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Monoacylglycerol acyltransferase 2 (MGAT2) is an important enzyme highly expressed in the human small intestine and liver for the regulation of triglyceride absorption and homeostasis. We report that treatment with BMS-963272, a potent and selective MGAT2 inhibitor, decreased inflammation and fibrosis in CDAHFD and STAM, two murine nonalcoholic steatohepatitis (NASH) models. In high-fat-diet-treated cynomolgus monkeys, in contrast to a selective diacylglycerol acyltransferase 1 (DGAT1) inhibitor, BMS-963272 did not cause diarrhea. In a Phase 1 multiple-dose trial of healthy human adults with obesity (NCT04116632), BMS-963272 was safe and well tolerated with no treatment discontinuations due to adverse events. Consistent with the findings in rodent models, BMS-963272 elevated plasma long-chain dicarboxylic acid, indicating robust pharmacodynamic biomarker modulation; increased gut hormones GLP-1 and PYY; and decreased body weight in human subjects. These data suggest MGAT2 inhibition is a promising therapeutic opportunity for NASH, a disease with high unmet medical needs.
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Hepatopatia Gordurosa não Alcoólica , Obesidade , Animais , Humanos , Camundongos , Peso Corporal , Inflamação/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Adulto , Ensaios Clínicos Fase I como AssuntoRESUMO
BACKGROUND: The phase 3 CheckMate 214 trial demonstrated higher response rates and improved overall survival with nivolumab plus ipilimumab versus sunitinib in first-line therapy for advanced clear-cell renal cell carcinoma (RCC). An unmet need exists to identify patients with RCC who are most likely to benefit from treatment with nivolumab plus ipilimumab. METHODS: In exploratory analyses, pretreatment levels of programmed death ligand 1 were assessed by immunohistochemistry. Genomic and transcriptomic biomarkers (including tumor mutational burden and gene expression signatures) were also investigated. RESULTS: Biomarkers previously associated with benefit from immune checkpoint inhibitor-containing regimens in RCC were not predictive for survival in patients with RCC treated with nivolumab plus ipilimumab. Analysis of gene expression identified an association between an inflammatory response and progression-free survival with nivolumab plus ipilimumab. CONCLUSIONS: The exploratory analyses reveal relationships between molecular biomarkers and provide supportive data on how the inflammation status of the tumor microenvironment may be important for identifying predictive biomarkers of response and survival with combination immunotherapy in patients with RCC. Further validation may help to provide biomarker-driven precision treatment for patients with RCC.
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Carcinoma de Células Renais , Neoplasias Renais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/patologia , Ensaios Clínicos Fase III como Assunto , Humanos , Ipilimumab/farmacologia , Ipilimumab/uso terapêutico , Neoplasias Renais/patologia , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Sunitinibe/uso terapêutico , Microambiente TumoralRESUMO
OBJECTIVE: SSc is a rheumatic autoimmune disease affecting roughly 20 000 people worldwide and characterized by excessive collagen accumulation in the skin and internal organs. Despite the high morbidity and mortality associated with SSc, there are no approved disease-modifying agents. Our objective in this study was to explore transcriptomic and model-based drug discovery approaches for SSc. METHODS: In this study, we explored the molecular basis for SSc pathogenesis in a well-studied mouse model of scleroderma. We profiled the skin and lung transcriptomes of mice at multiple timepoints, analysing the differential gene expression that underscores the development and resolution of bleomycin-induced fibrosis. RESULTS: We observed shared expression signatures of upregulation and downregulation in fibrotic skin and lung tissue, and observed significant upregulation of key pro-fibrotic genes including GDF15, Saa3, Cxcl10, Spp1 and Timp1. To identify changes in gene expression in responses to anti-fibrotic therapy, we assessed the effect of TGF-ß pathway inhibition via oral ALK5 (TGF-ß receptor I) inhibitor SB525334 and observed a time-lagged response in the lung relative to skin. We also implemented a machine learning algorithm that showed promise at predicting lung function using transcriptome data from both skin and lung biopsies. CONCLUSION: This study provides the most comprehensive look at the gene expression dynamics of an animal model of SSc to date, provides a rich dataset for future comparative fibrotic disease research, and helps refine our understanding of pathways at work during SSc pathogenesis and intervention.
