Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD – an apparatus that was designed for high-throughput susceptibility testing – allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.
Biofilms; Imaging, Three-Dimensional
Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up- vs. down-regulation, spot intensity, ρ-value, fold-change, cycle threshold (Ct), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and ρ-value) proves to be the most critical factor, with significant correlations of ρ>0.80 consistently observed when quality scores are applied.
Polymerase Chain Reaction; Microarray Analysis; Gene Expression; Nucleic Acid Amplification Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA
How the power required for bird flight varies as a function of forward speed can be used to predict the flight style and behavioral strategy of a bird for feeding and migration. A U-shaped curve was observed between the power and flight velocity in many birds, which is consistent to the theoretical prediction by aerodynamic models. In this article, we present a general genetic model for fine mapping of quantitative trait loci (QTL) responsible for power curves in a sample of birds drawn from a natural population. This model is developed within the maximum likelihood context, implemented with the EM algorithm for estimating the population genetic parameters of QTL and the simplex algorithm for estimating the QTL genotype-specific parameters of power curves. Using Monte Carlo simulation derived from empirical observations of power curves in the European starling (Sturnus vulgaris), we demonstrate how the underlying QTL for power curves can be detected from molecular markers and how the QTL detected affect the most appropriate flight speeds used to design an optimal migration strategy. The results from our model can be directly integrated into a conceptual framework for understanding flight origin and evolution.
QTL; Linkage Disequilibrium; Power Curve; Bird
In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work and reporters for transcriptional analyses. In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157. The deletion and replacement of the LEE4 operon of E. coli O157 is detailed to show the advantages and limitations of the technology.
Gene Deletion; Escherichia coli O157:H7
The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional.
Reverse Transcription; DNA, Complementary
Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods.
Hepatitis B Virus, Duck; Protein Interaction Mapping
Chromatin immunoprecipitation (ChIP) technique allows detection of proteins that bind to chromatin. While this technique has been applied extensively in cell-based studies, its tissue-based application remains poorly explored. We are specifically interested in examining estrogen-dependent transcriptional mechanism in respect of recruitment of estrogen receptor-alpha (ERα), a ligand-activated transcription factor, to uterine gene promoters in mice. Recent gene-array studies, utilizing ERα knock-out vs. wild-type mice, have revealed that estrogen regulates numerous uterine genes temporally and most importantly via ERα during the phase-II response, including three well characterized genes viz., lactoferrin (Ltf), progesterone receptor (Pgr) and cyclinD1 (Ccnd1). Here, utilizing systematic ChIP studies, we demonstrate endogenous recruitment of ERα to above uterine gene promoters following estradiol-17β (E2) injection in mice.
Chromatin; Immunoprecipitation; Estrogen Receptor Alpha
Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.
Green Fluorescent Proteins; Blue Fluorescent Protein, Aequorea Victoria
The application of naked DNA containing type I interferon (IFN) transgenes is a promising potential therapeutic approach for controlling chronic viral infections. Herein, we detail the application of this approach that has been extensively used to restrain ocular HSV-1 infection, for antagonizing vaginal HSV-2 infection. We show that application of IFN-α1, -α5, and –β transgenes to vaginal mouse lumen 24 hours prior to HSV-2 infection reduces HSV-2 mediated mortality by 2.5 to 3-fold. However, other type I IFN transgenes (IFN- α4, -α5, -α6, and –α9) are non effectual against HSV-2. We further show that the efficacy of IFN-α1 transgene treatment is independent of CD4+ T lymphocytes. However, in mice depleted of CD8+ T lymphocytes, the ability of IFN-α1 transgene treatment to antagonize HSV-2 was lost.
Virus; Interferon; T-lymphocyte; Transgene
The Chromatin Immunoprecipiation (ChIP) provides a powerful technique for identifying the in vivo association of transcription factors with regulatory elements. However, obtaining meaningful information for promoter interactions is extremely challenging when the promoter is a member of a class of highly homologous elements. Use of PCR primers with small numbers of mutations can limit cross-hybridization with non-targeted sequences and distinguish a pattern of binding for factors with the regulatory element of interest. In this report, we demonstrate the selective in vivo association of NF-κB, p300 and CREB with the human Iγ1 promoter located in the intronic region upstream of the Cγ1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology.
Gene Expression Regulation; Transcription Factors; Chromatin Immunoprecipitation; B cells
Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy.
Microtubules; Mitosis; Cell Death; Nocodazole
Electromyography (EMG) signals can be used for clinical/biomedical applications, Evolvable Hardware Chip (EHW) development, and modern human computer interaction. EMG signals acquired from muscles require advanced methods for detection, decomposition, processing, and classification. The purpose of this paper is to illustrate the various methodologies and algorithms for EMG signal analysis to provide efficient and effective ways of understanding the signal and its nature. We further point up some of the hardware implementations using EMG focusing on applications related to prosthetic hand control, grasp recognition, and human computer interaction. A comparison study is also given to show performance of various EMG signal analysis methods. This paper provides researchers a good understanding of EMG signal and its analysis procedures. This knowledge will help them develop more powerful, flexible, and efficient applications.
Electromyography; Fourier Analysis; Muscles; Nervous System
Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species.
Polymerase Chain Reaction; Leukocytes; Gene Expression; RNA, Messenger