Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens.

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.

Biomarkers kinetics in the assessment of ventilator-associated pneumonia response to antibiotics - results from the BioVAP study.

PURPOSE: Our aim was to evaluate the role of biomarker kinetics in the assessment of ventilator-associated pneumonia (VAP) response to antibiotics. MATERIALS AND METHODS: We performed a prospective, multicenter, observational study to evaluate in 37 microbiologically documented VAP, the kinetics of C-reactive protein (CRP), procalcitonin (PCT), mid-region fragment of pro-adrenomedullin (MR-proADM). The kinetics of each variable, from day 1 to 6 of therapy, was assessed with a time dependent analysis comparing survivors and non-survivors. RESULTS: During the study period kinetics of CRP as well as its relative changes, CRP-ratio, was significantly different between survivors and non-survivors (p=0.026 and p=0.005, respectively). On day 4 of antibiotic therapy, CRP of survivors was 47% of the initial value while it was 96% in non-survivors. The kinetics of other studied variables did not distinguish between survivors and non-survivors. In survivors the bacterial load also decreased markedly. Adequate initial antibiotic therapy was associated with lower mortality (p=0.025) and faster CRP decrease (p=0.029). CONCLUSIONS: C-reactive protein kinetics can be used to identify VAP patients with poor outcome as soon as four days after the initiation of treatment. (Trial registration - NCT02078999; registered 3 August 2012).