The document discusses using microRNA (miRNA) expression analysis to understand the effects of the testicular toxicant 2,5-hexanedione in rats. Rats were dosed for 14 days, and 8 miRNAs were found to be differentially expressed in the testis. Predicted miRNA targets and known targets were analyzed, both suggesting involvement of the FSH signaling pathway, important for Sertoli cell function and germ cell maturation. The results indicate miRNAs play important roles in regulating testicular function, and integrating miRNA profiling with pathway analysis can provide insights into miRNA-mRNA interactions and potential mechanistic biomarkers of toxicity.
The document describes a study that used bisulfite sequencing and the Bismark alignment program to determine cytosine methylation rates in the chloroplast and whole genomes of Zea mays (maize). False-positive methylation rates were determined using the unmethylated chloroplast genome. Whole genome methylation rates in CpG, CHG and CHH contexts were found to be 87.0-87.8%, 73.4-74.9% and 2.9% respectively, consistent with previous studies. The proposed workflow for bisulfite sequencing included quality control with Trim Galore and FastQC followed by alignment with Bismark.
1) The study explores how the transcription factor KLF1 regulates the gene encoding delta-aminolevulinic acid dehydratase (Alad), the first enzyme in the heme biosynthesis pathway.
2) Using a cell line model where KLF1 can be induced, the study finds that KLF1 significantly increases Alad mRNA and protein levels by increasing transcription from the Alad promoter.
3) In contrast, the study finds little effect of KLF1 on mRNA levels of the genes encoding two other early heme biosynthesis enzymes, ALAS2 and PBGD, suggesting KLF1 specifically regulates Alad transcription.
Phosphorylation of serine 526 in the activation loop of MEKK3 is required for its kinase activity and ability to activate downstream signaling pathways. Mutation of serine 526 to alanine abolished MEKK3 activity, while mutation to aspartic or glutamic acid created constitutively active mutants. Serine 526 is autophosphorylated and its phosphorylation is regulated by protein phosphatase 2A and its association with 14-3-3 proteins, which prevents dephosphorylation. Phosphorylation of endogenous MEKK3 on serine 526 occurs in response to osmotic stress.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
1. The ChampionChIP system allows for epigenetic analysis of histone modifications and transcription factor binding in a single day. It uses validated antibodies, primers, and qPCR arrays to analyze multiple genomic regions simultaneously.
2. The system was used to analyze dynamic bivalent histone modification patterns of pluripotency genes during mouse embryonic carcinoma cell differentiation induced by retinoic acid. Distinct patterns were observed for genes and heterochromatic regions.
3. The system correctly identified histone modification distribution and was used to map modifications around the CDKN1A gene. It also analyzed p53 binding and correlated gene expression changes in response to drug treatment in cancer cell lines.
This study analyzed the salicylic acid methyltransferase (SAMT) protein in Asclepias curassavica milkweed. The researcher extracted RNA from A. curassavica leaf tissue, amplified the SAMT gene, cloned it into a vector plasmid, and performed assays. Analysis of the SAMT amino acid sequence showed motifs predicting preference for salicylic acid over benzoic acid. Enzyme assays using GC-MS confirmed SAMT preferentially methylated salicylic acid. Statistical analysis supported the hypothesis that SAMT amino acid sequence correlates with substrate preference.
This document discusses translational models of drug-induced liver injury. It begins by outlining the integrated mechanistic drug safety approach, which investigates chemicals, patients, and the interaction between the two. A key part of this approach is understanding the mechanisms of drug bioactivation and the consequences for cellular toxicity. Paracetamol is used as a case study, as it is a common cause of drug-induced liver injury. The document explores the mechanisms of paracetamol toxicity, including how its reactive metabolite NAPQI causes oxidative stress and depletion of glutathione, leading to liver cell damage through both necrosis and inflammation. It also discusses the role of the Nrf2 transcription factor in regulating the antioxidant response and adapting cells to
Approach for limited cell ChIP-Seq on a semiconductor-based sequencing platformThermo Fisher Scientific
Dendritic cell (DC) lineages coordinate immune system activity
through functional specialization.
• Irf4, a transcription factor(TF), is required for CD11b+ DC
lineage development from bone marrow stem cells and has
been implicated in multiple inflammatory diseases, eg. asthma.
• The epigenetic consequences of immune specialization in
CD11b+ DCs and relation to inflammatory diseases remain
largely unexplored partly due to the difficulty of using highly
purified, and typically, limited populations of cells in ChIP-seq
(chromatin immunoprecipitation then sequencing) assays.
• A robust, multiplexed ChIP-seq protocol – using an input
control, TF (CTCF) and histone modification marks (H3K9me3-
methylation, H3K27ac-acetylation) - was developed using
limited amounts of K562 cells, for the Ion ProtonTM system.
• Peak-calling analysis was performed using using MACS2.
• Significant data correlations were observed with ENCODE.
• The Ion ProtonTM results are based on chromatin derived from
1 million(M) cells, making it viable for generating data from a
limited number of primary cells. This is in contrast to the 10M
cells recommended by ENCODE.
• The developed methodology was used to compare Irf4 genomic
binding sites generated from flow-sorted populations of 1, 3, 5,
and 20M CD11b+ lineage murine DCs.
• Comparable Irf4 ChIP-seq results were obtained from 5M
versus 20M cells, indicating that as low as 5M flow-sorted cells
can be used to acquire high quality(FDR: 10-19) data.
• We identified genomic Irf4 binding sites proximal to genes,
whose activity is consistent with CD11b+ DC lineage activity
and/or known to contribute to inflammatory disease.
• We examined Irf4 functional regulation of the identified gene
targets via RNA-seq analysis with CD11b+ DCs and a related
lineage, CD103+ DCs. Integrating expression analysis with
ChIP-seq indicates a unique CD11b+ DC gene expression
program concordant with Irf4 loci association in comparison to
CD103+ DC (data not shown).
The document describes a study that used bisulfite sequencing and the Bismark alignment program to determine cytosine methylation rates in the chloroplast and whole genomes of Zea mays (maize). False-positive methylation rates were determined using the unmethylated chloroplast genome. Whole genome methylation rates in CpG, CHG and CHH contexts were found to be 87.0-87.8%, 73.4-74.9% and 2.9% respectively, consistent with previous studies. The proposed workflow for bisulfite sequencing included quality control with Trim Galore and FastQC followed by alignment with Bismark.
1) The study explores how the transcription factor KLF1 regulates the gene encoding delta-aminolevulinic acid dehydratase (Alad), the first enzyme in the heme biosynthesis pathway.
2) Using a cell line model where KLF1 can be induced, the study finds that KLF1 significantly increases Alad mRNA and protein levels by increasing transcription from the Alad promoter.
