Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for panpathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4 + adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10 + ) airway-like and SPC + saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC -progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.

The dosage for GW9662 was incorrectly noted in Results, Methods, and the legend for Figure 6. The correct sentences appear below.Results: We treated a cohort of younger animals for 2 weeks at a dose of 4 mg/kg body weight and found that there was a 31% reduction in the expression of Cd36 ( Figure 6F) and a 48% reduction in liver TG content in JAK2L animals treated with GW9662 versus those treated with vehicle ( Figure 6G). Methods: Each day, an aliquot of stock drug was thawed and then resuspended in DMSO/saline at a final concentration of 4 mg/kg body weight in a final volume of 100 μl. The authors regret the error.

6Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been 3 7 successfully tested in proof-of-concept case studies in patients with acute illness of unknown 3 8 etiology, but to date has been largely confined to research settings. Here we developed and 3 9 validated an mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from 4 0 cerebrospinal fluid (CSF) in a licensed clinical laboratory. A clinical bioinformatics pipeline, 4 1 SURPI+, was developed to rapidly analyze mNGS data, automatically report detected 4 2 pathogens, and provide a graphical user interface for evaluating and interpreting results. We 4 3 established quality metrics, threshold values, and limits of detection of between 0.16 -313 4 4 genomic copies or colony forming units per milliliter for each representative organism type. 4 5 Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, a spiked 4 6 phage used as an internal control was a reliable indicator of sensitivity loss. Diagnostic test 4 7 accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% 4 8 sensitivity and 99% specificity compared to original clinical test results, with 81% positive 4 9

Prevention or treatment of lung diseases caused by the failure to form, or destruction of, existing alveoli, as observed in infants with bronchopulmonary dysplasia and adults with emphysema, requires understanding of the molecular mechanisms of alveolar development. In addition to its critical role in gas exchange, the pulmonary circulation also contributes to alveolar morphogenesis and maintenance by the production of paracrine factors, termed "angiocrines," that impact the development of surrounding tissue. To identify lung angiocrines that contribute to alveolar formation, we disrupted pulmonary vascular development by conditional inactivation of the Vegf-A gene during alveologenesis. This resulted in decreased pulmonary capillary and alveolar development and altered lung elastin and retinoic acid (RA) expression. We determined that RA is produced by pulmonary endothelial cells and regulates pulmonary angiogenesis and elastin synthesis by induction of VEGF-A and fibroblast growth factor (FGF)-18, respectively. Inhibition of RA synthesis in newborn mice decreased FGF-18 and elastin expression and impaired alveolarization. Treatment with RA and vitamin A partially reversed the impaired vascular and alveolar development induced by VEGF inhibition. Thus we identified RA as a lung angiocrine that regulates alveolarization through autocrine regulation of endothelial development and paracrine regulation of elastin synthesis via induction of FGF-18 in mesenchymal cells.