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Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals.
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Catepsina B/metabolismo , Elementos Facilitadores Genéticos , Eritema/genética , Duplicação Gênica , Regulação da Expressão Gênica , Ceratose/genética , Dermatopatias Genéticas/genética , Estudos de Casos e Controles , Catepsina B/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 8/genética , Variações do Número de Cópias de DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Epiderme/metabolismo , Epigenômica , Eritema/epidemiologia , Feminino , Marcadores Genéticos , Humanos , Queratinócitos/metabolismo , Ceratose/epidemiologia , Células MCF-7 , Masculino , Noruega/epidemiologia , Linhagem , Dermatopatias Genéticas/epidemiologia , África do Sul/epidemiologiaRESUMO
BACKGROUND: Macular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters. METHODS: A multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact. RESULTS: Ophthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity. CONCLUSION: We identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.
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Distrofias Hereditárias da Córnea/genética , Mutação , Sulfotransferases/genética , Adulto , Córnea/patologia , Distrofias Hereditárias da Córnea/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , África do Sul , Carboidrato SulfotransferasesRESUMO
BACKGROUND: Exact sample annotation in expression microarray datasets is essential for any type of pharmacogenomics research. RESULTS: Candidate markers were explored through the application of Hartigans' dip test statistics to a publically available human whole genome microarray dataset. The marker performance was tested on 188 serial samples from 53 donors and of variable tissue origin from five public microarray datasets. A qualified transcript marker panel consisting of three probe sets for human leukocyte antigens HLA-DQA1 (2 probe sets) and HLA-DRB4 identified sample donor identifier inconsistencies in six of the 188 test samples. About 3% of the test samples require root-cause analysis due to unresolvable inaccuracies. CONCLUSIONS: The transcript marker panel consisting of HLA-DQA1 and HLA-DRB4 represents a robust, tissue-independent composite marker to assist control donor annotation concordance at the transcript level. Allele-selectivity of HLA genes renders them good candidates for "fingerprinting" with donor specific expression pattern.
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MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
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MicroRNAs/genética , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250 µg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 µg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate.
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Anticorpos Monoclonais/farmacologia , Hepatócitos/transplante , Fígado/patologia , Adulto , Idoso , Animais , Anticorpos Monoclonais/administração & dosagem , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Genoma , Hepatócitos/citologia , Humanos , Antígeno Ki-67/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Análise de Componente Principal , Albumina Sérica/metabolismo , Análise de Sobrevida , Doadores de Tecidos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Receptor fas/imunologiaRESUMO
Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.
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Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Vias Biossintéticas , Farmacorresistência Fúngica , Regulação Fúngica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. METHODS: Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). RESULTS: Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. CONCLUSIONS: The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.
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BACKGROUND: Due to the unparalleled genetic diversity of its peoples, Africa is attracting growing research attention. Several African populations have been assessed in global initiatives such as the International HapMap and 1000 Genomes Projects. Notably excluded, however, is the southern Africa region, which is inhabited predominantly by southeastern Bantu-speakers, currently suffering under the dual burden of infectious and non-communicable diseases. Limited reference data for these individuals hampers medical research and prevents thorough understanding of the underlying population substructure. Here, we present the most detailed exploration, to date, of genetic diversity in 94 unrelated southeastern Bantu-speaking South Africans, resident in urban Soweto (Johannesburg). RESULTS: Participants were typed for ~4.3 million SNPs using the Illumina Omni5 beadchip. PCA and ADMIXTURE plots were used to compare the observed variation with that seen in selected populations worldwide. Results indicated that Sowetans, and other southeastern Bantu-speakers, are a clearly distinct group from other African populations previously investigated, reflecting a unique genetic history with small, but significant contributions from diverse sources. To assess the suitability of our sample as representative of Sowetans, we compared our results to participants in a larger rheumatoid arthritis case-control study. The control group showed good clustering with our sample, but among the cases were individuals who demonstrated notable admixture. CONCLUSIONS: Sowetan population structure appears unique compared to other black Africans, and may have clinical implications. Our data represent a suitable reference set for southeastern Bantu-speakers, on par with a HapMap type reference population, and constitute a prelude to the Southern African Human Genome Programme.
