RESUMO
The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.
Assuntos
Análise de Sequência de DNA/métodos , Leveduras/genética , Sistemas CRISPR-Cas , Biologia Computacional/métodos , DNA Fúngico/genética , Biblioteca GênicaRESUMO
BACKGROUND: Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. RESULTS: We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu's positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson's disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. CONCLUSIONS: A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations.
Assuntos
Cobre/metabolismo , Homeostase/genética , Leveduras/genética , Leveduras/metabolismo , Respiração Celular , Análise por Conglomerados , Cobre/deficiência , Meios de Cultura , Dissulfiram/farmacologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Homeostase/efeitos dos fármacos , Humanos , Hidrazinas/farmacologia , Concentração de Íons de Hidrogênio , Fenótipo , Vacúolos/genética , Vacúolos/metabolismo , Leveduras/efeitos dos fármacosRESUMO
BACKGROUND: The modified Glasgow Prognostic Score (mGPS), an inflammation-based prognostic score that uses thresholds of C-reactive protein (> 10 mg/L) and albumin (< 35 g/L), has been found to be independently prognostic of survival in patients with cancer. The objective of the current study was to establish whether the addition of a differential leukocyte count and a high-sensitivity C-reactive protein measurement enhanced the prognostic value of the mGPS. METHODS: A total of 12,119 patients who had an incidental blood sample taken between 2000 and 2007 for C-reactive protein, albumin, and a differential leukocyte count as well as a diagnosis of cancer made within 2 years were identified. This group was studied for the prognostic value of neutrophil, lymphocyte, and platelet counts. In addition 2742 patients whose blood was sampled after the introduction of high-sensitivity C-reactive protein measurements were studied for the prognostic value of different thresholds. RESULTS: Using cancer-specific survival as an endpoint, the prognostic value of the mGPS (hazard ratio [HR], 2.61; P < .001 [area under the receiver operating characteristic curve (AUC), 0.695]) was found to be improved by the addition of neutrophil and platelet counts (HR, 4.86; P < .001 [AUC, 0.734]) and a high-sensitivity C-reactive protein measurement (> 3 mg/L) (HR, 5.77; P < .001 [AUC, 0.734]). CONCLUSIONS: The results of the current study demonstrate that the addition of neutrophil and platelet counts, as well as a high-sensitivity C-reactive protein measurement, enhanced the prognostic value of the mGPS.
Assuntos
Proteína C-Reativa/análise , Inflamação/mortalidade , Inflamação/fisiopatologia , Contagem de Leucócitos , Neoplasias/diagnóstico , Contagem de Plaquetas , Idoso , Área Sob a Curva , Estudos de Coortes , Feminino , Humanos , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Neoplasias/mortalidade , Neoplasias/fisiopatologia , Neutrófilos , Valor Preditivo dos Testes , Prognóstico , Escócia , Albumina Sérica/análiseRESUMO
BACKGROUND: As the incidence of prostate cancer continues to rise steeply, there is an increasing need to identify more accurate prognostic markers for the disease. There is some evidence that a higher modified Glasgow Prognostic Score (mGPS) may be associated with poorer survival in patients with prostate cancer but it is not known whether this is independent of other established prognostic factors. Therefore the aim of this study was to describe the relationship between mGPS and survival in patients with prostate cancer after adjustment for other prognostic factors. METHODS: Retrospective clinical series on patients in Glasgow, Scotland, for whom data from the Scottish Cancer Registry, including Gleason score, Prostate Specific Antigen (PSA), C-reactive protein (CRP) and albumin, six months prior to or following the diagnosis, were included in this study. RESULTS: Seven hundred and forty four prostate cancer patients were identified; of these, 497 (66.8%) died during a maximum follow up of 11.9 years. Patients with mGPS of 2 had poorest 5-year and 10-year relative survival, of 32.6% and 18.8%, respectively. Raised mGPS also had a significant association with excess risk of death at five years (mGPS 2: Relative Excess Risk = 3.57, 95% CI 2.31-5.52) and ten years (mGPS 2: Relative Excess Risk = 3.42, 95% CI 2.25-5.21) after adjusting for age, socioeconomic circumstances, Gleason score, PSA and previous in-patient bed days. CONCLUSIONS: The mGPS is an independent and objective prognostic indicator for survival of patients with prostate cancer. It may be useful in determining the clinical management of patients with prostate cancer in addition to established prognostic markers.
Assuntos
Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Idoso , Terapia Combinada , Seguimentos , Humanos , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Neoplasias da Próstata/terapia , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
INTRODUCTION: Recent in-vitro studies have suggested that a critical checkpoint early in the inflammatory process involves the interaction between neutrophils and platelets. This confirms the importance of the innate immune system in the elaboration of the systemic inflammatory response. The aim of the present study was to examine whether a combination of the neutrophil and platelet counts were predictive of survival in patients with cancer. METHODS: Patients with histologically proven colorectal cancer who underwent potentially curative resection at a single centre between March 1999 and May 2013 (n = 796) and patients with cancer from the Glasgow Inflammation Outcome Study, who had a blood sample taken between January 2000 and December 2007 (n = 9649) were included in the analysis. RESULTS: In the colorectal cancer cohort, there were 173 cancer and 135 non-cancer deaths. In patients undergoing elective surgery, cancer-specific survival (CSS) at 5 years ranged from 97% in patients with TNM I disease and NPS = 0 to 57% in patients with TNM III disease and NPS = 2 (p = 0.019) and in patients undergoing elective surgery for node-negative colon cancer from 98% (TNM I, NPS = 0) to 65% (TNM II, NPS = 2) (p = 0.004). In those with a variety of common cancers there were 5218 cancer and 929 non-cancer deaths. On multivariate analysis, adjusting for age and sex and stratified by tumour site, incremental increase in the NPS was significantly associated with poorer CSS (p<0.001). CONCLUSION: The neutrophil-platelet score predicted survival in a variety of common cancers and highlights the importance of the innate immune system in patients with cancer.
