Degradation of spent craft brewer's yeast by caprine rumen hyper ammonia-producing bacteria.

UNLABELLED: Spent yeast from craft beers often includes more hops (Humulus lupulus L.) secondary metabolites than traditional recipes. These compounds include α- and ß- acids, which are antimicrobial to the rumen hyper ammonia-producing bacteria (HAB) that are major contributors to amino acid degradation. The objective was to determine if the hops acids in spent craft brewer's yeast (CY; ~ 3·5 mg g(-1) hops acids) would protect it from degradation by caprine rumen bacteria and HAB when compared to a baker's yeast (BY; no hops acids). Cell suspensions were prepared by harvesting rumen fluid from fistulated goats, straining and differential centrifugation. The cells were re-suspended in media with BY or CY. After 24 h (39°C), HAB were enumerated and ammonia was measured. Fewer HAB and less ammonia was produced from CY than from BY. Pure culture experiments were conducted with Peptostreptococcus anaerobiusBG1 (caprine HAB). Ammonia production by BG1 from BY was greater than from CY. Ammonia production was greater when exogenous amino acids were included, but similar inhibition was observed in CY treatments. These results indicate that rumen micro-organisms deaminated the amino acids in CY to a lesser degree than BY. SIGNIFICANCE AND IMPACT OF THE STUDY: Spent brewer's yeast has long been included in ruminant diets as a protein supplement. However, modern craft beers often include more hops (Humulus lupulus L.) than traditional recipes. These compounds include α- and ß- acids, which are antimicrobial to the rumen hyper ammonia-producing bacteria (HAB) that are major contributors to amino acid degradation. This study demonstrated that hops acids in spent craft brewer's yeast protected protein from destruction by HABin vitro. These results suggest that the spent yeast from craft breweries, a source of beneficial hops secondary metabolites, could have value as rumen-protected protein.

Thymol and related alkyl phenols activate the hTRPA1 channel.

BACKGROUND AND PURPOSE: Thymol, a major component of thyme and oregano, has medical uses in oral care products as an astringent and antibiotic. Its distinctive sharp odour and pungent flavour are considered aversive properties. The molecular basis of these aversive properties is not well understood. EXPERIMENTAL APPROACH: The ability of thymol to activate human transient receptor potential channel A1 (hTRPA1) expressed in stably transfected human embryonic kidney 293 (HEK293) cells was measured by membrane potential and calcium-sensitive dyes in a fluorescence-imaging plate reader (FLIPR) assay. Direct activation of hTRPA1 currents was measured by whole-cell voltage clamp recording. Intracellular calcium changes were measured using fura-2 dye. The FLIPR assay was also used to measure membrane potential changes elicited by thymol after pretreatment with camphor, a known TRPA1 inhibitor. The ability of related alkyl phenols to activate hTRPA1 was also determined. KEY RESULTS: Thymol potently activated a membrane potential response and intracellular calcium increase in hTRPA1-expressing HEK293 cells in a concentration-dependent manner. Activation by thymol desensitized hTRPA1 to further exposure to thymol or the known ligand allyl isothiocyanate (AITC). The related phenols 2-tert-butyl-5-methylphenol, 2,6-diisopropylphenol (propofol) and carvacrol also activated hTRPA1. Phenols with less bulky carbon substitutions and lower logP values were less potent in general. The response to thymol was blocked by camphor. CONCLUSIONS AND IMPLICATIONS: These results suggest a role for hTRPA1 activation in the reported pungent and aversive properties of some of these pharmaceutically important phenols.

Volumetric contraction in some tooth-coloured restorative materials.