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População Negra/genética , Genética Populacional , Polimorfismo de Nucleotídeo Único , Feminino , Técnicas de Genotipagem , Humanos , Masculino , Análise de Componente Principal , África do SulRESUMO
CONTEXT: Correct gender assignment in humans at the molecular level is crucial in many scientific disciplines and applied areas. MATERIALS AND METHODS: Candidate gender markers were identified through supervised statistical analysis of genome wide microarray expression data from human blood samples (N = 123, 58 female, 65 male) as a training set. The potential of the markers to predict undisclosed tissue donor gender was tested on microarray data from 13 healthy and 11 cancerous human tissue collections (internal) and external datasets from samples of varying tissue origin. The abundance of some genes in the marker panel was quantified by RT-PCR as alternative analytical technology. RESULTS: We identified and qualified predictive, gender-specific transcript markers based on a set of five genes (RPS4Y1, EIF1AY, DDX3Y, KDM5D and XIST). CONCLUSION: Gene expression marker panels can be used as a robust tissue- and platform-independent predictive approach for gender determination.
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Perfilação da Expressão Gênica , RNA Mensageiro/sangue , Análise para Determinação do Sexo/métodos , Biomarcadores/sangue , RNA Helicases DEAD-box/sangue , RNA Helicases DEAD-box/genética , Feminino , Histona Desmetilases/sangue , Histona Desmetilases/genética , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , RNA Longo não Codificante/sangue , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Proteínas Ribossômicas/sangue , Proteínas Ribossômicas/genética , TranscriptomaRESUMO
Anti-cancer therapy based on anthracyclines (DNA intercalating Topoisomerase II inhibitors) is limited by adverse effects of these compounds on the cardiovascular system, ultimately causing heart failure. Despite extensive investigations into the effects of doxorubicin on the cardiovascular system, the molecular mechanisms of toxicity remain largely unknown. MicroRNAs are endogenously transcribed non-coding 22 nucleotide long RNAs that regulate gene expression by decreasing mRNA stability and translation and play key roles in cardiac physiology and pathologies. Increasing doses of doxorubicin, but not etoposide (a Topoisomerase II inhibitor devoid of cardiovascular toxicity), specifically induced the up-regulation of miR-208b, miR-216b, miR-215, miR-34c and miR-367 in rat hearts. Furthermore, the lowest dosing regime (1 mg/kg/week for 2 weeks) led to a detectable increase of miR-216b in the absence of histopathological findings or alteration of classical cardiac stress biomarkers. In silico microRNA target predictions suggested that a number of doxorubicin-responsive microRNAs may regulate mRNAs involved in cardiac tissue remodeling. In particular miR-34c was able to mediate the DOX-induced changes of Sipa1 mRNA (a mitogen-induced Rap/Ran GTPase activating protein) at the post-transcriptional level and in a seed sequence dependent manner. Our results show that integrated heart tissue microRNA and mRNA profiling can provide valuable early genomic biomarkers of drug-induced cardiac injury as well as novel mechanistic insight into the underlying molecular pathways.
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Antibióticos Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , MicroRNAs/genética , Miocárdio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Doxorrubicina/farmacologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Masculino , MicroRNAs/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ativação Transcricional/efeitos dos fármacos , Transcriptoma , Regulação para Cima/efeitos dos fármacos , Vacúolos/efeitos dos fármacosRESUMO
With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.
