RESUMO
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
Assuntos
Células Epiteliais Alveolares/metabolismo , Enterócitos/metabolismo , Células Caliciformes/metabolismo , Interferon Tipo I/metabolismo , Mucosa Nasal/citologia , Peptidil Dipeptidase A/genética , Adolescente , Células Epiteliais Alveolares/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/fisiologia , COVID-19 , Linhagem Celular , Células Cultivadas , Criança , Infecções por Coronavirus/virologia , Enterócitos/imunologia , Células Caliciformes/imunologia , Infecções por HIV/imunologia , Humanos , Influenza Humana/imunologia , Interferon Tipo I/imunologia , Pulmão/citologia , Pulmão/patologia , Macaca mulatta , Camundongos , Mycobacterium tuberculosis , Mucosa Nasal/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/genética , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Análise de Célula Única , Tuberculose/imunologia , Regulação para CimaRESUMO
Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.
Assuntos
Cromotripsia , Proteoma , Humanos , Proteoma/genética , Proteoma/metabolismo , Proteômica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA/genéticaRESUMO
Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.
Assuntos
Plasticidade Celular , Células Epiteliais , Regeneração , Mucosa Respiratória , Células-Tronco , Animais , Feminino , Humanos , Masculino , Camundongos , Células Epiteliais/citologia , Células Epiteliais/patologia , Metaplasia/etiologia , Metaplasia/patologia , Mucosa Respiratória/citologia , Mucosa Respiratória/lesões , Mucosa Respiratória/patologia , Células-Tronco/citologia , Tretinoína/metabolismo , Tretinoína/farmacologia , Vitamina A/metabolismo , Vitamina A/farmacologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos C57BLRESUMO
The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Células Epiteliais/metabolismo , Animais , Asma/genética , Células Epiteliais/citologia , Feminino , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Humanos , Pulmão/citologia , Masculino , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Traqueia/citologiaRESUMO
The Hippo pathway is important for regulating tissue homeostasis, and its dysregulation has been implicated in human cancer. However, it is not well understood how the Hippo pathway becomes dysregulated because few mutations in core Hippo pathway components have been identified. Therefore, much work in the Hippo field has focused on identifying upstream regulators, and a complex Hippo interactome has been identified. Nevertheless, it is not always clear which components are the most physiologically relevant in regulating YAP/TAZ. To provide an overview of important Hippo pathway components, we created knockout cell lines for many of these components and compared their relative contributions to YAP/TAZ regulation in response to a wide range of physiological signals. By this approach, we provide an overview of the functional importance of many Hippo pathway components and demonstrate NF2 and RHOA as important regulators of YAP/TAZ and TAOK1/3 as direct kinases for LATS1/2.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Transdução de Sinais/genética , Aciltransferases , Proteínas de Ciclo Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Silenciamento de Genes , Células HEK293 , Via de Sinalização Hippo , Humanos , Neurofibromina 2 , Proteínas Nucleares , Fosforilação , Proteínas Serina-Treonina Quinases , Fatores de Transcrição , Proteínas Supressoras de Tumor , Proteína rhoA de Ligação ao GTPRESUMO
UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified 'mitochondrial proteins' as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44-targeting defects were rescued by reexpression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Proteínas Relacionadas à Autofagia/genética , Demência Frontotemporal/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação , Animais , Linhagem Celular , Modelos Animais de Doenças , Células HeLa , Humanos , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/genética , Proteômica/métodosRESUMO
Olfactory neuroblastoma (ONB, esthesioneuroblastoma) is a sinonasal cancer with an underdeveloped diagnostic toolkit, and is the subject of many incidents of tumor misclassification throughout the literature. Despite its name, connections between the cancer and normal cells of the olfactory epithelium have not been systematically explored and markers of olfactory epithelial cell types are not deployed in clinical practice. Here, we utilize an integrated human-mouse single-cell atlas of the nasal mucosa, including the olfactory epithelium, to identify transcriptomic programs that link ONB to a specific population of stem/progenitor cells known as olfactory epithelial globose basal cells (GBCs). Expression of a GBC transcription factor NEUROD1 distinguishes both low- and high-grade ONB from sinonasal undifferentiated carcinoma, a potential histologic mimic with a distinctly unfavorable prognosis. Furthermore, we identify a reproducible subpopulation of highly proliferative ONB cells expressing the GBC stemness marker EZH2, suggesting that EZH2 inhibition may play a role in the targeted treatment of ONB. Finally, we study the cellular states comprising ONB parenchyma using single-cell transcriptomics and identify evidence of a conserved GBC transcriptional regulatory circuit that governs divergent neuronal-versus-sustentacular differentiation. These results link ONB to a specific cell type for the first time and identify conserved developmental pathways within ONB that inform diagnostic, prognostic, and mechanistic investigation.
