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MOTIVATION: Functional interpretation of high-throughput metabolomic and transcriptomic results is a crucial step in generating insight from experimental data. However, pathway and functional information for genes and metabolites are distributed among many siloed resources, limiting the scope of analyses that rely on a single knowledge source. RESULTS: RaMP-DB 2.0 is a web interface, relational database, API and R package designed for straightforward and comprehensive functional interpretation of metabolomic and multi-omic data. RaMP-DB 2.0 has been upgraded with an expanded breadth and depth of functional and chemical annotations (ClassyFire, LIPID MAPS, SMILES, InChIs, etc.), with new data types related to metabolites and lipids incorporated. To streamline entity resolution across multiple source databases, we have implemented a new semi-automated process, thereby lessening the burden of harmonization and supporting more frequent updates. The associated RaMP-DB 2.0 R package now supports queries on pathways, common reactions (e.g. metabolite-enzyme relationship), chemical functional ontologies, chemical classes and chemical structures, as well as enrichment analyses on pathways (multi-omic) and chemical classes. Lastly, the RaMP-DB web interface has been completely redesigned using the Angular framework. AVAILABILITY AND IMPLEMENTATION: The code used to build all components of RaMP-DB 2.0 are freely available on GitHub at https://github.com/ncats/ramp-db, https://github.com/ncats/RaMP-Client/ and https://github.com/ncats/RaMP-Backend. The RaMP-DB web application can be accessed at https://rampdb.nih.gov/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Metabolómica , Programas Informáticos , Bases de Datos Factuales , Perfilación de la Expresión Génica , Bases del Conocimiento , ProteínasRESUMEN
Understanding the molecular underpinnings of disease severity and progression in human studies is necessary to develop metabolism-related preventative strategies for severe COVID-19. Metabolites and metabolic pathways that predispose individuals to severe disease are not well understood. In this study, we generated comprehensive plasma metabolomic profiles in >550 patients from the Longitudinal EMR and Omics COVID-19 Cohort. Samples were collected before (n = 441), during (n = 86), and after (n = 82) COVID-19 diagnosis, representing 555 distinct patients, most of which had single timepoints. Regression models adjusted for demographics, risk factors, and comorbidities, were used to determine metabolites associated with predisposition to and/or persistent effects of COVID-19 severity, and metabolite changes that were transient/lingering over the disease course. Sphingolipids/phospholipids were negatively associated with severity and exhibited lingering elevations after disease, while modified nucleotides were positively associated with severity and had lingering decreases after disease. Cytidine and uridine metabolites, which were positively and negatively associated with COVID-19 severity, respectively, were acutely elevated, reflecting the particular importance of pyrimidine metabolism in active COVID-19. This is the first large metabolomics study using COVID-19 plasma samples before, during, and/or after disease. Our results lay the groundwork for identifying putative biomarkers and preventive strategies for severe COVID-19.
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COVID-19 , Nucleótidos , Humanos , Quinurenina , Prueba de COVID-19 , Estudios Prospectivos , FosfolípidosRESUMEN
Understanding relationships between spontaneous cancer in companion (pet) canines and humans can facilitate biomarker and drug development in both species. Towards this end we developed an experimental-bioinformatic protocol that analyzes canine transcriptomics data in the context of existing human data to evaluate comparative relevance of canine to human cancer. We used this protocol to characterize five canine cancers: melanoma, osteosarcoma, pulmonary carcinoma, B- and T-cell lymphoma, in 60 dogs. We applied an unsupervised, iterative clustering method that yielded five co-expression modules and found that each cancer exhibited a unique module expression profile. We constructed cancer models based on the co-expression modules and used the models to successfully classify the canine data. These canine-derived models also successfully classified human tumors representing the same cancers, indicating shared cancer biology between canines and humans. Annotation of the module genes identified cancer specific pathways relevant to cells-of-origin and tumor biology. For example, annotations associated with melanin production (PMEL, GPNMB, and BACE2), synthesis of bone material (COL5A2, COL6A3, and COL12A1), synthesis of pulmonary surfactant (CTSH, LPCAT1, and NAPSA), ribosomal proteins (RPL8, RPS7, and RPLP0), and epigenetic regulation (EDEM1, PTK2B, and JAK1) were unique to melanoma, osteosarcoma, pulmonary carcinoma, B- and T-cell lymphoma, respectively. In total, 152 biomarker candidates were selected from highly expressing modules for each cancer type. Many of these biomarker candidates are under-explored as drug discovery targets and warrant further study. The demonstrated transferability of classification models from canines to humans enforces the idea that tumor biology, biomarker targets, and associated therapeutics, discovered in canines, may translate to human medicine.
