Comprehensive quality control utilizing the prehybridization third-dye image leads to accurate gene expression measurements by cDNA microarrays

BACKGROUND: Gene expression profiling using microarrays has become an important genetic tool. Spotted arrays prepared in academic labs have the advantage of low cost and high design and content flexibility, but are often limited by their susceptibility to quality control (QC) issues. Previously, we have reported a novel 3-color microarray technology that enabled array fabrication QC. In this report we further investigated its advantage in spot-level data QC. RESULTS: We found that inadequate amount of bound probes available for hybridization led to significant, gene-specific compression in ratio measurements, increased data variability, and printing pin dependent heterogeneities. The impact of such problems can be captured through the definition of quality scores, and efficiently controlled through quality-dependent filtering and normalization. We compared gene expression measurements derived using our data processing pipeline with the known input ratios of spiked in control clones, and with the measurements by quantitative real time RT-PCR. In each case, highly linear relationships (R(2)>0.94) were observed, with modest compression in the microarray measurements (correction factor<1.17). CONCLUSION: Our microarray analytical and technical advancements enabled a better dissection of the sources of data variability and hence a more efficient QC. With that highly accurate gene expression measurements can be achieved using the cDNA microarray technology

Pursuit of Genes Active in Defense of Upland Cotton Against Bacterial Blight

Biochemistry and Molecular Biolog

Phenotypic and Genotypic Models of Streptococcal Colonization of the Human Tonsil

Pharyngitis due to oropharyngeal infection with Streptococcus pyogenes is most commonly treated by using penicillin-derived antibiotics.While treatment failure in the 1950’s was reported in 4-8% of children, more recent studies have alarmingly found antibiotic failure as high as 20-40% providing the impetus to study this important pathogen. The contiguous mucosa along neighboring oropharyngeal surfaces is classically unaffected during Strep ”Throat” suggesting pathogen specificity for palatine tonsil epithelium. While recent studies are advancing the premise of pathogen-host microenvironment effects on streptococcal virulence, the specific interaction between S. pyogenes and the human tonsillar surface relative to neighboring tissues remains insufficiently understood. This thesis investigates the unique interaction between the Group A Streptococcus, GrAS, and the human tonsil surface. We were particularly interested in the transcriptional response exhibited by the bacterium during coculture with this tissue epithelium compared to epithelium from an anatomically neighboring sight to determine if phenotype might be informed by the genotypic profile. During the course of this work, we introduce new palatine tonsil tumorderived cell lines for their novel usefulness as a model for studying pharyngitis. Using primary tonsil epithelial cells from non-malignant patient samples to confirm our studies with the cancer cell line, we were successful in demonstrating a tissue-specific phenotype and have begun the process of elucidating the genotypic nuances that an M1 strain exhibited in direct response to the tonsillar environment. These and further studies should allow us to better understand the pathogenic adaptions exhibited by this bacterium at its preferred target niche for infection

Characterising the role of a putative mammalian aspartate dehydrogenase in hepatic lipogenesis and secretion of very low density lipoproteins

