Quantitative analysis of 3-OH oxylipins in fermentation yeast.

Despite the ubiquitous distribution of oxylipins in plants, animals, and microbes, and the application of numerous analytical techniques to study these molecules, 3-OH oxylipins have never been quantitatively assayed in yeasts. The formation of heptafluorobutyrate methyl ester derivatives and subsequent analysis with gas chromatography - negative chemical ionization - mass spectrometry allowed for the first determination of yeast 3-OH oxylipins. The concentration of 3-OH 10:0 (0.68-4.82 ng/mg dry cell mass) in the SMA strain of Saccharomyces pastorianus grown in laboratory-scale beverage fermentations was elevated relative to oxylipin concentrations in plant tissues and macroalgae. In fermenting yeasts, the onset of 3-OH oxylipin formation has been related to fermentation progression and flocculation initiation. When the SMA strain was grown in laboratory-scale fermentations, the maximal sugar consumption rate preceded the lowest concentration of 3-OH 10:0 by ∼4.5 h and a distinct increase in 3-OH 10:0 concentration by ∼16.5 h.

Construction of a highly thermostable 1,3-1,4-ß-glucanase by combinational mutagenesis and its potential application in the brewing industry.

OBJECTIVES: To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-ß-glucanase by combinational mutagenesis and to test its effect in congress mashing. RESULTS: A mutant ß-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg-1 and 189 s-1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml-1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively. CONCLUSIONS: The mutant ß-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

Improving uncertainty in Widmark equation calculations: Alcohol volume, strength and density.

The Widmark equation is probably the most commonly used calculation for medicolegal purposes. Recently the National Research Council (USA) and the Forensic Science Regulator (UK) have called for the uncertainty of all results to be given with all forensic measurements and calculations. To improve the uncertainty of measurement of results from Widmark calculations we have concentrated on the uncertainties of measurement involved in the calculation of amount of alcohol, that of the volume of alcohol, the concentration of alcohol and the density of alcohol as previous studies have investigated some of the other factors involved. Using experimental studies, the scientific literature and legal statutes, we have determined revised and improved uncertainties of the concentration of ethanol for Widmark calculations for both the USA and UK. Based on the calculations that we have performed we recommend the use of Monte Carlo Simulation for the determination of uncertainty of measurement for Widmark Calculations.

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing.

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF -ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360-460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF -ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360-460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.

Influence of different types of bedding material on the prevalence of pododermatitis in rabbits.

Pododermatitis is a disorder with a complex etiology, because potentially determined by various factors such as posture, nutrition and genetics. In the case of presence of pododermal lesions, it is often noticed that rabbits go to eat at the bowl less frequently, but an evident weight loss is not always perceptible. In fact, similarly to poultry, overweight animals seem to be prone to the onset of pododermatitis. An experiment was carried out to test the effect of different types of bedding material (straw vs. wood shavings vs. coarse bark mulch vs. cotton) on the prevalence of pododermatitis in 30 fattening Separator rabbits. Once a week, the four experimental runs were assessed for contamination (macroscopic contamination by faeces/urine/litter) as well as for lesion scoring of the footpad. In addition, the water and ammonia binding behaviour of bedding was tested. The most favourable values as to dry matter, NH3 (p < .05) and also scoring of the litter were generally observed when cotton was used as bedding material. This is probably due to the high water binding capacity, the faster water release and the structural effects of the material itself (softness). By contrast, the structure of the coarse bark mulch was not suitable for binding liquids (especially urine) and led to increased contamination of the barrels. These results suggest that rabbits with an increased tendency to sore soles should be kept on bedding materials with desirable physical properties (e.g. cotton litter).

Storage Temperature Impacts on Anthocyanins Degradation, Color Changes and Haze Development in Juice of "Merlot" and "Ruby" Grapes (Vitis vinifera).