BACKGROUND: Much of the concern about the setting contraction of tooth-coloured restorative materials has been focused on the composite resins. This study investigated setting contraction of a range of glassionomer materials and included, for comparison, products from other groups of restorative materials. METHODS: A deflecting disk method was used to determine the volumetric contraction of three conventional (non-light cured) glass-ionomer cements (GICs), two restorative, one "lining" consistency and one adhesive/lining consistency resin-modified glass-ionomers (RMGIs), two resin adhesives, three restorative composite resins and two compomers. The influence of powder:liquid ratio on two hand-mixed materials was also examined. RESULTS: The light-cured materials (including RMGIs) showed substantially greater per cent contraction at 5 minutes than did the three conventional GICs (not light cured) and a substantially greater proportion of the 30-minute contraction had occurred at 5 minutes for these light-cured materials. Their further contraction after 1 hour was generally less than 5 per cent of the 1 hour contraction. CONCLUSION: Although the conventional GICs contract more slowly in the first 5 minutes, by 30 minutes the current restorative GICs and RMGIs exhibit a volumetric setting contraction that is comparable with the composite resins and compomers and is generally in the range of 2-3 per cent.

Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.

Compounds capable of generating singlet oxygen represent a source of artifactual data in scintillation proximity assays measuring phosphopeptide binding to SH2 domains.

We developed scintillation proximity assays (SPA) to discover compounds which inhibit phosphopeptide binding to Src homology 2 (SH2) domain proteins Grb2 and Syk. An assay artifact is reported here as a caveat to others. The SPA used an antibody to couple glutathione-S-transferase SH2 domain fusion proteins to scintillant beads coated with protein A. A pyrazoloquinolone and indolocarbazole inhibited [3H]phosphopeptide binding in both assays. Their potency in the SPA increased with prolonged (2 to 24 h) assay exposure to ambient light. They were inactive in absence of light and in an alternate binding assay. Both compounds absorbed visible light and generated singlet oxygen based on 2-methylfuran-trapping experiments. Their inhibitory activity was suppressed by the singlet oxygen scavengers sodium azide and dithiothreitol. The results suggest that compounds, not previously considered photosensitizers, generated enough singlet oxygen to damage oxidant-sensitive SPA components. Therefore, this SPA should be protected from light to minimize occurrence of false positives.

Three-year evaluation of computer-machined ceramic inlays: influence of luting agent.

OBJECTIVE: Changes in the occlusal marginal adaptation of computer-machined inlay restorations were assessed over 3 years. METHOD AND MATERIALS: Each of 21 patients received two Cerec (Vita Mark II) two- or three-surface restorations, with margins entirely in enamel, placed using two of four luting agents: a homogeneous microfilled resin composite, a fine hybrid composite, a relatively coarse hybrid composite, and an encapsulated glass-ionomer cement. Replicas of the restorations were made at baseline 6 months or 1 year (93% of original restorations), 2 years (79%), and 3 years (83%). The percentage of continuous margin on the occlusal margin was evaluated for the enamel-luting agent and luting agent-porcelain interfaces using a scanning electron microscope. RESULTS: Mean continuous margin values were high for all groups at the enamel-luting agent interface; there were no significant differences among the luting agents. For each luting agent, the percentage of continuous margin decreased significantly at the luting agent-porcelain interface over 3 years; differences among the luting agents at 3 years were not significant (Kruskal-Wallis test). After 3 years, the wear of the luting agents was significantly different. The least wear occurred with the microfilled resin composite; the coarse hybrid composite showed the most wear. CONCLUSION: The occlusal margins of Cerec inlays exhibited consistently high-quality adaptation at 3 years. The microfilled resin composite luting agent was more wear resistant than the hybrid composites and the glass-ionomer cement.

The influence of modified cavity design and finishing techniques on the clinical performance of amalgam restorations: a 2-year clinical study.