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Enfermedades de los Perros/genética , Redes Reguladoras de Genes , Neoplasias/genética , Neoplasias/veterinaria , Animales , Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Neoplasias Óseas/veterinaria , Biología Computacional , Enfermedades de los Perros/clasificación , Perros , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/veterinaria , Linfoma de Células B/genética , Linfoma de Células B/veterinaria , Linfoma de Células T/genética , Linfoma de Células T/veterinaria , Melanoma/genética , Melanoma/veterinaria , Anotación de Secuencia Molecular , Terapia Molecular Dirigida , Neoplasias/clasificación , Oncogenes , Osteosarcoma/genética , Osteosarcoma/veterinaria , Especificidad de la Especie , Investigación Biomédica TraslacionalRESUMEN
To clarify how smoking leads to heart attack and stroke, we developed an endothelial cell model (iECs) generated from human induced Pluripotent Stem Cells (iPSC) and evaluated its responses to tobacco smoke. These iECs exhibited a uniform endothelial morphology, and expressed markers PECAM1/CD31, VWF/ von Willebrand Factor, and CDH5/VE-Cadherin. The iECs also exhibited tube formation and acetyl-LDL uptake comparable to primary endothelial cells (EC). RNA sequencing (RNA-Seq) revealed a robust correlation coefficient between iECs and EC (R = 0.76), whereas gene responses to smoke were qualitatively nearly identical between iECs and primary ECs (R = 0.86). Further analysis of transcriptional responses implicated 18 transcription factors in regulating responses to smoke treatment, and identified gene sets regulated by each transcription factor, including pathways for oxidative stress, DNA damage/repair, ER stress, apoptosis, and cell cycle arrest. Assays for 42 cytokines in HUVEC cells and iECs identified 23 cytokines that responded dynamically to cigarette smoke. These cytokines and cellular stress response pathways describe endothelial responses for lymphocyte attachment, activation of coagulation and complement, lymphocyte growth factors, and inflammation and fibrosis; EC-initiated events that collectively lead to atherosclerosis. Thus, these studies validate the iEC model and identify transcriptional response networks by which ECs respond to tobacco smoke. Our results systematically trace how ECs use these response networks to regulate genes and pathways, and finally cytokine signals to other cells, to initiate the diverse processes that lead to atherosclerosis and cardiovascular disease.
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Células Endoteliales/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Modelos Biológicos , Fumar Tabaco/efectos adversos , Citocinas/análisis , Células Endoteliales/patología , Humanos , Células Madre Pluripotentes Inducidas/patologíaRESUMEN
High-throughput screening (HTS) of small-molecule libraries accelerates the discovery of chemical leads to serve as starting points for probe or therapeutic development. With this approach, thousands of unique small molecules, representing a diverse chemical space, can be rapidly evaluated by biologically and physiologically relevant assays. The origins of numerous United States Food and Drug Administration-approved cancer drugs are linked to HTS, which emphasizes the value in this methodology. The National Institutes of Health Molecular Libraries Program made HTS accessible to the public sector, enabling the development of chemical probes and drug-repurposing initiatives. In this work, the impact of HTS in the field of oncology is considered among both private and public sectors. Examples are given for the discovery and development of approved cancer drugs. The importance of target validation is discussed, and common assay approaches for screening are reviewed. A rigorous examination of the PubChem database demonstrates that public screening centers are contributing to early-stage drug discovery in oncology by focusing on new targets and developing chemical probes. Several case studies highlight the value of different screening strategies and the potential for drug repurposing.
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Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Aprobación de Drogas , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMEN
A chemical genomics "Toxmatrix" method was developed to elucidate mechanisms of cytotoxicity using neuronal models. Quantitative high-throughput screening (qHTS) was applied to systematically screen each toxicant against a panel of 70 modulators, drugs or chemicals that act on a known target, to identify interactions that either protect or sensitize cells to each toxicant. Thirty-two toxicants were tested at 10 concentrations for cytotoxicity to SH-SY5Y human neuroblastoma cells, with results fitted to the Hill equation to determine an IC50 for each toxicant. Thirty-three toxicant:modulator interactions were identified in SH-SY5Y cells for 14 toxicants, as modulators that shifted toxicant IC50 values lower or higher. The target of each modulator that sensitizes cells or protects cells from a toxicant suggests a mode of toxicant action or cellular adaptation. In secondary screening, we tested modulator-toxicant pairs identified from the SH-SY5Y primary screening for interactions in three differentiated neuronal human cell lines: dSH-SY5Y, conditionally immortalized dopaminergic neurons (LUHMES), and neural stem cells. Twenty toxicant-modulator pairs showed pronounced interactions in one or several differentiated cell models. Additional testing confirmed that several modulators acted through their primary targets. For example, several chelators protected differentiated LUHMES neurons from four toxicants by chelation of divalent cations and buthionine sulphoximine sensitized cells to 6-hydroxydopamine and 4-(methylamino)phenol hemisulfate by blocking glutathione synthesis. Such modulators that interact with multiple neurotoxicants suggest these may be vulnerable toxicity pathways in neurons. Thus, the Toxmatrix method is a systematic high-throughput approach that can identify mechanisms of toxicity and cellular adaptation.