3) In contrast, the study finds little effect of KLF1 on mRNA levels of the genes encoding two other early heme biosynthesis enzymes, ALAS2 and PBGD, suggesting KLF1 specifically regulates Alad transcription.
Phosphorylation of serine 526 in the activation loop of MEKK3 is required for its kinase activity and ability to activate downstream signaling pathways. Mutation of serine 526 to alanine abolished MEKK3 activity, while mutation to aspartic or glutamic acid created constitutively active mutants. Serine 526 is autophosphorylated and its phosphorylation is regulated by protein phosphatase 2A and its association with 14-3-3 proteins, which prevents dephosphorylation. Phosphorylation of endogenous MEKK3 on serine 526 occurs in response to osmotic stress.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
1. The ChampionChIP system allows for epigenetic analysis of histone modifications and transcription factor binding in a single day. It uses validated antibodies, primers, and qPCR arrays to analyze multiple genomic regions simultaneously.
2. The system was used to analyze dynamic bivalent histone modification patterns of pluripotency genes during mouse embryonic carcinoma cell differentiation induced by retinoic acid. Distinct patterns were observed for genes and heterochromatic regions.
3. The system correctly identified histone modification distribution and was used to map modifications around the CDKN1A gene. It also analyzed p53 binding and correlated gene expression changes in response to drug treatment in cancer cell lines.
This study analyzed the salicylic acid methyltransferase (SAMT) protein in Asclepias curassavica milkweed. The researcher extracted RNA from A. curassavica leaf tissue, amplified the SAMT gene, cloned it into a vector plasmid, and performed assays. Analysis of the SAMT amino acid sequence showed motifs predicting preference for salicylic acid over benzoic acid. Enzyme assays using GC-MS confirmed SAMT preferentially methylated salicylic acid. Statistical analysis supported the hypothesis that SAMT amino acid sequence correlates with substrate preference.
This document discusses translational models of drug-induced liver injury. It begins by outlining the integrated mechanistic drug safety approach, which investigates chemicals, patients, and the interaction between the two. A key part of this approach is understanding the mechanisms of drug bioactivation and the consequences for cellular toxicity. Paracetamol is used as a case study, as it is a common cause of drug-induced liver injury. The document explores the mechanisms of paracetamol toxicity, including how its reactive metabolite NAPQI causes oxidative stress and depletion of glutathione, leading to liver cell damage through both necrosis and inflammation. It also discusses the role of the Nrf2 transcription factor in regulating the antioxidant response and adapting cells to
Approach for limited cell ChIP-Seq on a semiconductor-based sequencing platformThermo Fisher Scientific
Dendritic cell (DC) lineages coordinate immune system activity
through functional specialization.
• Irf4, a transcription factor(TF), is required for CD11b+ DC
lineage development from bone marrow stem cells and has
been implicated in multiple inflammatory diseases, eg. asthma.
• The epigenetic consequences of immune specialization in
CD11b+ DCs and relation to inflammatory diseases remain
largely unexplored partly due to the difficulty of using highly
purified, and typically, limited populations of cells in ChIP-seq
(chromatin immunoprecipitation then sequencing) assays.
• A robust, multiplexed ChIP-seq protocol – using an input
control, TF (CTCF) and histone modification marks (H3K9me3-
methylation, H3K27ac-acetylation) - was developed using
limited amounts of K562 cells, for the Ion ProtonTM system.
• Peak-calling analysis was performed using using MACS2.
• Significant data correlations were observed with ENCODE.
• The Ion ProtonTM results are based on chromatin derived from
1 million(M) cells, making it viable for generating data from a
limited number of primary cells. This is in contrast to the 10M
cells recommended by ENCODE.
• The developed methodology was used to compare Irf4 genomic
binding sites generated from flow-sorted populations of 1, 3, 5,
and 20M CD11b+ lineage murine DCs.
• Comparable Irf4 ChIP-seq results were obtained from 5M
versus 20M cells, indicating that as low as 5M flow-sorted cells
can be used to acquire high quality(FDR: 10-19) data.
• We identified genomic Irf4 binding sites proximal to genes,
whose activity is consistent with CD11b+ DC lineage activity
and/or known to contribute to inflammatory disease.
• We examined Irf4 functional regulation of the identified gene
targets via RNA-seq analysis with CD11b+ DCs and a related
lineage, CD103+ DCs. Integrating expression analysis with
ChIP-seq indicates a unique CD11b+ DC gene expression
program concordant with Irf4 loci association in comparison to
CD103+ DC (data not shown).
This document summarizes an evaluation seminar on cell signaling and signal transduction pathways presented by Mrutyunjay B Bellad of the Department of Pharmacology at H.S.K. College of Pharmacy in Bagalkot. The seminar covered various topics related to cell signaling including introduction, types of cell signaling, signal molecules and their actions, signaling through different receptor types, second messengers, G-protein coupled receptors, and signal transduction pathways. References included standard pharmacology textbooks.
A suppressor mutation counters the effects of an original mutation by restoring the wild-type phenotype. There are two main types of suppressor mutations: intragenic mutations occur within the same gene and restore function through alternate amino acid substitutions, while intergenic mutations occur elsewhere in the genome and restore function through interacting gene products. Suppressor mutations are useful for studying protein-protein interactions and dissecting biological pathways.
This document summarizes protein engineering techniques. It discusses:
1. The process of protein engineering involves diversification of genes through random mutation/recombination, selection of variants with desired properties, and amplification of selected variants.
2. Examples of techniques used for diversification include error-prone PCR and DNA shuffling. Screens and selections are used to identify variants with improved properties.
3. Multienzyme systems can be artificially synthesized through gene fusion, joining genes to create a single polypeptide with active sites from both enzymes. This allows proximity of enzymes to catalyze sequential reactions.
