Biotechnology annual review最新文献

Cells contain molecules, which become fluorescent when excited by UV/Vis radiation of suitable wavelength. This fluorescence emission, arising from endogenous fluorophores, is an intrinsic property of cells and is called auto-fluorescence to be distinguished from fluorescent signals obtained by adding exogenous markers. The majority of cell auto-fluorescence originates from mitochondria and lysosomes. Together with aromatic amino acids and lipo-pigments, the most important endogenous fluorophores are pyridinic (NADPH) and flavin coenzymes. In tissues, the extracellular matrix often contributes to the auto-fluorescence emission more than the cellular component, because collagen and elastin have, among the endogenous fluorophores, a relatively high quantum yield. Changes occurring in the cell and tissue state during physiological and/or pathological processes result in modifications of the amount and distribution of endogenous fluorophores and chemical-physical properties of their microenvironment. Therefore, analytical techniques based on auto-fluorescence monitoring can be utilized in order to obtain information about morphological and physiological state of cells and tissues. Moreover, auto-fluorescence analysis can be performed in real time because it does not require any treatment of fixing or staining of the specimens. In the past few years spectroscopic and imaging techniques have been developed for many different applications both in basic research and diagnostics.

Species identification represents a critical issue in food chain safety and quality control. Several procedures are available to detect animal proteins in cattle feed or to trace transgenic foods. The most effective approach is based on the use of DNA as a marker. Amplification of DNA provides rapid, sensitive and specific protocols. Several target genes can be used, but new insights come from the mitochondrial genome, which is naturally amplified in each cell and shows a remarkable resistance to degradation. These are key points when analysing complex matrices such as foods, animal feedstuff or environmental samples. Traceability is important to prevent BSE or to monitor novel foods, such as genetically modified organisms. Amplification is commonly performed, but it requires expertise and a molecular biology laboratory to perform restriction analysis, electrophoresis or gel staining for the visualisation of results. Hereby, we consider a strategy based on multiple nested amplification and reverse hybridisation assay that virtually requires only a thermocycler and a water bath. The protocol is rapid and simple and can simultaneously detect different species in a DNA sample. This promising approach allows microarray developments, opening up to further perspectives. An international application has been published under the patent cooperation treaty. Presently, a ban on feeding ruminants on cattle-derived proteins is in force in Europe and USA. The identification of metazoan traces in a sample is not only a mere preventive measure for BSE, but represents a possible screening system for monitoring biotechnology products and procedures, as well as a quality control strategy to assure consumer's rights.

Non-Hodgkin's lymphoma (NHL) remains an important complication of associated HIV infection despite advances in antiretroviral therapy (ART), and the optimum chemotherapy regimen for this disease remains to be defined. A dose-escalation trial was performed to determine the maximum tolerated doses of etoposide and doxorubicin as part of the 12-week VACOP-B regimen, supported by filgrastim (r-metHuG-CSF). Patients with aggressive histology HIV-related NHL who were previously untreated with chemotherapy, and who had no active opportunistic infection were eligible for the study. Chemotherapy consisted of cyclophosphamide 350 mg/m2, vincristine 2 mg, bleomycin 10 U/m2; and prednisone 100 mg q2 days x 12 weeks, with increasing doses of doxorubicin 25-50 mg/m2 and etoposide 25-50 mg/m2 intravenously and 50-100 mg/m2 orally. Central nervous system prophylaxis (intrathecal cytarabine 50 mg x 4 doses), antifungal, and Pneumocystis carinii prophylaxis were used, and filgrastim was administered to prevent neutropenic complications. One dose level was expanded to permit the concomitant use of ART. Endpoints were determination of maximum tolerated dose of doxorubicin and etoposide, treatment tolerability, and survival. Forty-seven patients were enrolled, most with diffuse large-cell or immunoblastic NHL. Protocol-defined maximum tolerated dose was not reached and the limits of dose-limiting toxicity were not exceeded, even in patients receiving ART. Thirty-two cycles (4.9%) were delayed >6 days because of toxicity; 30 patients (64%) completed all 12 weeks of treatment. After completion of therapy, 14 patients had a complete response (30%), and 4 had a partial response (8%). Median time to progression was 9 months. At 42 months, progression-free survival was 25% and overall survival was 28%.

