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Discovery of the archaeal chemical link between glycogen (starch) synthase families using a new mass spectrometry assay.

Starch and its analogue glycogen are biosynthesized by enzymes that have been classified by sequence similarities into two families that have no significant sequence overlap: the animal/fungal glycogen synthases and the plant/bacterial glycogen (starch) synthases. Recent gene sequence analysis of putative archaea enzymes implicates them as a third family that links the structural and functional features of the other two classes. Herein, we present the first rapid electrospray ionization mass spectrometry-based assay to quantify any carbohydrate-polymerizing activity, the first cloning and recombinant expression as well as verification of the putative function of a glycogen synthase from the hyperthermophilic archaea Pyrococcus furiosus, and the characterization of a variety of glycogen synthases with the new assay. The new assay allowed the determination of Km and Vmax values for the rabbit, yeast, and P. furiosus glycogen synthases. Most surprisingly, unlike the synthases from rabbit or yeast and in contradiction to what would be expected from structural studies of other nucleotide-sugar binding proteins, the synthase from the archaea source accepts both uridine- and adenine-diphosphate activated glucose competitively and with comparable affinities to form a glucose polymer. This loose substrate specificity implicates this protein as the chemical link between the two branches of glycogen synthases that have evolved to accept primarily one or the other nucleotide as well as a good source enzyme for polymer bioengineering efforts.

Adenosine Diphosphate Glucose↗

Validation of recombinant and bovine chymosin by mass spectrometry.

Mass spectrometry has been used to map chymosin from a fermentative source. The copresence of the two known genetic variants A (Asp(244)) and B (Gly(244)) was ascertained in bovine chymosin. By contrast, either the A or the B genetic variant occurred in the three commercial samples of recombinant calf chymosin (RCC). Specific biomarker proteins were searched to identify the enzyme source, in both bovine chymosin and RCC samples. Analyzing the derived tryptic peptides, evidence was provided that RCC and bovine chymosin are mainly formed by (1-323), (3-323), and (40p-323) (suffix "p" denotes residues in the pro-segment region of chymosin), whereas the minor components, (4-323), (5-323), and (6-323), were only detected in bovine chymosin. Additionally, the three commercial RCC samples contained the protein species (1-323), (38p-323), (39p-323), and (40p-323) and the shorter form (3-323). Differentiation of the natural and bioengineered enzyme is based upon the detection of these unique minor components by mass spectrometry.

Amino Acid Sequence↗

Bioactivity of anacardic acid against colorado potato beetle (Leptinotarsa decemlineata) larvae.

Anacardic acid (2-hydroxy-6-alkylbenzoic acid) produced and secreted from glandular trichomes of zonal geranium (Pelargonium x hortorum; Geraniaceae family) provides resistance to small pests (aphids and spider mites). To assess the potential bioactivity of anacardic acid against larger insect pests and to determine if an alternate mode of application (ingestion rather than the topical application) could impart resistance to pests, the effects of anacardic acid consumption on the development of Colorado potato beetle larvae were tested. Analysis of dose-response curves indicated a significant effect on weight gain and mortality. Assessment of food preference (treated versus untreated) indicated larvae avoid food containing anacardic acid and have a lower feeding rate on food containing anacardic acid. On the basis of these results, it is suggested that anacardic acid, applied as a chemical spray or through bioengineering production in crop plants, may provide a new tool in the arsenal to minimize damage to plants caused by pests.

Anacardic Acids↗

Low-temperature growth of uniform ZnO particles with controllable ellipsoidal morphologies and characteristic luminescence patterns.

Uniform ellipsoidal ZnO particles have been synthesized in an aqueous solution in the presence of triethonalamine (TEA) mediated by sonication at the temperature below 80 degrees C. Scanning electron microscopy observations reveal that the ellipsoidal particles are highly uniform with a hexagonal cross-section. The morphologies of the ZnO particles can be systematically controlled from elongated rugby ball-like ellipsoidal to half-ellipsoidal by increasing the TEA concentration. Spatial resolved cathodoluminescence measurements at room temperature show that the ellipsoidal ZnO particles are intrinsically encoded with barcode-like ultraviolet luminescence patterns, which are of either a wide stripe or a narrow stripe perpendicular to the length at the core of the particles depending on the growth temperature. Moreover, the luminescence spectra of the ellipsoidal particles can be tuned by heat treatments at elevated temperatures, while maintaining the luminescence patterns. We believe that the well-defined uniform ellipsoidal ZnO particles embedded with unique luminescence characteristics hold great potential for use in bioengineering and photonics, such as biological labeling and optical probes.