Crimson Publishers- CPG Methylation in G-Quadruplex and IMotif DNA StructuresCrimsonPublishers-SBB
Abberant hypomethylation in DNA regions with noncanonical folding potential (ncDNA motifs) is believed to predetermine tumor development - presumably, by facilitating G-quadruplex (G4) and/or i-motif (IM) formation via altering nucleosome positioning (stable G4s induce subsequent genomic rearrangements). We questioned whether CpG methylation per se affects the dsDNA-ncDNA equilibrium. Thermodynamic studies of genomic and model oligonucleotides with methylated CpG sites at different positions are reported. The genomic oligonucleotides analyzed in this work are DNA fragments with reportedly different methylation statuses in colorectal cancer and normal cells. Free energies of duplex, ncDNA formation from single strands were calculated based on melting curve analyses. Polyethylenglycole was used to imitate crowding effect. Our results suggest that CpG methylation may alter the energetic barrier for dsDNA-IM transitions.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to A...David W. Salzman
This document investigates the interaction between let-7a microRNA, Argonaute2 protein, and mRNA targets. It finds that recombinant Argonaute2 is sufficient to direct let-7a-guided cleavage of a fully complementary mRNA target in vitro. Additionally, it determines that the 5' terminal uracil of let-7a is critical for recruitment of the mRNA target to the let-7a-Argonaute2 complex. Mutation of this 5' uracil inhibits formation of the ternary let-7a-Argonaute2-mRNA complex, but does not affect formation of the binary let-7a-Argonaute2 complex. This suggests the 5' urac
This document discusses mutagenesis and genetic toxicology. It begins with an overview of DNA and the central dogma of biology. It then defines mutation and different types of mutations such as substitution, deletion, and insertion. It discusses chromosomal aberrations and the phenotypic expressions of mutations. It defines mutagenesis as the process of generating genetic mutations, which can be induced by mutagens. Common mutagens include chemicals, UV radiation, and ionizing radiation. Genetic toxicology identifies and analyzes agents that cause toxicity directed toward genetic material. Tests of genetic toxicology include the Ames assay, in vitro assays to detect chromosomal damage, and in vivo assays to detect effects on chromosomes. The comet assay is also discussed
This document describes the development of a label-free targeted LC-MS proteomic workflow to identify and validate biomarkers of kinase inhibition. The method was applied to characterize inhibitors of protein kinase CK2 (CK2). Eight commercially available CK2 inhibitors were evaluated in human cell lines. CX-4945 and inhibitor VIII were found to most effectively inhibit CK2. Elongation factor 1-delta and eukaryotic translation initiation factor 2B were identified as biomarkers of CK2 inhibition, with IF2B demonstrating superior ability to monitor cellular CK2 activity. The targeted proteomics approach provides a robust platform for studying kinase inhibitors without phosphopeptide enrichment and can be adapted to other kinase-inhibitor studies
1. This study examined how lipid nanoparticle (LNP) formulation and mRNA modifications impact delivery efficiency in normal versus Niemann-Pick C1 (NPC1)-deficient cells.
2. The results showed that an optimized LNP for mRNA delivery was less effective in NPC1-deficient cells, while the original LNP used for siRNA had higher efficacy in NPC1-deficient cells. This suggests the formulations impact trafficking pathways.
3. Modifying LNP composition, such as reducing PEG percentage or changing phospholipid, reversed the efficacy pattern by formulation. mRNA modifications also impacted trafficking.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
Austin Neurology & Neurosciences is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Neurology & Neurological Sciences.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Neurology & Neurological Sciences. Austin Neurology & Neurosciences accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of neurology & neurosciences.
Austin Neurology & Neurosciences strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group also brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
1) The study compares the effects of manganese toxicity in normal human B lymphocyte cell lines and cell lines containing a homozygous mutation in the parkin gene.
2) Results showed that manganese toxicity and cell death were similar in both cell lines, indicating cell death from manganese was not altered in cells lacking parkin activity.
3) However, manganese did inhibit mitochondrial function to a greater extent in cells lacking parkin, as shown by a decrease in ATP production, although mitochondrial membrane potential was unaffected. This suggests mutations in parkin can lead to changes in signaling pathways involved in manganese toxicity.
Genetic toxicology involves assessing the effects of physical and chemical agents on DNA and genetic processes in living cells. It examines the health impacts of genetic alterations in somatic and germ cells, mechanisms that induce alterations like DNA damage and repair, and formation of gene mutations. A variety of assays are used to detect genetic alterations, with goals of identifying mutagenic chemicals and repair mechanisms. These assays examine DNA damage, mutations in nonmammalian and mammalian models, and chromosomal aberrations. Germ cell mutagenesis is also evaluated through assays measuring gene mutations and chromosomal alterations.
This document summarizes a study that sequenced the THCA and CBDA synthase genes from marijuana samples seized by law enforcement. The study found:
1) There is significant variability in the sequences of these synthase genes among different Cannabis strains.
2) THCA synthase sequences are less variable and have more conserved single nucleotide polymorphisms than CBDA synthase sequences.
3) Many samples appeared to contain multiple copies of the synthase genes, some of which may be non-functional "pseudogenes".
4) No strong correlation was found between synthase gene sequence and THC or CBD levels in different samples. The results suggest distinguishing active from inactive synthase genes is complex.
The document summarizes the Human Genome Project. It discusses that the goal of the project was to sequence the entire human genome to better understand genetic factors in human disease. Key points include:
- The project was a large international collaboration that aimed to map all human genes by 2003, two years ahead of schedule.
- Sequencing the genome provides insights into disease diagnosis, treatment and prevention by identifying genetic risk factors.
- The project established methods for large-scale genome sequencing and mapped over 20,000 human genes.
- Understanding the genome is advancing fields like gene therapy, personalized medicine and cancer research.
This document discusses tyrosine kinases, which are enzymes that transfer phosphate groups and act as on-off switches in cellular functions. Tyrosine kinases are implicated in cancer development and progression. The document describes the structural classification, general characteristics, and mechanism of action of tyrosine kinases. It also discusses kinetic studies of tyrosine kinases like Bruton's tyrosine kinase and applications of tyrosine kinase inhibitors in cancer therapy and other diseases.
The PDE4 inhibitors roflumilast, rolipram, and apremilast were shown to inhibit TNF-α production in a dose-dependent manner, with roflumilast being the most potent. Rolipram shortened the duration of anesthesia recovery in mice, suggesting it may cause side effects like nausea, while roflumilast and apremilast did not have this effect. BCAR3 expression was found to be differentially expressed in ammonia-exposed mice, contributing to resistance to chemical-induced lung injury. The BCAR3 promoter region was cloned and shown to have promoter activity, indicating BCAR3 expression can be regulated by alternative promoters.
This is Part 2 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Nov. 2010. Part 1 is availabile here in ppt and as a webinar at the LinkedIn DABT CE group link
This document reports on a study that investigated the effects of oxidative damage to mRNA on translation. The researchers found that a single oxidative lesion, 8-oxoguanine (8-oxoG), drastically reduced the rate of peptide bond formation during translation by more than three orders of magnitude, regardless of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of tRNA selection. This suggests that the modification causes the translational machinery to stall. Consistent with these findings, mRNAs containing 8-oxoG were observed to accumulate and associate with polyribosomes in yeast strains where mRNA surveillance mechanisms were compromised, providing evidence that cells have evolved to cope with damaged mRNA.
