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Recent trends in protease-catalyzed peptide synthesis.

Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

Animals↗

[Possibilities of gene-technological production of drugs, especially also the interferons].

For a long time proteins have been used in Medicine as medication. Human Growth Hormone (isolated from Pituitaries of Cadavers) or insuline (isolated from porcine or bovine pancreas) are examples. Genetic engineering has essentially simplified the production of such well known proteins and on the other hand enabled search for and characterization of new human proteins. New active principles may thus be discovered, their physiological importance studied and if possible used for therapeutic purposes. Examples are thrombolytic proteins or interferon. Today more than 20 new proteins are already registered and more than 150 proteins are intensively tested in clinics.

Genetic Engineering↗

DIROM: an experimental design interactive system for directed mutagenesis and nucleic acids engineering.

A computer system DIROM for oligonucleotide-directed mutagenesis and artificial gene design has been designed for better experimental planning and control. DIROM permits searching for optimal oligonucleotides with respect to certain important parameters, namely sufficient energy of oligonucleotide-target hybridization, the secondary structure of oligonucleotide and target DNA, the presence of alternate binding sites in the target DNA and terminal G/C pairs. It can also be used to plan polymerase chain reaction experiments, for optimal primer selection, in sequencing, etc. DIROM enables one to search for both existing and potential restriction sites, to perform vector + target sequence construction. The system consists of a set of original algorithms that formalize the empirical knowledge of oligonucleotide action as primers.

Algorithms↗

Recent progress in biomolecular engineering.

During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.

Animals↗

Design of graph-based evolutionary algorithms: a case study for chemical process networks.

This paper describes the adaptation of evolutionary algorithms (EAs) to the structural optimization of chemical engineering plants, using rigorous process simulation combined with realistic costing procedures to calculate target function values. To represent chemical engineering plants, a network representation with typed vertices and variable structure will be introduced. For this representation, we introduce a technique on how to create problem specific search operators and apply them in stochastic optimization procedures. The applicability of the approach is demonstrated by a reference example. The design of the algorithms will be oriented at the systematic framework of metric-based evolutionary algorithms (MBEAs). MBEAs are a special class of evolutionary algorithms, fulfilling certain guidelines for the design of search operators, whose benefits have been proven in theory and practice. MBEAs rely upon a suitable definition of a metric on the search space. The definition of a metric for the graph representation will be one of the main issues discussed in this paper. Although this article deals with the problem domain of chemical plant optimization, the algorithmic design can be easily transferred to similar network optimization problems. A useful distance measure for variable dimensionality search spaces is suggested.

Algorithms↗

Diversifying carotenoid biosynthetic pathways by directed evolution.

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.

Bacteria↗

Surface and gas-phase resistances to the evaporation of droplets.

Attempt has been made to asses quantitatively the magnitudes of both the gas-phase resistance and the surface resistance to the evaporation of droplets and to determine the transition point at which the evaporation changes from a diffusion-controlled to a surface-controlled process. This transition point is strongly dependent upon the ambient pressure and the droplet size. It is found that at atmospheric pressure the particle radius at which the transition takes place is in the range of 1 - 10mu for water, 0.1mu for n-dibutylphthalate, 0.1 - 1.0mu for mercury, and 0.001mu for liquid helium. Furthermore, we have found that the surface resistance and the gas-phase resistance vary with the size of the droplet in opposite direction, leading to the existence of a maximum rate of evaporation at a certain value of the droplet radius, this radius being dependent upon the characteristics of the liquid concerned. This conclusion may be useful in the search for an optimum and most efficient method of combustion of fuel sprays in automotive engines, gas turbines, and oil-burning power plants.

Aerosols↗

Neuronal cell transplantation for Parkinson's and Huntington's diseases.

The brain constitutes a privileged transplantation site. Under appropriate conditions neuronal tissues can survive transplantation into the damaged brain, integrate with the host, and alleviate functional impairments associated with neurological disease. The experimental techniques have been developed to the point of clinical application with demonstrable benefit in Parkinson's disease, and similar applications in Huntington's disease appear to be imminent. Nevertheless, present techniques require use of embryonic/fetal tissues which will limit the availability of donors for the foreseeable future. There is an active search for alternative sources of tissue that are equally effective but more readily available, including engineered cells, expanded stem/precursor cells, and xenografts.

Adrenal Glands↗

High solubility of random-sequence proteins consisting of five kinds of primitive amino acids.

Searching for functional proteins among random-sequence libraries is a major challenge of protein engineering; the difficulties include the poor solubility of many random-sequence proteins. A library in which most of the polypeptides are soluble and stable would therefore be of great benefit. Although modern proteins consist of 20 amino acids, it has been suggested that early proteins evolved from a reduced alphabet. Here, we have constructed a library of random-sequence proteins consisting of only five amino acids, Ala, Gly, Val, Asp and Glu, which are believed to have been the most abundant in the prebiotic environment. Expression and characterization of arbitrarily chosen proteins in the library indicated that five-alphabet random-sequence proteins have higher solubility than do 20-alphabet random-sequence proteins with a similar level of hydrophobicity. The results support the reduced-alphabet hypothesis of the primordial genetic code and should also be helpful in constructing optimized protein libraries for evolutionary protein engineering.

Amino Acid Sequence↗

Crystallization of the cytotoxic domain of a ribosome-inactivating colicin in complex with its immunity protein.