Adsorption↗

Can the replacement of a single atom in the enzyme horseradish peroxidase convert it into a monoxygenase? A density functional study.

Density functional calculations on horseradish peroxidase mutants are presented, whereby one or two of the nitrogen atoms of the axial histidine ligand have been replaced by phosphorus atoms. Our calculations show that phosphorus entices a push effect on the oxoiron group, whereas a histidine side chain withdraws electrons. As a result, we predict that a phosphorus-substituted histidine ligand will convert the active form of a peroxidase into a monoxygenase. This subsitution may be useful for the bioengineering of commercially exploitable enzymes.

Histidine↗

Synthesis and characterization of partially biodegradable and thermosensitive hydrogel.

A partially biodegradable and thermosensitive hybrid hydrogel network (DAN series) based on dextran-allylisocyanate (Dex-AI) and poly(N-isopropylacrylamide) (PNIPAAm) was synthesized via UV photocrosslinking. These hybrid hydrogels were characterized in terms of their chemical structure, thermal, mechanical, morphological and temperature-induced swelling properties. The effect of the composition ratio of Dex-AI to PNIPAAm on such properties were examined. The differential scanning calorimetry data show that this Dex-AI/PNIPAAm hybrid network has an increased lower critical solution temperature (LCST) and glass transition temperature (Tg) with an increase in the Dex-AI content. The interior morphology of these hybrid hydrogels revealed a decreased porous microstructure with an increase in the Dex-AI content in the hybrid network. Furthermore, if the Dex-AI composition became too high, a distinctive network structure with two different microporous structures appeared. The mechanical properties of these hybrid hydrogels also increased with an increase in the Dex-AI content. The temperature dependence of the swelling ratio, the deswelling kinetics as well as the reswelling kinetics was also characterized by gravimetric method. When comparing with a normal PNIPAAm hydrogel, these Dex-AI/PNIPAAm hybrid networks, due to the presence of Dex-AI moiety, also show improved temperature-induced intelligent properties, such as the faster and controllable response dynamics, which may find promising applications in a wide variety of fields, such as biomedical and bioengineering fields.

Absorbable Implants↗

Efficacy of a barrier cream and its vehicle as protective measures against occupational irritant contact dermatitis.

The actual advantage of barrier creams over bland emollients for skin protection is still hotly debated. In a randomized, double-blinded study, a newly-introduced barrier cream and its moisturizing vehicle were compared regarding their skin compatibility, efficacy and resulting acceptance. Thus, 2 panels of 25 hospital nurses with mild signs of skin irritation were asked to use 1 of the test products provided (verum or vehicle) over a period of 4 weeks. Effects of both types of preparations were studied weekly by clinical examination and the instrumental assessment of bioengineering parameters. Results showed no significant differences between barrier cream and vehicle. In both groups, clinical skin status improved and stratum corneum hydration increased significantly during the study period. Both preparations were tolerated and accepted well, thus showing both skin protection and skin care. These results contribute to the debate as to whether a strict distinction between "skin care" and "skin protection" products is justified. The vehicle alone is capable of positively influencing skin status. Emphasis must be laid on regular, frequent, and correct application of a product for it to be effective.

Adult↗

Multicentre study for the development of an in vivo model to evaluate the influence of topical formulations on irritation.

Although skin protective products to prevent irritant skin reactions are in wide use, neither standardized test models to prove differences in efficacy exist, nor has the quality or the reproducibility of results been evaluated in a multicentre approach. This should be mandatory when developing or testing skin care products. Therefore, we have designed a multicentre study in an approach to find a standardized test procedure for the evaluation of skin protective products. In this irritation study, a repeated short-time occlusive irritation test (ROIT) with a standardized protocol has been evaluated in 2 phases (12 days and 5 days protocol) in 4 (n=20) respectively 6 (n=33) skilled centres. The skin reaction was induced by 2 irritants (0.5% aq. SLS and toluene, 2x a day for 30 min). Its modification by 3 different cream bases with different hydrophilicity was analyzed. The irritation was monitored by bioengineering methods (TEWL measurement, colorimetry) and by clinical scoring. The evaluation showed that significant results could already be achieved with the 5-day protocol. Furthermore, in spite of the expected inter-centre variations due to heterogeneity of the individual threshold of irritation, interpretation of clinical score, and inter-instrumental variability, the ranking of the vehicles regarding reduction of the irritant reaction was consistent in all centres.