The document discusses various causes of drug-induced liver injury including direct toxicity from drugs like acetaminophen and carbon tetrachloride as well as idiosyncratic reactions. Certain drugs are more likely to cause hepatotoxicity through both direct toxicity and idiosyncratic mechanisms. Supportive treatment measures for acetaminophen overdose-induced liver injury are also outlined. Herbal and dietary supplements can also potentially cause liver injury through mechanisms like pyrrolizidine alkaloid contamination.
This document proposes predictive toxicology and toxicogenomics services from Accommodator Consultancy Services. It discusses the need for predictive toxicology due to bans on animal testing and advances in computational modeling. The company has infrastructure for molecular modeling and data analysis. Services proposed include data processing, toxicology data integration, text mining, expert systems, and assisting with carcinogenicity tests. Benefits include reducing animal testing and speeding safety assessments. Challenges and trends in the field are also reviewed.
This document summarizes an evaluation seminar on cell signaling and signal transduction pathways presented by Mrutyunjay B Bellad of the Department of Pharmacology at H.S.K. College of Pharmacy in Bagalkot. The seminar covered various topics related to cell signaling including introduction, types of cell signaling, signal molecules and their actions, signaling through different receptor types, second messengers, G-protein coupled receptors, and signal transduction pathways. References included standard pharmacology textbooks.
A suppressor mutation counters the effects of an original mutation by restoring the wild-type phenotype. There are two main types of suppressor mutations: intragenic mutations occur within the same gene and restore function through alternate amino acid substitutions, while intergenic mutations occur elsewhere in the genome and restore function through interacting gene products. Suppressor mutations are useful for studying protein-protein interactions and dissecting biological pathways.
This document summarizes protein engineering techniques. It discusses:
1. The process of protein engineering involves diversification of genes through random mutation/recombination, selection of variants with desired properties, and amplification of selected variants.
2. Examples of techniques used for diversification include error-prone PCR and DNA shuffling. Screens and selections are used to identify variants with improved properties.
3. Multienzyme systems can be artificially synthesized through gene fusion, joining genes to create a single polypeptide with active sites from both enzymes. This allows proximity of enzymes to catalyze sequential reactions.
Crimson Publishers- CPG Methylation in G-Quadruplex and IMotif DNA StructuresCrimsonPublishers-SBB
Abberant hypomethylation in DNA regions with noncanonical folding potential (ncDNA motifs) is believed to predetermine tumor development - presumably, by facilitating G-quadruplex (G4) and/or i-motif (IM) formation via altering nucleosome positioning (stable G4s induce subsequent genomic rearrangements). We questioned whether CpG methylation per se affects the dsDNA-ncDNA equilibrium. Thermodynamic studies of genomic and model oligonucleotides with methylated CpG sites at different positions are reported. The genomic oligonucleotides analyzed in this work are DNA fragments with reportedly different methylation statuses in colorectal cancer and normal cells. Free energies of duplex, ncDNA formation from single strands were calculated based on melting curve analyses. Polyethylenglycole was used to imitate crowding effect. Our results suggest that CpG methylation may alter the energetic barrier for dsDNA-IM transitions.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to A...David W. Salzman
This document investigates the interaction between let-7a microRNA, Argonaute2 protein, and mRNA targets. It finds that recombinant Argonaute2 is sufficient to direct let-7a-guided cleavage of a fully complementary mRNA target in vitro. Additionally, it determines that the 5' terminal uracil of let-7a is critical for recruitment of the mRNA target to the let-7a-Argonaute2 complex. Mutation of this 5' uracil inhibits formation of the ternary let-7a-Argonaute2-mRNA complex, but does not affect formation of the binary let-7a-Argonaute2 complex. This suggests the 5' urac
This document discusses mutagenesis and genetic toxicology. It begins with an overview of DNA and the central dogma of biology. It then defines mutation and different types of mutations such as substitution, deletion, and insertion. It discusses chromosomal aberrations and the phenotypic expressions of mutations. It defines mutagenesis as the process of generating genetic mutations, which can be induced by mutagens. Common mutagens include chemicals, UV radiation, and ionizing radiation. Genetic toxicology identifies and analyzes agents that cause toxicity directed toward genetic material. Tests of genetic toxicology include the Ames assay, in vitro assays to detect chromosomal damage, and in vivo assays to detect effects on chromosomes. The comet assay is also discussed
This document describes the development of a label-free targeted LC-MS proteomic workflow to identify and validate biomarkers of kinase inhibition. The method was applied to characterize inhibitors of protein kinase CK2 (CK2). Eight commercially available CK2 inhibitors were evaluated in human cell lines. CX-4945 and inhibitor VIII were found to most effectively inhibit CK2. Elongation factor 1-delta and eukaryotic translation initiation factor 2B were identified as biomarkers of CK2 inhibition, with IF2B demonstrating superior ability to monitor cellular CK2 activity. The targeted proteomics approach provides a robust platform for studying kinase inhibitors without phosphopeptide enrichment and can be adapted to other kinase-inhibitor studies
1. This study examined how lipid nanoparticle (LNP) formulation and mRNA modifications impact delivery efficiency in normal versus Niemann-Pick C1 (NPC1)-deficient cells.
2. The results showed that an optimized LNP for mRNA delivery was less effective in NPC1-deficient cells, while the original LNP used for siRNA had higher efficacy in NPC1-deficient cells. This suggests the formulations impact trafficking pathways.
3. Modifying LNP composition, such as reducing PEG percentage or changing phospholipid, reversed the efficacy pattern by formulation. mRNA modifications also impacted trafficking.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
Austin Neurology & Neurosciences is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Neurology & Neurological Sciences.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Neurology & Neurological Sciences. Austin Neurology & Neurosciences accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of neurology & neurosciences.
Austin Neurology & Neurosciences strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group also brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
1) The study compares the effects of manganese toxicity in normal human B lymphocyte cell lines and cell lines containing a homozygous mutation in the parkin gene.
2) Results showed that manganese toxicity and cell death were similar in both cell lines, indicating cell death from manganese was not altered in cells lacking parkin activity.
3) However, manganese did inhibit mitochondrial function to a greater extent in cells lacking parkin, as shown by a decrease in ATP production, although mitochondrial membrane potential was unaffected. This suggests mutations in parkin can lead to changes in signaling pathways involved in manganese toxicity.
Genetic toxicology involves assessing the effects of physical and chemical agents on DNA and genetic processes in living cells. It examines the health impacts of genetic alterations in somatic and germ cells, mechanisms that induce alterations like DNA damage and repair, and formation of gene mutations. A variety of assays are used to detect genetic alterations, with goals of identifying mutagenic chemicals and repair mechanisms. These assays examine DNA damage, mutations in nonmammalian and mammalian models, and chromosomal aberrations. Germ cell mutagenesis is also evaluated through assays measuring gene mutations and chromosomal alterations.
