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PubMed2005

Artificial and engineered chromosomes: non-integrating vectors for gene therapy.

Non-integrating gene-delivery platforms demonstrate promise as potentially ideal gene-therapy vector systems. Although several approaches are under development, there is little consensus as to what constitutes a true 'artificial' versus an 'engineered' human chromosome. Recent progress must be evaluated in light of significant technical challenges that remain before such vectors achieve clinical utility. Here, we examine the principal classes of non-integrating vectors, ranging from episomes to engineered mini-chromosomes to true human artificial chromosomes. We compare their potential as practical gene-transfer platforms and summarize recent advances towards eventual applications in gene therapy. Although chromosome-engineering technology has advanced considerably within recent years, difficulties in establishing composition of matter and effective vector delivery currently prevent artificial or engineered chromosomes being accepted as viable gene-delivery platforms.

Chromosomes, Artificial, Human↗

PubMed2005

Volatile science? Metabolic engineering of terpenoids in plants.

Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites.

Biotechnology↗

PubMed2006

Using ecosystem engineers to restore ecological systems.

Ecosystem engineers affect other organisms by creating, modifying, maintaining or destroying habitats. Despite widespread recognition of these often important effects, the ecosystem engineering concept has yet to be widely used in ecological applications. Here, we present a conceptual framework that shows how consideration of ecosystem engineers can be used to assess the likelihood of restoration of a system to a desired state, the type of changes necessary for successful restoration and how restoration efforts can be most effectively partitioned between direct human intervention and natural ecosystem engineers.

Ecology↗

PubMed2004

Asbestos exposure from gaskets during disassembly of a medium duty diesel engine.

Diesel engines have historically used asbestos-containing gaskets leading to concerns of fiber release and mechanic exposure. Other published studies regarding asbestos fiber release during gasket removal have reported on short-duration events; were conducted under simulated work conditions; or had other limitations. There are no comprehensive studies relating to diesel engine gaskets under conditions similar to those reported herein, evaluating asbestos fiber release from gaskets during all facets of a complete disassembly and cleaning of a medium duty diesel engine in a busy repair and service shop by a journeyman mechanic. Asbestos content of all gaskets was identified; all disassembly tasks were described and timed; and personal and area air monitoring was conducted for each task. Twenty seven of thirty three gaskets contained chrysotile asbestos in concentrations that ranged from 5 to 70%. All but one air monitoring sample reported results below the limit of reliable detection even though plumes of visible dust were evident during various removal, cleaning, and buffing procedures. The detection limit for airborne asbestos fibers in this investigation was influenced by the presence of other shop dust in the air. Our investigation demonstrates that using shop-standard procedures in an established repair facility, a journeyman mechanic has very little potential for exposure to airborne asbestos fibers during disassembly of an engine, approximately 10% or less than that currently considered to be acceptable by OSHA.

Air Pollution, Indoor↗

PubMed2003

Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.

Bone marrow derived mesenchymal stem cells (MSC) have been shown to be progenitor cells for mesenchymal tissues. These cells may also provide a potential therapy for bone repair. Our previous studies showed that MSC engineered with the gene for bone morphogenetic protein 2 (BMP-2), a growth factor for bone cells, were capable of differentiating into osteoblast lineage and inducing autologous bone formation in several animal models. Culturing individual MSC for autologous implantation, however, remains problematic. The number of human MSC with osteogenic potential decreases with age, and, in certain diseases, the patient's marrow may be damaged or the healthy cells reduced in number. In this study, we used rats with a femoral segmental defect to investigate whether allogeneic BMP-2 engineered MSC would facilitate bone healing. We show that BMP-2 engineered allogeneic MSC can repair critical bone defects to the same degree as rats treated with BMP-2 engineered autologous MSC, if the allogeneic group receives short-term treatment with immunosuppressant FK506. We also show that allogeneic gene transferred MSC are directly involved in bone repair, in addition to acting as gene deliverers.

Animals↗

PubMed1995

Multiskilling--re-engineering work process.

Many North American companies have recognized the need to re-engineer their core processes to achieve breakthrough improvements in cost, service and efficiency. In fact, it is estimated that U.S. companies alone will spend millions on business re-engineering projects this year. But change experts say that most re-engineering is in name only, cautiously tackling only one process or department at a time. Even fewer hospitals have attempted this magnitude of change. Toronto's Sunnybrook Health Science Centre is the first institution of its size in Canada to embark on a multifaceted re-engineering strategy toward a model of patient focused care. The following is an overview of Sunnybrook's experience with the first of these strategies: multiskilling service workers. The concept of multiskilling provides for a focus on redesigning job classifications to broaden the scope of responsibility. For Sunnybrook, this entailed the amalgamation of six service positions--unit aide, health care aide, dietary aide, orderly, porter, housekeeper and attendant into one service assistant position.

