A Novel High-Throughput Screening Tool in Saccharomyces Cerevisiae

<p>The elucidation of drug targets and their biological effects can be aided by the identification of yeast deletion mutants that confer hypersensitivity to the drug. However, the biological activities of some compounds are reduced by the mechanisms of the pleotropic drug resistance (PDR) network. For this reason a PDR-deficient strain with deletions in the master transcriptional regulators of the PDR network, PDR1 and PDR3 was created. This double deletion mutant strain was robotically mass mated against the non-essential deletion mutant array (DMA) to create genome wide nonessential PDR-deficient DMA (PD-DMA). No phenotypic enhancements were observed with Δpdr1Δpdr3 double mutant and the various deletion strains of the DMA. Increased genome-wide sensitivity of the PDR-deficient mutants was demonstrated by screening pools of PD-DMA and the DMA against cycloheximide, a Pdr5p substrate and rapamycin which is not. Similar sensitivities were observed for the non-PDR substrate rapamycin for the two deletion mutant pools while sensitivity to Pdr5p substrate cycloheximide was significantly more sensitive in the PD-DMA. The genome-wide increased sensitivity of the PDR-deficient mutants was further assessed by screening the LOPAC library of pharmacologically active compounds against pools of PD-DMA and the wild-type background DMA. The DMA screen identified 5 of 1280 compounds having bioactivity whilst the PD-DMA screen identified 25 compounds including the 5 identified by the DMA. The 20 additional compounds identified in the PDR-deficient background were inactive at the concentrations used in the wild-type background. The PD-DMA was then used in chemical genetic profiling assays; namely solid-phase chemical genetic profiling and DNA barcode microarray experiments. The PD-DMA was screened against natural products, rapamycin and cycloheximide with well characterized chemical genetic profiles. The PDR-deficient strains hypersensitivity to rapamycin and cycloheximide were indicative of TORC1 pathway inhibition and translational elongation inhibition, respectively. This was consistent with literature as rapamycin is an inhibitor of TORC1 and it mimics nutrient starvation response and cycloheximide inhibits eukaryotic translational elongation. These results validated the utility of PD-DMA as a hypersensitive genome-wide deletion reagent for chemical genetic profiling. Following validation of the PD-DMA, biochemical assays were performed on latrunculin-A, a less well characterised marine natural product and Plakortide-T a marine natural product with novel activity. These inhibitory drugs were shown to be PDR substrates with biological activity at low nanomolar concentrations. Latrunculin-A, was ~28 fold more potent in the PDR-deficient strain. In contrast, plakortide-T was biologically active only in the PDR-deficient background and the PDR mediated efflux did not involve the major efflux transporters. DNA barcode microarray experiments performed with latrunculin-A identified several hypersensitive deletion mutants consistent with cytoskeletal disruption specifically, actin microfilament assembly. Latrunculin-A is known to bind monomeric G-actin and inhibit actin polymerisation. However, in novel findings tubulin cytoskeleton disassembly was also shown to be mediated by latrunculin-A. Plakortide-T belongs to a class of compounds that disrupts calcium homeostasis. DNA barcode microarray experiments performed identified 56 deletion mutants hypersensitive to Plakortide-T, however, none were involved in calcium homeostasis and the deletion mutants were not over represented in any of the GO terms. Plakortide-T caused hypersensitivity in several deletion mutants of genes encoding for mitochondrial proteins. This activity however, did not generate reactive oxygen species as increased oxidation of free thiols or induction of the oxidative stress response was not observed. Plakortide-T was shown to induce an increase in cytosolic calcium detected by the nuclear localisation of Crz1p, a transcription factor activated in response to increased cytosolic calcium. This activation was dependent on functional calcineurin which further validates this response is an increase in cytosolic calcium.</p>

