Miat and interacting protein Metadherin maintain a stem-like niche to promote medulloblastoma tumorigenesis and treatment resistance.

Long noncoding RNAs (lncRNAs) play essential roles in the development and progression of many cancers. However, the contributions of lncRNAs to medulloblastoma (MB) remain poorly understood. Here, we identify Miat as an lncRNA enriched in the sonic hedgehog group of MB that is required for maintenance of a treatment-resistant stem-like phenotype in the disease. Loss of Miat results in the differentiation of tumor-initiating, stem-like MB cells and enforces the differentiation of tumorigenic stem-like MB cells into a nontumorigenic state. Miat expression in stem-like MB cells also facilitates treatment resistance by down-regulating p53 signaling and impairing radiation-induced cell death, which can be reversed by therapeutic inhibition of Miat using antisense oligonucleotides. Mechanistically, the RNA binding protein Metadherin (Mtdh), previously linked to resistance to cytotoxic therapy in cancer, binds to Miat in stem-like MB cells. Like the loss of Miat, the loss of Mtdh reduces tumorigenicity and increases sensitivity to radiation-induced death in stem-like MB cells. Moreover, Miat and Mtdh function to regulate the biogenesis of several microRNAs and facilitate tumorigenesis and treatment resistance. Taken together, these data reveal an essential role for the lncRNA Miat in sustaining a treatment-resistant pool of tumorigenic stem-like MB cells.

Supervised machine learning models for classifying common causes of dizziness.

PURPOSE: The objective of this study was to use a supervised machine learning (ML) platform and a national dataset to identify factors important in classifying common types of dizziness. METHODS: Using established clinical criteria and responses to the balance and dizziness supplement from the 2016 National health Interview Survey (n = 33,028), case definitions for vestibular migraine (VM), benign paroxysmal positional vertigo (BPPV) Ménière's disease (MD), persistent postural-perceptual dizziness (PPPD), superior canal dehiscence (SCD), and bilateral vestibular hypofunction (BVH) were generated. One hundred thirty-six variables consisting of sociodemographic characteristics and medical comorbidities were used to develop decision tree models to predict these common types of dizziness. RESULTS: The one-year prevalence of dizziness in the U.S. was 16.8% (5562 respondents). VM was highly prevalent, representing 4.0% of the overall respondents (n = 1327). ML decision tree models were able to correctly classify all 6 dizziness subtypes with high accuracy (sensitivity range, 70-92%; specificity range, 89-99%) using responses to questions about functional limitations due to dizziness, such as falls due to dizziness and modification of social activities due to dizziness. CONCLUSIONS: In a large population-based dataset, supervised ML models accurately predicted dizziness subtypes according to responses to questions that do not pertain to dizziness symptoms alone.

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast.

BACKGROUND: CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis. RESULTS: We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. CONCLUSIONS: Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

Leukotriene C4 Potentiates IL-33-Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation.

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

Primary care providers' responses to pregnancy intention screening challenges: community-based participatory research at an urban community health centre.

BACKGROUND: Incorporating pregnancy intention screening into primary care to address unmet preconception and contraception needs may improve delivery of family planning services. A notable research gap exists regarding providers' experiences conducting this screening in primary care. OBJECTIVE: To explore primary care providers' perceived challenges in conducting pregnancy intention screening with women of reproductive age and to identify strategies to discuss this in primary care settings. METHODS: This qualitative study emerged from a 2017 community-based participatory research project. We conducted semi-structured, in-depth interviews with 10 primary care providers who care for women of reproductive age at an urban federally qualified health centre. Analysis consisted of interview debriefing, transcript coding and content analysis with the Community Advisory Board. RESULTS: Across departments, respondents acknowledged difficulties conducting pregnancy intention screening and identified strategies for working with patients' individual readiness to discuss pregnancy intention. Strategies included: linking patients' health concerns with sexual and reproductive health, applying a shared decision-making model to all patient-provider interactions, practicing goal setting and motivational interviewing, fostering non-judgmental relationships and introducing pregnancy intention in one visit but following up at later times when more relevant for patients. CONCLUSIONS: Opportunities exist for health centres to address pregnancy intention screening challenges, such as implementing routine screening and waiting room tools to foster provider and patient agency and sharing best practices with providers across departments by facilitating comprehensive training and periodic check-ins. Exploring providers' experiences may assist health centres in improving pregnancy intention screening in the primary care setting.