Administration, Topical↗

An overview of the efficacy of topical corticosteroids in experimental human nickel contact dermatitis.

We review controlled trials of corticosteroid effect in experimentally elicited acute nickel contact dermatitis in man, in the hope of clarifying optimal efficacy for clinical use. To maximize discrimination and objectivity, we focus on data with 1 well-characterized allergen, nickel, in studies utilizing bioengineering documentation. Higher potency corticosteroids are effective (unlike in experimental irritant contact dermatitis), but optimum schedules still require definition.

Administration, Topical↗

Natural vegetable fats in the prevention of irritant contact dermatitis.

Chronic irritant contact dermatitis (ICD) is one of the most pressing problems in occupational medicine and is common in the food processing industry. To date, protective creams that fulfil the special requirements in the foodstuffs industry have not been available. Therefore, we studied the efficacy of pre-exposure application of natural vegetable fats in the prevention of experimentally induced ICD. A panel of 20 healthy volunteers was tested with a repetitive irritation test using sodium lauryl sulfate (SLS) as a standard irritant in a randomized study. Application sites were assessed clinically and by the use of bioengineering techniques (evaporimetry, chromametry, and corneometry). Rape seed and palm fats showed significant protective potential. Gas-chromatographic analysis revealed differences in the fatty acid composition of the vegetable. Higher content of linoleic acid and lower content of oleic acid was associated with beneficial effects. Our results are a new approach in the prevention of ICD and towards the development of new protective preparations for workplaces in the foodstuffs industry.

Adult↗

Tacrolimus enhances irritation in a 5-day human irritancy in vivo model.

Tacrolimus (FK 506) is a macrolide discovered in 1984 as a metabolic product of Streptomyces tsukabaensis. It has been used successfully in treating atopic dermatitis, allergic contact dermatitis, lichen planus mucosae and pyoderma gangrenosum. In the present study, we evaluated the anti-inflammatory activity of FK 506 in 2 human skin inflammation models. FK 506 as Protopic(R) cream was tested (i) in a 4-day repetitive irritation test with 2 x daily application of sodium lauryl sulphate (SLS), and (ii) in a UVB erythema model. The effect was evaluated visually and quantified by non-invasive bioengineering methods, namely chromametry and tewametry (TEWL). When FK 506 was applied 30 min after SLS irritation, an increased inflammation in comparison to controls was observed with all 3 methods, with only the TEWL data reaching statistical significance. 1 x daily application of FK 506 for 5 days, starting at the end of the 4-day irritation period, was without any effect. Similarly, no effect of FK 506 was seen in the UVB model. In conclusion, FK 506 was shown to enhance experimentally induced irritant contact dermatitis and not to accelerate healing of irritant contact dermatitis and UVB erythema.

Adult↗

Effect of a lipid-rich emollient containing ceramide 3 in experimentally induced skin barrier dysfunction.

In the present study we compared the effect of a ceramide 3-containing emollient (Locobase(R) Repair) with a control emollient (vaselinum album/cremor lanette ana) and untreated damaged skin using clinical, bioengineering and immunohistochemical methods in two different models of experimentally induced skin barrier dysfunction. In model A (n = 13) skin barrier dysfunction was inflicted at three investigation sites by tape stripping. In model B (n = 13) the volunteers were patch tested at three investigation sites with sodium dodecyl sulphate (0.2%) for 4 h a day for 4 consecutive days. The investigation sites were treated once a day with the above-mentioned agents. Irritant reaction was assessed daily by erythema scoring and measurements of transepidermal water loss (TEWL). After 5D, punch biopsies were taken from all sites. Immunohistochemical assessment was carried out with respect to epidermal proliferation, epidermal differentiation and Langerhans cells. Tape stripping resulted in an erythematous reaction and an increase of TEWL associated with up-regulation of cycling cells, involucrin and expression of cytokeratin 16. At day 4, ceramide 3-containing emollient significantly decreased (p < 0.03) the erythema score, TEWL and cycling cells in comparison with the untreated site. Repetitive exposure to SDS induced a variable degree of erythema, gradual increase of TEWL, an increase of cycling cells, and up-regulation of involucrin, E-FABP and SKALP. The treatment with the control emollient significantly prevented erythema, increase of TEWL and cycling cells at day 4 compared to the untreated site. In summary, the present study demonstrated that both tested emollients improve skin barrier in different conditions compared to the untreated skin. There is some indication that formulations containing skin-related lipids might be of benefit in barrier disruption following tape stripping. Different models and clinical trials are needed to establish the usefulness in specific conditions of emollients containing skin-related lipids.