Use of modified cavo-surface angles (CSAs) and finishing techniques for amalgam restorations may influence the long term marginal fracture around these restorations. One operator placed 111 Tytin amalgam restorations in Class I and II preparations in the permanent teeth of 37 patients, each of whom received three restorations. One of these restorations was placed in a cavity prepared with a 'traditional' CSA and was finished more than 24 h after placement. Prepared cavities for the remaining two restorations for each patient were 'modified' with the aim of producing an occlusal CSA of 90-110 degrees. One restoration was carved-only and the other was finished more than 24 h after placement. Clinical evaluation of the restorations was carried out at baseline, 1 year and 2 years. At these times, the restorations were photographed and stone replicas were obtained from impressions taken at the cavity preparation stage to determine the cavo-surface angles. Careful application of the modified cavity design provided a mean cavo-surface angle of 109 degrees whereas a 'traditional' cavity design provided a mean cavo-surface angle of 126 degrees. Using the Mahler photographic method of evaluation, a combination of modification of the occlusal cavo-surface angle and finishing of the restoration was found to have an influence on the marginal fracture at 2 years. The clinical significance of these findings, with respect to the modified cavity design, is discussed.

A clinical evaluation of posterior composite resin restorations: 8-year findings.

OBJECTIVES: From a continuing investigation of the clinical performance of three different types of composite resin, the findings of the clinical evaluation at 8 years are presented. METHODS: One operator placed 330 restorations in Class I and Class II preparations in the posterior teeth of 72 patients. Every patient received at least one restoration of each type of material: a microfilled composite, a small particle hybrid, a relatively coarse particle hybrid, and an amalgam control. Restorations were evaluated using clinical criteria. RESULTS: Forty six patients attended the 8-year recall. Twenty-five of the 213 restorations (13.7% of the composites and 5.8% of the amalgams) originally placed in these 46 patients had previously failed or been lost from the study or were assessed as requiring replacement at the 8-year recall. Bulk fracture and secondary caries at the margin were the most common forms of failure in the composite restorations. Other failures or losses were associated with a non-margin defect in the composite, caries not associated with the restoration, pulpal considerations, extraction for orthodontics and reasons unknown. One-hundred and ninety-three restorations (including five that required replacement) were available for clinical evaluation at 8 years and these included 17 Class II restorations. Significantly fewer restorations placed with the coarse particle hybrid exhibited evidence of marginal deterioration. CONCLUSION: At 8 years, composite restorations in posterior teeth had failed at a rate two to three times that of amalgam restorations. The most common types of failure were bulk fracture and secondary caries at the margin.

Apparatus for precision calibration of skinfold calipers.

Despite the fact that skinfold calipers are widely used to measure subcutaneous adipose tissue, the current methods of calibration are quite crude. Methods such as hanging masses from the caliper jaws until they remain open, lack validity and reliability because the caliper jaws are stationary instead of dynamic, and the opening jaws give an upscale reading of the jaw gap not a downscale reading that occurs when the calipers are being used to measure skinfolds. This report describes how to build an apparatus capable of measuring static and dynamic, upscale and downscale jaw pressures of a variety of caliper types and also provides guidelines specifically for calibration and servicing of Harpenden calipers. The key areas to maintain are the caliper springs (which should have spring coefficients ranging 1.104-1.153 N.mm-1 ), the pivot joint (which should operate smoothly), the indicator mechanism (which should require only 0.78-0.88 N for full movement), and the jaw alignment which should be square to ensure that the full effective jaw surface area of 90 mm2 is applied to the skinfold. While there are insufficient data at this time to prescribe rigid calibration criteria, assessment of approximately 100 new and used Harpenden calipers indicates that, after servicing, the important dynamic downscale jaw pressure will range 7.7-8.4 g.mm-2 at 5 mm of jaw gap and 7.3-8.0 g.mm-2 at 40 mm of jaw gap. Dynamic upscale jaw pressure should be within 1.0-1.5 g.mm-2 of the corresponding dynamic downscale jaw pressure. Am. J. Hum. Biol. 10:689-697, 1998. © 1998 Wiley-Liss, Inc.

Microleakage of amalgam alloys: an update.