A Novel High-Throughput Screening Tool in Saccharomyces Cerevisiae

<p>The elucidation of drug targets and their biological effects can be aided by the identification of yeast deletion mutants that confer hypersensitivity to the drug. However, the biological activities of some compounds are reduced by the mechanisms of the pleotropic drug resistance (PDR) network. For this reason a PDR-deficient strain with deletions in the master transcriptional regulators of the PDR network, PDR1 and PDR3 was created. This double deletion mutant strain was robotically mass mated against the non-essential deletion mutant array (DMA) to create genome wide nonessential PDR-deficient DMA (PD-DMA). No phenotypic enhancements were observed with Δpdr1Δpdr3 double mutant and the various deletion strains of the DMA. Increased genome-wide sensitivity of the PDR-deficient mutants was demonstrated by screening pools of PD-DMA and the DMA against cycloheximide, a Pdr5p substrate and rapamycin which is not. Similar sensitivities were observed for the non-PDR substrate rapamycin for the two deletion mutant pools while sensitivity to Pdr5p substrate cycloheximide was significantly more sensitive in the PD-DMA. The genome-wide increased sensitivity of the PDR-deficient mutants was further assessed by screening the LOPAC library of pharmacologically active compounds against pools of PD-DMA and the wild-type background DMA. The DMA screen identified 5 of 1280 compounds having bioactivity whilst the PD-DMA screen identified 25 compounds including the 5 identified by the DMA. The 20 additional compounds identified in the PDR-deficient background were inactive at the concentrations used in the wild-type background. The PD-DMA was then used in chemical genetic profiling assays; namely solid-phase chemical genetic profiling and DNA barcode microarray experiments. The PD-DMA was screened against natural products, rapamycin and cycloheximide with well characterized chemical genetic profiles. The PDR-deficient strains hypersensitivity to rapamycin and cycloheximide were indicative of TORC1 pathway inhibition and translational elongation inhibition, respectively. This was consistent with literature as rapamycin is an inhibitor of TORC1 and it mimics nutrient starvation response and cycloheximide inhibits eukaryotic translational elongation. These results validated the utility of PD-DMA as a hypersensitive genome-wide deletion reagent for chemical genetic profiling. Following validation of the PD-DMA, biochemical assays were performed on latrunculin-A, a less well characterised marine natural product and Plakortide-T a marine natural product with novel activity. These inhibitory drugs were shown to be PDR substrates with biological activity at low nanomolar concentrations. Latrunculin-A, was ~28 fold more potent in the PDR-deficient strain. In contrast, plakortide-T was biologically active only in the PDR-deficient background and the PDR mediated efflux did not involve the major efflux transporters. DNA barcode microarray experiments performed with latrunculin-A identified several hypersensitive deletion mutants consistent with cytoskeletal disruption specifically, actin microfilament assembly. Latrunculin-A is known to bind monomeric G-actin and inhibit actin polymerisation. However, in novel findings tubulin cytoskeleton disassembly was also shown to be mediated by latrunculin-A. Plakortide-T belongs to a class of compounds that disrupts calcium homeostasis. DNA barcode microarray experiments performed identified 56 deletion mutants hypersensitive to Plakortide-T, however, none were involved in calcium homeostasis and the deletion mutants were not over represented in any of the GO terms. Plakortide-T caused hypersensitivity in several deletion mutants of genes encoding for mitochondrial proteins. This activity however, did not generate reactive oxygen species as increased oxidation of free thiols or induction of the oxidative stress response was not observed. Plakortide-T was shown to induce an increase in cytosolic calcium detected by the nuclear localisation of Crz1p, a transcription factor activated in response to increased cytosolic calcium. This activation was dependent on functional calcineurin which further validates this response is an increase in cytosolic calcium.</p>

Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes’ single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

A two-stage normalization method for robust differential expression analysis in microarray experiments.

