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After a decade of progress, an expanded role for metabolic engineering.

Over the past decade, metabolic engineering has emerged as an active and distinct discipline characterized by its over-arching emphasis on integration. In practice, metabolic engineering is the directed improvement of cellular properties through the application of modern genetic methods. Although it was applied on an ad hoc basis for several years following the introduction of recombinant techniques [1,2], metabolic engineering was formally defined as a new field approximately a decade ago [3]. Since that time, many creative applications, directed primarily to metabolite overproduction, have been reported [4]. In parallel, recent advances in the resolution and acquisition time of biological data, especially structural and functional genomics, has amplified interest in the systemic view of biology that metabolic engineering provides. To facilitate the burgeoning scientific exchange in this area on a more regular and convenient basis, a new conference series was launched in 1996 followed by a new journal in 1999.

Base Sequence↗

The engineering effects of fluids flow on freely suspended biological macro-materials and macromolecules.

The manufacture of many biotechnologically important products requires consideration of the physical breakage and biochemical degradation pathways at all stages during processing, storage and transportation. The engineering flow environment in most items of bioprocess equipment has long been recognised as a key factor in determining these pathways and is the focus of the present review. Because of its industrial significance, the detrimental effects of the engineering flow environment on freely suspended bioparticles have been the subject of many scientific investigations over the past few decades. There is a general consensus of opinion that fluid shear and elongational stresses are the two main breakage pathways of relevance to processing of most biomaterials. An additional degradation pathway has also been identified involving significant losses of biological activity of macromolecules at gas-liquid, gas-solid and liquid-liquid interfaces. In such cases, the engineering flow field is shown to have a secondary role in determining the kinetics of inactivation. An equally important consideration in the optimisation of the relevant unit operations is the biomechanical integrity of the flow sensitive material. The biomechanical and biorheological parameters that determine the integrity of biomaterials are poorly defined, their evaluations present future research challenges and are of immediate engineering significance.

Biocompatible Materials↗

Enhancing antitumor by immunization with fusion of dendritic cells and engineered tumor cells.

A novel approach for a dentritic cells (DCs)-based tumor vaccine was developed for the formation of hybrid-engineered J558 after fusion with DCs. To make the hybrid-tumor vaccine generate more efficient specific CTL cytotoxicity against wild-type tumor cells, we genetically engineered tumor cells with mIL-12 gene prior to the cell fusion. mIL-12 was detected at 870 +/- 60 pg/(10(5) cells/ml) in the culture supernatants and the fusion ratio was about 30% by the co-focal microscopic analysis. Vaccination of mice with DCs fused with engineered J558 induced more efficient tumor-specific CTL cytotoxicity against wild-type tumor cells in vitro and with efficient antitumor immunity in vivo. These results suggest that this approach of using DCs fused with engineered tumor cells could be applied in clinical settings of DCs-based cancer vaccines.

Animals↗

Preparation of severely curved simulated root canals using engine-driven rotary and conventional hand instruments.

This in vitro study evaluated the efficacy and safety of six different nickel-titanium engine-driven instruments used with a torque-controlled engine device and nickel-titanium hand and stainless steel hand instruments in preparation of curved canals. A total of 80 curved (36 degrees) simulated root canals were prepared. Images before and after were superimposed, and instrumentation areas were observed. Time of instrumentation, instrument failure, change in working length and weight loss were also recorded. Results show that stainless steel hand instruments cause significantly less transportation towards the inner wall of the canal than do nickel-titanium hand instruments. No instrument fracture occurred with hand instruments, but 30-60% breakage of instruments was recorded during instrumentation with the engine-driven devices. The working length was maintained by all types of instruments. Newly developed nickel-titanium rotary files were not able to prevent straightening of the severely curved canals when a torque-controlled engine-driven device was used.

Dental Alloys↗

Permeability issues in whole-cell bioprocesses and cellular membrane engineering.

Nutrient uptake and waste excretion are among the many important functions of the cellular membrane. While permitting nutrients into the cell, the cellular membrane system evolves to guide against noxious agents present in the environment from entering the intracellular milieu. The semipermeable nature of the membrane is at odds with biomolecular engineers in their endeavor of using microbes as cell factory. The cellular membrane often retards the entry of substrate into the cellular systems and prevents the product from being released from the cellular system for an easy recovery. Consequently, productivities of whole-cell bioprocesses such as biocatalysis, fermentation, and bioremediations are severely compromised. For example, the rate of whole-cell biocatalysis is usually 1-2 orders of magnitude slower than that of the isolated enzymes. When product export cannot keep pace with the production rate, intracellular product accumulation quickly leads to a halt of production due to product inhibition. While permeabilization via chemical or physical treatment of cell membrane is effective in small-scale process, large-scale implementation is problematic. Molecular engineering approach recently emerged as a much better alternative. Armed with increasingly sophisticated tools, biomolecular engineers are following nature's ingenuity to derive satisfactory solutions to the permeability problem. This review highlights these exciting molecular engineering achievements.

