Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Multi-trait assessment of early-in-life female, male and genomic measures for use in genetic selection to improve female reproductive performance of Brahman cattle

Early-in-life female and male measures with potential to be practical genetic indicators were chosen from earlier analyses and examined together with genomic measures for multi-trait use to improve female reproduction of Brahman cattle. Combinations of measures were evaluated on the genetic gains expected from selection of sires and dams for each of age at puberty (AGECL, i.e. first observation of a corpus luteum), lactation anoestrous interval in 3-year-old cows (LAI), and lifetime annual weaning rate (LAWR, i.e. the weaning rate of cows based on the number of annual matings they experienced over six possible matings). Selection was on an index of comparable records for each combination. Selection intensities were less than theoretically possible but assumed a concerted selection effort was able to be made across the Brahman breed. The results suggested that substantial genetic gains could be possible but need to be confirmed in other data. The estimated increase in LAWR in 10 years, for combinations without or with genomic measures, ranged from 8 to 12 calves weaned per 100 cows from selection of sires, and from 12 to 15 calves weaned per 100 cows from selection of sires and dams. Corresponding reductions in LAI were 60-103 days or 94-136 days, and those for AGECL were 95-125 or 141-176 days, respectively. Coat score (a measure of the sleekness or wooliness of the coat) and hip height in females, and preputial eversion and liveweight in males, were measures that may warrant wider recording for Brahman female reproduction genetic evaluation. Pregnancy-test outcomes from Matings 1 and 2 also should be recorded. Percentage normal sperm may be important to record for reducing LAI and scrotal size and serum insulin-like growth factor-I concentration in heifers at 18 months for reducing AGECL. Use of a genomic estimated breeding value (EBV) in combination with other measures added to genetic gains, especially at genomic EBV accuracies of 40%. Accuracies of genomic EBVs needed to approach 60% for the genomic EBV to be the most important contributor to gains in the combinations of measures studied.

Paramagnetic probes in membrane biophysics

The use of paramagnetic probes in membrane research is reviewed. Electron paramagnetic resonance studies on model and biological membranes doped with covalent and non-covalent spin-labels have been discussed with special emphasis on the methodology and the type of information obtainable on several important phenomena like membrane fluidity, lipid flip-flop, lateral diffusion of lipids, lipid phase separation, lipid bilayer phase transitions, lipid-protein interactions and membrane permeability. Nuclear magnetic resonance spectroscopy has also been effectively used to study the conformations of cation mediators across membranes and to analyse in detail the transmembrane ionic motions at the mechanistic level.

Employee green behavior: A theoretical framework, multilevel review, and future research agenda

We propose a conceptual model based on person–environment interaction, job performance, and motivational theories to structure a multilevel review of the employee green behavior (EGB) literature and agenda for future research. We differentiate between required EGB prescribed by the organization and voluntary EGB performed at the employees’ discretion. The review investigates institutional-, organizational-, leader-, team-, and employee-level antecedents and outcomes of EGB and factors that mediate and moderate these relationships. We offer suggestions to facilitate the development of the field, and call for future research to adopt a multilevel perspective and to investigate the outcomes of EGB.

Field evaluation of transgenic pineapple (Ananas comosus (L.) Merr.) cv. ‘Smooth Cayenne’ for resistance to blackheart under subtropical conditions

A comparative analysis of transgenic pineapple lines transformed with a polyphenol oxidase (PPO) gene (ppo) and the untransformed cultivar ‘Smooth Cayenne’ was made from plants grown in a series of field trials under cool subtropical conditions in southeast Queensland. In the four field trials where blackheart was recorded, all of the control lines expressed blackheart on each occasion and exhibited the greatest incidence (50%) and severity (34%) of symptoms. Irrespective of the gene transfer method or the gene construct used, 38% of the lines produced were regarded as blackheart resistant, having no blackheart symptoms in two or more trials. Five blackheart resistant transgenic lines consistently performed as well as or better than control plants in terms of fruit characteristics and quality.