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Neuronas Dopaminérgicas/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Genómica , Ensayos Analíticos de Alto Rendimiento , Células-Madre Neurales/efectos de los fármacos , Neurotoxinas/toxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Humanos , Células-Madre Neurales/metabolismoRESUMEN
Three-dimensional (3D) cell culture in vitro promises to improve representation of neuron physiology in vivo. This inspired development of a 3D culture platform for LUHMES (Lund Human Mesencephalic) dopaminergic neurons for high-throughput screening (HTS) of chemicals for neurotoxicity. Three culture platforms, adhesion (2D-monolayer), 3D-suspension, and 3D-shaken, were compared to monitor mRNA expression of seven neuronal marker genes, DCX, DRD2, ENO2, NEUROD4, SYN1, TH, and TUBB3. These seven marker genes reached similar maxima in all three formats, with the two 3D platforms showing similar kinetics, whereas several markers peaked earlier in 2D adhesion compared to both 3D culture platforms. The differentiated LUHMES (dLUHMES) neurons treated with ziram, methylmercury or thiram dynamically increased expression of metallothionein biomarker genes MT1G, MT1E and MT2A at 6 h. These gene expression increases were generally more dynamic in 2D adhesion cultures than in 3D cultures, but were generally comparable between 3D-suspension and 3D-u plate (low binding) platforms. Finally, we adapted 3D-suspension culture of dLUHMES and neural stem cells to 1536 well plates with a HTS cytotoxicity assay. This HTS assay revealed that cytotoxicity IC50 values were not significantly different between adhesion and 3D-suspension platforms for 31 of 34 (91%) neurotoxicants tested, whereas IC50 values were significantly different for at least two toxicants. In summary, the 3D-suspension culture platform for LUHMES dopaminergic neurons supported full differentiation and reproducible assay results, enabling quantitative HTS (qHTS) for cytotoxicity in 1536 well format with a Robust Z' score of 0.68.
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Neuronas Dopaminérgicas , Ensayos Analíticos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento/métodos , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Técnicas de Cultivo Tridimensional de Células/métodos , Biomarcadores/metabolismo , Compuestos de Metilmercurio/toxicidad , Neurotoxinas/toxicidad , Línea Celular , Células CultivadasRESUMEN
Epidemiology studies evaluate associations between the metabolome and disease risk. Urine is a common biospecimen used for such studies due to its wide availability and non-invasive collection. Evaluating the robustness of urinary metabolomic profiles under varying preanalytical conditions is thus of interest. Here we evaluate the impact of sample handling conditions on urine metabolome profiles relative to the gold standard condition (no preservative, no refrigeration storage, single freeze thaw). Conditions tested included the use of borate or chlorhexidine preservatives, various storage and freeze/thaw cycles. We demonstrate that sample handling conditions impact metabolite levels, with borate showing the largest impact with 125 of 1,048 altered metabolites (adjusted P < 0.05). When simulating a case-control study with expected inconsistencies in sample handling, we predicted the occurrence of false positive altered metabolites to be low (< 11). Predicted false positives increased substantially (³63) when cases were simulated to undergo alternate handling. Finally, we demonstrate that sample handling impacts on the urinary metabolome were markedly smaller than those in serum. While changes in urine metabolites incurred by sample handling are generally small, we recommend implementing consistent handling conditions and evaluating robustness of metabolite measurements for those showing significant associations with disease outcomes.