Biodegradation, Environmental↗

Tissue engineering heart valves: valve leaflet replacement study in a lamb model.

BACKGROUND: Valve replacements using either bioprosthetic or mechanical valves have the disadvantage that these structures are unable to grow, repair, or remodel and are both thrombogenic and susceptible to infection. These characteristics have significantly limited their durability and longevity. In an attempt to begin to overcome these shortcomings, we have tested the feasibility of constructing heart valve leaflets in lambs by seeding a synthetic polyglycolic acid fiber matrix in vitro with fibroblasts and endothelial cells. METHODS: Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from the fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold constructed from polyglycolic acid fibers was seeded with fibroblasts, which grew to form a tissue-like sheet. This tissue was subsequently seeded with endothelial cells, which formed a cellular monolayer coating around the leaflet. Using these constructs, autologous (n = 3) and allogenic (n = 4) tissue engineered leaflets were implanted in 7 animals. In each animal the right posterior leaflet of the pulmonary valve was resected and replaced with an engineered valve leaflet. RESULTS: All animals survived the procedure. Postoperative echocardiography demonstrated no evidence of stenosis and trivial pulmonary regurgitation in the autografts and moderate regurgitation in the allogenic valves. Collagen analysis of the constructs showed development of an extracellular matrix. Histologic evaluation of the constructs demonstrated appropriate cellular architecture. CONCLUSIONS: This preliminary experiment showed that a tissue engineered valve leaflet constructed from its cellular components can function in the pulmonary valve position. Tissue engineering of a heart valve leaflet is feasible, and these preliminary studies suggest that autograft tissue will probably be superior to allogenic tissue.

Animals↗

Human engineering environments for the aged.

Human engineering is generally concerned with the design of human tasks, systems, and environments so that people can work and live safely, comfortably, and effectively. As a product of World War II, human engineering has until recently been primarily concerned with military and aerospace systems. Lately, however, the field has been focussing on broader societal problems. Many human engineering principles can easily be generalized to cover the design of certain features of environments for the aged. However, the most effective use of human engineering in this area will require research to supply better information about the aged, their needs, capacities and limitations.

Journal Article↗

Evaluation of the driver's cab in the Rc5 engine.

The opinion of engine drivers concerning the design of the cab in a new engine (Rc5, ASEA, Sweden) is surveyed in the present study, which is aimed at recommendations for further improvements of the new cab. Fifty engine drivers from the northern part of Sweden (Ange, Vannas and Boden), who had been driving the new engine, were selected at random to answer a specially designed questionnaire. Most of the drivers considered the new cab as better than the old one (Rc4) in several respects. Particularly appreciated was an adjustable footrest, on which the safety regulator pedal was fitted, which enabled an individually adjusted position for driving. They also preferred the automatic brake valve located close to the driver and with its movements in a sagittal direction. A number of proposals for further improvement of the cab are presented in order to attain an optimally designed driver's cab.

Journal Article↗

Health care professional workstation: software system construction using DSSA scenario-based engineering process.

This paper describes a new method for the evolutionary determination of user requirements and system specifications called scenario-based engineering process (SEP). Health care professional workstations are critical components of large scale health care system architectures. We suggest that domain-specific software architectures (DSSAs) be used to specify standard interfaces and protocols for reusable software components throughout those architectures, including workstations. We encourage the use of engineering principles and abstraction mechanisms. Engineering principles are flexible guidelines, adaptable to particular situations. Abstraction mechanisms are simplifications for management of complexity. We recommend object-oriented design principles, graphical structural specifications, and formal components' behavioral specifications. We give an ambulatory care scenario and associated models to demonstrate SEP. The scenario uses health care terminology and gives patients' and health care providers' system views. Our goal is to have a threefold benefit. (i) Scenario view abstractions provide consistent interdisciplinary communications. (ii) Hierarchical object-oriented structures provide useful abstractions for reuse, understandability, and long term evolution. (iii) SEP and health care DSSA integration into computer aided software engineering (CASE) environments. These environments should support rapid construction and certification of individualized systems, from reuse libraries.

Computer Systems↗

Immunodetection of recombinant proteins based on antibodies directed against a metal binding peptide engineered for purification by immobilized metal affinity chromatography.