The complex between the ribonuclease domain of the ribosome-inactivating colicin E3 and its protein inhibitor, the cognate immunity Im3, has been crystallized and preliminary X-ray characterization has been performed. Single crystals of the 1:1 complex were grown from hanging-drop vapour-diffusion experiments using 2-propanol as a precipitant. The space group is P3(1)21 or P3(2)21, with unit-cell parameters a = b = 93.7, c = 76.2 A. When cryocooled, these crystals diffract to a resolution of 2.4 A. A search for suitable conventional heavy-atom derivatives was unsuccessful and so Im3 mutants containing engineered cysteine or methionine residues have been produced for mercury soaks and selenomethionine-labelling experiments, respectively.

Amino Acid Substitution↗

SGXPro: a parallel workflow engine enabling optimization of program performance and automation of structure determination.

SGXPro consists of four components. (i) A parallel workflow engine that was designed to automatically manage communication between the different processes and build systematic searches of algorithm/program/parameter space to generate the best possible result for a given data set. This is performed by offering the user a palette of programs and techniques commonly used in X-ray structure determination in an environment that lets the user choose programs in a mix-and-match manner, without worrying about inter-program communication and file formats, during the structure-determination process. The current SGXPro program palette includes 3DSCALE, SHELXD, ISAS, SOLVE/RESOLVE, DM, SOLOMON, DMMULTI, BLAST, AMoRe, EPMR, XTALVIEW, ARP/wARP and MAID. (ii) A client/server architecture that allows the user to utilize the best computing facility available. (iii) Plug-in-and-play design, which allows easily integration of new programs into the system. (iv) User-friendly interface.

Crystallography, X-Ray↗

Iditis: protein structure database.

The validation, enrichment and organization of the data stored in PDB files is essential for those data to be used accurately and efficiently for modelling, experimental design and the determination of molecular interactions. The Iditis protein structure database has been designed to allow the widest possible range of queries to be performed across all available protein structures. The Iditis database is the most comprehensive protein structure resource currently available, and contains over 500 fields of information describing all publicly deposited protein structures. A custom-written database engine and graphical user interface provide a natural and simple environment for the construction of searches for complex sequence- and structure-based motifs. Extensions and specialized interfaces allow the data generated by the database to used in conjunction with a wide range of applications.

Database Management Systems↗

Making bone: implant insertion into tissue-engineered bone for maxillary sinus floor augmentation-a preliminary report.

Autologous, allogenic and alloplastic materials for bony reconstruction in the cranio-maxillofacial area have many drawbacks thus stimulating the on-going search for new (bio-)materials. Whereas cultivated skin and mucosa are already in clinical routine use in head and neck reconstruction, so far there has been no successful clinical application to the best of our knowledge of periosteum-derived, tissue-engineered bone for augmentation of the edentulous posterior maxilla. In a pilot study, augmentation of the posterior maxilla was carried out using a bone matrix derived from mandibular periosteal cells on a polymer fleece. This paper demonstrates fabrication of the matrix, clinical application, and the histological results in two patients. The results suggest that periosteum-derived osteoblasts on a suitable matrix form lamellar bone within 4 months which allows reliable implant insertion.

Alveolar Ridge Augmentation↗

Exploring development and disease through germ-line genetic engineering in the mouse.

New developments in mouse genetics have transformed the field of genetics from a reactive to a proactive science. Current technologies in transgenesis, chromosome engineering, and saturation mutagenesis are introduced. These approaches permit the creation of new mutations and consequent mutant phenotypes that facilitate both the understanding of functions of existing genes and the search for previously unidentified genes affecting important phenotypes in all mammals.

Animals↗

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.

Adolescent↗

Engineering blood: synthetic substitutes from fluorinated compounds.

Concerns about blood safety and the logistical problems associated with conventional transfusion have fuelled the search for effective alternatives (so-called blood substitutes). Such materials include hemoglobin derivatives and those based on synthetic, highly fluorinated, inert organic compounds called perfluorochemicals (PFCs). PFCs dissolve large volumes of oxygen and other gases, are unreactive in the body, and are excreted primarily as a vapor by exhalation. Liquid PFCs are immiscible with blood and other body fluids, but can be injected safely into the bloodstream as submicron emulsions. Emulsified PFCs have been evaluated in clinical trials as temporary, intravascular tissue-oxygenating fluids. One such emulsion, a commercial perflubron-based, phospholipid-stabilized formulation, is in advanced clinical trials as an alternative to transfusing donated (allogeneic) blood during surgery. Basic and clinical studies have shown that this emulsion can adequately maintain tissue oxygenation during acute blood loss with no abnormal hemodynamic changes. The use of PFC emulsions as an efficacious, short-term transfusion alternative underpins the longer term objective of producing a totally synthetic, bioengineered blood substitute.

Animals↗

Multiobjective satisfaction within an interactive evolutionary design environment.

The paper introduces the concept of an Interactive Evolutionary Design System (IEDS) that supports the engineering designer during the conceptual/preliminary stages of the design process. Requirement during these early stages relates primarily to design search and exploration across a poorly defined space as the designer's knowledge base concerning the problem area develops. Multiobjective satisfaction plays a major role, and objectives are likely to be ill-defined and their relative importance uncertain. Interactive evolutionary search and exploration provides information to the design team that contributes directly to their overall understanding of the problem domain in terms of relevant objectives, constraints, and variable ranges. This paper describes the development of certain elements within an interactive evolutionary conceptual design environment that allows off-line processing of such information leading to a redefinition of the design space. Such redefinition may refer to the inclusion or removal of objectives, changes concerning their relative importance, or the reduction of variable ranges as a better understanding of objective sensitivity is established. The emphasis, therefore, moves from a multiobjective optimization over a preset number of generations to a relatively continuous interactive evolutionary search that results in the optimal definition of both the variable and objective space relating to the design problem at hand. The paper describes those elements of the IEDS relating to such multiobjective information gathering and subsequent design space redefinition.

Algorithms↗