Assuntos
Estesioneuroblastoma Olfatório , Neoplasias Nasais , Neoplasias dos Seios Paranasais , Humanos , Camundongos , Animais , Estesioneuroblastoma Olfatório/diagnóstico , Estesioneuroblastoma Olfatório/metabolismo , Estesioneuroblastoma Olfatório/patologia , Neoplasias dos Seios Paranasais/patologia , Neurônios/patologia , Neoplasias Nasais/genética , Neoplasias Nasais/diagnóstico , Cavidade Nasal/metabolismo , Cavidade Nasal/patologiaRESUMO
tRNAscan-SE has been widely used for transfer RNA (tRNA) gene prediction for over twenty years, developed just as the first genomes were decoded. With the massive increase in quantity and phylogenetic diversity of genomes, the accurate detection and functional prediction of tRNAs has become more challenging. Utilizing a vastly larger training set, we created nearly one hundred specialized isotype- and clade-specific models, greatly improving tRNAscan-SE's ability to identify and classify both typical and atypical tRNAs. We employ a new comparative multi-model strategy where predicted tRNAs are scored against a full set of isotype-specific covariance models, allowing functional prediction based on both the anticodon and the highest-scoring isotype model. Comparative model scoring has also enhanced the program's ability to detect tRNA-derived SINEs and other likely pseudogenes. For the first time, tRNAscan-SE also includes fast and highly accurate detection of mitochondrial tRNAs using newly developed models. Overall, tRNA detection sensitivity and specificity is improved for all isotypes, particularly those utilizing specialized models for selenocysteine and the three subtypes of tRNA genes encoding a CAU anticodon. These enhancements will provide researchers with more accurate and detailed tRNA annotation for a wider variety of tRNAs, and may direct attention to tRNAs with novel traits.
Assuntos
RNA de Transferência/genética , Análise de Sequência de DNA/métodos , Software , Genes Arqueais , Genes Bacterianos , Genes FúngicosRESUMO
RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.
Assuntos
Cardiomegalia/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Guanilato Ciclase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Aorta , Autofagia/fisiologia , Benzoatos/metabolismo , Compostos de Bifenilo/metabolismo , Constrição Patológica , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Cisteína/metabolismo , Endotelina-1/farmacologia , Ativação Enzimática , Everolimo/farmacologia , Técnicas de Introdução de Genes , Hidrocarbonetos Fluorados/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Estresse Oxidativo , Fosforilação , Pressão , Proteostase , Ratos , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
The immaturity of pluripotent stem cell (PSC)-derived tissues has emerged as a universal problem for their biomedical applications. While efforts have been made to generate adult-like cells from PSCs, direct benchmarking of PSC-derived tissues against in vivo development has not been established. Thus, maturation status is often assessed on an ad-hoc basis. Single cell RNA-sequencing (scRNA-seq) offers a promising solution, though cross-study comparison is limited by dataset-specific batch effects. Here, we developed a novel approach to quantify PSC-derived cardiomyocyte (CM) maturation through transcriptomic entropy. Transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation, and other potential batch effects. With this new model, we analyzed over 45 scRNA-seq datasets and over 52,000 CMs, and established a cross-study, cross-species CM maturation reference. This reference enabled us to directly compare PSC-CMs with the in vivo developmental trajectory and thereby to quantify PSC-CM maturation status. We further found that our entropy-based approach can be used for other cell types, including pancreatic beta cells and hepatocytes. Our study presents a biologically relevant and interpretable metric for quantifying PSC-derived tissue maturation, and is extensible to numerous tissue engineering contexts.
Assuntos
Benchmarking , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Análise de Célula Única/métodos , Transcriptoma , Expressão Gênica , Hepatócitos/citologia , Humanos , Células Secretoras de Insulina/citologia , Análise de Sequência de RNA/métodos , Engenharia TecidualRESUMO
Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration â¼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.
Assuntos
Cardiomegalia/tratamento farmacológico , Nefroesclerose/tratamento farmacológico , Canal de Cátion TRPC6/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Fibrose , Células HEK293 , Coração/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , CamundongosRESUMO
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.
Assuntos
Proteínas de Transporte/genética , Contração Miocárdica/genética , Miocárdio/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Coração/diagnóstico por imagem , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Fosforilação , Sarcômeros/metabolismoRESUMO
Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as "likely" or "real." This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.