This study evaluated the degradation kinetics of selected anthocyanins and the change in polymeric color, browning index, and haze development of grape juices from "Merlot" and "Ruby" grape cultivars stored at 5, 25, and 35°C for up to 360 days. Five major anthocyanins namely malvidin-3-O-glucoside (M3G), delphinidin-3-O-glucoside (D3G), petunidin-3-O-glucoside (Pt3G), peonidin-3-O-glucoside (Pn3G), and cyanidin-3-O-glucoside (C3G) were identified. Juice from "Merlot" had significantly higher (p < 0.05) content of all individual anthocyanins as compared to "Ruby." During the long-term storage, total, and individual anthocyanins from both cultivars degraded following first-order reaction kinetics at the rate strongly dependent on temperature. At the end of the storage, noticeably higher loss of anthocyanins (95-99.9%) was observed at 25 and 35°C as compared to storage at 5°C [50-60% ("Merlot"); 74-81% ("Ruby")]. Considerably lower rate of decay was observed at 5°C (k = 0.01-0.04) as compared to 25 (k = 0.04-0.14) and 35°C (k = 0.05-0.14) storage temperatures. The most temperature sensitive anthocyanin compounds were C3G (Ea = 66.5 kJ/mol) and D3G (Ea = 63.3 kJ/mol). At higher storage temperatures, significant (p < 0.05) and strong negative correlations were observed between anthocyanin concentrations and the levels of haze, polymeric and brown color development during storage. Storing grape juice, at lower temperature conditions could reduce the continuous loss of biologically active anthocyanins as well as the development of haze and brown color.

Compositional, ultrastructural and nanotechnological characterization of the SMA strain of Saccharomyces pastorianus: Towards a more complete fermentation yeast cell analysis.

Nano scanning Auger microscopy (NanoSAM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) have been used in materials science research for some time, but NanoSAM, in particular, has only recently been applied to biological specimens. Here, the first concurrent utilization of NanoSAM, TOF-SIMS and microscopic techniques for the examination of a standard beverage fermentation strain of Saccharomyces pastorianus uncovered the presence of intracellular networks of CO2 in fermenting cells. Respiring cells produced few bubbles and instead had large internal vacuolar structures. Transmission electron microscopy analysis also showed osmiophilic layers at the cell exterior of fermenting cells that became more prevalent with fermentation duration, while osmiophilic layers were largely absent in respiring cells. TOF-SIMS analysis showed a compositional difference at the exterior and interior of SMA cells and between fermenting and respiring cells. Fermenting cells also appeared to have different 3-OH oxylipin profiles compared to respiring cells based upon examination with immunofluorescence microscopy. The results of this work and further study using these materials science techniques will substantially enhance our understanding of the chemical, ultrastructural and metabolic changes that occur in fermentation yeasts.

Hamstrings and psoas lengths during normal and crouch gait: implications for muscle-tendon surgery.

Crouch gait, one of the most common movement abnormalities among children with cerebral palsy, is characterized by persistent flexion of the knee during the stance phase. Short hamstrings are thought to be the cause of crouch gait; thus, crouch gait is often treated by surgical lengthening of the hamstrings. In this study, a graphics-based model of the lower extremity was used in conjunction with three-dimensional kinematic data obtained from gait analysis to estimate the lengths of the hamstrings and psoas muscles during normal and crouch gaits. Only three of 14 subjects with crouch gait (four of 20 limbs with knee flexion of 20 degrees or more throughout stance) had hamstrings that were shorter than normal by more than 1 SD during walking. Most (80%) of the subjects with crouch gait had hamstrings of normal length or longer, despite persistent knee flexion during stance. This occurred because the excessive knee flexion was typically accompanied by excessive hip flexion throughout the gait cycle. All of the subjects with crouch gait had a psoas that was shorter than normal by more than 1 SD during walking. These results emphasize the need to consider the geometry and kinematics of multiple joints before performing surgical procedures aimed at correcting crouch gait.

Modeling of orthokinetic flocculation of Saccharomyces cerevisiae.

This study examined the flocculation behavior of two Saccharomyces cerevisiae strains expressing either Flo1 (LCC1209) genotype or NewFlo (LCC125) phenotype in a laminar flow field by measurement of the fundamental flocculation parameter, the orthokinetic capture coefficient. This orthokinetic capture coefficient was measured as a function of shear rate (5.95-223 s(-1)) and temperature (5-45 degrees C). The capture coefficients of these suspensions were directly proportional to the inverse of shear rate, and exhibited an increase as the temperature was increased to 45 degrees C. The capture coefficient of pronase-treated cells was also measured over similar shear rate and temperature range. A theory, which predicts capture coefficient values due to zymolectin interactions, was simplified from that developed by Long et al. [Biophys. J. 76: (1999) 1112]. This new modified theory uses estimates of: (1) cell wall densities of zymolectins and carbohydrate ligands; (2) cell wall collision contact area; and (3) the forward rate coefficient of binding to predict theoretical capture coefficients. A second model that involves both zymolectin interactions and DLVO forces was used to describe the phenomenon of yeast flocculation at intermediate shear ranges, to explain yeast flocculation in laminar flow.

Multivariate changes in coordination of postural control following spaceflight.