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Serum total immunoglobulin E levels (total IgE) capture the state of the immune system in relation to allergic sensitization. High levels are associated with airway obstruction and poor clinical outcomes in pediatric asthma. Inconsistent patient response to anti-IgE therapies motivates discovery of molecular mechanisms underlying serum IgE level differences in children with asthma. To uncover these mechanisms using complementary metabolomic and transcriptomic data, abundance levels of 529 named metabolites and expression levels of 22,772 genes were measured among children with asthma in the Childhood Asthma Management Program (CAMP, N=564) and the Genetic Epidemiology of Asthma in Costa Rica Study (GACRS, N=309) via the TOPMed initiative. Gene-metabolite associations dependent on IgE were identified within each cohort using multivariate linear models and were interpreted in a biochemical context using network topology, pathway and chemical enrichment, and representation within reactions. A total of 1,617 total IgE-dependent gene-metabolite associations from GACRS and 29,885 from CAMP met significance cutoffs. Of these, glycine and guanidinoacetic acid (GAA) were associated with the most genes in both cohorts, and the associations represented reactions central to glycine, serine, and threonine metabolism and arginine and proline metabolism. Pathway and chemical enrichment analysis further highlighted additional related pathways of interest. The results of this study suggest that GAA may modulate total IgE levels in two independent pediatric asthma cohorts with different characteristics, supporting the use of L-Arginine as a potential therapeutic for asthma exacerbation. Other potentially new targetable pathways are also uncovered.
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UNLABELLED: High-throughput technologies can identify genes whose expression profiles correlate with specific phenotypes; however, placing these genes into a biological context remains challenging. To help address this issue, we developed nested Expression Analysis Systematic Explorer (nEASE). nEASE complements traditional gene ontology enrichment approaches by determining statistically enriched gene ontology subterms within a list of genes based on co-annotation. Here, we overview an open-source software version of the nEASE algorithm. nEASE can be used either stand-alone or as part of a pathway discovery pipeline. AVAILABILITY: nEASE is implemented within the Multiple Experiment Viewer software package available at http://www.tm4.org/mev. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Algoritmos , Perfilación de la Expresión Génica/métodos , Programas Informáticos , Humanos , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Vocabulario ControladoRESUMEN
High throughput screening (HTS) is widely used in drug discovery and chemical biology to identify and characterize agents having pharmacologic properties often by evaluation of large chemical libraries. Standard HTS data can be simply plotted as an x-y graph usually represented as % activity of a compound tested at a single concentration vs compound ID, whereas quantitative HTS (qHTS) data incorporates a third axis represented by concentration. By virtue of the additional data points arising from the compound titration and the incorporation of logistic fit parameters that define the concentration-response curve, such as EC50 and Hill slope, qHTS data has been challenging to display on a single graph. Here we provide a flexible solution to the rapid plotting of complete qHTS data sets to produce a 3-axis plot we call qHTS Waterfall Plots. The software described here can be generally applied to any 3-axis dataset and is available as both an R package and an R shiny application.
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Canine osteosarcoma is increasingly recognized as an informative model for human osteosarcoma. Here we show in one of the largest clinically annotated canine osteosarcoma transcriptional datasets that two previously reported, as well as de novo gene signatures devised through single sample Gene Set Enrichment Analysis (ssGSEA), have prognostic utility in both human and canine patients. Shared molecular pathway alterations are seen in immune cell signaling and activation including TH1 and TH2 signaling, interferon signaling, and inflammatory responses. Virtual cell sorting to estimate immune cell populations within canine and human tumors showed similar trends, predominantly for macrophages and CD8+ T cells. Immunohistochemical staining verified the increased presence of immune cells in tumors exhibiting immune gene enrichment. Collectively these findings further validate naturally occurring osteosarcoma of the pet dog as a translationally relevant patient model for humans and improve our understanding of the immunologic and genomic landscape of the disease in both species.
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Neoplasias Óseas , Osteosarcoma , Humanos , Animales , Perros , Pronóstico , Transcriptoma , Genómica , Osteosarcoma/genética , Osteosarcoma/veterinaria , Neoplasias Óseas/genética , Neoplasias Óseas/veterinariaRESUMEN
BACKGROUND: As new infectious diseases (ID) emerge and others continue to mutate, there remains an imminent threat, especially for vulnerable individuals. Yet no generalizable framework exists to identify the at-risk group prior to infection. Metabolomics has the advantage of capturing the existing physiologic state, unobserved via current clinical measures. Furthermore, metabolomics profiling during acute disease can be influenced by confounding factors such as indications, medical treatments, and lifestyles. METHODS: We employed metabolomic profiling to cluster infection-free individuals and assessed their relationship with COVID severity and influenza incidence/recurrence. FINDINGS: We identified a metabolomic susceptibility endotype that was strongly associated with both severe COVID (ORICUadmission = 6.7, p-value = 1.2 × 10-08, ORmortality = 4.7, p-value = 1.6 × 10-04) and influenza (ORincidence = 2.9; p-values = 2.2 × 10-4, ßrecurrence = 1.03; p-value = 5.1 × 10-3). We observed similar severity associations when recapitulating this susceptibility endotype using metabolomics from individuals during and after acute COVID infection. We demonstrate the value of using metabolomic endotyping to identify a metabolically susceptible group for two-and potentially more-IDs that are driven by increases in specific amino acids, including microbial-related metabolites such as tryptophan, bile acids, histidine, polyamine, phenylalanine, and tyrosine metabolism, as well as carbohydrates involved in glycolysis. INTERPRETATIONS: These metabolites may be identified prior to infection to enable protective measures for these individuals. FUNDING: The Longitudinal EMR and Omics COVID-19 Cohort (LEOCC) and metabolomic profiling were supported by the National Heart, Lung, and Blood Institute and the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health.