Using an in vitro air pressure test, the authors found a wide variation in the microleakage characteristics of amalgam restorations made from 33 recently manufactured precapsulated alloys. Spherical particle alloys as a group exhibited the highest microleakage values. When the authors changed the mercury/alloy ratios to produce 1 percent more mercury in the capsules by weight, microleakage decreased significantly. Because extensive microleakage of amalgam restorations has been related to increased postoperative sensitivity, the authors suggest that practitioners should be alert to this potential problem.

Ophirapstanol trisulfate, a new biologically active steroid sulfate from the deep water marine sponge Topsentia ophiraphidites.

Ophirapstanol trisulfate [1], a new steroid trisulfate related to sokotrasterol trisulfate was isolated from a deep water marine sponge Topsentia ophiraphidites. Compound 1 exhibited significant inhibition in the guanosine diphosphate/G-protein RAS exchange assay. The structure elucidation of 1 and ophirapstanol [2] by nmr spectroscopy is described.

A clinical evaluation of posterior composite resin restorations.

There is a trend towards manufacturers seeking to provide dentists with a single, all-purpose composite resin, usually of the small particle hybrid type. This three-year clinical study compared the clinical performance of three different types of composite resin used in posterior teeth and identified several modes of failure. Of the 330 restorations (three composite resins and one amalgam control) initially placed in 72 patients, 223 restorations in 48 patients were available for evaluation at three years. Modified clinical criteria for assessing the restorations were able to discriminate among the composite resins. A microfilled composite and a small particle hybrid exhibited increasing evidence of marginal fracture (crevice) with time. In addition, the small particle hybrid showed evidence of wear at the margins more frequently than the other materials. Of the restorations available for assessment, four restorations of each of these two types of composite resin required replacement during the study. Coarse particle hybrid restorations showed evidence of wear but little evidence of marginal fracture.

The assessment of apical leakage of root-filled teeth by the electrochemical technique.

Since its inception by Jacobson and von Fraunhofer in 1976, several studies have used the electrochemical method to assess the leakage occurring around root canal fillings. This study has examined aspects of the electrochemical technique and the effects that this test method may have on leakage results. The findings have raised concerns about the application of the technique. It is evident that electrochemical leakage testing over 30 days can influence the electrochemical leakage reading at 30 days and may influence the linear dye penetration recorded at 30 days. It is suggested that testing for electrochemical leakage should not precede the assessment of linear dye penetration.

The adaptation achieved by four root canal filling techniques as assessed by three methods.

Four root canal filling techniques--lateral condensation, McSpadden compaction, ultrasonic activation (Enac), and thermoplastic injection (Ultrafil)--were assessed for adaptation of the filling material to the canal wall. The adaptation and leakage were examined quantitatively using an electrochemical method and a linear dye penetration method, and qualitatively by radiographic evaluation. Using the electrochemical method, differences among obturation techniques were relatively small, although a greater proportion of the teeth that had been filled by lateral condensation exhibited no leakage. Radiographically, the techniques appeared to exhibit similar adaptation in the apical 6 mm. The techniques of lateral condensation and ultrasonic activation showed superior control of length of the root canal filling.

Occlusal margin defects around different types of composite resin restorations in posterior teeth.

Using stone replicas of individual restorations from a clinical study investigating three types of posterior composite resins, four types of defects were identified on the occlusal margins. It was observed that particular types of defects were commonly associated with each type of composite resin. The microfilled composite usually exhibited crevice formation (marginal fracture), the small-particle hybrid showed evidence of both wear and crevice formation, and a coarse composite resin most noticeably exhibited wear. Fracture of excess composite was associated with the fine-textured composites.

Finishing techniques for amalgam restorations: clinical assessment at three years.