Assuntos
Fibras Musculares de Contração Lenta , Doenças Musculares , Humanos , Fibras Musculares de Contração Rápida , Músculo Esquelético , ProteômicaRESUMO
BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab (IFX) levels in inflammatory bowel disease (IBD) patients can help to guide dose adjustments or changes to therapy for selected patients in remission or with secondary loss of response (LOR). AIMS: To determine how IFX TDM is utilised in a real-life clinical setting and to quantify the potential for TDM to reduce the unnecessary use of IFX. METHODS: Data from all public IBD IFX level testing performed across Australia were prospectively collected from June 2016 to July 2017 to assess physician-reported for testing indications (induction, in remission or LOR) and associated results. The hypothetical influence of IFX TDM was based on an optimal therapeutic range of 6-10 mg/L for mucosal healing. RESULTS: Secondary LOR (reactive TDM) was the most common indication for TDM. These patients have consistently lower median IFX levels: 3.02 mg/L (IQR 1.14-6.67 mg/L) versus 5.22 mg/L (IQR 2.70-8.12 mg/L), P = 0.0001 compared with patients in remission (proactive TDM). TDM helped to identify unnecessary use of IFX in 30.6% of the TDM tests performed in luminal Crohn disease and ulcerative colitis patients, with an associated drug cost saving of $531.38 per IFX TDM test episode. Unnecessary IFX use was identified in 38.9% (96/247) of reactive IFX TDM tests performed and in 19.3% (35/181) of proactive testing. CONCLUSION: Use of both reactive and proactive IFX TDM is cost-effective for IBD management as it informs the clinician where unnecessary use of IFX can be stopped.
Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Austrália , Colite Ulcerativa/tratamento farmacológico , Redução de Custos , Monitoramento de Medicamentos , Fármacos Gastrointestinais/uso terapêutico , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab/uso terapêuticoRESUMO
Transfer RNAs (tRNAs) are ubiquitous across the tree of life. Although tRNA structure is highly conserved, there is still significant variation in sequence features between clades, isotypes and even isodecoders. This variation not only impacts translation, but as shown by a variety of recent studies, nontranslation-associated functions are also sensitive to small changes in tRNA sequence. Despite the rapidly growing number of sequenced genomes, there is a lack of tools for both small- and large-scale comparative genomics analysis of tRNA sequence features. Here, we have integrated over 150 000 tRNAs spanning all domains of life into tRNAviz, a web application for exploring and visualizing tRNA sequence features. tRNAviz implements a framework for determining consensus sequence features and can generate sequence feature distributions by isotypes, clades and anticodons, among other tRNA properties such as score. All visualizations are interactive and exportable. The web server is publicly available at http://trna.ucsc.edu/tRNAviz/.
Assuntos
RNA de Transferência/química , Software , Sequência de Bases , Gráficos por Computador , Sequência Consenso , RNA Arqueal/química , RNA Bacteriano/química , RNA de Transferência/classificação , Análise de Sequência de RNARESUMO
Neurons in the murine olfactory epithelium (OE) differ by the olfactory receptor they express as well as other molecular phenotypes that are regionally restricted. These patterns can be precisely regenerated following epithelial injury, suggesting that spatial cues within the tissue can direct neuronal diversification. Nonetheless, the permanency and mechanism of this spatial patterning remain subject to debate. Via transplantation of stem and progenitor cells from dorsal OE into ventral OE, we demonstrate that, in mice of both sexes, nonautonomous spatial cues can direct the spatially circumscribed differentiation of olfactory sensory neurons. The vast majority of dorsal transplant-derived neurons express the ventral marker OCAM (NCAM2) and lose expression of NQO1 to match their new location. Single-cell analysis also demonstrates that OSNs adopt a fate defined by their new position following progenitor cell transplant, such that a ventral olfactory receptor is expressed after stem and progenitor cell engraftment. Thus, spatially constrained differentiation of olfactory sensory neurons is plastic, and any bias toward an epigenetic memory of place can be overcome.SIGNIFICANCE STATEMENT Spatially restricted differentiation of olfactory sensory neurons is both key to normal olfactory function and a challenging example of biological specificity. That the stem cells of the olfactory epithelium reproduce the organization of the olfactory periphery to a very close approximation during lesion-induced regeneration begs the question of whether stem cell-autonomous genomic architecture or environmental cues are responsible. The plasticity demonstrated after transfer to a novel location suggests that cues external to the transplanted stem and progenitor cells confer neuronal identity. Thus, a necessary prerequisite is satisfied for using engraftment of olfactory stem and progenitor cells as a cellular therapeutic intervention to reinvigorate neurogenesis whose exhaustion contributes to the waning of olfaction with age.