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Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Fungos/química , Isocumarinas/farmacologia , Lisina-tRNA Ligase/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Antimaláricos/isolamento & purificação , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Isocumarinas/isolamento & purificação , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidoresRESUMO
External access to scientific technology plays an increasingly important part in pharmaceutical R&D. One advantage of accessing technology externally is the avoidance of costs associated with purchase and the reduced time required for developing new methods; in addition, access to external scientific expertise can be beneficial. However, few conceptual frameworks exist for achieving an optimal mix of internal and external technology access. In this review, we describe the virtuous technology cycle (VTC) concept and exemplify its application to next-generation sequencing (NGS). Based on selected examples, we show that the VTC concept can greatly enhance the number of technologies accessed and thus significantly increase flexibility and efficiency in drug discovery. We also discuss the challenges of externally accessing NGS technologies.
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Descoberta de Drogas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Tecnologia FarmacêuticaRESUMO
Recent developments in high-density genotyping and statistical analysis methods that have enabled genome-wide association studies in humans can also be applied to outbred mouse populations. Increased recombination in outbred populations is expected to provide greater mapping resolution than traditional inbred line crosses, improving prospects for identifying the causal genes. We carried out genome-wide association mapping by using 288 mice from a commercially available outbred stock; NMRI mice were genotyped with a high-density single-nucleotide polymorphism array to map loci influencing high-density lipoprotein cholesterol, systolic blood pressure, triglyceride levels, glucose, and urinary albumin-to-creatinine ratios. We found significant associations (P < 10(-5)) with high-density lipoprotein cholesterol and identified Apoa2 and Scarb1, both of which have been previously reported, as candidate genes for these associations. Additional suggestive associations (P < 10(-3)) identified in this study were also concordant with published quantitative trait loci, suggesting that we are sampling from a limited pool of genetic diversity that has already been well characterized. These findings dampen our enthusiasm for currently available commercial outbred stocks as genetic mapping resources and highlight the need for new outbred populations with greater genetic diversity. Despite the lack of novel associations in the NMRI population, our analysis strategy illustrates the utility of methods that could be applied to genome-wide association studies in humans.
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BACKGROUND: The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2'-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels. METHODS AND FINDINGS: Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins. CONCLUSIONS: In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.
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Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metilação de DNA , Epigênese Genética , MicroRNAs/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Decitabina , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Large-scale molecular profiling technologies have assisted the identification of disease biomarkers and facilitated the basic understanding of cellular processes. However, samples collected from human subjects in clinical trials possess a level of complexity, arising from multiple cell types, that can obfuscate the analysis of data derived from them. Failure to identify, quantify, and incorporate sources of heterogeneity into an analysis can have widespread and detrimental effects on subsequent statistical studies.We describe an approach that builds upon a linear latent variable model, in which expression levels from mixed cell populations are modeled as the weighted average of expression from different cell types. We solve these equations using quadratic programming, which efficiently identifies the globally optimal solution while preserving non-negativity of the fraction of the cells. We applied our method to various existing platforms to estimate proportions of different pure cell or tissue types and gene expression profilings of distinct phenotypes, with a focus on complex samples collected in clinical trials. We tested our methods on several well controlled benchmark data sets with known mixing fractions of pure cell or tissue types and mRNA expression profiling data from samples collected in a clinical trial. Accurate agreement between predicted and actual mixing fractions was observed. In addition, our method was able to predict mixing fractions for more than ten species of circulating cells and to provide accurate estimates for relatively rare cell types (<10% total population). Furthermore, accurate changes in leukocyte trafficking associated with Fingolomid (FTY720) treatment were identified that were consistent with previous results generated by both cell counts and flow cytometry. These data suggest that our method can solve one of the open questions regarding the analysis of complex transcriptional data: namely, how to identify the optimal mixing fractions in a given experiment.