In this research, we introduce an approach to improve the reliability of genetic data analysis. Consistency of the results obtained from microarray data analysis strongly relies on elimination of non-biological variations during data normalization process. Instability in Housekeeping Gene (HKG) expression after performing common normalization methods might be an indication of inefficiency potentially resulting in sampling bias in differential expression analysis. This research aims to reduce the sampling bias in microarray experiments proposing a two-stage normalization algorithm. Proposed approach consists of non-linear Quantile normalization at the first stage and linear HKG based normalization at the second stage. We tested the efficiency of the two-stage normalization method using publicly available microarray datasets obtained from the experiments mainly in the field of reproductive biology. Results show that combined Robust Multiarray Average (RMA) and HKG normalization method reduces the sampling bias in experiments when variations in HKG expression is observed after RMA normalization.

A two-stage normalization method for robust differential expression analysis in microarray experiments.

In this research, we introduce an approach to improve the reliability of genetic data analysis. Consistency of the results obtained from microarray data analysis strongly relies on elimination of non-biological variations during data normalization process. Instability in Housekeeping Gene (HKG) expression after performing common normalization methods might be an indication of inefficiency potentially resulting in sampling bias in differential expression analysis. This research aims to reduce the sampling bias in microarray experiments proposing a two-stage normalization algorithm. Proposed approach consists of non-linear Quantile normalization at the first stage and linear HKG based normalization at the second stage. We tested the efficiency of the two-stage normalization method using publicly available microarray datasets obtained from the experiments mainly in the field of reproductive biology. Results show that combined Robust Multiarray Average (RMA) and HKG normalization method reduces the sampling bias in experiments when variations in HKG expression is observed after RMA normalization.

miRNA Profiling in the Chicken Liver under the Influence of Early Microbiota Stimulation with Probiotic, Prebiotic, and Synbiotic

Epigenetic regulation of gene expression is a form of interaction of the external environment on reading and transcription of genetic information encoded in nucleic acids. We provided evidence that early stimulation of the chicken microbiota with in ovo delivered synbiotics influenced gene expression and DNA methylation in the liver. Therefore, we hypothesize that the stimulation of microbiota by administering bioactive substances in ovo also affects the activity of miRNA in liver. For the analysis of miRNA activity, RNA was isolated from liver of adult broiler chicken and native chicken breed. The animals received a prebiotic, probiotic and synbiotic in ovo on day 12 of egg incubation. The analysis of miRNA expression was performed using the LNA method on a miRNA panel selected on the basis of previous microarray experiments. We have found increased miRNA expression activity after probiotic and synbiotic administration, especially in native chicken breed. Our results suggest that prebiotics reduce or do not affect miRNA activity. We have also shown that miRNA activity is regulated by the substance and genotype of the chicken. We can conclude that miRNAs constitute an important component of the molecular mechanism of host–probiotic interaction in liver.

Euler diagrams drawn with ellipses area-proportionally (Edeap)

BackgroundArea-proportional Euler diagrams are frequently used to visualize data from Microarray experiments, but are also applied to a wide variety of other data from biosciences, social networks and other domains.ResultsThis paper details Edeap, a new simple, scalable method for drawing area-proportional Euler diagrams with ellipses. We use a search-based technique optimizing a multi-criteria objective function that includes measures for both area accuracy and usability, and which can be extended to further user-defined criteria. The Edeap software is available for use on the web, and the code is open source. In addition to describing our system, we present the first extensive evaluation of software for producing area-proportional Euler diagrams, comparing Edeap to the current state-of-the-art; circle-based method, venneuler, and an alternative ellipse-based method, eulerr.ConclusionsOur evaluation—using data from the Gene Ontology database via GoMiner, Twitter data from the SNAP database, and randomly generated data sets—shows an ordering for accuracy (from best to worst) of eulerr, followed by Edeap and then venneuler. In terms of runtime, the results are reversed with venneuler being the fastest, followed by Edeap and finally eulerr. Regarding scalability, eulerr cannot draw non-trivial diagrams beyond 11 sets, whereas no such limitation is present in Edeap or venneuler, both of which draw diagrams up to the tested limit of 20 sets.