Lee Larcombe

The rational development of molecularly imprinted polymer-based sensors for protein detection

The detection of specific proteins as biomarkers of disease, health status, environmental monitoring, food quality, control of fermenters and civil defence purposes means that biosensors for these targets will become increasingly more important. Among the technologies used for building specific recognition properties, molecularly imprinted polymers (MIPs) are attracting much attention. In this critical review we describe many methods used for imprinting recognition for protein targets in polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors (277 references).

Passive control of quorum sensing: prevention of Pseudomonas aeruginosa biofilm formation by imprinted polymers.

Here we present the first molecular imprinted polymer (MIP) that is able to attenuate the biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa through specific sequestration of its signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-AHL). The MIP was rationally designed using computational modeling, and its capacity and specificity and that of a corresponding blank polymer toward signal molecule of P. aeruginosa (3-oxo-C(12)-AHL) and its analogue were tested. The biofilm formation in the presence of polymers and without polymers was studied using scanning confocal laser microscopy. Staining with crystal violet dye was used for the quantification of the biofilm formation. A significant reduction of the biofilm growth was observed in the presence of MIP (>80%), which was superior to that of the resin prepared without template, which showed a reduction of 40% in comparison with biofilm, which was grown without polymer addition. It was shown that 3-oxo-C(12)-AHL-specific MIP prevented the development of quorum-sensing-controlled phenotypes (in this case, biofilm formation) from being up-regulated. The developed MIP could be considered as a new tool for the elimination of life-threatening infections in a multitude of practical applications; it could, for example, be grafted on the surface of medical devices such as catheters and lenses, be a component of paints, or be used as a wound adsorbent.

Temporal and spatial changes in the microbial bioaerosol communities in green-waste composting.

In this study, the microbial community within compost, emitted into the airstream, downwind and upwind from a composting facility was characterized and compared through phospholipid fatty acid analysis and 16S rRNA gene analysis using denaturing gradient gel electrophoresis and bar-coded pyrosequencing techniques. All methods used suggested that green-waste composting had a significant impact upon bioaerosol community composition. Daily variations of the on-site airborne community showed how specific site parameters such as compost process activity and meteorological conditions affect bioaerosol communities, although more data are required to qualify and quantify the causes for these variations. A notable feature was the dominance of Pseudomonas in downwind samples, suggesting that this genus can disperse downwind in elevated abundances. Thirty-nine phylotypes were homologous to plant or human phylotypes containing pathogens and were found within compost, on-site and downwind microbial communities. Although the significance of this finding in terms of potential health impact was beyond the scope of this study, it clearly illustrated the potential of molecular techniques to improve our understanding of the impact that green-waste composting emissions may have on the human health.

Microarray Meta-Analysis: From Data to Expression to Biological Relationships

Since the introduction of microarray technology, it has become the workhorse for mRNA expression profiling. Its application ranges from investigating gene function, regulation, and co-expression, to clinical use in diagnosis and prognosis. Over the last decade, a large number of microarray experiments have become available in public repositories often addressing similar or related hypotheses. The large compendia of gene expression data provide the opportunity to conduct meta-analyses by combining data from various independent but related studies. Such data integration has the potential to enhance the reliability and generalizability of the results of individual microarray studies.

Meta-analysis of expression of l(3)mbt tumor-associated germline genes supports the model that a soma-to-germline transition is a hallmark of human cancers.

Evidence is starting to emerge indicating that tumorigenesis in metazoans involves a soma‐to‐germline transition, which may contribute to the acquisition of neoplastic characteristics. Here, we have meta‐analyzed gene expression profiles of the human orthologs of Drosophila melanogaster germline genes that are ectopically expressed in l(3)mbt brain tumors using gene expression datasets derived from a large cohort of human tumors. We find these germline genes, some of which drive oncogenesis in D. melanogaster, are similarly ectopically activated in a wide range of human cancers. Some of these genes normally have expression restricted to the germline, making them of particular clinical interest. Importantly, these analyses provide additional support to the emerging model that proposes a soma‐to‐germline transition is a general hallmark of a wide range of human tumors. This has implications for our understanding of human oncogenesis and the development of new therapeutic and biomarker targets with clinical potential.