Administration, Cutaneous↗

Dynamic modeling and jaw biomechanics.

Bioengineered simulations of dynamic events in the human masticatory system are relatively new. A primary advantage is their ability to integrate structure and function in cause-and-effect scenarios. By permitting detailed analyses of these interactions, and the prototyping of prosthetic additions, the models generate working hypotheses. Significant issues in their use include the importation and measurement of structural geometry, the choice of parameters affecting dynamics (e.g. inertial properties and viscoelasticities) and the nature of the modeling process (e.g. whether models are kinetically driven by muscle contraction, or kinematically defined by movement channels). Presently, there are few accepted standards or conventions for managing these computational data in the human jaws, and the data used are often derived from multiple and disparate sources. This review focuses on the approaches, assumptions, and key applications of dynamic modeling in the human masticatory system. It considers the role of imaging, the restrictions imposed by assumptions of unknown or unverifiable data, and how modeling can be a useful research technique despite these hurdles. The review concludes with a comment on creating virtual models for educational purposes.

Biomechanical Phenomena↗

1.4 Research and the dental student.

There has been significant concern that the dental curriculum and system of clinical education, in particular, is not designed to take advantage of the explosion in knowledge in biomedical science and its application to the health of the public. Although there are some examples of innovations in dental education on a global scale that have the capacity to increase the assimilation of basic and clinical knowledge, most of the dental education models are mired in the traditional '2 + 2' approach to education. This can be seen in North America and the European '2 + 3' model or the stomatological '4 + 2' approach. In each of these systems, the basic and behavioural science courses continue to be perceived as hurdles over which students must leap in order to reach the clinical programmes where there is little opportunity to use basic science information to advance patient care and treatment. Examples of issues that are not well represented include: innovations in imaging; diagnosis; bio-materials; science-based approaches to clinical practice; novel approaches to therapeutics; interactions between the oral, dental and craniofacial complex and systemic health and disorders; the role of oral infections and systemic disease; the increasing appreciation of chronic diseases and disorders such as osteoporosis and diabetes that affect oral tissues; the promise of bioengineering, tissue engineering and biomimetics; the potential use of saliva as a diagnostic tool; the understanding of oral complications of cancer treatment; the treatments of HIV/AIDS diseases and hepatitis; the use of dental and dental hygiene staff on health-care teams to deal with issues such as birth defects, orofacial trauma, head and neck cancer, chronic pain management and so on. There seems to be an excessive emphasis on restorative dentistry and, to a lesser extent, on the more biological approaches to diagnosis, prevention and therapeutics. This continued lack of integration of basic and clinical sciences in the curriculum continues to foster a dental workforce that is highly technically competent to provide specific clinical services but poorly equipped to evaluate and implement new biological approaches to diagnosis, therapeutics and intervention. Unfortunately, after many attempts by organized dental symposia aimed at the integration of basic and clinical sciences, there has been little discernible curricular change. It appears that there is an opportunity through this global congress to identify the best practices in the various global curricula that could change this paradigm in dental education and lead us toward the education of a more scientifically orientated practitioner-one who can take advantage of innovations in new and emerging technologies in their application to patient care. It is the challenge of this section to try to ascertain the best method or methods by which dental education promotes research to the dental student and what research represents in terms of critical thinking and evidence-based approaches to dental education and clinical practice.

Competency-Based Education↗

Optical coherence tomography in dermatology: a review.