Clinical Competence↗

PubMed1998

Biological monitoring of young workers exposed to polycyclic aromatic hydrocarbons in engine repair workshops.

Several polycyclic aromatic hydrocarbons (PAHs) are carcinogenic in rodents, while exposure to these compounds has been associated with various types of human cancer. Engine exhaust and used engine oils are major PAH sources in engine repair workshops. Urinary 1-hydroxypyrene (1-OHP) levels and some genotoxicity parameters (sister-chromatid exchange, SCE; micronucleus, MN) have been compared among engine repair workers (n = 61, age: between 13 and 18) and workers not occupationally exposed to PAH (n = 30, age: between 13 and 19). The mean value (+/- S.E.) of urinary 1-OHP excretions of workers was 4.71 +/- 0.53 micromol/mol creatinine and also mean urinary 1-OHP for controls was found to be as 1.55 +/- 0.28 micromol/mol creatinine. The mean values (+/- S.E.) of SCE frequency per cell in peripheral lymphocytes from the workers and controls were 4.47 +/- 0.09 and 4.06 +/- 0.16, respectively (p < 0.05). The mean (+/- S.E.) MN (per thousand) frequencies in peripheral lymphocytes from the workers and controls were 1.87 +/- 0.04 and 1.56 +/- 0.06, respectively (p < 0.0001). The effect of smoking for the above parameters were also evaluated.

Adolescent↗

PubMed1990

Structure of a thermostable disulfide-bridge mutant of phage T4 lysozyme shows that an engineered cross-link in a flexible region does not increase the rigidity of the folded protein.

A disulfide bond introduced between amino acid positions 9 and 164 in phage T4 lysozyme has been shown to significantly increase the stability of the enzyme toward thermal denaturation [Matsumura, M., Becktel, W.J., Levitt, M., & Matthews, B. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6562-6566]. To elucidate the structural features of the engineered disulfide, the crystal structure of the disulfide mutant has been determined at 1.8-A resolution. Residue 9 lies in the N-terminal alpha-helix, while residue 164 is located at the extreme C terminus of T4 lysozyme, which is the most mobile part of the molecule. The refined structure shows that the formation of the disulfide bond is accompanied by relatively large (approximately 2.5 A) localized shifts in C-terminal main-chain atoms. Comparison of the geometry of the engineered disulfide with those of naturally observed disulfides in proteins shows that the engineered bridge adopts a left-handed spiral conformation with a typical set of dihedral angles and C alpha-C alpha distance. The geometry of the engineered disulfide suggests that it is slightly more strained than the disulfide of oxidized dithiothreitol but that the strain is within the range observed in naturally occurring disulfides. The wild-type and cross-linked lysozymes have very similar overall crystallographic temperature factors, indicating that the introduction of the disulfide bond does not impose rigidity on the folded protein structure. In particular, residues 162-164 retain high mobility in the mutant structure, consistent with the idea that stabilization of the protein is due to the effect of the disulfide cross-link on the unfolded rather than the folded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗

PubMed2004

Effect of engine load on diesel soot particles.

This study concentrates on characterization of nonvolatile fraction of diesel particles. These particles have an impact on earth's radiation balance as well as on health effects of vehicle emissions. In addition to composition and size distribution of particles, an important factor affecting their health effects and properties and lifetimes in the atmosphere is their morphology. The effect of engine parameters on soot particle size distributions and also on particle morphology has been studied. It was found that the shape of the size distribution and also the structure of diesel particles depend on engine load. The number distributions were found to obey log-normal assumption. The width of the distribution increased with increasing engine load. The geometric standard deviations of measured distributions varied from 1.7 to 2.1. Simultaneously, the fractal dimension of particles decreased with increasing engine load. The values for mass fractal dimensions based on sealing of particle mass and mobility size were between 2.6 and 2.8. Both electron microscopy and measurements of aerodynamic size versus mobility size suggest that the morphology of particles in different size regimes vary, with the large particles being less compact than the small ones.

Air Movements↗

PubMed2004

In-line laser holography and video analysis of eroded floc from engineered and estuarine sediments.

Microphytobenthic polymers mediate intertidal sediment erosion processes, through biostabilization and modifying the nature of eroded floc material. The latter is of key importance with respect to sediment transport dynamics, including floc aggregation and particle deposition. In this study, eroded floc material was analyzed by video imaging, alongside novel application of in-line laser holography (ILH). The erosion of engineered sediment was compared to that of natural estuarine sediments. Both video and holography showed an increase in floc size eroded from engineered cohesive clay sediment as a function of sediment dewatering and sediment polymer content. Estuarine sediment showed a curvilinear increase in floc size as a function of both microphytobenthic biomass and sediment colloidal polymer content when measured by video analysis. Holography did not show these functions for floc size due to temporal limitations of the current ILH methodology. An interaction of sediment polymer binding and sediment desiccation was observed for engineered sediments and, most notably, for estuarine cohesive sediments. In conclusion, engineered sediments were not accurate analogues for natural intertidal sediments, failing to reproduce eroded floc material similar to that from estuarine cohesive sediment. The size of eroded floc from estuarine sediments is a function of the complex interaction between biological and physicochemical processes, primarily algal colloidal polymer and desiccation. Holography demonstrated an excellent potential for the high-resolution imaging of eroded material but is limited by temporal constraints; the solution to this would be the development of real-time holographic video.

Colloids↗

PubMed2004

Development and application of a mobile laboratory for measuring emissions from diesel engines. 2. Sampling for toxics and particulate matter.

Limited data are available on the emission rates of speciated volatile and semivolatile organic compounds, as well as the physical and chemical characteristics of fine particulate matter (PM) from mobile, in-use diesel engines operated on the road. A design for the sampling of these fractions and the first data from in-use diesel sources are presented in this paper. Emission rates for carbonyls, 1,3-butadiene, benzene, toluene, xylene, PM, and elemental and organic carbon (EC and OC) are reported for a vehicle driven while following the California Air Resources Board (ARB) four-mode heavy heavy-duty diesel truck (HHDDT) cycle and while transiting through a major transportation corridor. Results show that distance specific emission rates are substantially greater in congested traffic as compared with highway cruise conditions. Specifically, emissions of toxic compounds are 3-15 times greater, and PM is 7 times greater under these conditions. The dependence of these species on driving mode suggests that health and source apportionment studies will need to account for driving patterns in addition to emission factors. Comparison of the PM/NOx ratios obtained for the above tests provides insight into the presence and importance of "off-cycle" emissions during on-road driving. Measurements from a stationary source (operated and tested at constant engine speed) equipped with an engine similar to that in the HHDDT yielded a greater understanding of the relative dependence of emissions on load versus engine transients. These data are indicative of the type of investigations made possible by the development of this novel laboratory.

Air Pollutants↗

PubMed1999

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.

We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.

Animals↗

PubMed2026

Engineered genomic attachment sites for site-specific recombinases enable high-efficiency integration in plants and human cells.

Here we developed a DNA-centric strategy for optimizing site-specific recombination by rationally engineering chimeric attachment sites. The high-activity att variants enhance Bxb1-mediated integration efficiency in human cells and plants. Among these att variants, the engineered attB(V111) site achieved 51.9% integration efficiency in HEK293T cells (1.7-fold versus wild-type attB) and 35.6% in rice protoplasts (4.4-fold versus wild-type attB). When paired with an engineered single protein mutant in the Bxb1 catalytic domain, the optimized system achieved targeted integration efficiencies of 31% for a CD19 chimeric antigen receptor cassette and 25% for an ornithine transcarbamylase expression cassette in human cells. In rice, these engineered variants enabled integration of a 5.8 kb herbicide-resistance cassette at a targeted genomic locus, with stable integration detected in 24% of regenerated plants. Oxford Nanopore-based long-read sequencing of edited plants reveals complete and precise insertion with high specificity. Propagation of edited seedlings to T1 plants confirms heritable editing to future generations. This approach provides a safe, broadly applicable approach for recombinase-based genome editing.

Journal Article↗

PubMed2000

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Flow Cytometry↗

PubMed1998

Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL).

Fas ligand is a type II transmembrane protein which can induce apoptosis in Fas-expressing cells. Recent reports indicate that expression of FasL in transplanted cells may cause graft rejection and, on the other hand, tumor cells may lose their tumorigenicity when they are engineered to express FasL. These effects could be related to recruitment of neutrophils by FasL with activation of their cytotoxic machinery. In this study we investigated the antitumor effect of allogenic fibroblasts engineered to express FasL. Fibroblasts engineered to express FasL (PA317/FasL) did not exert toxic effects on transformed liver cell line (BNL) or colon cancer cell line (CT26) in vitro, but they could abrogate their tumorigenicity in vivo. Histological examination of the site of implantation of BNL cells mixed with PA317/FasL revealed massive infiltration of polymorphonuclear neutrophils and mononuclear cells. A specific immune protective effect was observed in animals primed with a mixture of BNL or CT26 and PA317/FasL cells. Rechallenge with tumor cells 14 or 100 days after priming resulted in protection of 100 or 50% of animals, respectively. This protective effect was due to CD8+ cells since depletion of CD8+ led to tumor formation. In addition, treatment of pre-established BNL tumors with a subcutaneous injection of BNL and PA317/FasL cell mixture at a distant site caused significant inhibition of tumor growth. These data demonstrate that allogenic cells engineered with FasL are able to abolish tumor growth and induce specific protective immunity when they are mixed with neoplastic cells.

Animals↗

PubMed2001

Apical extrusion of debris and irrigants using two hand and three engine-driven instrumentation techniques.

AIM: To evaluate the weight of debris and irrigant volume extruded apically from extracted teeth in vitro after endodontic instrumentation using the balanced force technique, a hybrid hand instrumentation technique, and three engine-driven techniques utilizing nickel-titanium instruments (ProFile .04, Quantec 2000 and Pow-R). METHODOLOGY: Five groups of 20 extracted human teeth with single canals were instrumented using one or other of five techniques: balanced force, hybrid, Quantec 2000, ProFile .04, or Pow-R. Debris extruded from the apical foramen during instrumentation were collected into preweighed 1.5 mL tubes. Following instrumentation, the volume of extruded irrigant fluid was determined by visual comparison to control centrifuge tubes filled with 0.25 mL increments of distilled water. The weight of dry extruded dentine debris was also established. RESULTS: Overall, the engine-driven techniques extruded less debris than the manual ones. However, there was no statistical difference between the balanced force technique and the engine-driven methods. The volume of irrigant extruded through the apex was directly associated with the weight of extruded debris, except within the ProFile group. The hybrid technique was associated with the greatest extrusion of both debris and irrigant. CONCLUSIONS: Overall, the engine-driven nickel-titanium systems were associated with less apical extrusion.

Dental High-Speed Technique↗

PubMed2002

Tissue engineering of biological cardiovascular system surrogates.

Cardiovascular diseases are common in ageing communities globally. This fact is most striking in the industrialised world where the aged population makes up a large proportion of society. Elderly patients are frequently treated surgically with grafts to replace damaged tissues and vessels. The number of human-donated components is insufficient and synthetic surrogates are sought. These might be wholly mechanical, wholly biological, or tissue engineered complexes of cells and their products growing in a scaffold. At present, many such composites exist with potential for use as substitutes for specific blood vessels. The challenges of producing tissue engineered heart valves are now being widely explored. Neotissues must provide an effective, durable, non-thrombogenic and non-immunogenic substitute that will fulfil the purpose of the natural tissue. The aims and scope of this paper are to review current and novel concepts in the field of tissue engineering of biological cardiovascular system surrogates. Mechanical stresses and strains on cardiovascular cells in vitro have been recognised and can be measured by a culture force monitor. Physiological stresses can be generated by a tensioning culture force monitor and applied to engineered tissue, aligning the cells and mimicking arterial wall architecture. The hydrostatic forces a vessel experiences and mechanical parameters of blood vessels can be studied in the tubular culture system of a multi-cue bioreactor.

Journal Article↗

PubMed2003

Consumer perception of risk associated with eating genetically engineered soybeans is less in the presence of a perceived consumer benefit.

OBJECTIVE: To determine whether perceived benefit alters personal risk perception associated with eating genetically engineered soybeans, consumer desire for labeling, preferred phrase on a label symbol, and desired information in an educational brochure. DESIGN: Comparison of responses of two consumer groups who completed one of two survey versions. SUBJECTS/SETTINGS: One hundred fifty supermarket shoppers, age 21 years and older, for each survey or n=300 total. Focus groups and a pilot test were used to develop the final survey in which consumers read a description of a genetically engineered soybean with either no obvious consumer benefit or an obvious consumer benefit and then completed a set of attitude questions and evaluated a voluntary label design and educational brochure content. Main outcome measures were mean opinion scores of personal risk and desire for labeling and ranking of desired label phrase and brochure topics. STATISTICAL ANALYSIS: Chi;(2) and t Tests were used. RESULTS: Consumers reading about the soybean with obvious consumer benefit were significantly more comfortable eating these than those reading about the soybean with no obvious consumer benefit (2.9+/-1.1 vs 3.4+/-1.0, respectively; P</=.001). However, the groups did not differ in desire for labeling of foods made with these soybeans or preferred brochure content. They did differ significantly in preferred phrase on the symbol (P</=.05). APPLICATIONS/CONCLUSIONS: Dietitians can use descriptions of genetic engineering applications such as those in this study to help consumers assess these applications. Dietitians can play a critical role in explaining labeling terms and designing educational materials when the FDA finalizes voluntary labeling regulations for genetically engineered foods.

Adult↗