The validity of the GENEActiv wrist-worn accelerometer for measuring adult sedentary time in free living

OBJECTIVES Based on self-reported measures, sedentary time has been associated with chronic disease and mortality. This study examined the validity of the wrist-worn GENEactiv accelerometer for measuring sedentary time (i.e. sitting and lying) by posture classification, during waking hours in free living adults. DESIGN Fifty-seven participants (age=18-55 years 52% male) were recruited using convenience sampling from a large metropolitan Australian university. METHODS Participants wore a GENEActiv accelerometer on their non-dominant wrist and an activPAL device attached to their right thigh for 24-h (00:00 to 23:59:59). Pearson's Correlation Coefficient was used to examine the convergent validity of the GENEActiv and the activPAL for estimating total sedentary time during waking hours. Agreement was illustrated using Bland and Altman plots, and intra-individual agreement for posture was assessed with the Kappa statistic. RESULTS Estimates of average total sedentary time over 24-h were 623 (SD 103) min/day from the GENEActiv, and 626 (SD 123) min/day from the activPAL, with an Intraclass Correlation Coefficient of 0.80 (95% confidence intervals 0.68-0.88). Bland and Altman plots showed slight underestimation of mean total sedentary time for GENEActiv relative to activPAL (mean difference: -3.44min/day), with moderate limits of agreement (-144 to 137min/day). Mean Kappa for posture was 0.53 (SD 0.12), indicating moderate agreement for this sample at the individual level. CONCLUSIONS The estimation of sedentary time by posture classification of the wrist-worn GENEActiv accelerometer was comparable to the activPAL. The GENEActiv may provide an alternative, easy to wear device based measure for descriptive estimates of sedentary time in population samples

The systematics of Endoraecium in Australia

Endoraecium (Raveneliaceae, Pucciniales) is a genus of rust that infects several species of Acacia (Fabaceae) in Australia, south-east Asia and Hawaii. Thirteen species of Endoraecium have been described, including seven species that are endemic to Australia, one species to south-east Asia and five to Hawaii. This study investigated the systematics of Endoraecium from 50 specimens in Australia and south-east Asia with a combined morphological and molecular approach. Phylogenetic analyses were conducted on combined datasets of the SSU, ITS and LSU regions of rDNA. The recovered phylogeny (i) supported a recent division of Endoraecium digitatum into five separate species based on morphology and host specificity and (ii) found lineages that did not correspond with known species.

Networks of stored grain diseases and pests: Strategies for sampling and mitigation

Pathogens and pests of stored grains move through complex dynamic networks linking fields, farms, and bulk storage facilities. Human transport and other forms of dispersal link the components of this network. A network model for pathogen and pest movement through stored grain systems is a first step toward new sampling and mitigation strategies that utilize information about the network structure. An understanding of network structure can be applied to identifying the key network components for pathogen or pest movement through the system. For example, it may be useful to identify a network node, such as a local grain storage facility, through which grain from a large number of fields will be accumulated and move through the network. This node may be particularly important for sampling and mitigation. In some cases more detailed information about network structure can identify key nodes that link two large sections of the network, such that management at the key nodes will greatly reduce the risk of spread between the two sections. In addition to the spread of particular species of pathogens and pests, we also evaluate the spread of problematic subpopulations, such as subpopulations with pesticide resistance. We present an analysis of stored grain pathogen and pest networks for Australia and the United States.

Gut-inhabiting yeasts from the gut of Australian passalid beetles

Passalidae (Polyphaga, Coleoptera) is a family of beetles with approximately 960 species that are distributed worldwide. Preliminary studies that characterized ascomycete and basidiomycete yeasts in the gut of these wood-eating beetles from the USA, Guatemala, and Thailand, demonstrated associations between certain yeast taxa and passalids. We extended the study to include yeasts and beetles from tropical forests near Cairns and Brisbane, Queensland, Australia. We isolated more than 1000 yeast strains from about 150 beetles belonging to 10 species. LSU and ITS rRNA markers were used to identify a subset of 250 yeast strains, which revealed that the gut of Australian passalids contained undescribed ascomycetes in the Debaryomyces, Dipodascus, Kazachstania, Ogataea, Scheffersomyces, Sugiyamaella, Spathaspora, Torulaspora, and Zygowilliopsis clades, as well as basidiomycetes in the genera Cryptococcus and Trichosporon. A close relative of Candida subhashii (Spathaspora clade) and the xylose-fermenting yeast Scheffersomyces stipitis were the most common species isolated in Queensland. These results agree with those of previous studies that showed a common association of xylose-fermenting yeasts in the gut of lignicolous insects. Species and higher taxa of yeasts, however, vary between Queensland passalids and those previously collected in distant regions.