This document summarizes a study that sequenced the THCA and CBDA synthase genes from marijuana samples seized by law enforcement. The study found:
1) There is significant variability in the sequences of these synthase genes among different Cannabis strains.
2) THCA synthase sequences are less variable and have more conserved single nucleotide polymorphisms than CBDA synthase sequences.
3) Many samples appeared to contain multiple copies of the synthase genes, some of which may be non-functional "pseudogenes".
4) No strong correlation was found between synthase gene sequence and THC or CBD levels in different samples. The results suggest distinguishing active from inactive synthase genes is complex.
The document summarizes the Human Genome Project. It discusses that the goal of the project was to sequence the entire human genome to better understand genetic factors in human disease. Key points include:
- The project was a large international collaboration that aimed to map all human genes by 2003, two years ahead of schedule.
- Sequencing the genome provides insights into disease diagnosis, treatment and prevention by identifying genetic risk factors.
- The project established methods for large-scale genome sequencing and mapped over 20,000 human genes.
- Understanding the genome is advancing fields like gene therapy, personalized medicine and cancer research.
This document discusses tyrosine kinases, which are enzymes that transfer phosphate groups and act as on-off switches in cellular functions. Tyrosine kinases are implicated in cancer development and progression. The document describes the structural classification, general characteristics, and mechanism of action of tyrosine kinases. It also discusses kinetic studies of tyrosine kinases like Bruton's tyrosine kinase and applications of tyrosine kinase inhibitors in cancer therapy and other diseases.
The PDE4 inhibitors roflumilast, rolipram, and apremilast were shown to inhibit TNF-α production in a dose-dependent manner, with roflumilast being the most potent. Rolipram shortened the duration of anesthesia recovery in mice, suggesting it may cause side effects like nausea, while roflumilast and apremilast did not have this effect. BCAR3 expression was found to be differentially expressed in ammonia-exposed mice, contributing to resistance to chemical-induced lung injury. The BCAR3 promoter region was cloned and shown to have promoter activity, indicating BCAR3 expression can be regulated by alternative promoters.
This is Part 2 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Nov. 2010. Part 1 is availabile here in ppt and as a webinar at the LinkedIn DABT CE group link
This document reports on a study that investigated the effects of oxidative damage to mRNA on translation. The researchers found that a single oxidative lesion, 8-oxoguanine (8-oxoG), drastically reduced the rate of peptide bond formation during translation by more than three orders of magnitude, regardless of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of tRNA selection. This suggests that the modification causes the translational machinery to stall. Consistent with these findings, mRNAs containing 8-oxoG were observed to accumulate and associate with polyribosomes in yeast strains where mRNA surveillance mechanisms were compromised, providing evidence that cells have evolved to cope with damaged mRNA.
The document discusses various causes of drug-induced liver injury including direct toxicity from drugs like acetaminophen and carbon tetrachloride as well as idiosyncratic reactions. Certain drugs are more likely to cause hepatotoxicity through both direct toxicity and idiosyncratic mechanisms. Supportive treatment measures for acetaminophen overdose-induced liver injury are also outlined. Herbal and dietary supplements can also potentially cause liver injury through mechanisms like pyrrolizidine alkaloid contamination.
This document proposes predictive toxicology and toxicogenomics services from Accommodator Consultancy Services. It discusses the need for predictive toxicology due to bans on animal testing and advances in computational modeling. The company has infrastructure for molecular modeling and data analysis. Services proposed include data processing, toxicology data integration, text mining, expert systems, and assisting with carcinogenicity tests. Benefits include reducing animal testing and speeding safety assessments. Challenges and trends in the field are also reviewed.
Toxicogenomics uses genomic technologies to study the effects of toxicants like drugs and chemicals on human health. It provides information on their molecular-level effects and potential toxicities. While this field shows promise to enhance risk assessments, more coordinated efforts are needed to generate data, study existing data in new ways, and address challenges. A large public database and initiatives like a proposed Human Toxicogenomics Initiative could help realize its potential to improve predictive toxicology and public health decisions.
In vitro data and in silico models for predictive toxicologyEFSA EU
The SEURAT project is a 7-year, 50 million Euro collaboration between the European Commission and Cosmetics Europe to develop non-animal approaches for repeated dose systemic toxicity testing. It involves over 70 research partners across 16 countries. The project aims to adopt a toxicological mode-of-action framework and use this knowledge to develop complementary in vitro and computational models that can predict toxicity endpoints needed for safety assessment. Key activities include developing genetically engineered cell lines, multi-scale models of organ toxicity, and an adverse outcome pathway knowledgebase to structure toxicity information. The models and data generated will be stored in online repositories to support regulatory safety evaluation.
This document summarizes a study that evaluated gene expression profiling of 84 human drug metabolizing enzymes using a pathway-focused PCR array. Researchers treated human hepatocytes with 3 drugs and used the PCR array to measure mRNA expression. They found high intra- and inter-laboratory reproducibility of the PCR array results based on correlation of ΔCT and ΔΔCT values and overlap of differentially expressed genes between experiments. Comparison to TaqMan data also showed high concordance, validating the PCR array as a reliable tool for quantifying drug metabolizing gene expression.
Systemic analysis of data combined from genetic qtl's and gene expression dat...Laurence Dawkins-Hall
Elucidating changes in gene expression by Micro array genomic sweeps of genetic QTLs linked to Tryp resistance in WT cattle to identify putative candidates underpinning pathophysiology
Genomic gene expression changes resulting from Trypanosomiasis: a horizontal study Examining expression changes elucidated by micro arrays in seminal tissues associated with the pathophysiology of Trypanosomiasis during disease progression
This document provides an overview of using gene expression profiling to evaluate drug metabolism-induced toxicity. It discusses how drug metabolism can lead to toxicity and the need to systematically evaluate this in pre-clinical studies. It then describes using Qiagen's RT2 Profiler PCR Arrays, which allow profiling of 84 drug metabolizing enzyme genes, to detect abnormalities in drug metabolism and identify mechanisms of toxicity using human hepatocytes treated with different compounds as an example application. The results showed induction and inhibition of various metabolizing enzyme genes in response to the compounds tested.