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Bleomicina , Escleroderma Sistêmico , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Perfilação da Expressão Gênica , Humanos , Pulmão/patologia , Camundongos , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Pele/patologiaRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use. METHODS: We previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform. RESULTS: Comparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥ ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma (fold change 1.92, FDR 0.006) and significantly correlated (Pearson r = - 0.38, p = 0.022) to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis. CONCLUSIONS: Our study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.
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Proteínas Sanguíneas/análise , Perfilação da Expressão Gênica , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/genética , Pulmão/química , Proteoma , Proteômica , Transcriptoma , Biomarcadores/sangue , Estudos de Casos e Controles , Humanos , Fibrose Pulmonar Idiopática/diagnósticoRESUMO
BACKGROUND: Nivolumab is an immune checkpoint inhibitor targeting the programmed death-1 receptor that improves survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). In contrast to other tumor types that respond to immunotherapy, factors such as programmed death ligand-1 (PD-L1) status and tumor mutational burden show limited predictive utility in ccRCC. To address this gap, we report here the first molecular characterization of nivolumab response using paired index lesions, before and during treatment of metastatic ccRCC. METHODS: We analyzed gene expression and T-cell receptor (TCR) clonality using lesion-paired biopsies provided in the CheckMate 009 trial and integrated the results with their PD-L1/CD4/CD8 status, genomic mutation status and serum cytokine assays. Statistical tests included linear mixed models, logistic regression models, Fisher's exact test, and Kruskal-Wallis rank-sum test. RESULTS: We identified transcripts related to response, both at baseline and on therapy, including several that are amenable to peripheral bioassays or to therapeutic intervention. At both timepoints, response was positively associated with T-cell infiltration but not associated with TCR clonality, and some non-Responders were highly infiltrated. Lower baseline T-cell infiltration correlated with elevated transcription of Wnt/ß-catenin signaling components and hypoxia-regulated genes, including the Treg chemoattractant CCL28. On treatment, analysis of the non-responding patients whose tumors were highly T-cell infiltrated suggests association of the RIG-I-MDA5 pathway in their nivolumab resistance. We also analyzed our data using previous transcriptional classifications of ccRCC and found they concordantly identified a molecular subtype that has enhanced nivolumab response but is sunitinib-resistant. CONCLUSION: Our study describes molecular characteristics of response and resistance to nivolumab in patients with metastatic ccRCC, potentially impacting patient selection and first-line treatment decisions. TRIAL REGISTRATION NUMBER: NCT01358721.
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Biomarcadores Tumorais , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Antígeno B7-H1/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Antígenos CD4/genética , Antígenos CD8/genética , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Citocinas/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Neoplasias Renais/sangue , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Mutação , Nivolumabe/efeitos adversos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.
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Idiopathic pulmonary fibrosis (IPF), the scarring of lung parenchyma resulting in the loss of lung function, remains a fatal disease with a significant unmet medical need. Patients with severe IPF often develop acute exacerbations resulting in the rapid deterioration of lung function, requiring transplantation. Understanding the pathophysiological mechanisms contributing to IPF is key to develop novel therapeutic approaches for end-stage disease. We report here RNA-sequencing analyses of lung tissues from a cohort of patients with transplant-stage IPF (n=36), compared with acute lung injury (ALI) (n=11) and nondisease controls (n=19), that reveal a robust gene expression signature unique to end-stage IPF. In addition to extracellular matrix remodelling pathways, we identified pathways associated with T-cell infiltration/activation, tumour development, and cholesterol homeostasis, as well as novel alternatively spliced transcripts that are differentially regulated in the advanced IPF lung versus ALI or nondisease controls. Additionally, we show a subset of genes that are correlated with percent predicted forced vital capacity and could reflect disease severity. Our results establish a robust transcriptomic fingerprint of an advanced IPF lung that is distinct from previously reported microarray signatures of moderate, stable or progressive IPF and identifies hitherto unknown candidate targets and pathways for therapeutic intervention in late-stage IPF as well as biomarkers to characterise disease progression and enable patient stratification.