Recently we reported (Evans, D.B., Tarpley W.G. and Sharma, S.K. (1991) Expression and characterization of chimeric rDNA proteins engineered for purification and cleavage. Protein Expr. Purif. 2, 205-213) a genetically engineered metal binding peptide (mbp) for the purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). Therefore, we have been interested in developing mbp-based immunodetection methods for these engineered proteins. To this end, the following linker peptide containing the mbp (His-Asp-His-Asp-His) was designed, synthesized and conjugated to porcine thyroglobulin: Ac-Cys-Gly-Glu-Glu-His-Asp-His-Asp-His-Pro-Phe-His-Leu. Rabbits immunized with this conjugate developed antibodies that cross-react with peptides containing the mbp sequence. A number of chimeric recombinant proteins, expressed in E. coli, with and without the mbp portion (His-Asp-His-Asp-His) of the fusion peptide (His-Asp-His-Asp-His-Pro-Phe-His-Leu) were analyzed by ELISA and immunoblotting. Results from these studies show that the anti-mbp antibodies detect chimeric proteins containing the mbp, while chimeric proteins lacking this pentapeptide were negative in both immunodetection techniques. The usefulness of this approach has also been demonstrated in following IMAC purification and enzymatic cleavage of the mbp. These immunodetection techniques utilizing anti-mbp antibodies should be applicable to other proteins engineered to contain the mbp for IMAC purification.

Amino Acid Sequence↗

Efficacy of apical seal of Engine Plugger condensed root canal fillings--leakage to dyes.

A new rotatory instrument, the Engine Plugger, that is claimed to be less fragile than the McSpadden Compactor, has been introduced since the recognition of thermomechanical compaction as a valid technique for root canal filling. The purpose of this study was to test the efficacy of the apical seal afforded by the Engine Plugger in comparison with that of an established technique, lateral condensation. Eighty roots with round canals prepared to size 45 were randomly divided into two groups: one compacted with the Engine Plugger (Group 1) and the other with the lateral condensation technique (Group 2). AH-26 sealer was used in half the specimens. Four additional roots served as controls. The roots were cleared following immersion in Procion brilliant green, and the maximal depth of penetration of the dye was measured with a stereomicroscope. Severe leakage occurred in both groups when sealer was not used (mean depth: 3.17 mm in Group 1; 4.3 mm in Group 2). Addition of sealer reduced leakage significantly (p less than 0.001). There was no statistically significant difference in the depth of penetration of the dye between the two groups when AH-26 was used, although a trend in favor of the Engine Plugger method (0.15 mm versus 0.425 mm for lateral condensation) was noted.

Coloring Agents↗

Toward development of an implantable tissue engineered liver.

Hepatocyte transplantation on implantable devices is a tissue engineering approach to improve the treatment of liver disease and the efficacy of ex vivo gene therapy. Diverse physiological functions and high metabolic activity of the liver represent significant challenges to engineering implantable devices that provide long-term hepatic support. Liver tissue engineering research has explored alternatives to direct hepatocyte injection that include hepatocyte attachment to microcarriers, encapsulation and transplantation on biodegradable polymer scaffolds. Successful function of hepatocytes transplanted on implantable devices in animal models has been documented by production of albumin and other liver-specific markers, and clearance of bilirubin and urea metabolites. Strategies used to achieve these successes are reviewed, with particular emphasis on biodegradable polymer scaffolds, and two areas of investigation that may improve the function of implantable tissue engineered liver devices are highlighted.

Animals↗

Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus.

Protein engineering has been employed to investigate the effect of specific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus xylosus. Three different variants, corresponding to a 101 amino acid region of the major glycoprotein (G protein) of human respiratory syncytial virus (RSV), were generated in which multiple hydrophobic phenylalanine residues were either substituted or deleted. The different G protein fragments were expressed as one part of recombinant receptors designed for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engineered fragment, efficiently targeted to the outer cell surface of recombinant S. xylosus cells as determined by different methods, including fluorescence-activated cell sorting. In addition, immunization of mice with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The present strategy of hydrophobic engineering should be of general interest in surface-display applications and for secretion of proteins otherwise difficult to translocate through host cell membranes.

Amino Acid Sequence↗

DNA adduct formation in mice following treatment with used engine oil and identification of some of the major adducts by 32P-postlabelling.

