Browsing by Subject "Microarray"

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The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants
(2010-08) Syrett, Heather Angel; Ellington, Andrew D.; Kitto, George B.; Willets, Katherine A.; Iyer, Vishwanath R.; Yin, Yuhui W.
The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun.
Development of wireless DNA microarray sensors
(2010-08) Chow, Kwok-Fan; Crooks, Richard M. (Richard McConnell); Bard, Allen J.; Bielawski, Christopher; Manthiram, Arumugam; Stevenson, Keith
The development of wireless DNA microelectrochemical microarray sensors is described. The operational principles of these sensors are based on bipolar electrochemistry. Bipolar electrodes are used to fabricate the wireless microarrays in this work. The systems are configured so that DNA sensing is carried out at the cathodic end of a bipolar electrode (BPE) and the result of the sensing experiment is reported at the anodic end of the BPE. There are two types of reporting platforms developed in this study. The first type relies on the emission of electrogenerated chemiluminescence (ECL). The system is configured so that ECL is emitted at the anodic end of the BPE when the target DNA is hybridized to the capture probe DNA immobilized on the cathodic end of the BPE. However, when there is no hybridization reaction occurs, there is no ECL emission on the electrode surface. The second type of reporting platform developed is based on silver electrodissolution at the anodic end of a BPE. When a reduction reaction occurs at the cathodic end of a BPE, it triggers oxidation and dissolution of silver deposited at the anodic end of the BPE. The loss of silver can easily be detected by the naked eye. This detection principle is used for DNA detection: when the target DNA is hybridized to capture probe DNA on the BPE, the BPE becomes shorter. However, if target DNA does not hybridize to the electrode surface, the length of the BPE remains the same. The BPE microarrays described in this work eliminate the need for complicated microfabrication procedures and instrumentation. For example, as many as 1000 BPEs can be simultaneously controlled using just two driving electrodes and a simple power supply. To fully utilize BPE microarrays for specific sensing tasks, a method based on robotic spotting was developed to modify the cathodic end of each BPE in the array. Because each BPE in a microarray is individually addressable, this development allows each BPE to perform a particular sensing operation.
Genetic basis for ichthyotoxicity and osmoregulation in the euryhaline haptophyte, Prymnesium parvum N. Carter
(2014-05) Talarski, Aimee Elizabeth; La Claire, John W., 1951-
There is limited information currently available regarding the underlying physiological responses and molecular mechanisms of osmoregulation, acetate metabolism [in relation to the synthesis of glycerolipids, polyunsaturated fatty acids (PUFA), and ichthyotoxins], and transport in Prymnesium parvum N. Carter, a microalga that causes devastating harmful algal blooms (HAB) worldwide. This dissertation examines gene expression under environmental conditions that are associated with HAB formation, including phosphate limitation and low salinity, using microarrays and RNA sequencing (RNA-Seq). A comparative fatty acid methyl ester (FAME) analysis at 30 vs. 5 practical salinity units (psu) was performed to gain additional insight into acetate metabolism. The RNA-Seq analysis included a de novo assembly of the P. parvum transcriptome, generating 47,289 transcripts, of which 35.4% were identifiable. This permitted the evaluation of the expression of many more genes compared with the microarray analysis, which examined ~3,500 genes. Relevant candidate genes identified included those whose products are involved in osmolyte production, salinity stress, and ion transport. With respect to the putative synthesis of polyketide ichthyotoxins, 32 different polyketide synthase (PKS) transcripts were identified in the transcriptome assembly, none of which were differentially expressed. Hemolysin and monogalactosyldiacylglycerol synthase were downregulated at 30 vs. 5 psu, suggesting the increased presence of additional ichthyotoxins at the lower salinity. Evidence for several PUFA synthesis pathways was also revealed. Fatty acid compositions were largely similar at the two salinities, containing relatively prominent quantities of the PUFA stearidonic acid, but compositions varied among strains. The transcription of genes whose products are associated with vesicular transport was elevated, and higher levels of extracellular prymnesins were observed in HAB-forming conditions. Thus, with regard to acetate metabolism, I have revealed evidence for the post-transcriptional regulation of the production of prymnesins and the contributory effects of hemolysin, monogalactosyldiacylglycerol, and PUFA towards ichthyotoxicity. Further, I propose that toxin transport is triggered in HAB-forming conditions, in which the toxins are actively being excreted. Collectively, these data shed light on the transcriptional responses that occur following alterations in phosphate availability and salinity, including those associated with the synthesis and delivery of a number of potential ichthyotoxins from P. parvum.