A systematic literature review of the enabling environment elements to improve implementation of water safety plans in high-income countries.

Effective risk management helps ensure safe drinking water and protect public health. Even in high-income countries, risk management sometimes fails and waterborne disease, including outbreaks, occur. To help reduce waterborne disease, the WHO Guidelines for Drinking Water Quality recommend water safety plans (WSPs), a systematic preventive risk management strategy applied from catchment to consumer. Since the introduction of WSPs, international guidelines, national and state legislation, and local practices have facilitated their implementation. While various high-income OECD countries have documented successes in improving drinking water safety through implementing WSPs, others have little experience. This review synthesizes the elements of the enabling environment that promoted the implementation of WSPs in high-income countries. We show that guidelines, regulations, tools and resources, public health support, and context-specific evidence of the feasibility and benefits of WSPs are elements of the enabling environment that encourage adoption and implementation of WSPs in high-income countries. These findings contribute to understanding the ways in which to increase the uptake and extent of WSPs throughout high-income countries to help improve public health.

Water safety plans: bridges and barriers to implementation in North Carolina.

First developed by the World Health Organization, and now used in several countries, water safety plans (WSPs) are a multi-step, preventive process for managing drinking water hazards. While the beneficial impacts of WSPs have been documented in diverse countries, how to successfully implement WSPs in the United States remains a challenge. We examine the willingness and ability of water utility leaders to implement WSPs in the US state of North Carolina. Our findings show that water utilities have more of a reactive than preventive organizational culture, that implementation requires prioritization of time and resources, perceived comparative advantage to other hazard management plans, leadership in implementation, and identification of how WSPs can be embedded in existing work practices. Future research could focus on whether WSP implementation provides benefits such as decreases in operational costs, and improved organization of records and communication.

Increased ILC2s in the eosinophilic nasal polyp endotype are associated with corticosteroid responsiveness.

Group 2 innate lymphoid cells (ILC2s) have recently been identified in human nasal polyps, but whether numbers of ILC2s differ by polyp endotype or are influenced by corticosteroid use is unknown. Here, we show that eosinophilic nasal polyps contained double the number of ILC2s vs. non-eosinophilic polyps. Polyp ILC2s were also reduced by 50% in patients treated with systemic corticosteroids. Further, using a fungal allergen challenge mouse model, we detected greatly reduced Th2 cytokine-producing and Ki-67+ proliferating lung ILC2s in mice receiving dexamethasone. Finally, ILC2 Annexin V staining revealed extensive apoptosis after corticosteroid treatment in vivo and in vitro. Thus, ILC2s are elevated in the eosinophilic nasal polyp endotype and systemic corticosteroid treatment correlated with reduced polyp ILC2s. Finally, allergen-challenged mice showed reduced ILC2s and increased ILC2 apoptosis after corticosteroid treatment suggesting that ILC2 may be responsive to corticosteroids in eosinophilic respiratory disease.

Innate type 2 response to Alternaria extract enhances ryegrass-induced lung inflammation.

BACKGROUND: Exposure to the fungal allergen Alternaria alternata as well as ryegrass pollen has been implicated in severe asthma symptoms during thunderstorms. We have previously shown that Alternaria extract induces innate type 2 lung inflammation in mice. We hypothesized that the innate eosinophilic response to Alternaria extract may enhance lung inflammation induced by ryegrass. METHODS: Mice were sensitized to ryegrass allergen and administered a single challenge with A. alternata extract before or after final ryegrass challenges. Levels of eosinophils, neutrophils, Th2 cells, innate lymphoid cells (ILC2), interleukin (IL)-5 and IL-13 in bronchoalveolar lavage (BAL) as well as inflammation and mucus were assessed. RESULTS: Mice receiving ryegrass sensitization and challenge developed an eosinophilic lung response. A single challenge with Alternaria extract given 3 days before or 3 days after ryegrass challenges resulted in increased eosinophils, peribronchial inflammation and mucus production in the airways compared with ryegrass-only challenges. Type 2 ILC2 and Th2 cell recruitment to the airways was increased after Alternaria extract exposure in ryegrass-challenged mice. Innate immune challenges with Alternaria extract induced BAL eosinophilia, Th2 cell recruitment as well as ILC2 expansion and proliferation. CONCLUSIONS: A single exposure to Alternaria extract in ryegrass-sensitized and -challenged mice enhances the type 2 lung inflammatory response, including airway eosinophilia, peribronchial infiltrate, and mucus production, possibly through Th2 cell recruitment and ILC2 expansion. If translated to humans, exposure to both grass pollen and Alternaria may be a potential cause of thunderstorm-related asthma.