Assuntos
Plaquetas , Neoplasias Colorretais/sangue , Neoplasias Colorretais/mortalidade , Neoplasias/sangue , Neoplasias/mortalidade , Neutrófilos , Idoso , Idoso de 80 Anos ou mais , Plaquetas/imunologia , Estudos de Coortes , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Imunidade Inata , Estimativa de Kaplan-Meier , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias/imunologia , Neutrófilos/imunologia , Contagem de Plaquetas , Valor Preditivo dos Testes , PrognósticoRESUMO
INTRODUCTION: Markers of the systemic inflammatory response, including C-reactive protein and albumin (combined to form the modified Glasgow Prognostic Score), as well as neutrophil, lymphocyte and platelet counts have been shown to be prognostic of survival in patients with cancer. The aim of the present study was to examine the prognostic relationship between these markers of the systemic inflammatory response and all-cause, cancer, cardiovascular and cerebrovascular mortality in a large incidentally sampled cohort. METHODS: Patients (n = 160 481) who had an incidental blood sample taken between 2000 and 2008 were studied for the prognostic value of C-reactive protein (>10mg/l, albumin (>35mg/l), neutrophil (>7.5×109/l) lymphocyte and platelet counts. Also, patients (n = 52 091) sampled following the introduction of high sensitivity C-reactive protein (>3mg/l) measurements were studied. A combination of these markers, to make cumulative inflammation-based scores, were investigated. RESULTS: In all patients (n = 160 481) C-reactive protein (>10mg/l) (HR 2.71, p<0.001), albumin (>35mg/l) (HR 3.68, p<0.001) and neutrophil counts (HR 2.18, p<0.001) were independently predictive of all-cause mortality. These associations were also observed in cancer, cardiovascular and cerebrovascular mortality before and after the introduction of high sensitivity C-reactive protein measurements (>3mg/l) (n = 52 091). A combination of high sensitivity C-reactive protein (>3mg/l), albumin and neutrophil count predicted all-cause (HR 7.37, p<0.001, AUC 0.723), cancer (HR 9.32, p<0.001, AUC 0.731), cardiovascular (HR 4.03, p<0.001, AUC 0.650) and cerebrovascular (HR 3.10, p<0.001, AUC 0.623) mortality. CONCLUSION: The results of the present study showed that an inflammation-based prognostic score, combining high sensitivity C-reactive protein, albumin and neutrophil count is prognostic of all-cause mortality.
Assuntos
Proteína C-Reativa/análise , Inflamação/patologia , Albumina Sérica/análise , Idoso , Idoso de 80 Anos ou mais , Plaquetas/citologia , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/mortalidade , Transtornos Cerebrovasculares/diagnóstico , Transtornos Cerebrovasculares/mortalidade , Estudos de Coortes , Feminino , Seguimentos , Humanos , Contagem de Leucócitos , Linfócitos/citologia , Masculino , Pessoa de Meia-Idade , Neoplasias/mortalidade , Neoplasias/patologia , Neutrófilos/citologia , Contagem de Plaquetas , Prognóstico , Análise de SobrevidaRESUMO
Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
Assuntos
Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Resistência a Medicamentos/genética , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Haploinsuficiência , Humanos , Farmacogenética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
INTRODUCTION: Components of the systemic inflammatory response, combined to form inflammation-based prognostic scores (modified Glasgow Prognostic Score (mGPS), Neutrophil Lymphocyte Ratio (NLR), Platelet Lymphocyte Ratio (PLR), Prognostic Index (PI), Prognostic Nutritional Index (PNI)) have been associated with cancer specific survival. The aim of the present study was to compare the prognostic value of these scores. METHODS: Patients (n=27,031) who had an incidental blood sample taken between 2000 and 2007 for C-reactive protein, albumin, white cell, neutrophil, lymphocyte and platelet counts, as well as a diagnosis of cancer (Scottish Cancer Registry) were identified. Of this group 8759 patients who had been sampled within two years following their cancer diagnosis were studied. RESULTS: On follow up, there were 5163 deaths of which 4417 (86%) were cancer deaths. The median time from blood sampling to diagnosis was 1.7 months. An elevated mGPS, NLR, PLR, PI and PNI were predictive of a reduced cancer specific survival independent of age, sex and deprivation and tumour site (all p<0.001). The area under the receiver operator curves was greatest for mGPS and PI. Specifically, in colorectal cancer, an elevated mGPS and PI were predictive of a reduced cancer specific survival independent of age, sex, deprivation and tumour stage (both p<0.001). CONCLUSION: The results of the present study show that systemic inflammation-based scores, in particular the mGPS and PI, have prognostic value in cancer independent of tumour site. Based on the present results and the existing validation literature, the mGPS should be included in the routine assessment of all patients with cancer.
Assuntos
Inflamação/sangue , Neoplasias/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Proteína C-Reativa/análise , Estudos de Coortes , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Neoplasias/mortalidade , Neoplasias/patologia , Avaliação Nutricional , Contagem de Plaquetas , Prognóstico , Análise de SobrevidaRESUMO
The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.