Use of the optimum finishing technique for an amalgam restoration may enhance the marginal integrity of the restoration and discourage its unnecessary early replacement. Two hundred and twenty-eight high copper amalgam restorations in 56 patients were evaluated, using clinical assessment criteria, up to three years after placement. Each patient had received at least one carved-only amalgam, at least one immediately finished restoration, and at least one amalgam that was polished at a subsequent appointment. Regardless of the finishing technique, the restorations exhibited similar marginal integrity up to three years after placement. Polished restorations were found to have substantially superior surface texture and less likelihood of surface discoloration. No evidence was found to support the use of immediate finishing techniques. The clinical significance of these findings, with respect to the need to polish amalgam restorations, is discussed.

Finishing of amalgam restorations: a three-year clinical study.

Class I and Class II amalgam restorations were placed in the permanent teeth of 66 patients, each of whom received a minimum of three restorations. At least one of these restorations was carved-only, at least one was immediately finished (using a prophylaxis paste on rubber cups at low speed, 8 or 10 min after trituration), and at least one restoration was polished (using finishing burs and polishing points, more than 24 h after placement). The number of restorations available for assessment of marginal fracture using the 11-unit photographic scale developed by Mahler was 253 at baseline, 249 at 1-year and 2-year recalls and 228 at the 3-year recall. Although polished restorations exhibited greater initial (baseline) marginal fracture, at 1-year, 2-year and 3-year recalls the restorations were found to exhibit a similar amount of marginal fracture regardless of the finishing technique. The amount of perceived marginal fracture increased steadily after 1 year. The immediate finishing of amalgam restorations showed no long-term benefit over the other techniques and has little to commend it. The clinical finishing of amalgam restorations is discussed in the light of this and other research.

Actions of a 5-lipoxygenase inhibitor, Sch 40120, on acute inflammatory responses.

Sch 40120 (10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzo[b] [1,8]naphthyridin-5(7H)-one) is an inhibitor of the 5-lipoxygenase enzyme in rat neutrophils, human neutrophils and the the MC9 murine mast cell clone with IC50 values of 8, 4 and 7 microM, respectively. The drug was examined for its effects on acute inflammatory responses in the paw and pleural cavity of rats. The drug suppressed paw inflammation triggered by a reverse passive Arthus reaction or a subplantar injection of the polysaccharide carrageenan with p.o. ED50 values of 0.2 and 1.5 mg/kg, respectively. In reverse passive Arthus reaction and carrageenan pleurisy models, Sch 40120 was found to suppress both the cellular and fluid components of the acute inflammation. The p.o. ED50 values for inhibition of cells and fluid in pleurisy models were in the range of 0.1 to 0.7 mg/kg. When applied locally to the ears of mice, the drug blocked an arachidonic acid-induced and leukotriene-mediated ear inflammation with an ED50 of 0.072 mg/ear. These findings suggest that Sch 40120 is a potent anti-inflammatory agent that may be particularly useful in the treatment of inflammatory diseases such as psoriasis in which leukotrienes appear to be major mediators of the pathological symptoms that characterize the disease state.

Direct posterior composite resin restorations: a review. 2. Clinical technique.

Despite the increased use of composite resin to restore posterior teeth, there is evidence that clinicians should be selective in their use of these restorations. This paper describes the clinical technique--preparatory procedures, preparation of the cavity, preparation for placement of composite resin, placement of composite resin and finishing of the restoration--for the relatively conservative use of composite resin in posterior teeth and reviews the literature to discuss briefly many of the controversial aspects of the technique.

Direct posterior composite resin restorations: a review. 1. Factors influencing case selection.

The principal factors that influence case selection for direct composite resin restorations in posterior teeth are discussed. These include the perceived preference for tooth-coloured aesthetics, survival rate and replacement of posterior composites, clinical problems and concerns such as occlusal surface defects and the numerous effects of the material's polymerization contraction, and the availability of alternative tooth-coloured techniques for restoring posterior teeth. Specific guidelines in case selection are suggested. A subsequent paper reviews controversial aspects of the restorative technique for posterior composites and, on this basis, defines important principles in the clinical technique.