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Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease affecting ~5 million people globally. We have constructed an accurate model of IPF disease status using elastic net regularized regression on clinical gene expression data. Leveraging whole transcriptome microarray data from 230 IPF and 89 control samples from Yang et al. (2013), sourced from the Lung Tissue Research Consortium (LTRC) and National Jewish Health (NJH) cohorts, we identify an IPF gene expression signature. We performed optimal feature selection to reduce the number of transcripts required by our model to a parsimonious set of 15. This signature enables our model to accurately separate IPF patients from controls. Our model outperforms existing published models when tested with multiple independent clinical cohorts. Our study underscores the utility of elastic nets for gene signature/panel selection which can be used for the construction of a multianalyte biomarker of disease. We also filter the gene sets used for model input to construct a model reliant on secreted proteins. Using this approach, we identify the preclinical bleomycin rat model that is most congruent with human disease at day 21 post-bleomycin administration, contrasting with earlier timepoints suggested by other studies.
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Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fibrose Pulmonar Idiopática/metabolismo , Modelos Biológicos , Transcriptoma , Animais , Biomarcadores/metabolismo , Bleomicina/efeitos adversos , Bleomicina/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Masculino , RatosRESUMO
Precision-cut liver tissue slice (PCLS) contains all major cell types of the liver parenchyma and preserves the original cell-cell and cell-matrix contacts. It represents a promising ex vivo model to study liver fibrosis and test the antifibrotic effect of experimental compounds in a physiological environment. In this study using RNA sequencing, we demonstrated that various pathways functionally related to fibrotic mechanisms were dysregulated in PCLSs derived from rats subjected to bile duct ligation. The activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib each reversed a subset of genes dysregulated in fibrotic PCLSs, and of those genes we identified 608 genes whose expression was reversed by all three compounds. These genes define a molecular signature characterizing many aspects of liver fibrosis pathology and its attenuation in the model. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were further validated as efficacy end points for the evaluation of antifibrotic activity of experimental compounds. Finally, we showed that blockade of αV-integrins with a small molecule inhibitor attenuated the fibrotic phenotype in the model. Overall, our results suggest that the rat fibrotic PCLS model may represent a valuable system for target validation and determining the efficacy of experimental compounds. NEW & NOTEWORTHY We investigated the antifibrotic activity of three compounds, the activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib, in a rat fibrotic precision-cut liver tissue slice model using RNA sequencing analysis. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were then established as efficacy end points to validate the antifibrotic activity of the αV-integrin inhibitor CWHM12. This study demonstrated the value of the rat fibrotic PCLS model for the evaluation of antifibrotic drugs.
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Imidazóis/farmacologia , Indóis/farmacologia , Cirrose Hepática/tratamento farmacológico , Fígado/efeitos dos fármacos , Quinoxalinas/farmacologia , Animais , Biomarcadores/metabolismo , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Fígado/metabolismo , Cirrose Hepática/metabolismo , Masculino , Ratos Sprague-DawleyRESUMO
The translation of novel pulmonary fibrosis therapies from preclinical models into the clinic represents a major challenge demonstrated by the high attrition rate of compounds that showed efficacy in preclinical models but demonstrated no significant beneficial effects in clinical trials. A precision-cut lung tissue slice (PCLS) contains all major cell types of the lung and preserves the original cell-cell and cell-matrix contacts. It represents a promising ex vivo model to study pulmonary fibrosis. In this study, using RNA sequencing, we demonstrated that transforming growth factor-ß1 (TGFß1) induced robust fibrotic responses in the rat PCLS model, as it changed the expression of genes functionally related to extracellular matrix remodeling, cell adhesion, epithelial-to-mesenchymal transition, and various immune responses. Nintedanib, pirfenidone, and sorafenib each reversed a subset of genes modulated by TGFß1, and of those genes we identified 229 whose expression was reversed by all three drugs. These genes define a molecular signature characterizing many aspects of pulmonary fibrosis pathology and its attenuation in the rat PCLS fibrosis model. A panel of 12 genes and three secreted biomarkers, including procollagen I, hyaluronic acid, and WNT1-inducible signaling pathway protein 1 were validated as efficacy end points for the evaluation of antifibrotic activity of experimental compounds. Finally, we showed that blockade of αV-integrins suppressed TGFß1-induced fibrotic responses in the rat PCLS fibrosis model. Overall, our results suggest that the TGFß1-induced rat PCLS fibrosis model may represent a valuable system for target validation and to determine the efficacy of experimental compounds.