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Algoritmos , Sangue/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Transcrição Gênica/genética , Adulto , Linhagem Celular , Feminino , Humanos , Análise dos Mínimos Quadrados , Modelos Lineares , RNA Mensageiro/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: This short communication focuses the on articular cartilage and the subchondral bone, both of which play important roles in the development of osteoarthritis (OA). There are indications that estrogen-deficiency, as the post-menopausal state, accelerate the development of OA. FINDINGS: We investigated, which extracellular matrix (ECM) protein, proteases and different pro-inflammatory factors was up- or down-regulated in the knee joint tissue in response to estrogen-deficiency in rats induced by ovariectomy. These data support previous findings that several metalloproteinases (MMPs) and cysteine proteases are co-regulated with numerous collagens and proteoglycans that are important for cartilage integrity. Furthermore quite a few pro-inflammatory cytokines were regulated by estrogen deprivation. CONCLUSION: We found multiple genes where regulated in the joint by estrogen-deficiency, many of which correspond well with our current knowledge of the pathogenesis of OA. It supports that estrogen-deficiency (e.g. OVX) may accelerate joint deterioration. However, there are also data that draw attention the need for better understanding of the synergy between proteases and tissue turnover.
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The European InnoMed-PredTox project was a collaborative effort between 15 pharmaceutical companies, 2 small and mid-sized enterprises, and 3 universities with the goal of delivering deeper insights into the molecular mechanisms of kidney and liver toxicity and to identify mechanism-linked diagnostic or prognostic safety biomarker candidates by combining conventional toxicological parameters with "omics" data. Mechanistic toxicity studies with 16 different compounds, 2 dose levels, and 3 time points were performed in male Crl: WI(Han) rats. Three of the 16 investigated compounds, BI-3 (FP007SE), Gentamicin (FP009SF), and IMM125 (FP013NO), induced kidney proximal tubule damage (PTD). In addition to histopathology and clinical chemistry, transcriptomics microarray and proteomics 2D-DIGE analysis were performed. Data from the three PTD studies were combined for a cross-study and cross-omics meta-analysis of the target organ. The mechanistic interpretation of kidney PTD-associated deregulated transcripts revealed, in addition to previously described kidney damage transcript biomarkers such as KIM-1, CLU and TIMP-1, a number of additional deregulated pathways congruent with histopathology observations on a single animal basis, including a specific effect on the complement system. The identification of new, more specific biomarker candidates for PTD was most successful when transcriptomics data were used. Combining transcriptomics data with proteomics data added extra value.
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Ciclosporinas/toxicidade , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos/genética , Gentamicinas/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Proteômica/métodos , Animais , Biomarcadores/análise , Relação Dose-Resposta a Droga , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and beta2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.
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Biomarcadores Farmacológicos/urina , Clusterina/urina , Cistatina C/urina , Testes de Função Renal/métodos , Microglobulina beta-2/urina , Animais , Biomarcadores Farmacológicos/metabolismo , Distribuição de Qui-Quadrado , Clusterina/genética , Clusterina/metabolismo , Creatinina/sangue , Creatinina/metabolismo , Cistatina C/genética , Cistatina C/metabolismo , Perfilação da Expressão Gênica , Histocitoquímica , Rim/química , Rim/efeitos dos fármacos , Rim/lesões , Rim/patologia , Nefropatias/diagnóstico , Nefropatias/patologia , Glomérulos Renais/patologia , Túbulos Renais Proximais/patologia , Masculino , Prognóstico , Proteinúria/urina , Curva ROC , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.
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Biomarcadores Farmacológicos , Cistatina C/sangue , Nefropatias/diagnóstico , Testes de Função Renal/métodos , Animais , Biomarcadores Farmacológicos/sangue , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Farmacológicos/urina , Nitrogênio da Ureia Sanguínea , Carbapenêmicos/toxicidade , Creatinina/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Gentamicinas/toxicidade , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Curva ROC , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
We describe cell type-specific significance analysis of microarrays (csSAM) for analyzing differential gene expression for each cell type in a biological sample from microarray data and relative cell-type frequencies. First, we validated csSAM with predesigned mixtures and then applied it to whole-blood gene expression datasets from stable post-transplant kidney transplant recipients and those experiencing acute transplant rejection, which revealed hundreds of differentially expressed genes that were otherwise undetectable.