Non biopsy diagnosis of acute rejection of Renal allograftBakshish Singh
This document discusses non-invasive methods for diagnosing acute rejection in renal transplant patients. Currently, graft biopsy is the gold standard but it is invasive and detects rejection at a late stage. The presentation evaluates various potential biomarkers being studied through genomics, proteomics, and metabolomics approaches. Several individual studies are highlighted that found biomarkers like urinary MCP-1, VEGF, cytokines, and certain metabolites that showed potential for detecting early acute rejection non-invasively. Magnetic resonance imaging is also discussed as a non-invasive imaging technique being researched. In summary, the ideal non-invasive biomarker has yet to be identified but a combination of markers may provide a more realistic approach for different clinical scenarios.
The document discusses the growing problem of antibiotic resistance and potential new approaches to addressing it. It notes that 2 million nosocomial infections occur in the US each year, 70% of which are resistant to at least one drug. 90,000 people die from these infections annually, a nearly 600% increase since 1992. The document then examines RecA, a bacterial protein involved in DNA repair, as a potential new target for antibiotics. It summarizes research investigating various compounds that inhibit RecA's function and could help combat antibiotic resistance.
This study analyzed various gene expression, microRNA, and chromatin immunoprecipitation sequencing datasets to investigate the transcriptional regulation of autophagy during adipocyte differentiation. Differentially expressed genes and autophagy-related genes were identified across time points of differentiation. The mTOR pathway was most activated in differentiating adipocytes while the Erb pathway was activated in maturing adipocytes. Regulatory networks were constructed linking microRNAs, transcription factors, and autophagy genes. Finally, mouse phenotypes and diseases associated with dysregulation of these networks were identified. The coordinated regulation of autophagy allows it to function appropriately at different stages of adipocyte differentiation.
This document summarizes a study that investigated the complexes containing the RNA helicase DDX6, which is a key component of P-bodies involved in posttranscriptional regulation. The researchers identified DDX6 complexes using tandem affinity purification coupled with mass spectrometry. They found that DDX6 was present in three main complexes: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. Investigation of P-body assembly under various conditions identified three proteins required for assembly in all conditions: DDX6, 4E-T, and LSM14A. The results reveal that P-body assembly involves different pathways that nevertheless share these three key factors connecting
Genomics and proteomics in drug discovery and developmentSuchittaU
This document discusses the role of genomics and proteomics in drug discovery and development. It explains that genomics and proteomics technologies can help identify new drug targets by comparing gene and protein expression between healthy and diseased cells. Proteomics in particular analyzes changes in protein levels and can quantify individual proteins using techniques like 2D gel electrophoresis and mass spectrometry. The integration of genomics and proteomics provides a more comprehensive understanding of biological systems and is improving the drug discovery process.
1) The document describes the design and synthesis of new (bis)ureidopropyl and (bis)thioureidopropyl diamine compounds as inhibitors of the histone demethylase LSD1.
2) Key compounds featured 3-5-3 and 3-6-3 carbon backbone architectures. Several compounds displayed single-digit micromolar IC50 values against recombinant LSD1 in vitro.
3) Compound 6d showed low micromolar cell viability IC50 values against lung and breast cancer cell lines. It also increased mRNA expression of silenced tumor suppressor genes in lung cancer cells.
oral presentation; Systematic approach for dissecting the molecular
mechanisms of transcriptional regulation in bacteria.
SIGNIFICANCE
Organisms must constantly make regulatory decisions in
response to a change in cellular state or environment. However, while the catalog of genomes expands rapidly, we
remain ignorant about how the genes in these genomes are
regulated. Here, we show how a massively parallel reporter
assay, Sort-Seq, and information-theoretic modeling can be
used to identify regulatory sequences. We then use chromatography and mass spectrometry to identify the regulatory
proteins that bind these sequences. The approach results in
quantitative base pair-resolution models of promoter mechanism and was shown in both well-characterized and unannotated promoters in Escherichia coli. Given the generality of the
approach, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in a wide range of
bacteria.
Identification of the Enrichment Pathways of Catalase in Multiple Primary Tumorsdaranisaha
Reactive oxygen species (ROS) has been detected in almost all cancers, which it involves many aspects of carcinogenesis and tumor progression. We previously reported a novel oncogenic role of manganese superoxide dismutase (MnSOD; SOD2) in invasive lung adenocarcinoma (LUAD) by upregulating forkhead box protein M1 (FOXM1) and matrix metalloproteinase-2 (MMP2) expression. In this study, we used a comprehensive analysis and further evaluated the effects of a hydrogen peroxide (H2O2) scavenger, catalase (CAT) in The Cancer Genome Atlas (TCGA) datasets of the different cancer types.
This document summarizes a study that analyzed gene expression in barley and Arabidopsis plants infected with powdery mildew and Turnip mosaic virus, respectively. The researchers extracted total RNA and polysome-associated (actively translated) RNA from infected and uninfected plants. Microarray analysis identified genes differentially expressed in total and polysomal RNA in response to infection. In barley, 3505 genes were differentially expressed in resistant plants infected with powdery mildew, but no genes were differentially expressed in susceptible plants. In Arabidopsis, 958 genes were differentially expressed in response to Turnip mosaic virus infection. Gene ontology analysis showed that differentially expressed genes were enriched for specific biological functions.
This document summarizes recent research on the pharmacogenetics of drug transporters. It focuses on two classes of membrane transporter proteins: ATP-binding cassette (ABC) transporters and solute carriers (SLC). The document reviews studies on genetic variants in ABCB1 (P-glycoprotein) and their effects on the pharmacokinetics and toxicity of substrate drugs. While in vitro studies show some variants affect protein expression and function, in vivo studies have found inconsistent relationships between ABCB1 variants and the disposition of drugs like digoxin, morphine, docetaxel, and irinotecan. The document suggests haplotype analysis may provide better insight than analyzing variants individually.
An Enrichment Analysis For Cardiometabolic Traits Suggests Non-Random Assignm...Mandy Brown
This document summarizes a study that investigated whether genes associated with cardiometabolic disorders tend to be regulated by specific microRNAs (miRNAs) or by miRNAs randomly. The study retrieved 520 single-nucleotide polymorphisms (SNPs) linked to cardiometabolic traits from genome-wide association studies, which corresponded to 304 genes. Enrichment analysis found that these cardiometabolic genes were over-represented among the predicted target genes of several miRNAs, providing evidence that miRNAs non-randomly regulate sets of functionally related genes involved in cardiometabolic traits.
The document discusses using PCR arrays to profile gene expression and epigenetics. PCR arrays allow researchers to analyze expression of up to 84 genes related to a pathway or disease using real-time PCR. They include controls to check for genomic DNA contamination and assay performance. As an example, the document describes how a researcher could use a PCR array to compare gene expression between metastatic and non-metastatic breast tumor samples.
