Development of whole-cell and cell-free biosensors for the detection and differentiation of organic and inorganic forms of copper.

The modern world has seen exposure of bacterial communities to toxic metals at selective levels. This manifests itself both intentionally, through medicines and un-intentionally through waste streams. There is growing concern that selective exposure to metals may be linked to microbial resistance to antibiotics.

Gene expression profiling of copper-resistant Caco-2 clones.

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper.

Primary Culture of Cornea-Limbal Epithelial Cells In Vitro.

The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.

LC-MS proteomic profiling of Caco-2 human intestinal cells exposed to the copper-chelating agent, triethylenetetramine: A preliminary study.

Homeostasis of metal micronutrients such as copper is tightly regulated to ensure deficiency does not occur while restricting damage resulting from excess accumulation. Using LC-MS the effect on the proteome of intestinal Caco-2 cells of exposure to the chelator triethylenetetramine (TETA) was investigated. Continuous exposure of TETA at 25 μM to Caco-2 cells caused decreased cell yields and morphological changes.

Characterisation and proteomic profiling of continuously exposed Cu-resistant variants of the Caco-2 cell line.

Studies in hepatic systems identify multiple factors involved in the generation of copper resistance. As the intestine is the route of exposure to dietary copper, we wanted to understand how intestinal cells overcome the toxic effects of high copper and what mechanisms of resistance develop. Using the intestinal cell line Caco-2, resistance was developed by serial subculture in 50 μM copper in inorganic (CuSO) or organic (Cu proteinate) forms.

Copper -Dipyridoquinoxaline Is a Potent DNA Intercalator that Induces Superoxide-Mediated Cleavage via the Minor Groove.

Herein, we report the synthesis, characterisation, X-ray crystallography, and oxidative DNA binding interactions of the copper artificial metallo-nuclease [Cu(DPQ)(NO)](NO), where DPQ = dipyrido[3,2-:2',3'-]quinoxaline. The cation [Cu(DPQ)] (Cu-DPQ), is a high-affinity binder of duplex DNA and presents an intercalative profile in topoisomerase unwinding and viscosity experiments. Artificial metallo-nuclease activity occurs in the absence of exogenous reductant but is greatly enhanced by the presence of the reductant Na--ascorbate.

Oxidative DNA Cleavage with Clip-Phenanthroline Triplex-Forming Oligonucleotide Hybrids.

A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN-TFO hybrids is based on application of the alkyne-azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5'- or 3'-ends or in the middle of the TFO stretch.

A nutrient cocktail prevents lipid metabolism alterations induced by 20 days of daily steps reduction and fructose overfeeding: result from a randomized study.

Physical inactivity and sedentary behaviors are independent risk factors for numerous diseases. We examined the ability of a nutrient cocktail composed of polyphenols, omega-3 fatty acids, vitamin E, and selenium to prevent the expected metabolic alterations induced by physical inactivity and sedentary behaviors. Healthy trained men ( n = 20) (averaging ∼14,000 steps/day and engaged in sports) were randomly divided into a control group (no supplementation) and a cocktail group for a 20-day free-living intervention during which they stopped exercise and decreased their daily steps (averaging ∼3,000 steps/day).

Proteome-wide Adaptations of Mouse Skeletal Muscles during a Full Month in Space.

The safety of space flight is challenged by a severe loss of skeletal muscle mass, strength, and endurance that may compromise the health and performance of astronauts. The molecular mechanisms underpinning muscle atrophy and decreased performance have been studied mostly after short duration flights and are still not fully elucidated. By deciphering the muscle proteome changes elicited in mice after a full month aboard the BION-M1 biosatellite, we observed that the antigravity soleus incurred the greatest changes compared with locomotor muscles.

Correlating transcriptional networks to breast cancer survival: a large-scale coexpression analysis.

Weighted gene coexpression network analysis (WGCNA) is a powerful 'guilt-by-association'-based method to extract coexpressed groups of genes from large heterogeneous messenger RNA expression data sets. We have utilized WGCNA to identify 11 coregulated gene clusters across 2342 breast cancer samples from 13 microarray-based gene expression studies. A number of these transcriptional modules were found to be correlated to clinicopathological variables (e.

A simple and direct pre-embedding technique for ultrastructure of scarce biological specimens.

A simple and rapid technique for pre-embedding scarce biological specimens for Transmission electron microscopy (TEM) is reported. It is based on pre-embedding biological samples in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized with paraformaldehyde, glutaraldehyde, ammonium persulfate and Temed. Pre-embedding in BSA and BA offers several advantages over traditional pre-embedding techniques for TEM including the ability to visualize the sample and a more resistant matrix.

Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity.

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence.

Development of a robust microtiter plate-based assay method for assessment of bioactivity.

A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples. The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis. The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound.

Morphological quantification of pellets in Streptomyces hygroscopicus var. geldanus fermentation broths using a flatbed scanner.

An image analysis technique has been developed to allow high throughput morphological characterisation of microbial fermentation broths containing spherical pellets greater than 100 microm in diameter. Images of stained Streptomyces hygroscopicus var. geldanus culture samples at three different inoculum levels were captured using a flatbed scanner, at a resolution of 21 microm per pixel (1200 dots per inch) and subsequently analysed leading to the generation of a morphological profile of each sample.