An essential mesenchymal function for miR-143/145 in intestinal epithelial regeneration.

Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

Rag-Ragulator is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway.

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.

ZSWIM8 destabilizes many murine microRNAs and is required for proper embryonic growth and development.

MicroRNAs (miRNAs) pair to sites in mRNAs to direct the degradation of these RNA transcripts. Conversely, certain RNA transcripts can direct the degradation of particular miRNAs. This target-directed miRNA degradation (TDMD) requires the ZSWIM8 E3 ubiquitin ligase. Here, we report the function of ZSWIM8 in the mouse embryo. Zswim8 -/- embryos were smaller than their littermates and died near the time of birth. This highly penetrant perinatal lethality was apparently caused by a lung sacculation defect attributed to failed maturation of alveolar epithelial cells. Some mutant individuals also had heart ventricular septal defects. These developmental abnormalities were accompanied by aberrant accumulation of more than 50 miRNAs observed across 12 tissues, which often led to enhanced repression of their mRNA targets. These ZSWIM8-sensitive miRNAs were preferentially produced from genomic miRNA clusters, and in some cases, ZSWIM8 caused a switch in the dominant strand or isoform that accumulated from a miRNA hairpin-observations suggesting that TDMD provides a mechanism to uncouple coproduced miRNAs from each other. Overall, our findings indicate that the regulatory influence of ZSWIM8, and presumably TDMD, in mammalian biology is widespread and consequential, and posit the existence of many yet-unidentified transcripts that trigger miRNA degradation.

Abate and switch: miR-145 in stem cell differentiation.

MicroRNAs have been implicated as regulators of embryonic stem (ES) cell self-renewal and pluripotency. In this issue, Xu et al. (2009) demonstrate that miR-145 facilitates ES cell differentiation by repressing the core pluripotency factors OCT4, SOX2, and KLF4, thereby silencing the self-renewal program.

Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.

Tumor suppression by miR-26 overrides potential oncogenic activity in intestinal tumorigenesis.

Down-regulation of miR-26 family members has been implicated in the pathogenesis of multiple malignancies. In some settings, including glioma, however, miR-26-mediated repression of PTEN promotes tumorigenesis. To investigate the contexts in which the tumor suppressor versus oncogenic activity of miR-26 predominates in vivo, we generated miR-26a transgenic mice. Despite measureable repression of Pten, elevated miR-26a levels were not associated with malignancy in transgenic animals. We documented reduced miR-26 expression in human colorectal cancer and, accordingly, showed that miR-26a expression potently suppressed intestinal adenoma formation in Apc(min/+) mice, a model known to be sensitive to Pten dosage. These studies reveal a tumor suppressor role for miR-26 in intestinal cancer that overrides putative oncogenic activity, highlighting the therapeutic potential of miR-26 delivery to this tumor type.

Evidence-Based Management of the Critically Ill Adult With SARS-CoV-2 Infection.

Human infection by the novel viral pathogen SARS-CoV-2 results in a clinical syndrome termed Coronavirus Disease 2019 (COVID-19). Although the majority of COVID-19 cases are self-limiting, a substantial minority of patients develop disease severe enough to require intensive care. Features of critical illness associated with COVID-19 include hypoxemic respiratory failure, acute respiratory distress syndrome (ARDS), shock, and multiple organ dysfunction syndrome (MODS). In most (but not all) respects critically ill patients with COVID-19 resemble critically ill patients with ARDS due to other causes and are optimally managed with standard, evidence-based critical care protocols. However, there is naturally an intense interest in developing specific therapies for severe COVID-19. Here we synthesize the rapidly expanding literature around the pathophysiology, clinical presentation, and management of COVID-19 with a focus on those points most relevant for intensivists tasked with caring for these patients. We specifically highlight evidence-based approaches that we believe should guide the identification, triage, respiratory support, and general ICU care of critically ill patients infected with SARS-CoV-2. In addition, in light of the pressing need and growing enthusiasm for targeted COVID-19 therapies, we review the biological basis, plausibility, and clinical evidence underlying these novel treatment approaches.