CancerMA: a web-based tool for automatic meta-analysis of public cancer microarray data

The identification of novel candidate markers is a key challenge in the development of cancer therapies. This can be facilitated by putting accessible and automated approaches analysing the current wealth of ‘omic’-scale data in the hands of researchers who are directly addressing biological questions. Data integration techniques and standardized, automated, high-throughput analyses are needed to manage the data available as well as to help narrow down the excessive number of target gene possibilities presented by modern databases and system-level resources. Here we present CancerMA, an online, integrated bioinformatic pipeline for automated identification of novel candidate cancer markers/targets; it operates by means of meta-analysing expression profiles of user-defined sets of biologically significant and related genes across a manually curated database of 80 publicly available cancer microarray datasets covering 13 cancer types. A simple-to-use web interface allows bioinformaticians and non-bioinformaticians alike to initiate new analyses as well as to view and retrieve the meta-analysis results. The functionality of CancerMA is shown by means of two validation datasets. Database URL: http://www.cancerma.org.uk

Detection and imaging the expression of the trans-membrane protein CD44 in RT112 cells by use of enzyme-labeled antibodies and SECM.

The deposition of human RT112 cells in a patterned fashion onto glass substrates and subsequent imaging of the expression of the trans-membrane protein CD44 have been studied using scanning electrochemical microscopy (SECM). Patterns of RT112 cells derived from a transitional cell carcinoma of the bladder could be deposited on amino-modified glass substrates by cytospinning. These were then treated with horseradish peroxidase (HRP) labeled secondary antibodies to the trans-membrane protein CD44. Expression of CD44 protein by the cells directly leads to immobilisation of the labeled antibodies. The presence of the enzyme substrate (hydrogen peroxide) along with a hydroquinone mediator then allowed an enzymatic reaction to proceed, generating benzoquinone. Reduction of benzoquinone gave rise to positive feedback between the substrate and the SECM microelectrode tip. Control samples such as blank slides or slides not treated with HRP-labeled antibody showed negative feedback effects. Patterns of RT112 cells could be assembled and their expression of the target protein imaged whereas control samples showed minimal activity.

Polyphenol control of cell spreading on glycoprotein substrata

Cell–surface contacts are vital for many eukaryotic cells. The surface provides anchorage (facilitating spreading and proliferation), is involved in sensation, i.e., via mechano-, osmo- and chemoreceptors, and in addition nutrients may also be supplied via vessels adjacent to the basal lamina. Hence, the ability to manipulate the surface characteristics provides a mechanism for directly influencing cell behaviour. Applications such as medical implants and tissue engineering require biocompatible, stable surfaces for controlling cell behaviour. Mucin-coated surfaces inhibit cell spreading compared with poly(L-lysine) in vitro; here, we show that a composite layer assembled from mucin-EGCg aggregates counters the inhibition. Although the anti-spreading effects of the glycoprotein substratum on cell behaviour are similar to those observed for pure polysaccharide surfaces, the reversal of cell spreading inhibition by the admixture of polyphenol/glycoprotein substrata is remarkable and unexpected. Possible applications for a composite glycoprotein–polyphenol layer include medical devices, in particular for those operating at mucosal interfaces such as the oral, tracheal or gastrointestinal tract cavities, wound healing, cancer control and the controlled growth of therapeutic cell cultures.

An in silico study of the differential effect of oxidation on two biologically relevant G-quadruplexes: possible implications in oncogene expression.

G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential “hot-spots” for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.

Cancer germline gene activation: friend or foe?

The human male germ line is passed on via the production of haploid sperm, which, upon fusion with the female gamete, the ovum, will form a diploid zygote that will ultimately become a genetically unique individual. Male gametogenesis occurs throughout the lifespan of adult males. Sperm production is restricted to the seminiferous tubules of the testis, and this process is continually fed by the mitotic proliferation of germline stem cells (GSCs). The GSCs are a sub-group of spermatagonial cells, which are found at the basal layer of the seminiferous tubules. Upon receipt of differentiation signals, spermatagonial cells will undergo differentiation, first maturing to primary spermatocytes and then ultimately through to fully differentiated spermatozoa. During this cellular differentiation, meiosis occurs, reducing the chromosomal content from diploid to haploid status and driving genetic variation.