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COVID-19 , Enfermedades Transmisibles , Gripe Humana , Humanos , Metaboloma , Estudios Prospectivos , Gripe Humana/epidemiología , Metabolómica , Enfermedades Transmisibles/etiologíaRESUMEN
Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.
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Cuerpos Embrioides , Células Madre Pluripotentes , Humanos , Cuerpos Embrioides/metabolismo , Reproducibilidad de los Resultados , Organoides , Diferenciación CelularRESUMEN
Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax-a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a "species" DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.
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Bacillus anthracis/genética , Evolución Molecular , Genoma Bacteriano , Bacillus anthracis/patogenicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , VirulenciaRESUMEN
Ovarian cancer is one of the most lethal gynecological malignancies. Recurrence or acquired chemoresistance is the leading cause of ovarian cancer therapy failure. Overexpression of ATP-binding cassette subfamily B member 1 (ABCB1), commonly known as P-glycoprotein, correlates closely with multidrug resistance (MDR). However, the mechanism underlying aberrant ABCB1 expression remains unknown. Using a quantitative high-throughput combinational screen, we identified that terfenadine restored doxorubicin sensitivity in an MDR ovarian cancer cell line. In addition, RNA-seq data revealed that the Ca2+-mediated signaling pathway in the MDR cells was abnormally regulated. Moreover, our research demonstrated that terfenadine directly bound to CAMKIID to prevent its autophosphorylation and inhibit the activation of the cAMP-responsive element-binding protein 1 (CREB1)-mediated pathway. Direct inhibition of CAMKII or CREB1 had the same phenotypic effects as terfenadine in the combined treatment, including lower expression of ABCB1 and baculoviral IAP repeat-containing 5 (BIRC5, also known as survivin) and increased doxorubicin-induced apoptosis. In this study, we demonstrate that aberrant regulation of the Ca2+-mediated CAMKIID/CREB1 pathway contributes to ABCB1 over-expression and MDR creation and that CAMKIID and CREB1 are attractive targets for restoring doxorubicin efficacy in ABCB1-mediated MDR ovarian cancer.
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Human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs), hold great promise for drug discovery, toxicology studies, and regenerative medicine. Here, we describe standardized protocols and experimental procedures that combine automated cell culture for scalable production of hPSCs with quantitative high-throughput screening (qHTS) in miniaturized 384-well plates. As a proof of principle, we established dose-response assessments and determined optimal concentrations of 12 small molecule compounds that are commonly used in the stem cell field. Multi-parametric analysis of readouts from diverse assays including cell viability, mitochondrial membrane potential, plasma membrane integrity, and ATP production was used to distinguish normal biological responses from cellular stress induced by small molecule treatment. Collectively, the establishment of integrated workflows for cell manufacturing, qHTS, high-content imaging, and data analysis provides an end-to-end platform for industrial-scale projects and should leverage the drug discovery process using hPSC-derived cell types.
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Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento/métodos , HumanosRESUMEN
This chapter was inadvertently published with an error, the ninth point under 'Materials' section must read.
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BACKGROUND: Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. RESULTS: Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while ß-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased in vivo. CONCLUSIONS: Global proteomic profiling of SD1 comparing in vivo vs. in vitro proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under in vivo conditions, lending credence to their candidacy as potential vaccine targets.
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Proteínas Bacterianas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas , Proteoma/análisis , Shigella dysenteriae/química , Shigella dysenteriae/fisiología , Factores de Virulencia/biosíntesis , Animales , Electroforesis en Gel Bidimensional , Metabolismo Energético , Espectrometría de Masas , Shigella dysenteriae/crecimiento & desarrollo , Shigella dysenteriae/patogenicidad , Porcinos , VirulenciaRESUMEN
Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42â¯577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.