Endotoxin detection in a competitive electrochemical assay: synthesis of a suitable endotoxin conjugate

A biotin-lipopolysaccharide (biotin-LPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng(-1). The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotin-LPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidin-horseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotin-LPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml(-1), equivalent to 0.07 EU ml(-1).     

Microfluidics in biotechnology

Microfluidics enables biotechnological processes to proceed on a scale (microns) at which physical processes such as osmotic movement, electrophoretic-motility and surface interactions become enhanced. At the microscale sample volumes and assay times are reduced, and procedural costs are lowered. The versatility of microfluidic devices allows interfacing with current methods and technologies. Microfluidics has been applied to DNA analysis methods and shown to accelerate DNA microarray assay hybridisation times. The linking of microfluidics to protein analysis techologies, e.g. mass spectrometry, enables picomole amounts of peptide to be analysed within a controlled micro-environment. The flexibility of microfluidics will facilitate its exploitation in assay development across multiple biotechnological disciplines.     

Chemogenomics and biotechnology

A robust knowledge of the interactions between small molecules and specific proteins aids the development of new biotechnological tools and the identification of new drug targets, and can lead to specific biological insights. Such knowledge can be obtained through chemogenomic screens. In these screens, each small molecule from a chemical library is applied to each cell type from a library of cells, and the resulting phenotypes are recorded. Chemogenomic screens have recently become very common and will continue to generate large amounts of data. The interpretation of this data will occupy biologists and chemists alike for some time to come. This review discusses methods for the acquisition and interpretation of chemogenomic data, in addition to possible applications of chemogenomics in biotechnology.     

Bioethics and biotechnology

Biotechnology is at the intersection of science and ethics. Technological developments are shaped by an ethical vision, which in turn is shaped by available technology. Much in biotechnology can be celebrated for how it benefits humanity. But technology can have a darker side. Biotechnology can produce unanticipated consequences that cause harm or dehumanise people. The ethical implications of proposed developments must be carefully examined. The ethical assessment of new technologies, including biotechnology, requires a different approach to ethics. Changes are necessary because new technology can have a more profound impact on the world; because of limitations with a rights-based approach to ethics; because of the importance and difficulty of predicting consequences; and because biotechnology now manipulates humans themselves. The ethical questions raised by biotechnology are of a very different nature. Given the potential to profoundly change the future course of humanity, such questions require careful consideration. Rather than focussing on rights and freedoms, wisdom is needed to articulate our responsibilities towards nature and others, including future generations. The power and potential of biotechnology demands caution to ensure ethical progress.     

Nantotechniques and approaches in biotechnology

Nanotechnology has enabled the development of an amazing variety of methods for fabricating nanotopography and nanopatterned chemistry in recent years. Some of these techniques are directed towards producing single component particles, as well as multi-component assembly or self-assembly. Other methods are aimed at nanofeaturing and patterning surfaces that have a specific chemistry or topography. This article concentrates mainly on surface-directed nanobiotechnologies because they are nearer to commercial realisation, such as use in tissue engineering, control of biofouling and cell culture, than those directed at producing nanoparticles.     

An efficient method for the detection and elimination of systematic error in high-throughput screening

MOTIVATION: High-throughput screening (HTS) is an early-stage process in drug discovery which allows thousands of chemical compounds to be tested in a single study. We report a method for correcting HTS data prior to the hit selection process (i.e. selection of active compounds). The proposed correction minimizes the impact of systematic errors which may affect the hit selection in HTS. The introduced method, called a well correction, proceeds by correcting the distribution of measurements within wells of a given HTS assay. We use simulated and experimental data to illustrate the advantages of the new method compared to other widely-used methods of data correction and hit selection in HTS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Endotoxin and cancer chemo-prevention

Reduced rates of lung cancer have been observed in several occupational groups exposed to high levels of organic dusts contaminated by endotoxin. The underlying anti-neoplastic mechanism of endotoxin may be an increased secretion of endogenous anti-neoplastic mediators and activation of the toll-like receptors (TLR). A detoxified endotoxin derivative, Monophosphoryl Lipid A (MPL^®) is marketed in Europe since 1999 as part of the adjuvant systems in allergy vaccines for treatment of allergic rhino-conjunctivitis and allergic asthma. Over 200,000 patients have used them to date (nearly 70% in Germany). Since detailed exposure (MPL^® dose and timing of administration) and individual data are potentially available, an observational follow-up study could be conducted in Germany to investigate the protective effect of MPL^® against cancer, comparing cancer incidence in two groups of patients with allergic rhinitis: those treated with allergoids plus MPL^® and those treated with a vaccine including the same allergoids but not MPL^®. The protective effect of MPL^® could be quantified in ever and never smokers. If this proposed observational study provides evidence of protective effects, MPL^® could be immediately used as a chemo-preventive agent since it is already in use as adjuvant in human vaccines against cancer.     

Endotoxin and macrophage-migration inhibition

Endotoxin inhibits the in vitro migration of macrophages. Macrophages which have been stimulated by intraperitoneal oil (Marcol) are more sensitive to the endotoxin than are nonstimulated, normal macrophages. Other factors appear to affect the sensitivity of macrophages and there is great variation between individual animals. This effect is not due to toxicity since the macrophages remain viable. Furthermore, it can be reversed by the addition of polymyxin B. This action appears to be a direct effect on macrophages since it is still evident with viable, enriched populations. The action of endotoxin can be potentiated by exposure of macrophages to lymphocyte supernatants containing migration-inhibition factor. The action is not potentiated by periodate treatment. In this situation the two effects are additive. It is suggested that some of the variability in the migration-inhibition factor assay might be due to contaminating endotoxin. Endotoxin has been found to contaminate most biological materials. The degree of contamination might well influence the level of activation of the macrophage, and thus the responsiveness to migration-inhibition factor. This work supports the concept that the macrophage needs to be at a certain level of activation to respond to migration-inhibition factor. It is clear that the presence of contaminating endotoxin needs to be considered, and prevented, in all work on migration-inhibition factor.     

Cellulases and related enzymes in biotechnology

Basic and applied research on microbial cellulases, hemicellulases and pectinases has not only generated significant scientific knowledge but has also revealed their enormous potential in biotechnology. At present, cellulases and related enzymes are used in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and for research purposes. Indeed, the demand for these enzymes is growing more rapidly than ever before, and this demand has become the driving force for research on cellulases and related enzymes. The present article is an overview of the biotechnological state-of-the-art for cellulases and related enzymes.     

Microbiology and biotechnology education in India

Editorial

Microbiology and biotechnology education in India     

Magnetic separations in biotechnology

Magnetic separation techniques provide probably the most rapid and convenient method of separating certain particles from dilute suspensions, especially those that might block columns or filters. This and other applications of magnetism, including cell sorting and product recovery are discussed.