An exome-first approach to aid in the diagnosis of primary ciliary dyskinesia.

Unlike disorders of primary cilium, primary ciliary dyskinesia (PCD) has a much narrower clinical spectrum consistent with the limited tissue distribution of motile cilia. Nonetheless, PCD diagnosis can be challenging due to the overlapping features with other disorders and the requirement for sophisticated tests that are only available in specialized centers. We performed exome sequencing on all patients with a clinical suspicion of PCD but for whom no nasal nitric oxide test or ciliary functional assessment could be ordered. Among 81 patients (56 families), in whom PCD was suspected, 68% had pathogenic or likely pathogenic variants in established PCD-related genes that fully explain the phenotype (20 variants in 11 genes). The major clinical presentations were sinopulmonary infections (SPI) (n = 58), neonatal respiratory distress (NRD) (n = 2), laterality defect (LD) (n = 6), and combined LD/SPI (n = 15). Biallelic likely deleterious variants were also encountered in AKNA and GOLGA3, which we propose as novel candidates in a lung phenotype that overlaps clinically with PCD. We also encountered a PCD phenocopy caused by a pathogenic variant in ITCH, and a pathogenic variant in CEP164 causing Bardet-Biedl syndrome and PCD presentation as a very rare example of the dual presentation of these two disorders of the primary and motile cilia. Exome sequencing is a powerful tool that can help "democratize" the diagnosis of PCD, which is currently limited to highly specialized centers.

A Conserved Distal Lung Regenerative Pathway in Acute Lung Injury.

Improved tools have led to a burgeoning understanding of lung regeneration in mice, but it is not yet known how these insights may be relevant to acute lung injury in humans. We report in detail two cases of fulminant idiopathic acute lung injury requiring extracorporeal membrane oxygenation in previously healthy young adults with acute respiratory distress syndrome, one of whom required lung transplantation. Biopsy specimens showed diffuse alveolar injury with a striking paucity of alveolar epithelial regeneration, rare hyaline membranes, and diffuse contiguous airspace lining by macrophages. This novel constellation was termed diffuse alveolar injury with delayed epithelization. In addition, mirroring data from murine models of lung injury/regeneration, peribronchiolar basaloid pods (previously described as squamous metaplasia) and ciliated bronchiolarization were identified in these patients and in 39% of 57 historical cases with diffuse alveolar damage. These findings demonstrate a common and clinically relevant human disease correlate for murine models of severe acute lung injury. Evidence suggests that peribronchiolar basaloid pods and bronchiolarization are related spatially and temporally and likely represent overlapping sequential stages of the response to severe distal airway injury.

Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway.

Although activating mutations in RAS oncogenes are known to result in aberrant signaling through multiple pathways, the role of microRNAs (miRNAs) in the Ras oncogenic program remains poorly characterized. Here we demonstrate that Ras activation leads to repression of the miR-143/145 cluster in cells of human, murine, and zebrafish origin. Loss of miR-143/145 expression is observed frequently in KRAS mutant pancreatic cancers, and restoration of these miRNAs abrogates tumorigenesis. miR-143/145 down-regulation requires the Ras-responsive element-binding protein (RREB1), which represses the miR-143/145 promoter. Additionally, KRAS and RREB1 are targets of miR-143/miR-145, revealing a feed-forward mechanism that potentiates Ras signaling.

Circular reasoning: microRNAs and cell-cycle control.

MicroRNAs (miRNAs) have attracted considerable attention because of their important roles in development, normal physiology, and disease states including cancer. Recent studies have identified specific miRNAs that regulate the cell cycle and have documented that the loss or gain of miRNA-mediated cell-cycle control contributes to malignancy. miRNAs regulate classic cell-cycle control pathways by directly targeting proteins such as E2F transcription factors, cyclin-dependent kinases (Cdks), cyclins and Cdk inhibitors. Moreover, from recent findings, it has been suggested that miRNAs themselves might be subject to cell-cycle dependent regulation. Together, these observations indicate that the reciprocal control of RNA silencing and the metazoan cell cycle impacts cellular behavior and disease.

Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation.

Direct control of microRNA (miRNA) expression by oncogenic and tumor suppressor networks results in frequent dysregulation of miRNAs in cancer cells and contributes to tumorigenesis. We previously demonstrated that activation of the c-Myc oncogenic transcription factor (Myc) broadly influences miRNA expression and in particular leads to widespread miRNA down-regulation. miRNA transcripts repressed by Myc include several with potent tumor suppressor activity such as miR-15a/16-1, miR-34a, and let-7 family members. In this study, we have investigated mechanisms downstream of Myc that contribute to miRNA repression. Consistent with transcriptional down-regulation, Myc activity results in the decreased abundance of multiple miRNA primary transcripts. Surprisingly, however, primary transcripts encoding several let-7 miRNAs are not reduced in the high Myc state, suggesting a posttranscriptional mechanism of repression. The Lin-28 and Lin-28B RNA binding proteins were recently demonstrated to negatively regulate let-7 biogenesis. We now show that Myc induces Lin-28B expression in multiple human and mouse tumor models. Chromatin immunoprecipitation and reporter assays reveal direct association of Myc with the Lin-28B promoter resulting in transcriptional transactivation. Moreover, we document that activation of Lin-28B is necessary and sufficient for Myc-mediated let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation. These findings highlight an important role for Lin-28B in Myc-driven cellular phenotypes and uncover an orchestration of transcriptional and posttranscriptional mechanisms in Myc-mediated reprogramming of miRNA expression.

Rethinking the Physician-Scientist Pathway.

Physician-scientists have the potential to generate fundamental as well as translational breakthroughs. But many trainees who intend to pursue a hybrid career in research and patient care ultimately leave one or the other behind. In this Invited Commentary, the authors draw from their experience as early-career physician-scientists to frame physician-scientist training as having 2 phases: first, learning to think like a physician-scientist; second, learning to act like a physician-scientist. These phases roughly correspond to (1) clinical training (from medical school through residency or fellowship) that incorporates research exposure, and (2) a structured period of graduated research independence once the physician-scientist has become clinically autonomous. There are many effective ways to pursue each phase; what matters most is flexibility in the first phase and sustained support in the second. Accordingly, the authors suggest many potential reforms, including at the levels of the National Institutes of Health, private funders, as well as universities and research hospitals. The authors argue that rethinking physician-scientist training to support individualized paths to an independent hybrid career can help recruit and retain physician-scientists for years to come.

Cystic Fibrosis and the Cells of the Airway Epithelium: What Are Ionocytes and What Do They Do?

Cystic fibrosis (CF) is caused by defects in an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Recently, a new airway epithelial cell type has been discovered and dubbed the pulmonary ionocyte. Unexpectedly, these ionocytes express higher levels of CFTR than any other airway epithelial cell type. However, ionocytes are not the sole CFTR-expressing airway epithelial cells, and CF-associated disease genes are in fact expressed in multiple airway epithelial cell types. The experimental depletion of ionocytes perturbs epithelial physiology in the mouse trachea, but the role of these rare cells in the pathogenesis of human CF remains mysterious. Ionocytes have been described in diverse tissues(kidney and inner ear) and species (frog and fish). We draw on these prior studies to suggest potential roles of airway ionocytes in health and disease. A complete understanding of ionocytes in the mammalian airway will ultimately depend on cell type-specific genetic manipulation.

Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery.

To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.

Lung Histopathology in Coronavirus Disease 2019 as Compared With Severe Acute Respiratory Sydrome and H1N1 Influenza: A Systematic Review.