Sanitation: a global estimate of sewerage connections without treatment and the resulting impact on MDG progress.

Progress toward the sanitation component of Millennium Development Goal (MDG) Target 7c was reassessed to account for the need to protect communities and the wider population from exposure to human excreta. We classified connections to sewerage as "improved sanitation" only if the sewage was treated before discharge to the environment. Sewerage connection data was available for 167 countries in 2010; of these, 77 had published data on sewage treatment prevalence. We developed an empirical model to estimate sewage treatment prevalence for 47 additional countries. We estimate that in 2010, 40% of the global population (2.8 billion people) used improved sanitation, as opposed to the estimate of 62% (4.3 billion people) from the WHO/UNICEF Joint Monitoring Programme (JMP), and that 4.1 billion people lacked access to an improved sanitation facility. Redefining sewerage-without-treatment as "unimproved sanitation" in MDG monitoring would raise the 1990 baseline population using unimproved sanitation from 53% to 64% and the corresponding 2015 target from 27% to 32%. At the current rate of progress, we estimate a shortfall of 28 percentage points (1.9 billion people) in 2010 and a projected 27 percentage point shortfall in 2015.

Impaired induction of allergic lung inflammation by Alternaria alternata mutant MAPK homologue Fus3.

The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies.

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency. IMPORTANCE An effective HIV-1 vaccination strategy will need to stimulate rare precursor B cells of multiple bNAb lineages and affinity mature them along desired pathways. Here, we searched for V2-apex germ line-targeting Envs among a large set of diverse primate lentiviruses and identified minimally modified versions of one chimpanzee SIV Env that bound several human V2-apex bNAb precursors and stimulated one of these in a V2-apex bNAb precursor-expressing knock-in mouse. We also generated chimeric simian-chimpanzee immunodeficiency viruses and showed that they elicit low-titer V2-directed heterologous plasma breadth in six of seven infected rhesus macaques. Characterization of this antibody response identified a new class of weakly cross-reactive neutralizing antibodies that target the V2-apex, but only in occluded-open Env trimers. The existence of this cryptic epitope, which in some Env backgrounds is immunodominant, needs to be considered in immunogen design.

A Unique Spectrum of Spontaneous Tumors in Dino Knockout Mice Identifies Tissue-Specific Requirements for Tumor Suppression.

Here, we report that Dino, a lncRNA required for p53 signaling, suppresses spontaneous tumorigenesis in mice. Dino-/- mice develop significantly more malignant tumors than Dino+/+ littermate controls, consisting predominantly of sarcomas, B cell lymphomas and additional rare tumors. While the prevalence of lymphomas and sarcomas in Dino-/- mice is similar to that of mice with p53 loss, important distinctions emerged. p53-null mice predominantly develop T cell lymphomas; however, no spontaneous T cell lymphoma was observed in Dino-/- mice. Rather than being a phenocopy of the p53-null tumor spectrum, spontaneous tumors in Dino-/- mice resemble the spectrum of human cancers in which DINO is recurrently silenced by methylation in a manner that is mutually exclusive with TP53 alterations, suggesting that similar tissues in human and mouse require DINO for tumor suppression. Consistent with a tissue-specific role for Dino in tumor suppression, loss of Dino had no impact on the development of radiation-induced T cell lymphoma and oncogene-driven medulloblastoma, tumors that are accelerated by the loss of p53. Taken together, these data indicate that Dino serves as a potent tumor suppressor molecule specific to a select subset of tissues in mice and humans.

RNA-binding protein RBM3 intrinsically suppresses lung innate lymphoid cell activation and inflammation partially through CysLT1R.

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3-/- and Rbm3-/-Rag2-/- mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3-/- ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3-/- lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3-/-Cyslt1r-/- mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.