BACKGROUND/AIMS: Optical coherence tomography (OCT) is a non-invasive technique for morphological investigation of tissue. Since its development in the late 1980s it is mainly used as a diagnostic tool in ophthalmology. For examination of a highly scattering tissue like the skin, it was necessary to modify the method. Early studies on the value of OCT for skin diagnosis gave promising results. METHODS: The OCT technique is based on the principle of Michelson interferometry. The light sources used for OCT are low coherent superluminescent diodes operating at a wavelength of about 1300 nm. OCT provides two-dimensional images with a scan length of a few millimeters (mm), a resolution of about 15 microns and a maximum detection depth of 1.5 mm. The image acquisition can be performed nearly in real time. The measurement is non-invasive and with no side effects. RESULTS: The in vivo OCT images of human skin show a strong scattering from tissue with a few layers and some optical inhomogeneities. The resolution enables the visualization of architectural changes, but not of single cells. In palmoplantar skin, the thick stratum comeum is visible as a low-scattering superficial well defined layer with spiral sweat gland ducts inside. The epidermis can be distinguished from the dermis. Adnexal structures and blood vessels are low-scattering regions in the upper dermis. Skin tumors show a homogenous signal distribution. In some cases, tumor borders to healthy skin are detectable. Inflammatory skin diseases lead to changes of the OCT image, such as thickening of the epidermis and reduction of the light attenuation in the dermis. A quantification of treatment effects, such as swelling of the horny layer due to application of a moisturizer, is possible. Repeated measurements allow a monitoring of the changes over time. CONCLUSION: OCT is a promising new bioengineering method for investigation of skin morphology. In some cases it may be useful for diagnosis of skin diseases. Because of its non-invasive character, the technique allows monitoring of inflammatory diseases over time. An objective quantification of the efficacy and tolerance of topical treatment is also possible. Due to the high resolution and simple application, OCT is an interesting addition to other morphological techniques in dermatology.

Dermatology↗

Efficacy of corticosteroids in acute experimental irritant contact dermatitis?

BACKGROUND/AIMS: Topical corticoids are used to treat irritant contact dermatitis (ICD) in humans. However, their clinical efficacy remains sub judice. This study was designed to assess the efficacy of low- and medium-potency corticosteroids on irritant dermatitis. METHODS: We induced an acute ICD via open application of sodium lauryl sulphate (SLS) on the hands of subjects. The dorsal side of hands was irritated with 10% SLS five times in one day. Once on day 1 and twice daily on days 2-5, 1% hydrocortisone, 0.1% betamethasone-17-valerate and vehicle cream (petrolatum) were applied subsequently. Visual grading, bioengineering techniques and squamometry were used to quantify skin response. RESULTS: Corticosteroids were found ineffective in treating the surfactant-induced irritant dermatitis when compared with the vehicle and with the untreated control. CONCLUSION: The counterintuitive result (in a relatively realistic and robust model) should be interpreted with caution until verified with other irritants of varying physicochemical properties.

Adult↗

Human vasculogenesis ex vivo: embryonal aorta as a tool for isolation of endothelial cell progenitors.

SUMMARY: Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11- to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1]/vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD31-), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34+/CD31+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34+/CD31- endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.

Antigens, CD34↗

Extracellular electron transfer via microbial nanowires.

Microbes that can transfer electrons to extracellular electron acceptors, such as Fe(iii) oxides, are important in organic matter degradation and nutrient cycling in soils and sediments. Previous investigations on electron transfer to Fe(iii) have focused on the role of outer-membrane c-type cytochromes. However, some Fe(iii) reducers lack c-cytochromes. Geobacter species, which are the predominant Fe(iii) reducers in many environments, must directly contact Fe(iii) oxides to reduce them, and produce monolateral pili that were proposed, on the basis of the role of pili in other organisms, to aid in establishing contact with the Fe(iii) oxides. Here we report that a pilus-deficient mutant of Geobacter sulfurreducens could not reduce Fe(iii) oxides but could attach to them. Conducting-probe atomic force microscopy revealed that the pili were highly conductive. These results indicate that the pili of G. sulfurreducens might serve as biological nanowires, transferring electrons from the cell surface to the surface of Fe(iii) oxides. Electron transfer through pili indicates possibilities for other unique cell-surface and cell-cell interactions, and for bioengineering of novel conductive materials.

Biotechnology↗