Assuntos
Fibrose/tratamento farmacológico , Indóis/farmacologia , Pulmão/efeitos dos fármacos , Piridonas/farmacologia , Animais , Biomarcadores/metabolismo , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismoRESUMO
There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, we establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders antifungal-resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating the protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for treating fungal infectious disease that evades resistance.
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Antifúngicos/farmacologia , Depsipeptídeos/farmacologia , Fungos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/química , Depsipeptídeos/síntese química , Depsipeptídeos/química , Farmacorresistência Fúngica/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Fungos/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , Micoses/microbiologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/química , Serina-Treonina Quinases TOR/metabolismoRESUMO
The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.
Assuntos
Fibrose Cística/genética , Fungos/genética , Microbiota , Neuregulina-1/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Escarro/microbiologia , Adaptação Biológica , Farmacorresistência Fúngica/genética , Humanos , Microbiota/fisiologia , Mutação/genética , Neuregulina-1/genética , Pseudomonas aeruginosa/genéticaRESUMO
Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalone technology with potential utility in a clinical environment to aid in medical decision-making.
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The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.
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Antifúngicos/uso terapêutico , Calcineurina , Farmacorresistência Fúngica/genética , Proteínas de Choque Térmico HSP90 , Aneuploidia , Azóis/farmacologia , Benzoquinonas/farmacologia , Calcineurina/genética , Calcineurina/metabolismo , Inibidores de Calcineurina , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Combinação de Medicamentos , Ergosterol/farmacologia , Evolução Molecular , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tacrolimo/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps â¼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved -1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.DOI:http://dx.doi.org/10.7554/eLife.00078.001.
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Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Haloferax volcanii/genética , Histonas/genética , Nucleossomos/química , Fatores de Transcrição/genética , Evolução Biológica , Montagem e Desmontagem da Cromatina , Replicação do DNA , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Perfilação da Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/metabolismo , Histonas/metabolismo , Anotação de Sequência Molecular , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
BACKGROUND: Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens. RESULTS: Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens. CONCLUSION: Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.
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Testes Genéticos/métodos , Genômica/métodos , Animais , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , Controle de Qualidade , Interferência de RNA , Saccharomyces cerevisiae/genética , SoftwareRESUMO
Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach.
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Sistemas de Liberação de Medicamentos , Genômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Antifúngicos/farmacologia , Desenho de Fármacos , Perfilação da Expressão Gênica/métodos , Genoma Fúngico , Humanos , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity. RESULTS: We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO) collection used for screens of pooled yeast (Saccharomyces cerevisiae) deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 microm features sizes independently identified the primary target of tunicamycin to be ALG7. CONCLUSION: We show that the data obtained with 5 microm feature size is of comparable quality to the 30 microm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density.
Assuntos
Hibridização Genômica Comparativa , Genômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Fúngico/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , TunicamicinaRESUMO
OBJECTIVE: To evaluate the influence of acute autologous blood transfusion on postcardiopulmonary bypass coagulation disturbances evaluated by thromboelastography (TEG) as a point-of-care test. METHODS: This prospective randomized controlled study included consecutive patients who underwent elective cardiac surgery with cardiopulmonary bypass. The patients in group A underwent acute autologous blood transfusion with acute normovolemic haemodilution and those in group H received homologous blood, if needed, and served as controls. RESULTS: A total of 62 patients, from 68 enrolled, completed the study: 27 in group A and 35 in group H. Both groups had similar prolongation of prothrombin time and partial thromboplastin time, decreased platelets count and changes in postoperative thromboelastographic variables. There were no differences between them for postoperative bleeding, blood transfusions or haemoglobin values. There was significant prolongation of the R value of TEG (without heparinase) in both groups at 4 h after surgery compared with the immediate postoperative values: from 11.3 +/- 4.2 to 12.3 +/- 5.5 mm, P < 0.05 for group A and from 9.9 +/- 3.7 to 12.5 +/- 5.4 mm, P < 0.01 for group H. The R values of TEG with and without heparinase differed significantly (P < 0.05) at 4 h postoperatively. CONCLUSION: Autologous blood transfusion of 15% estimated blood volume did not affect postcardiopulmonary bypass coagulopathy, nor did it decrease blood loss or homologous blood and its products transfusion in the early postoperative period. TEG is a valuable measure for detecting coagulation dysfunction with a potential role in the postoperative management of cardiac patients.