BACKGROUND: Patients with severe coronavirus disease 2019 (COVID-19) have respiratory failure with hypoxemia and acute bilateral pulmonary infiltrates, consistent with ARDS. Respiratory failure in COVID-19 might represent a novel pathologic entity. RESEARCH QUESTION: How does the lung histopathology described in COVID-19 compare with the lung histopathology described in SARS and H1N1 influenza? STUDY DESIGN AND METHODS: We conducted a systematic review to characterize the lung histopathologic features of COVID-19 and compare them against findings of other recent viral pandemics, H1N1 influenza and SARS. We systematically searched MEDLINE and PubMed for studies published up to June 24, 2020, using search terms for COVID-19, H1N1 influenza, and SARS with keywords for pathology, biopsy, and autopsy. Using PRISMA-Individual Participant Data guidelines, our systematic review analysis included 26 articles representing 171 COVID-19 patients; 20 articles representing 287 H1N1 patients; and eight articles representing 64 SARS patients. RESULTS: In COVID-19, acute-phase diffuse alveolar damage (DAD) was reported in 88% of patients, which was similar to the proportion of cases with DAD in both H1N1 (90%) and SARS (98%). Pulmonary microthrombi were reported in 57% of COVID-19 and 58% of SARS patients, as compared with 24% of H1N1 influenza patients. INTERPRETATION: DAD, the histologic correlate of ARDS, is the predominant histopathologic pattern identified in lung pathology from patients with COVID-19, H1N1 influenza, and SARS. Microthrombi were reported more frequently in both patients with COVID-19 and SARS as compared with H1N1 influenza. Future work is needed to validate this histopathologic finding and, if confirmed, elucidate the mechanistic underpinnings and characterize any associations with clinically important outcomes.

Quantification of global microRNA abundance by selective isotachophoresis.

We here present and demonstrate a novel technique based on isotachophoresis (ITP) for the quantification of global microRNA (miRNA) abundance in total RNA. We leverage the selectivity of ITP to concentrate miRNA and exclude longer RNA molecules from the focused zone. We designed a novel ITP strategy where we initially establish three contiguous zones of sieving polymer, electrolyte, and denaturant concentrations. This allows for successive preconcentration, selection, and detection of miRNA. We optimized chemistry in each zone for high sensitivity and exquisite selectivity for miRNA. This technique allows for the measurement of the total miRNA content in a sample and its comparison between different cell types and tissues. We demonstrated and validated the efficacy of this technique by comparing global miRNA abundance in subconfluent and confluent cell cultures.

The Fast Literature Assessment and Review (FLARE) Initiative. A Collaborative Effort for Timely Literature Appraisal during the Coronavirus Disease Pandemic.

The emergence and worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused major disruptions to the healthcare system and medical education. In response, the scientific community has been acquiring, releasing, and publishing data at a remarkable pace. At the same time, medical practitioners are taxed with greater professional duties than ever before, making it challenging to stay current with the influx of medical literature.To address the above mismatch between data release and provider capacity and to support our colleagues, physicians at the Massachusetts General Hospital have engaged in an electronic collaborative effort focused on rapid literature appraisal and dissemination regarding SARS-CoV-2 with a focus on critical care.Members of the Division of Pulmonary and Critical Care, the Division of Cardiology, and the Department of Medicine at Massachusetts General Hospital established the Fast Literature Assessment and Review (FLARE) team. This group rapidly compiles, appraises, and synthesizes literature regarding SARS-CoV-2 as it pertains to critical care, relevant clinical questions, and anecdotal reports. Daily, FLARE produces and disseminates highly curated scientific reviews and opinion pieces, which are distributed to readers using an online newsletter platform.Interest in our work has escalated rapidly. FLARE was quickly shared with colleagues outside our division, and, in a short time, our audience has grown to include more than 4,000 readers across the globe.Creating a collaborative group with a variety of expertise represents a feasible and acceptable way of rapidly appraising, synthesizing, and communicating scientific evidence directly to frontline clinicians in this time of great need.

A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.