharyngeal, oropharyngeal, and/or blood specimens. SARS-CoV-2 viral load, ACE2 RNA levels, and serology for SARS-CoV-2 were quantified. Results A total of 192 children (mean age, 10.2 ± 7.0 years) were enrolled. Forty-nine children (26%) were diagnosed with acute SARS-CoV-2 infection; an additional 18 children (9%) met the criteria for MIS-C. Only 25 children (51%) with acute SARS-CoV-2 infection presented with fever; symptoms of SARS-CoV-2 infection, if present, were nonspecific. Nasopharyngeal viral load was highest in children in the first 2 days of symptoms, significantly higher than hospitalized adults with severe disease (P = .002). Age did not impact viral load, but younger children had lower angiotensin-converting enzyme 2 expression (P = .004). Immunoglobulin M (IgM) and Immunoglobulin G (IgG) to the receptor binding domain of the SARS-CoV-2 spike protein were increased in severe MIS-C (P Conclusions This study reveals that children may be a potential source of contagion in the SARS-CoV-2 pandemic despite having milder disease or a lack of symptoms; immune dysregulation is implicated in severe postinfectious MIS-C.

Genetic mutations in TAR DNA-binding protein 43 (TARDBP, also known as TDP-43) cause amyotrophic lateral sclerosis (ALS), and an increase in the presence of TDP-43 (encoded by TARDBP) in the cytoplasm is a prominent histopathological feature of degenerating neurons in various neurodegenerative diseases. However, the molecular mechanisms by which TDP-43 contributes to ALS pathophysiology remain elusive. Here we have found that TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Disease-associated mutations increase TDP-43 mitochondrial localization. In mitochondria, wild-type (WT) and mutant TDP-43 preferentially bind mitochondria-transcribed messenger RNAs (mRNAs) encoding respiratory complex I subunits ND3 and ND6, impair their expression and specifically cause complex I disassembly. The suppression of TDP-43 mitochondrial localization abolishes WT and mutant TDP-43-induced mitochondrial dysfunction and neuronal loss, and improves phenotypes of transgenic mutant TDP-43 mice. Thus, our studies link TDP-43 toxicity directly to mitochondrial bioenergetics and propose the targeting of TDP-43 mitochondrial localization as a promising therapeutic approach for neurodegeneration.

Genome instability is a potential limitation to the research and therapeutic application of induced pluripotent stem cells (iPSCs). Observed genomic variations reflect the combined activities of DNA damage, cellular DNA damage response (DDR), and selection pressure in culture. To understand the contribution of DDR on the distribution of copy number variations (CNVs) in iPSCs, we mapped CNVs of iPSCs with mutations in the central DDR gene ATM onto genome organization landscapes defined by genome-wide replication timing profiles. We show that following reprogramming the early and late replicating genome is differentially affected by CNVs in ATM-deficient iPSCs relative to wild-type iPSCs. Specifically, the early replicating regions had increased CNV losses during retroviral (RV) reprogramming. This differential CNV distribution was not present after later passage or after episomal reprogramming. Comparison of different reprogramming methods in the setting of defective DDR reveals unique vulnerability of early replicating open chromatin to RV vectors.

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Keywords: hPSC, Differentiation, Definitive endoderm, Heterogeneity, Single cell, RNA sequencing

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin-Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications., Generating organized kidney tissues from human pluripotent stem cell is a major challenge. Here, Freedman et al. describe a differentiation system forming spheroids and tubular structures, characteristic of these kidney structures, and using CRISPR/Cas9, delete PKD1/2, to model polycystic kidney disease.

A newborn boy had a profound hypoxic-ischemic insult at birth. He developed rhythmic myoclonus within 1 hour, which was initially assumed to be seizures, but was unresponsive to escalating antiepileptic therapy. On careful examination, it was evident that myoclonus could be induced and suppressed. Amplitude-integrated (figure) and video EEG (video on the Neurology ® Web site at [Neurology.org][1]) demonstrated a suppressed background with intermittent rhythmic pattern correlating with the movements. Laterality of this pattern changed with head positioning, suggesting artifact from pressure on dependent electrodes. Care was ultimately redirected when neuroimaging confirmed severe injury. [1]: http://Neurology.org