Hepatic lipogenesis and the secretion of very low-density lipoproteins are severely perturbed in orotic-acid fed rats. Proteomic analysis of this rodent fatty liver identified a protein enriched in the microsomal fraction. The protein contains a domain of unknown function 108 (DUF 108) and is found to compose a complete protein in archaebacteria and mammalian species, but a single domain in Caenorhabditis elegans protein Q19527. The cDNA for human protein A6ND91 was cloned and stable rat hepatoma cell lines, expressing recombinant A6ND91-FLAG tagged protein were produced. Significant increases in de-novo lipogenesis and secretion of triglyceride-enriched lipoproteins were observed. This thesis considers A6ND91 protein in terms of structure and proposed function. Previous work has indicated that the homologous protein Thermatoga maritime 1643 (TM1643) possesses NAD+ or NADP+-dependent dehydrogenase activity towards L-Aspartate, producing NADH or NADPH plus iminoaspartate, a substrate that rapidly degrades into oxaloacetate (OAA) and ammonia. In this thesis, analyses of primary and predicted secondary structure of A6ND91 and TM1643 revealed that residues binding NAD+/NADP+ and the proposed active catalytic residue, Histidine198, are conserved. Northern-blot analysis identified A6ND91 is highly expressed in human liver, moderately in kidney, and at low levels in the brain. Western-blot analysis identified that A6ND91 is predominately localised to the cytosol, with minimal amounts in the microsomal fraction. Proteomic analysis revealed that A6ND91 contains a phosphorylated residue, Serine168. The kinase potentially responsible for phosphorylation of the residue is Glycogen Synthase Kinase 3 (GSK3), a well-characterised enzyme involved in regulation of glycogen metabolism and acetyl-CoA production. Microarray analyses indicate that over-expression of A6ND91 perturbs activity of several metabolic pathways involved in carboxylic acid metabolism, cholesterol synthesis and complement activation. Closer inspection of the perturbed metabolic pathways revealed an overlap through the substrates OAA/pyruvate/acetyl-CoA. Assays were performed to establish the NAD+/NADH and NADP+/NADPH redox state in cell lines expressing A6ND91. Results established that NAD+/NADH ratio was significantly reduced, suggesting that A6ND91 may operate as a dehydrogenase. Collectively, results suggest that L-Aspartate dehydrogenase activity of TM1643 is conserved in human A6ND91, playing a key role in hepatic lipid and energy metabolism through the production of OAA and its subsequent conversion to pyruvate/acetyl-CoA and cholesterol

Integrated microbioreactors for rapid screening and analysis of bioprocesses

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Includes bibliographical references (leaves 131-140).(cont.) strains to characterize the bioreactor environment. The ability to reproducibly sacrifice microbioreactors mid-run is exploited to demonstrate the feasibility of linking microbioreactors to genome-wide expression studies using DNA microarrays. The potential of the microbioreactor for investigating different growth conditions is confirmed by comparing bacterial growth, as evaluated by the measured parameters, under conditions of different medium and oxygen concentration. It is shown that statistical differences can be observed, and that these differences are similar to those observed at a larger scale. The demonstrated functionality of the microbioreactor could potentially have a large impact in the numerous fields in which fermentations are used. In bioprocess development, the batch microbioreactor could be used to select strains at all stages of metabolic engineering and to explore and optimize growth conditions during scale-up. The microbioreactor could also be an effective tool in screening applications ranging from toxicology studies that use bacterial reporter strains, to studies that attempt to elucidate metabolic pathways, to intensification of genome-wide expression profiling using either direct links to DNA microarrays or screens of libraries carrying transcription reporters.This thesis presents the design, fabrication, and characterization of a batch microbioreactor with integrated, automated sensors and aeration through a permeable polymer membrane as a step towards establishing high-throughput bioprocessing platforms. In particular, the thesis demonstrates the feasibility of culturing bacterial cells in microliter volumes and obtaining reproducible results similar to those shown at larger scales. A microbioreactor designed to provide sufficient oxygen to a growing culture is fabricated out of PDMS and glass. Models are developed to understand oxygen transport and consumption as well as the kinetics of growth within the microbioreactor. Sensors are integrated to measure the growth parameters optical density (OD), dissolved oxygen (DO), and pH. Based on these measurements as well as cell morphology and total and viable cell counts, reproducibility is established and comparisons to bench-scale bioreactors are made. It is demonstrated that the behavior of bacteria at the two scales is very similar. It is further demonstrated that off-line analysis of the medium can be carried out by serial sacrifice of microbioreactors operating under identical conditions. The test case of HPLC analysis of the fermentation medium to measure glucose consumption and organic acid production is used. Additional sensing capabilities in the form of in situ measurements for luminescence and fluorescence are demonstrated, and a potential glucose sensor is modeled to explore feasibility. Once reproducibility in fabrication, experimental protocol, and experimental results is established, the microbioreactor is used for several applications. The ability to monitor luminescence and fluorescence on-line enables the use of bacterial reporterby Andrea Zanzotto.Ph.D

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

Host and pathogen transcriptional profiles of acute Brucella melitensis infection