Glycomics : integration of lectin and gene expression microarray data
(2011-08) Pilobello, Kanoelani Takaishi; Mahal, Lara K.; Anslyn, Eric V., 1960-
Glycomics is the systematic study of glycosylation in the context of a whole cell or organism. Glycosylated proteins are estimated to make up 50% of all proteins and cover the outside of the cell. Functional roles in glycosylation have been noted in pathogenesis, metastasis, and embryogenesis. However, the structure of these carbohydrates has been difficult to study due to the chemical nature of carbohydrates. Lectins, carbohydrate binding proteins excluding antibodies and enzymes, can be utilized to study glycosylation in a high throughput manner using a microarray format. Glycans, the carbohydrates attached to a protein or lipid, are not synthesized from a template. They are added co- or post-translationally by a concerted set of enzymes in the secretory pathway. In addition, the glycan structures may be altered by metabolism or trafficking. Cell type specific glycosylation has long been hypothesized due to observations of bacteria homing to tissues. We use lectin microarray technology to define the glycosylation in a subset of the NCI-60, a set of cell lines from different tissues. Using a customized gene expression microarray, we identify cell type dependent glycosylation genes and observe evidence of cell type dependent spliceforms for an O-glycosylated mucin. Data from the lectin microarray and a published gene expression data set were integrated using Generalized Singular Value Decomposition (GSVD), a linear matrix decomposition method. We have successfully decomposed the data into 3 cell type dependent meta patterns that segregate by glycosylation family. Correlation projection of the genes and subsequent gene ontology enrichment suggests that genes in different pathways covary with the types of glycosylation. An inverse relationship was revealed for the N- glycosylation pattern between the SVD of the lectins and the GSVD of the genes and lectins together. Whereas, the relationship was correlative for O-glycosylation, which was clearly illustrated in biplots. This work argues that types of glycosylation are regulated by different mechanisms in different cell types.
Investigating cellular responses to mutations in the glutathione and thioredoxin pathways of Escherichia coli
(2009-12) Chrysostomou, Constantine; Georgiou, George; Isaac, Sanchez
Inhibition of disulfide bond formation in Escherichia coli implicates an intricate collaboration of proteins which comprise the glutathione and thioredoxin reducing pathways. Bioengineers have successfully engineered E. coli possessing mutated reducing pathways that promote, rather than inhibit, disulfide bond formation in the cytoplasm. The transcriptome of six such mutant E. coli strains have been characterized using Microarray technology. We find that all mutant strains, exhibit a unique response to oxidative stress, not observed in wild type. Statistical analyses revealed the expression of more than 200 genes that are affected by mutations within the reducing pathways. Significantly up-regulated biological processes include cysteine biosynthesis, histidine biosynthesis, NADH Dehydrogenase I biosynthesis, sugar catabolic processes, and activation of stress responses . The second part of this work describes the construction of an E. coli strain that promotes the complete conversion of glutathione into its seemingly dormant derivative, glutathionylspermidine. This engineered strain can be used in assays designed to evaluate the effectiveness of glutathionylspermidine as a substitute for glutathione and, hopefully, allude to its true metabolic function.
Multi-analyte biosensing : the integration of sensing elements into a photolithographically constructed hydrogel based biosensor platform
(2005-05) Schmid, Matthew John; Willson, C. G. (C. Grant), 1939-
The genome sequencing programs have identified hundreds of thousands of genetic and proteomic targets for which there are presently no ascribed functions. The challenge for researchers now is to characterize them, as well as identify and characterize their natural variants. Historically, this has meant studying each individual target separately. However, due to the recent development of multi-analyte microarray devices, these characterizations can be performed in a combinatorial manner in which a single experiment provides information on thousands of targets at a time. In the past decade, microarray technology has settled in on two major designs. The first entails spotting individual receptor types onto a functionalized glass substrate. This is a simple and inexpensive process; however, due to the limited resolution of the mechanical devices used to do the spotting, the densities of these arrays are relatively low. Moreover, receptor preparation requires substantial time and effort. The second variety of microarray uses photolithographic techniques adapted from the semi-conductor industry to chemically synthesize the receptor elements in situ on the sensing surface. Because lithographic patterning is spatially very precise, these arrays achieve very high densities, with as many as one million features per square centimeter. Although these arrays obviate the necessity for laborious "off chip" probe preparation, they are expensive to produce and are limited to two types of receptors (oligonucleotides and peptides). This dissertation presents the development work performed on a hydrogel-based biosensor platform which provides a high density and low cost alternative to the two aforementioned designs. The array features are fabricated lithographically from a liquid pre-polymer doped with biologically active sensing elements at sizes as small as 50[micrometer]. Each of the feature types is uniquely shaped, which enables the features to be mass-produced in batches, pooled together and then assembled into randomly ordered arrays using highly-parallelized self-assembly techniques. The three-dimensional hydrogel features accommodate a wide variety of sensing elements, such as enzymes, antibodies and cells, which cannot be deployed using the traditional designs. This dissertation presents methods developed to integrate cellular and oligonucleotide sensing elements into the hydrogel features which preserve their biological activity and optimize the sensor's performance.