The 310 Helical Conformation of the Amino Terminal Decapeptide of Suzukacillin. 270 MHz 'H NMR Evidence for Eight Intramolecular Hydrogen Bonds

The amino terminal suzukacillin decapeptide fragment, Boc-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aitbh-eO Me, two pentapeptides Boc-AibPrc-Val-AibVal-OMe and Boc-Ala-AibAla-AibAibOMe, and the tripeptide Boc-Ala-AibAibOMe have been studied by 270-MHz 'H NMR spectroscopy. By use of solvent dependence of chemical shifts in a CDC13-(CD3),S0 system and temperature dependence of amide NH chemical shifts in (CD3),S0, the intramolecularly hydrogen bonded NH groups in these peptides have been identified. The tripeptide possesses one hydrogen bond, both pentapeptides show evidence for three intramolecular hydrogen bonds, and the decapeptide has eight NH groups participating in hydrogen bonding. An Ala( 1)-Aib(2) @ turn is proposed for the tripeptide. Both pentapeptides favor 310 helical conformations composed of three consecutive B turns. The decapeptide adopts a 310 helical conformation with some flexibility at the Va1(5)-Ala(6) segment. The proposed conformations are consistent with the known stereochemical preferences of Aib residues.

Populational reasoning

This section focuses on systems of reasoning that imagine youth as a unified whole, one that can be researched, talked about, planned for, and managed. Even research that focuses on individuals or specific contexts depends on and reproduces ideas of youth as an identifiable population. This section interrogates the rules and scaffolding of discourses that construct the social spaces in which we problematize and study youth in society. This introduction will set the agenda by addressing four elements of this process: the first addresses the rise of some of the crucial elements of contemporary governance, the instrument and practices through which the notion of the population was able to take shape. The second examines the rise of the personage of “the child,” and how new forms of governance not only utilized this new identity for the purposes of ongoing social management, but also organized its differentiation into a growing array of new social and administrative categories. The third specifically addresses “youth,” examining its various predecessors as targets for moral concern, as well as some of the recent cultural triggers for its formation. Finally, there is an assessment of the contemporary governance of populations of youth, based as it is around its twin existence as a governmental object, a target for an almost endless array of social, educational, legal, and psychological concerns and interventions, but also as an identity, a set of practices of the self.

The structures of ethyl 1,2,2-tricyano-3,3-dimethylcyclopropane-1-carboxylate and ethyl 2',3,3'-tricyanocyclohexanespirocyclopropane-2'-carboxylate

The crystal and molecular structures of C ,,H,IN302 (I) and C14HIsN302 (II) have been determined by direct methods using three-dimensional X-ray diffractometer data. Crystals of (I) are orthorhombic, space group Pna21, with a = 14.662(6), b = 10.492(5), c = 7.375 (3)A, Z = 4, V = 1134.5 A 3, D O = 1.25 (by flotation), D e = 1.269 Mgm -a, g(MoKa) = 0.085 mm -1. Crystals of (II) are monoclinic, space group P21/a, with a = 7.886 (5), b = 22.011 (8), c = 8.100 (3) A, fl = 103.12 (5) °, Z = 4, V = 1369.2 A 3, D O = 1.23 (by flotation), D e = 1.255 Mg m -3, g(Mo Kct) = 0.080 mm -1. Least-squares full-matrix refinement based on 782 (I) and 1400 independent reflections (II) converged at R = 0.040 (I) and 0.042 (II). The effect of electron-withdrawing substituents on the geometry of the cyclopropane ring is discussed.