1. • Discovery Compound Differentiation using
Toxicogenomics
• Investigative miRNA Expression Analysis
Molecular Toxicology in
Drug Discovery at
AstraZeneca
Joe Milano
6/16/2011
2. Outline
Introduction to our approach to microarray analysis
Differentiating compounds based on renal transcript
profiles to support drug discovery project progression
Establish miRNA analysis capability in AstraZeneca Safety
Assessment
miRNA expression and target prediction to understand 2,5-
hexanedione testicular effects
3. Analysis Approach
Rat 230 2.0 Array
31000
Transcripts
ANOVA
p=0.05
Statistics
Final
Gene
List
Filter Low
Expressing Genes
Signal Detection
Algorithm
List Analysis
Ontology Enrichment
Pathways Analysis
4. Pathway Analysis
Toxicity Analysis Workflow tool helps to analyze the
dataset(s) in view of toxicogenomics and drug response
information contained in the MetaCore database
GeneGo toxic pathology biomarkers
GeneGo toxicity processes
GeneGo toxicity maps
GO Processes
GO Molecular functions
GO Localizations
5. GeneGo Ontology Distribution Output
X-axis
-log(pValue) – the
statistical likelihood that a
subset of genes in an
ontology would appear in
a gene list.
Y-axis
Rank order by
significance for the
ontology.
p=0.05
6. Pathways and networks
Pathway – well established
biochemical or signal
transduction map. Eg. Insulin
pathway, apoptosis pathway
Network – an interactive map
that is drawn based on
interactions that have been
curated from the literature
7. Outline
Introduction to our approach to microarray analysis
Differentiating compounds based on renal transcript
profiles to support drug discovery project progression
Establish miRNA analysis capability in AstraZeneca Safety
Assessment
miRNA expression and target prediction to understand 2,5-
hexanedione testicular effects
8. Discovery Phase Compound
Differentiation
Compound AZ123 has entered development with known
kidney tox.
Presence of intracytoplasmic hyaline droplets
Increase in urine volume, urine protein and urine NAG
Indicative of renal tubular injury
Follow-up investigative compounds A, B and C are being
evaluated using a 14-day rat tox study
Similar structures and pharmacology
One of these will be selected for further development
9. Solution (or at least part of the solution)
Applied transcript profile analysis to differentiate
compounds and support a selection decision
Results evaluated with standard pathology, clinical
chemistry and urine protein biomarkers of nephrotoxicity
(Kim1, NGAL, aGST etc.)
Based on these analyses nephrotoxicity rank ordering
Compound C > Compound A ≈ Compound B
Selection of Compound A supported by toxicogenomics
analysis
10. Experimental Workflow
Male rats dosed daily for 14 days p.o. N=3
Total RNA isolated from whole kidneys
Transcript abundance assayed using
Affymetrix Rat 230 2.0 array
Pathway analysis on gene lists performed in GeneGo’s MetaCore
Data analyzed using GeneSpring GX
11. Affymetrix Data Analysis
GeneSpring GX 10.0
PLIER16 used for probe summarization
Data was filtered based on low raw signal
Statistical analyses (t-test or ANOVA) p=0.05
Lethality at high dose for 2 compounds made statistical
analysis difficult
Gene lists were analyzed in MetaCore’s Toxicity
Analysis Workflow using 1.3-fold threshold
12. Analysis Workflow
Compound A
Compound B
Compound C
Toxicity Analysis – Kidney
focus comparing all
compound transcript lists.
Compound differentiation
analyzing individual
compound lists
Gain understanding of the
relationships of individual
genes.
13. Toxicity Workflow- Compare All Lists
Each show about the
same number of gene
changes
Compound C shows
twice the number of
genes within 1.3-fold
threshold
14. Toxicity Analysis Workflow : All Gene Lists
p= 0.05
Gene lists analyzed in MetaCore
using Toxicity Analysis – kidney
focus
Most process not shared by all
kidney transcript profiles
Suggestion that there is a effect on
cell division and xenobiotic
metabolism
Need higher resolution to understand
similarities and differences
Compounds differentiated by
analyzing individual compound
transcript profiles
16. Toxicity Analysis Workflow: Single
Compound Lists
Compounds A and B ontology
distributions show enrichment for the
same top 4 endpoints
Several genes induced in the top 3
ontologies for both Compounds A
and B show significant overlap with
CAR and PXR related pathways
Compound C ontology distribution
shows
Common endpoint-CAR mediated
regulation kidney
Ontologies related to cell cycle
progression
Transcriptional response similar
for compounds A and B different
for Compound C
Compound A
Compound B
Compound Cp=0.05
17. CAR Regulation of Xenobiotic Metabolism
ABCC4 is induced by all 3
compounds
Both transporters and
Phase II genes induced
by compounds A and B
Compound C shows
induction of transporters
Expresssion profiles
overlap but with variable
effect on CAR (PXR)
controlled genes.
1- A Low Dose
2-A High Dose
3-B Low Dose
4- B High Dose
5-C Low Dose
6-C High Dose
18. CAR Regulation of Xenobiotic Metabolism:
Individual Transcripts
Most transcripts show induction less than 2-fold
Not impressive changes when examined individually
Biological relevance may be extracted from transcripts
that show low induction when put into biological context
19. Compound C genes that
are enriched for processes
involved in cell cycle
progression
This suggests a mitogenic
response in the kidney
Pathology did not show
increased mitotic index
Cell Cycle Progression of Mitosis
Compound C Specific Network
21. GO Molecular Functions
Both Compounds A and B show enrichments for
transcripts involved in glucuronosyltransferase
and glutathione functions
Compound C shows enrichment for genes
involved in multidrug transporter activity and
xenobiotic transporter activity
Driven by increase ABCC4, ABCC2 and MDR1
High dose AUC is 3x higher than compounds A or B
Suggests potential for drug accumulation at 14 days
Compound C
Compound A
p=0.05
Compound B
22. Pathology
Kidney pathology findings note intracytoplasmic
hyaline droplets (arrow) for all compounds.
Also seen with development compound AZ123
Presumed to be a2 -globulin specific to the male rat
Not used for human risk assessment
No difference between compounds
23. Toxic Pathology Biomarkers
Most ontologies for Compounds A and B
are not statistically significant.
Compound C enrichments strongly imply
kidney tubular injury/necrosis
Kidney hyaline droplet ontologies driven by
the expression of Slc11A2 and ALT.
Compound BCompound A
Compound C p=0.05
24. Clinical Chemistry
Data from all compounds show increase in LDH,
albumin, aGST and GSTYb1 in urine
Indicators of tubular damage
Increased albumin has been associated with renal hyaline
droplets
Also seen at same time point with AZ123
Compound C data show Kim1 protein increase in
remaining high dose animal and robust induction of
Kim1 transcript at both doses
25. Data Summary
Compounds A and B behave similarly with respect to toxicity networks
Induction of Xenobiotic Response genes, UGTs, GST reductase
Compound C is different from the other 2
Induction of genes involved in cell cycle control suggesting a mitogenic
response – regenerative?