The parallel gene expression profiles of Brucella melitensis and the host have not been elaborated. In this study, I analyze and discuss the transcriptional profiles of B. melitensis invasive-associated genes, the expression profile of intracellular B. melitensis and B. melitensis-infected non-phagocytic cells in the first 12 h post-infection (PI), and the in vivo temporal global transcriptome of both B. melitensis and the infected bovine host in the first 4 h PI. The initial study found that B. melitensis at late-log phase of growth were more invasive in non-phagocytic cells than at early-log or stationary growth phase. Microarray-based studies identified 454 Brucella genes differentially expressed between the most and the least invasive growth phases. Additionally, B. melitensis strains with transposon interrupted in loci BMEII0380 (acrA) and BMEI1538 (hypothetical protein) were found to be deficient in internalization compare with the wild-type strain. A second experiment was designed with the goal of characterizing host and pathogen transcriptome in parallel. For detecting intracellular Brucella gene expression, a combined protocol consisting of a linear amplification of sense-stranded RNA biased to pathogen transcripts to the previously enriched host:pathogen RNA mixed sample, was developed. RNA samples were hybridized on human and Brucella cDNA microarrays, which analysis revealed a common down-regulation transcriptional profile at 4 h PI that was reverse at 12 h PI. The integrity of B. melitensis virB operon and the expression of host MAPK1 were confirmed as critical for early B. melitensis intracellular survival and replication in non-phagocytic cells. Finally, a temporal morphological and molecular characterization of the initial B. melitensis:bovine host interaction using a calf ileal loop model was performed. B. melitensis was isolated from intestinal Peyer’s patches as soon as 15 min and from systemic blood after 30 min postintra luminal inoculation. Microarray results revealed a common transcriptional profile in Brucella, but two different transcriptional profiles were identified in the host in the first 4 h PI. The importance of differentially expressed biological processes, pathways and individual genes in the initial Brucella pathogenesis is discussed

Transcriptomic and proteomic analysis of lycopene-overproducing Escherichia coli strains

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.Systems biology represents a powerful method to describe and manipulate phenotypes of interest by incorporating biological information from various levels of cellular organization. Such an approach is illustrated from a library of both rationally-directed and combinatorial gene knockout strains of E. coli recombinantly producing the small molecule lycopene. Global genomic and proteomic expression changes associated with increased lycopene production of mutant E. coli constructs were discovered using whole-genome DNA microarrays and a novel LC-MS technique, respectively. While most genes and proteins showed few expression changes, key differences were identified, including targets distal to the non-mevalonate and precursorsupplying pathways. Based upon the expression data sets, it was hypothesized that the following may be associated with lycopene overproduction: histidine biosynthesis (hisH); the quinone pool (wrbA); acid resistance (ydeO and gadE); the glyoxylate pathway (iclR); NADPH redox balance (pntB); growth rate reduction; and membrane composition. In the pre-engineered background strain, deleting pntB (~20-25%) and ydeO (~30%) each led to moderately increased production; overexpressing wrbA led to 50-100% more production at 8 hours and 5-15% more production at later time points; deleting iclR caused small production increases (~5-10%); and supplementing media with histidine caused the parental and mutant strains to have similar production.(cont.) From these observations, several themes emerged. First, reduced cellular growth and energy conservation appear to be important tradeoffs for increasing lycopene production. Second, reducing overflow metabolism to acetate and corresponding acid stress as well as providing a gluconeogenic flux to increase lycopene precursors appeared beneficial. Next, NADPH availability and balance seemed to be critical production factors. The sS factor is known to affect lycopene accumulation, and it was observed to have far-reaching effects on both the transcriptomic and proteomic data sets. While expression changes were not strictly additive between the five mutant strains examined in comparison to the pre-engineered background strain, a number of these common factors appear to be responsible for the high lycopeneproduction phenotype. This work serves as an important example of incorporating multiple layers of complementary biological information to define a basis for an observed phenotype, demonstrating a powerful paradigm for realizing production increases via systems metabolic engineering.by Brian E. Mickus.Ph.D