Social Odors Conveying Dominance and Reproductive Information Induce Rapid Physiological and Neuromolecular Changes in a Cichlid Fish
(BioMed Central, 2015-02) Simoes, Jose M.; Barata, Eduardo N.; Harris, Rayna M.; O'Connell, Lauren A.; Hofmann, Hans A.; Oliveira, Rui F.; Harris, Rayna M.; Hofmann, Hans A.
Social plasticity is a pervasive feature of animal behavior. Animals adjust the expression of their social behavior to the daily changes in social life and to transitions between life-history stages, and this ability has an impact in their Darwinian fitness. This behavioral plasticity may be achieved either by rewiring or by biochemically switching nodes of the neural network underlying social behavior in response to perceived social information. Independent of the proximate mechanisms, at the neuromolecular level social plasticity relies on the regulation of gene expression, such that different neurogenomic states emerge in response to different social stimuli and the switches between states are orchestrated by signaling pathways that interface the social environment and the genotype. Here, we test this hypothesis by characterizing the changes in the brain profile of gene expression in response to social odors in the Mozambique Tilapia, Oreochromis mossambicus. This species has a rich repertoire of social behaviors during which both visual and chemical information are conveyed to conspecifics. Specifically, dominant males increase their urination frequency during agonist encounters and during courtship to convey chemical information reflecting their dominance status.
A systems pharmacology approach to discovery of drugs to ameliorate oxidant stress in human endothelial cells
(2015-05) Bynum, James Andrew, Jr.; Stavchansky, Salomon; Bowman, Phillip D; Kerwin, Sean M; Cui, Zhengrong; Williams, Robert O
Ischemia is characterized by reduced blood flow to an area of the body which can then cause cellular injury through the generation of reactive oxygen species (ROS), activation of inflammation, and induction of apoptosis. Although rapid reestablishment of flow is required to prevent organ death, the reperfusion phase of this injury can cause its own deleterious effects often exacerbating the initial insult. The combined action of the two injuries is termed ischemia/reperfusion (I/R) injury. Oxidative stress that results from ischemia/reperfusion injury is a common pathological condition that accompanies many human diseases including stroke, heart attack and traumatic injury. In addition, neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s disease appear to involve oxidative stress. Although actively investigated by the medical and pharmaceutical industry; limited progress has been made to ameliorate I/R injury and to date there is no drug approved for treatment for I/R injury. Therapeutic approaches to treat I/R injury have included the administration of compounds to scavenge ROS or induce protective pathways or genetic responses. It was previously reported that caffeic acid phenethyl ester (CAPE), a plant-derived polyphenol, displayed cytoprotective effects against menadione (MD)-induced oxidative stress in human umbilical vein endothelial cells (HUVEC), and the induction of heme oxygenase-1 (HMOX1), a phase II enzyme, played an important role for CAPE cytoprotection. In an effort to improve this cytoprotection, other phase II enzyme inducers were investigated and, 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) and 2-cyano-3,12-dioxooleana-1,9-dien-28-oyl methyl ester (CDDO-Me), were found to be potent inducers with a rapid onset of action. CDDO-Im and CDDO-Me, synthetic olenane triterpenoids, developed as anticancer agents were compared to CAPE revealing that CDDO-Im was a more potent inducer of Phase II enzymes including HMOX1 and provided better cytoprotection than CAPE. Gene expression profiling showed that CDDO-Im was more potent inducer of protective genes like HMOX1 than CAPE and additionally induced heat shock proteins. To better understand the mechanism of action of CDDO-IM, a gene expression time-course was undertaken to identify early initiators of the transcriptional response preceding cytoprotection. Application of systems pharmacology identified molecular networks of cell mediating processes.