An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells.

BACKGROUND & AIMS: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). METHODS: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. RESULTS: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. CONCLUSIONS: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Recent Levels of Evidence in Otolaryngology Journals.

OBJECTIVE: The development of evidence-based medicine has contributed to improved patient outcomes. This study aims to identify the trends in levels of evidence in otolaryngology journals over time, as represented by the 4 most widely circulated peer-reviewed otolaryngology journals. METHODS: A review of all articles from 2007, 2010, 2013, 2016, and 2019, in 4 major otolaryngology journals. Data points included journal source, year of publication, country of origin, first author sex, and subspecialty category within otolaryngology. Level of evidence was determined based on the study's primary research question and was graded on a scale of 1 (strongest) to 4 (weakest) based on the Oxford Centre of Evidence-based Medicine - Levels of Evidence guideline. Comparison of levels of evidence was performed using Kruskal-Wallis analysis of variance for ordinal data. RESULTS: About 4297 articles were identified over 12 years. The number of research articles remained consistent over the 12 years of this study. Clinical research increased from 78.6% to 85.1%. Female first authorship increased from 20.3% in 2007 to 31.0% in 2019. Of 3558 articles that constituted clinical research from 2007 to 2019, level 1 studies increased from 0.9% to 3.6%, with level 4 studies remaining stable at an overall rate of 60.3%. Randomized controlled trials remained stable at 4.6% of all studies. Systematic reviews increased from 3.2% to 8.4%. CONCLUSION: This article provides an update on the levels of evidence to allow for an honest self-assessment of otolaryngology as a scientific field.

Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53.

Coronavirus disease 2019 (COVID-19) remains a significant public health threat due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here, we use our recently developed integrative DNA And Protein Tagging methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.

Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53.

COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.

Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer.

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.

Psychiatric Boarding in the Pediatric Inpatient Medical Setting: A Retrospective Analysis.

BACKGROUND AND OBJECTIVES: Psychiatric concerns are a common presenting problem for pediatric providers across many settings, particularly on inpatient medical services. The volume of youth requiring intensive psychiatric treatment outnumbers the availability of psychiatric placements, and as a result many youth must board on pediatric medical units while awaiting placement. As the phenomenon of boarding in the inpatient pediatric setting increases, it is important to understand trends in boarding volume and characteristics of pediatric psychiatric boarders (PBs) and understand the supports they receive while boarding. METHODS: A retrospective chart review of patients admitted as PBs to a medical inpatient unit at a large northeastern US pediatric hospital during 2013. RESULTS: Four hundred thirty-seven PBs were admitted to the medical service from January to December 2013, representing a more than 50% increase from PB admissions in 2011 and 2012. Most PBs were admitted for suicidal attempt and/or ideation. Average length of boarding was 3.11 ± 3.34 days. PBs received a wide range of mental health supports throughout their admissions. PBs demonstrated modest but statistically significant clinical improvements over the course of their stay, with only a small proportion demonstrating clinical deterioration. CONCLUSIONS: Psychiatric boarding presents many challenges for families, providers, and the health care system, and PBs have complex psychiatric histories and needs. However, boarding may offer a valuable opportunity for psychiatric intervention and stabilization among psychiatrically vulnerable youth.

A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation.

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.

Vorinostat eliminates multicellular resistance of mesothelioma 3D spheroids via restoration of Noxa expression.

When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies.