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Photoimmobilization of biomolecules within a 3-dimensional hydrogel matrix.

It has been recognized that a three-dimensional cell invasive scaffold that provides both topographical and chemical cues is desirable in regenerative tissue engineering to encourage cell attachment, migration, regrowth and ultimately tissue repair. Carbohydrate hydrogels are attractive for such applications because they are generally biocompatible and able to match the mechanical properties of most soft tissues. Although carbohydrate hydrogels have been previously modified with cell adhesive peptides and proteins, complicated hydrogel matrix activation was required prior to biomolecule coupling and, perhaps more importantly, the overall immobilization yield was low at approximately 1%. In this study, we report the photo-immobilization of a model biomolecule, ovalbumin (OVA), to agarose gel. We describe two methods of modification where the photoactive moiety is coupled to either the protein (i.e. OVA) or the matrix (i.e. agarose) prior to immobilization. We found that the photo-immobilization yield depends on the location of the photoactive moiety. Using photoactive OVA, 1.8% of the OVA initially incorporated into the agarose gel is immobilized; using photoactive agarose, 9.3% of the OVA initially mixed with the agarose is immobilized. The latter is a significant improvement over previous yields and may be useful in attaining our goal of immobilizing a biomolecule gradient for guided tissue regeneration.

Benzophenones↗

Using landscape ecology to focus ecological risk assessment and guide risk management decision-making.

Ecological risk assessment (EcoRA) generally suffers from limited application of ecological knowledge in the definition and characterization of real-world sites. Not surprisingly, most remediation decisions, which follow, have little or no relationship to the valued ecological resources of the site or the broader region. The practice has evolved to favor engineering-based mitigation strategies, which eliminate excess chemical concentrations at sites, or otherwise break exposure pathways, but which may not be ecologically beneficial. The heavy emphasis of EcoRA on toxicity threshold levels tends to focus dollars on clean up of small areas or volumes with high concentrations. Moreover, intrusive remediation technologies often render an area uninhabitable to the very species that were to be protected. Infusion of ecological knowledge into EcoRA has been difficult. Most professional ecologists choose not to venture into the messy applied fields, leaving their impressive knowledge untapped. Moreover, narrowly defined responsibilities within government circles can limit cooperation and coordination. The realization that land use activities often have greater adverse consequences to wildlife than do chemicals provides an opportunity to change attitudes and practices. We are developing procedures that incorporate landscape features into the environmental management process. Specifically, we are using an iterative approach to: a) identify scenarios where habitat value is important in EcoRAs; b) guide selection of appropriate assessment species, i) keyed to wildlife distribution ranges; ii) keyed to a database of habitat suitability models; iii) cross-linked with the EPA exposure handbook species; iv) referenced to wildlife distributions (e.g., breeding bird survey); c) define data collection needs for reconnaissance-, screening-, and definitive-level characterization of habitat quality for potential assessment species; d) generate spatially explicit descriptions of habitat quality for various assessment species; and e) allocate exposure estimates using both habitat quality and spatial variations in chemical concentration. These refinements in theEcoRA process are expected to improve risk estimates and provide valuable information to be used instructuring risk management options. The approach can guide the planning process so that an assessment considers the most relevant species of the area and defines the relevant parameters to be measured. In risk characterization, these data are used to calculate more realistic exposure assessments. In guiding remediation, the approach logically considers a wider range of land management options than are considered at most sites today. For example, habitat enhancement can be used to draw animals away from contaminated zones. Contaminated localities that also have poor-quality habitat may be allowed to go through a slower, less costly bioremediation process until the risk level is lowered to acceptable levels. And direct comparisons of lost resources stemming from destructive remediation options can be assessed instead of merely focusing on the lowering of contaminant concentrations. This paper presents the conceptual foundation for incorporating landscape ecology into the risk assessment process.

Animals↗

Microbial attachment and feed digestion in the rumen.

Direct microscopic examination of the rumen and its contents shows microbial populations largely attached to feed particles in the digesta. Most feeds contain a surface layer that is resistant to attachment and therefore to digestion. Infiltration of these recalcitrant epidermal layers through damage sites or through focused enzymatic attack is essential for initiation of the digestive process. Proliferation of primary colonizing cells produces glycocalyx-enclosed microcolonies. Secondary colonizers from the ruminal fluid associate with microcolonies, resulting in the formation of multispecies microbial biofilms. These metabolically related organisms associate with their preferred substrates and produce the myriad of enzymes necessary for the digestion of chemically and structurally complex plant tissues. Upon accessing the internal, enzyme-susceptible tissues, microbial "digestive consortia" attach to a variety of nutrients, including protein, cellulose, and starch and digest insoluble feed materials from the inside out. Substances that prevent microbial attachment or promote detachment (e.g., condensed tannins, methylcellulose) can completely inhibit cellulose digestion. As the microbial consortium matures and adapts to a particular type of feed, it becomes inherently stable and its participant microorganisms are notoriously difficult to manipulate due to the impenetrable nature of biofilms. Properties of feed that place constraints on microbial attachment and biofilm formation can have a profound effect on both the rate and extent of feed digestion in the rumen. Developments in feed processing (i.e., chemical and physical), plant breeding, and genetic engineering (both of ruminal microorganisms and plants) that overcome these constraints through the promotion of microbial attachment and biofilm formation could substantially benefit ruminant production.

Animal Feed↗

Effectiveness of a glutaraldehyde formulation in decontamination of dental unit water systems.

AIM: Dental-medical devices may transmit infections caused by bacteria that are usually found in water distribution systems, and which are difficult to treat and control. High bacterial contamination in the water systems of dental units is due to the presence of biofilm inside the pipes. Our study evaluated the efficacy of glutaraldehyde formulated with quarternary ammonium salts (Sanicide T4) examined in a previous study, employing a series of assays to confirm or otherwise the results obtained previously. METHODS: A purification protocol for the dental unit water system, together with a protocol for daily maintenance treatment, were tested on two dental units (in the Departments of Conservative and Prosthetic Dentistry) taking specimens from the turbine, micro-engine and air-water gun. The chemical substance, at a concentration of 20 cc per litre of water, was allowed to act when the department closed, for a total of 15 days. The Sanicide T4 was handled with protective gloves and the dental units were fitted with two safety devices to avoid accidental ingestion. RESULTS: Laboratory results enabled us to compare values for bacterial load at 36 degrees C and at 22 degrees C and for Pseudomonas aeruginosa, before and after applying the test protocol. Data obtained are satisfactory except for Pseudo-monas in the fountain in the Department of Conservative Dentistry; the value was in any case below the safety level set by the American Dental Association, confirming the results obtained in our previous study. CONCLUSIONS: The two protocols may now be considered an excellent solution to control the development of biofilm. Should the product be found to be effective for a longer period of use, each dental unit should be provided with: an anti-reflux valve to stop accidental ingestion of disinfectant; a hydraulic mechanism to pump the product to the handpieces and other water supply devices; possibility of discriminating the use of the public water system from the use of that in the disinfectant circuit; an automatic mechanism whereby the disinfectant can be flushed out with drinking water every morning.

Decontamination↗

Clinical experience with a recombinant DNA hepatitis B vaccine.

The clinical testing of EngerixR-B, the hepatitis B vaccine produced by SmithKline Biologicals using recombinant DNA technology, started in February 1984. Since extensive pre-clinical laboratory work had established that the polypeptide (HBsAg) expressed in genetically engineered yeast cells was after purification--physically, chemically and antigenically similar to the viral surface antigen particles found in the blood of chronic carriers, the aims of the clinical trials were to compare the safety, reactogenicity, immunogenicity and protective efficacy of yeast-derived (YDV) and plasma-derived (PDV) vaccines. By September 1987, 89 studies had been initiated involving a total of 10,545 subjects aged from birth to 82 years. This extensive experience has established that the risk of hypersensitivity to yeast-derived contaminants is negligible since no hypersensitivity reaction has been observed in any vaccinee, the incidence and severity of local reactions have not increased after repeated inoculations and no anti-yeast antibodies were produced by vaccination. Reactogenicity has been comparable to that of PDV's consisting essentially of transient mild irritation at the site of injection presumably caused by the aluminium hydroxide used as adjuvant. The anti-HBs responses to YDV and PDV's were quantitatively (seroconversion rates, peak antibody levels and persistence) as well as qualitatively (epitope specificity and affinity) similar. The expected protective effect of the immune response to the vaccine was confirmed in a challenge study in chimpanzees and in vaccinated human populations (male homosexuals, institutionalized mentally retarded patients, neonates of carrier women) with historically a high infection rate.

Adult↗

[Prevalence and etiology of occupational dermatoses in workers of railway transport in 1958-1992].

Dermatoses are among the most prevalent occupational diseases on the railway transport; in 1961-1975 their share in total morbidity was 40%. The structure of diseases was as follows (%): contact dermatosis 87.4, eczema 6.8, allergic drug-induced dermatitis 1.4, folliculitis, toxicoderma, chemical burns, radial dermatitis and other kinds of dermatosis 0.84. The main causes of diseases were the use of many chemicals in transport processes and of Diesel engines on trains.

Dermatitis, Occupational↗

Directed evolution of biosynthetic pathways. Recruitment of cysteine thioethers for constructing the cell wall of Escherichia coli.

We report that expansion of thioether biosynthesis in Escherichia coli generates sulfur-containing amino acids that can replace meso-diaminopimelate, the essential amino acid used for cross-linking the cell wall. This was accomplished by jointly overexpressing the metB gene coding for L-cystathionine gamma-synthase and disrupting the metC gene, whose product, L-cystathionine beta-lyase, is responsible for the destruction of L-cystathionine and other L-cysteine thioethers. As a result, meso-lanthionine and L-allo-cystathionine were produced endogenously and incorporated in the peptidoglycan, thereby enabling E. coli strains auxotrophic for diaminopimelate to grow in its absence. Thus, current techniques of metabolic engineering can be applied to evolving the chemical constitution of living cells beyond its present state.

Alanine↗

Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen cells.

Recently, there has been great concern about the potential of industrial chemicals to act as endocrine disrupters. In this report, we conducted a pilot study to validate the use of AR-EcoScreen cells for tier 1 screening of androgen receptor (AR) agonist and antagonist activities. From 253 test compounds, we identified two AR agonists and nine antagonists. The two agonists, 2-tert-butylanthraquinone and benzoanthrone, were relatively weak (10% maximal activation of the positive control, 5alpha-dihydrotestosterone, at 2.54x10(-7) and 4.46x10(-6) M, respectively). The most potent antagonist was 3,3'-dichlorobenzidine dihydrochloride (IC50 = 2.28x10(-7) M). The order of the anti-androgenic activities was 3,3'-dichlorobenzidine dihydrochloride>4-diethylaminobenzaldehyde>4,4'-[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol]>2,4,6-trichlorophenylhydrazine = 4-(phenylpropyl)pyridine>2-hydroxy-4-methoxybenzophenone>2,2-bis(4-cyanophenyl)propane>4-methoxy-2-methyldiphenylamine = 2,4-diphenyl-4-methylpentene-1. These results suggest that AR-EcoScreen cell line has the potential to be used as a tool for the large scale tier 1 screening of chemicals for androgen receptor agonist and antagonist activity.

Androgen Antagonists↗

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology.

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.

3-Hydroxybutyric Acid↗

Electrochemical sensor toolkit for simultaneous glutamate detection at edge of cleft and peri-soma.

Simultaneously monitoring glutamate (Glu) dynamic at edge of synaptic cleft and peri-soma is crucial for understanding Glu-related pathology. Here, we created an electrochemical Glu sensors toolkit with spatial resolution of ∼60 nm, combining biologically engineered Glu binding protein for specifically capturing Glu together with chemically designed ferrocene groups for signal labeling. Modulation conjugation approach between GluR and ferrocene significantly improved sensitivity up to 32-folds. More importantly, protein engineering of residue mutation and linker peptides flexibility expanded linear range from 10 μM to 6 mM, accelerated on/off times down to 35/40 ms. This toolkit realized real-time quantifying of Glu both at edge of cleft and peri-soma, we discovered that Glu was almost released through SLC7A11 channels in calyx of held synapse upon oxygen-glucose-deprivation, while Glu was mainly released through hemichannels upon β-amyloid42 stimulation. Our work provided a methodology for investigating Glu release and reuptake and offered insights for Glu related pathology.

Glutamic Acid↗

Strategies for protein-based nanofabrication: Ni2+-NTA as a chemical mask to control biologically imposed symmetry.

BACKGROUND: Technologies that improve control of protein orientation on surfaces or in solution, through designed molecular recognition, will expand the range of proteins that are useful for biosensors, molecular devices and biomaterials. A limitation of some proteins is their biologically imposed symmetry, which results in indistinguishable recognition surfaces. Here, we have explored methods for modifying the symmetry of an oligomeric protein that exhibits useful self-assembly properties. RESULTS: Escherichia coli glutamine synthetase (GS) contains 24 solvent-exposed histidines on two symmetry-related surfaces. These histidines drive a metal-dependent self-assembly of GS tubes. Immobilization of GS on the affinity resin Ni2+-NTA followed by on-column modification with diethyl pyrocarbonate affords asymmetrically modified GS that self-assembles only to the extent of 'short' dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy. The utility of Ni2+-NTA as a chemical mask was also demonstrated for asymmetric modification of engineered cysteines adjacent to the natural histidines. CONCLUSIONS: Current genetic methods do not provide distinguishable recognition elements on symmetry-related surfaces of biologically assembled proteins. Ni2+-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional His-tags. This strategy may be extended to modification of a wide range of amino acids with a myriad of reagents.

Biocompatible Materials↗

Microbial cellulose as a specialty chemical.

Microbial polysaccharides are extensively used commercially as gelling or suspending agents, as protective colloids or as thickening agents. Until recently, microbial cellulose producing systems such as Acetobacter xylinum, had been used largely as model systems for the study of cellulose biosynthesis. Current advances in molecular biology and biochemical engineering promise to usher microbial cellulose into the specialty chemical market. This review will highlight some of the recent progress made in our understanding of microbial cellulose biochemistry and biosynthesis, describe some of its inherent virtues and identify current unique applications of this versatile biopolymer.

Journal Article↗

Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.

Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.

Animals↗

Integrating genomics and metabolomics for engineering plant metabolic pathways.

Plant metabolites are characterized by an enormous chemical diversity, every plant having its own complex set of metabolites. This variety poses analytical challenges, both for profiling multiple metabolites in parallel and for the quantitative analysis of selected metabolites. We are only just starting to understand the roles of these metabolites, many of them being involved in adaptations to specific ecological niches and some finding beneficial use (e.g. as pharmaceuticals). Spectacular advances in plant metabolomics offer new possibilities, together with the aid of systems biology, to explore the extraordinary complexity of the plant biochemical capacity. State-of-the art genomics tools can be combined with metabolic profiling to identify key genes that could be engineered for the production of improved crop plants.

Arabidopsis↗

Engineered enzymes for improved organic synthesis.

Recent developments to modify enzymes for use in organic synthesis have targeted several areas. These include altering the reaction mechanism of the enzyme to catalyse new reactions, switching substrate specificity, expanding substrate specificity, and improving substrate specificity, such as enantioselectivity in kinetic resolutions. Such modifications can be achieved either by rational redesign, which requires knowledge of the enzyme structure, or by random mutagenesis methods followed by screening. Both strategies of enzyme engineering can be successful and are very useful for improving the utility of enzymes for applied catalysis. Several examples illustrating these concepts in a variety of enzyme classes have appeared recently.

Catalysis↗

A genetically engineered, mutant human alpha-1-proteinase inhibitor is more resistant than the normal inhibitor to oxidative inactivation by chemicals, enzymes, cells, and cigarette smoke.

Alpha-1-proteinase inhibitors (alpha 1PI) containing methionine (Met-358) or valine (Met----Val-358) at the reactive center were synthesized in and purified to homogeneity from recombinant yeast. The pure proteins were exposed to 1 of 4 different oxidizing systems: N-chlorosuccinimide (chemical oxidation), myeloperoxidase plus peroxide and halide (enzymatic oxidation), activated neutrophils (cellular oxidation), or gas-phase cigarette smoke. The effect of these treatments on the leukocyte elastase inhibitory function of both proteins was then assessed. After brief exposures, substantial inactivation of the normal inhibitor occurred, whereas the mutant inhibitor remained fully active. More prolonged exposures led to complete inactivation of the normal protein and partial inactivation of the mutant inhibitor. These results suggest that the reactive center methionyl residue in alpha 1PI is more rapidly affected by oxidants than are other oxidizable residues in the inhibitor; however, Met-358 is not the only residue in alpha 1PI whose modification can lead to the inactivation of the elastase inhibitory capacity of this protein.

Binding Sites↗