Postural and gait instabilities in astronauts returning from spaceflight are thought to result from in-flight adaptation of central nervous system processing of sensory inputs from the vestibular, proprioceptive, and visual systems. We hypothesized that reorganization of posture control relying on these multiple inputs would result in not only greater amounts of sway, but also changes in interjoint coordination. We tested this hypothesis by examining the multivariate characteristics of postural sway and comparing the postural control gain used for maintenance of upright stance during the altered sensory conditions of the Sensory Organization Test (EquiTest, Neurocom Intl.). We used the covariance of hip and ankle kinematics as a measure of joint motion and interjoint coordination, and then utilized discriminant analysis to further examine these characteristics in a group of 10 first-time astronauts. In five of the six conditions, the most important difference was an increased relative utilization of the hip strategy, which would not be evident using conventional balance measures such as peak or root-mean-square sway. This finding was supported by indications of increased hip torque gains relative to lower extremity and neck motion in at least four conditions (p < 0.05). In contrast, ankle torque gains to these motions did not appear to change. These results suggest that after spaceflight, astronauts exhibit significant multivariate changes in multijoint coordination, of which increased sway is only one component. These changes are consistent with reweighting of vestibular inputs and changes in control strategy in a multivariable control system.

Contributions of altered sensation and feedback responses to changes in coordination of postural control due to aging.

We used multivariate kinematics and joint torque measurements during dynamic posturography to determine the relative contributions of changes in overall control gain, relative weighting of sensors, and noise-like effects on posture control in the elderly. Our results show that sway coordination and amplitude both change with age, but that changes in overall feedback gains do not explain these differences. We propose that increased sway of elderly subjects in platform sway-referenced conditions is due to sensory noise or decreased ability to detect small motions of the platform, while increased sway during visual sway-referencing is due to re-weighting of the various sensors.

Labyrinthectomy versus vestibular neurectomy: long-term physiologic and clinical outcomes.

OBJECTIVE: To determine whether long-term vestibular compensation (VC) and clinical outcomes differ after transmastoid labyrinthectomy (TML) versus retrolabyrinthine vestibular neurectomy (RVNS). STUDY DESIGN: Prospective, observational study. SETTING: Tertiary care, university hospital. PATIENTS: Twenty-one subjects were studied several years after they were relieved of spontaneous episodic vertigo caused by peripheral vestibular disease by TML or RVNS. INTERVENTIONS: All patients had undergone TML or RVNS more than 2.5 years before the study and returned for physiologic and functional studies of vestibular compensation. MAIN OUTCOME MEASURES: Completeness of physiologic VC, as assessed by electronystagmography and rotational chair testing; performance on computerized dynamic posturography; pure-tone and speech audiometry; self-assessment of balance and hearing function with validated survey instruments. RESULTS: There were no differences in the incidence of physiologic VC or functional recovery between the TML and RVNS subjects. Although a majority of subjects in each group had evidence of incomplete vestibular compensation, there was no difference in self-assessment of balance or hearing handicap at long-term follow-up. CONCLUSIONS: Long-term clinical balance and hearing outcomes are equivalent when TML and RVNS successfully cure spontaneous, episodic vertigo. There is a high incidence of incomplete VC after both procedures, though this does not usually produce a significant balance handicap.

EquiTest modification with shank and hip angle measurements: differences with age among normal subjects.

The Sensory Organization Test protocol of the EquiTest system (NeuroCom International, Clackamas Oregon) tests utilization of visual, vestibular, and proprioceptive sensors by manipulating the accuracy of visual and/or somatosensory inputs during quiet stance. In the standard Sensory Organization Test, both manipulation of sensory input (sway-referencing) and assessment of postural sway are based on ground reaction forces measured from a forceplate. The purpose of our investigation was to examine the use of kinematic measurements to provide a more direct feedback signal for sway-referencing and for assessment of sway. We compared three methods of sway-referencing: the standard EquiTest method based on ground reaction torque, kinematic feedback based on servo-controlling to shank motion, and a more complex kinematic feedback based on servo-controlling to follow position of the center of mass (COM) as calculated from a two-link biomechanical model. Fifty-one normal subjects (ages 20-79) performed the randomized protocol. When using either shank or COM angle for sway-referencing feedback as compared to the standard EquiTest protocol, the Equilibrium Quotient and Strategy Score assessments were decreased for all age groups in the platform sway-referenced conditions (SOT 4, 5, 6). For all groups of subjects, there were significant differences in one or more of the kinematic sway measures of shank, hip, or COM angle when using either of the alternative sway-referencing parameters as compared to the standard EquiTest protocol. The increased sensitivities arising from use of kinematics had the effect of amplifying differences with age. For sway-referencing, the direct kinematic feedback may enhance ability to reduce proprioceptive information by servo-controlling more closely to actual ankle motion. For assessment, kinematics measurements can potentially increase sensitivity for detection of balance disorders, because it may be possible to discriminate between body sway and acceleration and to determine the phase relationship between ankle and hip motion.