While no difference was found by pathology, Compound C data show
induction of Kim1, enrichment for transcripts associated with renal tubular
damage and up-regulation of cell cycle control genes
Nephrotoxicity rank ordering
Compound C > Compound A Compound B
Compound B later found to be a mutagen (Ames) and clastogen (rat
micronucleus)
These data support selection of Compound A to move forward
26. Outline
Introduction to our approach to microarray analysis
Differentiating compounds based on renal transcript
profiles to support drug discovery project progression
Establish miRNA analysis capability in AstraZeneca Safety
Assessment
miRNA expression and target prediction to understand 2,5-
hexanedione testicular effects
27. MicroRNAs (miRNAs)
miRNAs
Highly conserved, single stranded
RNAs (~22 nucleotides)
Reduce protein expression by
reducing mRNA translation
miRNA expression profiles can be
influenced by the cellular
environments
Emerging serum-based
biomarkers in various biological
and toxicological processes
Dicer – an endoribonuclease
cleaves pre-miRNA to mature
miRNA
RISC – RNA induced silencing
complex
Nucleus
Cytoplasm
mRNAmRNA
Protein-coding gene miRNA gene
Pre-miRNA
Pri-miRNA
Dicer
Mature miRNA
RISC
RISC
AAAA
Translational inhibition / mRNA degradation
Ribosome
ORF
28. Using miRNAs for Mechanistic
Investigation and Biomarker Assessment
miRNAs are known to have specific tissue expression
Promising tissue specific biomarkers of toxicity
Little is know about actual gene silencing targets and
regulated pathways for many miRNAs.
Used 2,5-hexanedione, Sertoli cell-specific toxicant, to
examine whether miRNAs might be potential biomarkers of
testicular toxicity.
Compared predicted target pathway ontologies to known
target pathway ontologies to confirm roles of miRNAs in
testis.
29. Experimental Design and Outcome
14-day rat study using the testicular toxicant, 2,5-hexanedione in
drinking water ad libitum
Left testis was taken for RNA isolation and miRNA analysis on ABI
Taqman miRNA array
8 miRNAs were differentially regulated
Applied two analysis strategies for miRNA evaluation
miRNAs were entered into a publicly available target prediction
algorithm (miRDB) which generated a list of 375 predicted targets
Analyzed in GeneGo’s MetaCore database for known targets and
interaction network construction yielding a list of 74 genes.
Both analysis strategies suggest miRNA targets involved FSH
signaling, cell cycle and cell adhesion pathways.
30. Approaches for identifying miRNA targets,
and potential mechanistic biomarkers
Marshall Thomas et al. Nature Structural & Molecular Biology 17 ,1169 (2010)
In vitro
In vivo
In silico
Toxicants
31. Male rats dose 14 days with 2,5-HD via ad libitum drinking water n = 3
RNA isolated from whole testis were analyzed by ABI rodent miRNA TaqMan low density
array card A
Statistical analysis yielded 8 differentially expressed miRNAs
Predicted miRNA targets
determined using miRDB
Experimentally determined miRNA targets mined
from GeneGo’s MetaCore
Network generation
Ontology enrichment analysis using genes
from network generation
Ontology enrichment analysis using genes
from target prediction
Experimental Workflow
32. Dysregulated miRNAs
All are down regulated
miRNAs were entered into GeneGo’s MetaCore database
and used to build an interaction network
Network consists of 75 genes
Each miRNA was entered into miRDB for target prediction
375 putative targets were pooled and entered into MetaCore for
Enrichment Analysis
33. Network Build with Differentially
Expressed miRNAs
Differentially expressed miRNAs were used in network build
Experimentally determined miRNA targets are highlighted by bold red
edges
Two-step interactions with known targets are included in this network
34. Ontology Enrichment Analyses
Predicted Targets Empirical Targets
Enrichment analyses show the FSH-β signaling network in common
and suggest involvement of cell cycle, cell adhesion and signal
transduction pathways.
35. Hypothylamic-Pituitary-Gonadal Axis
FSH stimulates the maturation
of germ cells by stimulation of
Sertoli cells.
Induces Sertoli cells to secrete
inhibin as part of a negative
feedback loop.
36. Toxic Pathology Comparison
Testis related toxic
pathologies
Predicted targets – 23 of 50
Empirical targets – 25 of 50
Strongly relates miRNAs to
testis function.
37. Common Predicted and Empirical
miRNA Targets
FOG2 (Friend of GATA) – transcription factor
Important regulator of hematopoiesis and cardiogenesis in mammals
Also has a role in gonadal differentiation and sex determination
Found in multiple cell lineages in both the ovary and testis
TCF8 (Transcription factor 8) – transcriptional repressor
Known to be an FSH-regulated gene in the ovary
TGFβ2 – receptor ligand
Plays an important role in multiple developmental processes
Known to block Inhibin A binding
Wee1 – protein kinase
Negative regulator of entry into mitosis – G2/M transition
Known to control the activity of M-phase promoting factor – CyclinB/Cdc2
by inhibitory phosphorylation
Transcript is decreased in the testis of men with spermatogenic failure
38. Conclusions
Predicted targets for 8 dysregulated miRNAs in the testis
show enrichment for biologically relevant pathways related
to 2,5-hexanedione toxicity
Two strategies for assessing the biological context of 8
dysregulated miRNAs in the testis show enrichment for
genes involved in FSH-β signaling, a pathway critical to
Sertoli cells stimulation and Germ cell maturation.
In silico approach demonstrates that miRNAs play
important roles in regulation of testicular function
Combining miRNA profiling with interactome/pathway
analysis is a promising approach for identifying
biological/toxicologically-relevant miRNA-mRNA
interactions, and potential mechanistic miRNAs biomarkers
Further study of miRNAs in plasma and testis is ongoing
40. The PLIER Algorithm
PLIER produces an improved signal (a summary value for a
probe set) by accounting for experimentally observed patterns
for feature behavior.
Quantile normalization
Raw intensity values are preprocessed to create equally distributed
data between chips.
Estimation of background
The intensity of the MM probe is treated as background and is
subtracted from PM probe.
41. GeneGo Process Networks
Reproduction FSH-beta signaling pathway
Effect of FSH on sertoli cell function
BMP_TGF beta signaling
Overlap with FSH signaling pathway – activin and BMP
Cell cycle_G2-M
Cell adhesion – cadherin, catenin and ephrin
p=0.05