Assuntos
Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/fisiologia , Animais , Diferenciação Celular/fisiologia , Sinais (Psicologia) , Epigênese Genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Moléculas de Adesão de Célula Nervosa/biossíntese , Moléculas de Adesão de Célula Nervosa/genética , Células-Tronco Neurais , Neurogênese/fisiologia , Plasticidade Neuronal , Transplante de Células-TroncoRESUMO
The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, whereas Ser2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. We found that Ssu72 dephosphorylates pSer5 effectively but only has low activities toward pSer7 and pSer2 The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ß-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. Our results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II that occur at different stages of transcription.
Assuntos
Proteínas de Drosophila/química , Proteínas Tirosina Fosfatases/química , RNA Polimerase II/química , Motivos de Aminoácidos , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
The remarkable capacity of the adult olfactory epithelium (OE) to regenerate fully both neurosensory and nonneuronal cell types after severe epithelial injury depends on life-long persistence of two stem cell populations: the horizontal basal cells (HBCs), which are quiescent and held in reserve, and mitotically active globose basal cells. It has recently been demonstrated that down-regulation of the ΔN form of the transcription factor p63 is both necessary and sufficient to release HBCs from dormancy. However, the mechanisms by which p63 is down-regulated after acute OE injury remain unknown. To identify the cellular source of potential signaling mechanisms, we assessed HBC activation after neuron-only and sustentacular cell death. We found that ablation of sustentacular cells is sufficient for HBC activation to multipotency. By expression analysis, next-generation sequencing, and immunohistochemical examination, down-regulation of Notch pathway signaling is coincident with HBC activation. Therefore, using HBC-specific conditional knockout of Notch receptors and overexpression of N1ICD, we show that Notch signaling maintains p63 levels and HBC dormancy, in contrast to its suppression of p63 expression in other tissues. Additionally, Notch1, but not Notch2, is required to maintain HBC dormancy after selective neuronal degeneration. Taken together, our data indicate that the activation of HBCs observed after tissue injury or sustentacular cell ablation is caused by the reduction/elimination of Notch signaling on HBCs; elimination of Jagged1 expressed by sustentacular cells may be the ligand responsible.
Assuntos
Células-Tronco Neurais/citologia , Mucosa Olfatória/citologia , Fosfoproteínas/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transativadores/metabolismo , Animais , Morte Celular , Biologia Computacional , Genótipo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , TranscriptomaRESUMO
BACKGROUND: Shoulder instability in young athletes is a complex problem with higher recurrence, higher reoperation, and lower return to sport (RTS) rates after arthroscopic shoulder stabilization compared with adults. METHODS: This is a prospective case series of young athletes with anterior shoulder instability after arthroscopic stabilization surgery. Primary outcomes were RTS and revision surgery, minimum follow-up was 24 months. Exclusion criteria were more than 3 preoperative episodes of instability, significant bone loss, or primary posterior instability. Demographic data, recurrent instability, revision surgery, sports pre- and postsurgery, patient satisfaction, level of RTS, time to RTS, and Single Assessment Numeric Evaluation (SANE) scores were analyzed. RESULTS: Sixty-seven athletes met inclusion criteria, 19 females and 48 males, with a mean age of 17.5 years (range, 13-21 years). Fifty-nine (88%) athletes returned to sport at an average of 7.1 months (standard deviation, ±1.8); 50 (75%) returned to the same level or higher. Football and lacrosse were the most common sports. Four of 67 athletes (6%), all male, underwent revision stabilization at 11-36 months for recurrent instability. The overall mean SANE score was 88. CONCLUSION: This study demonstrates that when the high-risk athlete, 21 years old or younger, is appropriately selected for arthroscopic shoulder stabilization by excluding those with 3 or more preoperative shoulder instability episodes and those with off-track and engaging instability patterns, excellent outcomes can be achieved with low revision surgery rates, high RTS rates, and high patient satisfaction.
Assuntos
Artroscopia , Instabilidade Articular/cirurgia , Reoperação/estatística & dados numéricos , Volta ao Esporte , Luxação do Ombro/cirurgia , Articulação do Ombro/cirurgia , Adolescente , Adulto , Feminino , Seguimentos , Humanos , Masculino , Estudos Prospectivos , Adulto JovemRESUMO
Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.