Neat1 is a p53-inducible lincRNA essential for transformation suppression.

The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. We identify Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1-/- mice, we examined the functional role of Neat1 in the p53 pathway. We found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in KrasG12D-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.

Alterations in Spanish Language Interpretation During Pediatric Critical Care Family Meetings.

OBJECTIVES: To characterize alterations in Spanish language medical interpretation during pediatric critical care family meetings. DESIGN: Descriptive, observational study using verbatim transcripts of nine PICU family meetings conducted with in-person, hospital-employed interpreters. SETTING: A single, university-based, tertiary children's hospital. SUBJECTS: Medical staff, family members, ancillary staff, and interpreters. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Interpreted speech was compared with original clinician or family speech using the qualitative research methods of directed content analysis and thematic analysis. Alterations occurred in 56% of interpreted utterances and included additions, omissions, substitutions, editorializations, answering for the patient/clinician, confessions, and patient advocacy. Longer utterances were associated with more alterations. CONCLUSIONS: To minimize interpreter alterations during family meetings, physicians should speak in short utterances (fewer than 20 words) and ask interpreters to interrupt in order to facilitate accurate interpretation. Because alterations occur, physicians may also regularly attempt to assess the family's understanding.

Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.

How Anesthesiologists Experience and Negotiate Ethical Challenges from Drug Shortages.

BACKGROUND: In the face of ongoing drug shortages, anesthesiologists have been described as having to become "Iron Chefs, challenged to create safe patient outcomes with missing ingredients. Unfortunately, developing responsive ethical guidance for how anesthesiologists should best handle ethical concerns with ongoing and mutable drug shortages is limited by the dearth of studies examining how bedside clinicians actually experience drug shortages and what ethical challenges they encounter. In order to better understand what ethical concerns individual anesthesiologists experience around drug shortages and how they negotiate them, we undertook this qualitative interview study. Methods: We conducted semi-structured interviews with anesthesiologists at three field sites: an academic tertiary care adult hospital that performs approximately 46,000 anesthetics annually comprising cases ranging across all surgical disciplines; the affiliated veteran's affairs hospital that performs 12,000 anesthetics annually; and, the affiliated children's hospital that performs 20,500 anesthetics annually. Results: 29 anesthesiologists were interviewed (17 adult and 12 pediatric anesthesiologists), representing a spectrum of practice areas in clinical anesthesia: general, pediatric, regional, pain, critical care, obstetrics, liver transplant, and palliative medicine. Three themes emerged: (1) uncertainty about responsibility for clinical decisions made in consequence to a shortage; (2) creativity, and its limits, in choosing anesthetic plans; and, (3) disclosure of concerns about shortages (to patients and colleagues). Conclusions: Our data suggests anesthesiologists have unmet needs for ethical guidance on how to approach drug shortages. First is managing responsibility for decisions stemming from a drug shortage. Second, interviewees struggled with disclosure of their concerns, both to patients and to surgical colleagues. A formal shared decision making approach may present the best solution, since the act of structuring a shared decision making conversation or creating a decision making tool will have to incorporate the views of all stakeholders around shortages and their potential clinical consequences.

A step beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL).

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the Förster radius, commonly less than 14 nm, FUEL can occur at distances up to µm or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.