Used engine oil from a petrol-powered vehicle was fractionated by column chromatography into seven parts for which the major polycyclic aromatic hydrocarbon (PAH) components were determined by GC. Topical treatment of mice with the fractions and 32P-postlabelling of the skin DNA resulted in the detection of multiple adduct spots on TLC for some, but not all, of the fractions. The majority of the DNA binding capacity of the used engine oil was possessed by the first three fractions, (equivalent to 25, 15 and 14.5%, respectively) of the adduct forming ability of the unfractionated oil. The chromatographic mobilities of the adduct spots induced by these fractions were compared to those produced by unfractionated used engine oil. In addition, mice were also treated topically with reference PAHs, either singly or as mixtures, dissolved in unused oil at the concentrations at which they were present in the used oil. Comparisons were made between the chromatographic mobilities of the adducts formed in mouse skin DNA by synthetic mixtures with those formed by the used oil. From these data, some of the major adducts produced by treatment with used engine oil are suggested to be formed by reactive metabolites of benzo[b]naphtho[1,2-d]thiophene, benzo[c]phenanthrene, benzo[g,h,i]fluoranthene, chrysene, benzo[a]pyrene and benzo[g,h,i]perylene.

Animals↗

Forming and maintaining a heat engine for quantum biology.

Chemical reactions upholding biological functions and structures are the process of measurement taking place among the participating chemical reactants. Chemical reactions occurring in thermal environments are either endothermic or exothermic. In particular, exothermic reactions that can live with temperature gradients of exogenous origin could potentially be competent enough to synthesize a robust quantum as a heat engine. Molecular organizations leading to the origin of the phenomenon of life might have been associated with the emergence of a quantum coherence embodied in a robust heat engine feeding on quantum decoherence. Evolutionary maintenance of a robust quantum heat engine, once appeared, can further be empowered by the build-up of temperature gradients of endogenous origin. Biology enriches the repertoire of quantum mechanics so as to include a robust heat engine as a legitimate member of a quantum in addition to the already established member of a quantum including an atom, molecule, and macromolecule.

Adenosine Triphosphate↗

Development of an HL7 interface engine, based on tree structure and streaming algorithm, for large-size messages which include image data.

A basic assumption of Health Level Seven (HL7) protocol is 'No limitation of message length'. However, most existing commercial HL7 interface engines do limit message length because they use the string array method, which is run in the main memory for the HL7 message parsing process. Specifically, messages with image and multi-media data create a long string array and thus cause the computer system to raise critical and fatal problem. Consequently, HL7 messages cannot handle the image and multi-media data necessary in modern medical records. This study aims to solve this problem with the 'streaming algorithm' method. This new method for HL7 message parsing applies the character-stream object which process character by character between the main memory and hard disk device with the consequence that the processing load on main memory could be alleviated. The main functions of this new engine are generating, parsing, validating, browsing, sending, and receiving HL7 messages. Also, the engine can parse and generate XML-formatted HL7 messages. This new HL7 engine successfully exchanged HL7 messages with 10 megabyte size images and discharge summary information between two university hospitals.

Algorithms↗

Use of a risk communication model to evaluate dietetics professionals' viewpoints on genetically engineered foods and crops.

The complex issues surrounding the application of genetic engineering to food and agriculture have generated a contentious debate among diverse interest groups. One pervasive dimension in the resultant discourse is the varying perceptions of the risks and benefits of genetically engineered foods and crops. In the risk communication model, technical information is evaluated within the context of an individual's values and perceptions. The purpose of this study was to explore how dietetics professionals respond to a complex set of interrelated issues associated with genetically engineered foods and crops and to identify what varying viewpoints may exist. Participants were asked to sort a total of 48 statements distributed across eight issue areas according to level of agreement and disagreement. Using Q methodology, a total of 256 sortings were analyzed using the centroid method and varimax rotation in factor analysis. Three distinct viewpoints emerged: Precautionary (R(2)=43%), Discerning Supporter (R(2)=11%), and Promoting (R(2)=5%). Across all viewpoints, respondents agreed that dietetics professionals should employ critical thinking skills to communicate the social, economic, environmental, ethical, and technical aspects of genetically engineered foods and crops. The findings have implications for how dietetics professionals can foster an open interchange of information among diverse groups.

Attitude of Health Personnel↗

A systems engineering perspective on the human-centered design of health information systems.

The discipline of systems engineering, over the past five decades, has used a structured systematic approach to managing the "cradle to grave" development of products and processes. While elements of this approach are typically used to guide the development of information systems that instantiate a significant user interface, it appears to be rare for the entire process to be implemented. In fact, a number of authors have put forth development lifecycle models that are subsets of the classical systems engineering method, but fail to include steps such as incremental hazard analysis and post-deployment corrective and preventative actions. In that most health information systems have safety implications, we argue that the design and development of such systems would benefit by implementing this systems engineering approach in full. Particularly with regard to bringing a human-centered perspective to the formulation of system requirements and the configuration of effective user interfaces, this classical systems engineering method provides an excellent framework for incorporating human factors (ergonomics) knowledge and integrating ergonomists in the interdisciplinary development of health information systems.

Artificial Intelligence↗