Phage display is an established technology that has been successfully applied, in the last fifteen years, to projects aimed at deciphering biological processes and/or at the isolation of molecules of practical value in several diverse applications. Bacteriophage lambda, representing a molecular cloning and expression tool widely utilized since decades, has also been exploited to develop vectors for the display of libraries on its capsid. In the last few years, lambda display approach has been consistently offering new enthralling perspectives of technological application, such as domain mapping, antigen discovery, and protein interaction studies or, more generally, in functional genomics.

The release of sequence data, particularly from a number of medically and biotechnologically important genomes, is increasing in an exponential fashion. In light of this, elucidating the structure and function of proteins, particularly in a "high throughput" manner, is an important quest. The production of recombinant proteins however is not always straightforward, with a number of proteins falling prey to low expression problems, a high susceptibility to proteolysis and the often despised production of inclusion bodies. Whilst expression as inclusion bodies can often be advantageous, their solubilization and renaturation is often a time consuming and empirical process. In this review, we aim to outline some of the more common approaches that have been applied to a variety of proteins and address issues associated with their handling.

Antibodies that react with many different molecular species of protein and non-protein nature are widely studied in biology and have particular utilities, but the precise epitopes recognized are seldom well defined. The definition of epitopes by X-ray crystallography of the antigen-antibody complex, the gold standard procedure, has shown that most antibody epitopes are conformational and specified by interactions with topographic determinants on the surface of the antigenic molecule. Techniques available for the definition of such epitopes are limited. Phage display using either gene-specific libraries, or random peptide libraries, provides a powerful technique for an approach to epitope identification. The technique can identify amino acids on protein antigens that are critical for antibody binding and, further, the isolation of peptide motifs that are both structural and functional mimotopes of both protein and non-protein antigens. This review discusses techniques used to isolate such mimotopes, to confirm their specificity, and to characterize peptide epitopes. Moreover there are direct practical applications to deriving epitopes or mimotopes by sequence, notably the development of new diagnostic reagents, or therapeutic agonist or antagonist molecules. The techniques developed for mapping of antibody epitopes are applicable to probing the origins of autoimmune diseases and certain cancers by identifying "immunofootprints" of unknown initiating agents, as we discuss herein, and are directly applicable to examination of a wider range of receptor-ligand interactions.

Proteome research has recently been stimulated by important technological advances in the field of recombinant protein expression. One major breakthrough was the development of a new generation of cell-free transcription/translation systems. The open and flexible character of these systems allows direct control over expression conditions via the addition of supplements to the expression reaction. The possibility of working with linear expression templates instead of cloned plasmids and the ease of downstream processing, circumventing the need for cell-lysis, makes them ideally suited for high-throughput screening applications. Among these novel cell-free systems, the Rapid Translation System (RTS) developed by Roche is the first one that is scalable from micrograms to milligrams of protein. This review describes the basic principles of RTS which differentiate it from traditional in vitro expression technologies, starting from template generation to high-end applications like labeling for structural biology research. Recent results obtained by RTS users from different institutions are presented to illustrate each step of a novel cell-free protein expression workflow and its benefits compared to traditional cell-based expression.

Biosimulation software is being used in pharmaceutical drug development to mimic diseases. Virtual clinical trials of new developing pharmaceutical drugs can be conducted on computers running disease simulations. Using virtual patients instead of clinical research patients can save both time and money for pharmaceutical companies in their search to discover new drugs and bring them to market. In the future, this type of research will be commonplace.

With the introduction of the common technical document (CTD), many writers in the biotech and pharmaceutical industries are now required to submit dossiers in this format. The format of the CTD is not extremely difficult from the familiar documents of the Biologic License Application (BLA) or New Drug Application (NDA). The CTD can be mapped to existing areas of the BLA or NDA. The components of the CTD are discussed and references to the current guidance worldwide are provided to assist the writer.

Anti-erythropoietin (EPO)-induced pure red cell aplasia (PRCA) is an uncommon, potentially life-threatening condition in which the bone marrow stops manufacturing red blood cells. In the past few years, reports of drug-induced, anti-EPO antibody-mediated PRCA have increased substantially, with most cases attributed to the use of one erythropoiesis-stimulating protein, Eprex. A literature review was undertaken to document the reports of drug-induced PRCA, with all drugs and drug regimens. The sudden increase in reports of antibody-mediated PRCA is discussed.