Insertional mutagenesis of Listeria monocytogenes 568 reveals genes that contribute to enhanced thermotolerance.

The objectives of this study were to identify molecular mechanisms of thermotolerance using transposon mutants of Listeria monocytogenes 568, serotype 1/2a, and to compare their thermal death kinetics at 52, 56 and 60 degrees C. Sixteen Tn917 transposon mutants with enhanced heat resistance were acquired from a library of 4300 mutants following a multi-step screening process. Genetic regions with Tn917 insertions encompassed a broad range of functionalities including; transport, metabolism, replication and repair, general stress, and structural properties. Modeling of the heat inactivation data using the Geeraerd et al. and Whiting (Fermi) models showed that the mutants' enhanced thermal resistance was manifested mostly through a significant (p

Time-temperature studies of gellan polysaccharide-high sugar mixtures: effect of sodium ions on structure formation.

Changes to the viscoelastic storage and loss moduli were measured as a function of temperature and oscillatory frequency for 0.5% (w/w) gellan:80% (w/w) cosolute dispersions with added Na(+) (40 to 160 mM). Isothermal frequency (0.15 to 15 Hz) and thermal scans (at 0.15 Hz) were performed over a decreasing then increasing temperature range of 85 to 5 degrees C and 5 to 85 degrees C, respectively. Moduli were found to increase in magnitude with decreasing temperature and increasing levels of Na(+) during cooling, then remained relatively thermally irreversible upon heating. Isothermal frequency (ITF) data were described using the time-temperature superposition (TTS) principle and the modified Cole-Cole (MCC) analysis. Both TTS and the MCC analyses successfully described the behavior of samples containing 40 mM added Na(+) during cooling and heating, and at the 100-mM Na(+) level during cooling. TTS superposed ITF data over the entire temperature range, whereas successful superposition was restricted to lower temperatures in the MCC analysis, where the viscoelastic response was dominated by the long-range relaxation of gellan chains between junction zones. Failure of both analyses was attributed to the formation of junction zones composed of polymer-polymer associations. It is proposed that the addition of Na(+) promotes the formation of a weakly cross-linked gellan network.

Density of lectin-like receptors in the FLO1 phenotype of Saccharomyces cerevisiae.

An improved lectin assay was recently developed to determine the density of lectin sites present on the surface of a haploid strain 1209 of Saccharomyces cerevisiae exhibiting the Flo1 phenotype. The assay is based on the use of FITC-avidin which contains a mannose side chain which functions as a binding site to the cell wall lectins termed zymolectins. An examination of the effect of culture time on the zymolectin density indicated no significant effect (P > 0.05) confirming other findings that Flo1 acts in a constitutive manner. Zymolectin densities for this strain averaged 5.4 x 10(6) sites cell-1.

Effect of altered sensory conditions on multivariate descriptors of human postural sway.

Multivariate descriptors of sway were used to test whether altered sensory conditions result not only in changes in amount of sway but also in postural coordination. Eigenvalues and directions of eigenvectors of the covariance of shnk and hip angles were used as a set of multivariate descriptors. These quantities were measured in 14 healthy adult subjects performing the Sensory Organization test, which disrupts visual and somatosensory information used for spatial orientation. Multivariate analysis of variance and discriminant analysis showed that resulting sway changes were at least bivariate in character, with visual and somatosensory conditions producing distinct changes in postural coordination. The most significant changes were found when somatosensory information was disrupted by sway-referencing of the support surface (P = 3.2 x 10(-10)). The resulting covariance measurements showed that subjects not only swayed more but also used increased hip motion analogous to the hip strategy. Disruption of vision, by either closing the eyes or sway-referencing the visual surround, also resulted in altered sway (P = 1.7 x 10(-10)), with proportionately more motion of the center of mass than with platform sway-referencing. As shown by discriminant analysis, an optimal univariate measure could explain at most 90% of the behavior due to altered sensory conditions. The remaining 10%, while smaller, are highly significant changes in posture control that depend on sensory conditions. The results imply that normal postural coordination of the trunk and legs requires both somatosensory and visual information and that each sensory modality makes a unique contribution to posture control. Descending postural commands are multivariate in nature, and the motion at each joint is affected uniquely by input from multiple sensors.