Plan Ekaterinoslavskoĭ gubernii Krivorozhskogo okruga Mikhaĭlovskogo raĭona na uchastok zemli otgranichennoĭ v pori︠a︡dke ustroĭstva kolonizatsionnogo fonda

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Probiotic Bacillus amyloliquefaciens Strain H57 Improves the Performance of Pregnant and Lactating Ewes Fed a Diet Based on Palm Kernel Meal

Probiotic supplements are single or mixed strain cultures of live microorganisms that benefit the host by improving the properties of the indigenous microflora (Seo et al 2010). In a pilot study at the University of Queensland, Norton et al (2008) found that Bacillus amyloliquefaciens Strain H57 (H57), primarily investigated as an inoculum to make high-quality hay, improved feed intake and nitrogen utilisation over several weeks in pregnant ewes. The purpose of the following study was to further challenge the potential of H57 -to show it survives the steam-pelleting process, and that it improves the performance of ewes fed pellets based on an agro-industrial by-product with a reputation for poor palatability, palm kernel meal (PKM), (McNeill 2013). Thirty-two first-parity White Dorper ewes (day 37 of pregnancy, mean liveweight = 47.3 kg, mean age = 15 months) were inducted into individual pens in the animal house at the University of Queensland, Gatton. They were adjusted onto PKM-based pellets (g/kg drymatter (DM): PKM, 408; sorghum, 430; chick pea hulls, 103; minerals and vitamins; Crude protein, 128; ME: 11.1MJ/kg DM) until day 89 of pregnancy and thereafter fed a predominately pelleted diet incorporating with or without H57 spores (10 9 colony forming units (cfu)/kg pellet, as fed), plus 100g/ewe/day oaten chaff, until day 7 of lactation. From day 7 to 20 of lactation the pelleted component of the diet was steadily reduced to be replaced by a 50:50 mix of lucerne: oaten chaff, fed ad libitum, plus 100g/ewe/day of ground sorghum grain with or without H57 (10 9 cfu/ewe/day). The period of adjustment in pregnancy (day 37-89) extended beyond expectations due to some evidence of mild ruminal acidosis after some initially high intakes that were followed by low intakes. During that time the diet was modified, in an attempt to improve palatability, by the addition of oaten chaff and the removal of an acidifying agent (NH4Cl) that was added initially to reduce the risk of urinary calculi. Eight ewes were removed due to inappetence, leaving 24 ewes to start the trial at day 90 of pregnancy. From day 90 of pregnancy until day 63 of lactation, liveweights of the ewes and their lambs were determined weekly and at parturition. Feed intakes of the ewes were determined weekly. Once lambing began, 1 ewe was removed as it gave birth to twin lambs (whereas the rest gave birth to a single lamb), 4 due to the loss of their lambs (2 to dystocia), and 1 due to copper toxicity. The PKM pellets were suspected to be the cause of the copper toxicity and so were removed in early lactation. Hence, the final statistical analysis using STATISTICA 8 (Repeated measures ANOVA for feed intake, One-way ANOVA for liveweight change and birth weight) was completed on 23 ewes for the pregnancy period (n = 11 fed H57; n = 12 control), and 18 ewes or lambs for the lactation period (n = 8 fed H57; n = 10 control). From day 90 of pregnancy until parturition the H57 supplemented ewes ate 17 more DM (g/day: 1041 vs 889, sed = 42.4, P = 0.04) and gained more liveweight (g/day: 193 vs 24.0, sed = 25.4, P = 0.0002), but produced lambs with a similar birthweight (kg: 4.18 vs 3.99, sed = 0.19, P = 0.54). Over the 63 days of lactation the H57 ewes ate similar amounts of DM but grew slower than the control ewes (g/day: 1.5 vs 97.0, sed = 21.7, P = 0.012). The lambs of the H57 ewes grew faster than those of the control ewes for the first 21 days of lactation (g/day: 356 vs 265, sed = 16.5, P = 0.006). These data support the findings of Norton et al (2008) and Kritas et al (2006) that certain Bacillus spp. supplements can improve the performance of pregnant and lactating ewes. In the current study we particularly highlighted the capacity of H57 to stimulate immature ewes to continue to grow maternal tissue through pregnancy, possibly through an enhanced appetite, which appeared then to stimulate a greater capacity to partition nutrients to their lambs through milk, at least for the first few weeks of lactation, a critical time for optimising lamb survival. To conclude, H57 can survive the steam pelleting process to improve feed intake and maternal liveweight gain in late pregnancy, and performance in early lactation, of first-parity ewes fed a diet based on PKM.