Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

Structure and Absolute Configuration of Kongiidiazadione,a New Phytotoxic 3‐Substituted‐5‐Diazenylcyclopentendione Produced by Diaporthe Kongii

A new 3‐substituted‐5‐diazenylcyclopentendione named kongiidiazadione was isolated from culture filtrates of Diaporthe kongii, associated with stem cankers on sunflower in Australia. Kongiidiazadione was characterized by spectroscopic (essentially nuclear magnetic resonance [NMR] and high‐resolution, electrospray ionization, mass spectrometry [HRESIMS]) methods as (?)‐5‐diazenyl‐3‐hydroxymethyl‐cyclopent‐3‐en‐1,2‐dione. The stereochemistry of the diazenyl group was determined by IR spectroscopy, while the (R) absolute configuration at C(5) was assigned by computational analysis of its electronic circular dichroism (ECD) spectrum. When assayed on leaf disks of different plant species at 5 mM, the kongiidiazadione had a differential impact, causing clear necrosis, in particular to Helianthus annuus. Moreover, kongiidiazadione proved to have a weak antibacterial activity against gram‐positive Bacillus amyloliquefaciens. Chirality 27:557–562, 2015. © 2015 Wiley Periodicals, Inc.     

Milt characteristics and plasma levels of gonadal steroids in greenback flounder Rhombosolea tapirina following treatment with exogenous hormones

Fish were treated with exogenous hormones, and milt and blood samples were collected for up to 96 h post‐treatment. Blood plasma samples were assayed for the gonadal steroids testosterone (T), 11‐ketotestosterone (11KT) and 17,20ß‐dihydroxy‐4‐pregnen‐3‐one (17.20ßP). Milt volume, spermatocrit and sperm motility were measured from milt samples. Non‐spermiated fish showed increased plasma T and 11KT in response to human chorionic gonadotropin (hCG) but not luteinising hormone releasing hormone analogue (LHRHa). Fish did not become spermiated in response to treatment with hCG, LHRHa, 11KT, 17‐hydroxyprogesterone (17P) or 17,20ßP. Spermiated fish showed an increase in milt volume in response to hCG and LHRHa but not exogenous steroids. Sperm motility declined to zero over 120 s and was not affected by hormone treatment or sampling time. Increased milt volume was accompanied by increased plasma T and 11KT, but not 17.20ßP levels. In a separate experiment, LHRHa delivered by injection or pellet was equally effective at increasing milt volume but had no effect on plasma steroid levels. Spermatocrit declined with stripping but was not affected by hormone treatment, nor was sperm motility. Co‐treatment of fish with 17P plus LHRHa had no additive effect on plasma steroid concentrations or milt volume. The results suggest that as in other teleosts, gonadotropin mediates milt production in greenback flounder.     

Crystal structure of a novel two domain GH78 family α‐rhamnosidase from Klebsiella oxytoca with rhamnose bound

The crystal structure of the GH78 family α‐rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non‐proline cis‐peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays α‐rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function. Proteins 2015; 83:1742–1749. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Homeoviscous and functional adaptations of mitochondrial membranes to growth temperature in soybean seedlings

The present study investigated whether the cold‐sensitive character of soybean is reflected at the level of mitochondrial membranes. When exposed to an increase of temperature (from 25 to 35 °C), mitochondrial membranes were characterized by a higher phosphatidylcholine : phosphatidylethanolamine ratio and a lower content in 18 : 3 fatty acid. After a reduction of temperature (from 25 to 18 °C) the opposite changes were found. Lipid lateral diffusion and local microviscosity appeared to be comparable in mitochondria from plantlets grown at 25 or 35 °C when assayed at the respective growth temperatures. Some functional aspects (cytochrome c oxidase activity or membrane conductance) tended to this behaviour whereas others (respiration rate or maximum membrane potential) did not. On the other hand, membranes from plants grown at 18 °C were more rigid. Moreover, as illustrated by cytochrome c oxidase activity or respiration rate, functional measurements suggested that these membranes were less active at this temperature. Thus the dynamic characteristics and functional properties measured in mitochondrial membranes were in favour of an adaptive trend at 35 °C, but not at 18 °C despite changes in lipid composition, in accordance with the cold‐sensitive character of the plant.     

Biphenyl Derivatives from the Aerial Parts of Oenanthe javanica and Their COX‐2 Inhibitory Activities

Four new biphenyl derivatives ( 1 – 4 ), along with six known biphenyl derivatives ( 5 – 10 ) were isolated and elucidated by their detailed analyses of spectroscopic data and references from the aerial parts of Oenanthe javanica for the first time. Compounds ( 1 – 10 ) were assayed for their activities about the inhibition of COX‐2 enzyme in vitro for the first time. Compounds 1 , 2 , 4 , and 6 showed inhibitory activities against COX‐2 with IC₅₀ values ranging from 22.18±0.29 to 108.54±0.42 μm .     

Induction of trap formation in nematode‐trapping fungi by bacteria‐released ammonia

A total of 11 bacterial strains were assayed for bacteria‐induced trap formation in the nematode‐trapping fungus Arthrobotrys oligospora YMF1·01883 with two‐compartmented Petri dish. These strains were identified on the basis of their 16S rRNA gene sequences. Volatile organic compounds (VOCs) of eight isolates were extracted using solid‐phase micro‐extraction (SPME) and their structures were identified based on gas chromatography‐mass spectrometry (GC‐MS). At the same time, all isolates were used for quantitative measurement of ammonia by the indophenol blue method. The effects of pure commercial compounds on inducement of trap formation in A. oligospora were tested. Taken together, results demonstrated that the predominant bacterial volatile compound inducing trap formation was ammonia. Meanwhile, ammonia also played a role in other nematode‐trapping fungi, including Arthrobotrys guizhouensis YMF1·00014, producing adhesive nets; Dactylellina phymatopaga YMF1·01474, producing adhesive knobs; Dactylellina cionopaga YMF1·01472, producing adhesive columns and Drechslerella brochopaga YMF1·01829, producing constricting rings.     

Age at reproductive maturity and effect of age and time of day on sex attraction in the potato psyllid Bactericera cockerelli

Abstract The potato psyllid, Bactericera (= Paratrioza) cockerelli (?ulc) (Hemiptera: Triozidae), is a major pest of potato. Studies were conducted to determine the age at which both males and females reach reproductive maturity and the effect of age and time of day on sex attraction. Adult B. cockerelli reach reproductive maturity within 48 h post‐eclosion, with females being mature on the day of eclosion and males at 1 day post‐eclosion. Oviposition generally began 2 days after mating but was delayed when females mated within 2 days post‐eclosion. In laboratory olfactometer assays, the age of females used as odor sources and the age of males assayed to these odors did not affect sex attraction, with both younger (1–4‐day‐old) and older (8–10‐day‐old) males being attracted to females, regardless of female age (1–4‐day‐old or 8–10‐day‐old). Males assayed to live females at different times during the photophase (between 8:00 and 20:00 hours) were attracted to females between 11:00 and 17:00 hours, showing a temporal periodicity in sex attraction with B. cockerelli at least during the photophase.     

Survival and persistence of nonspore-forming biothreat agents in water

Aims: To determine whether nonspore‐forming biothreat agents can survive and persist in potable water that does not contain a disinfectant. Methods and Results: Autoclaved, de‐chlorinated Atlanta municipal water was inoculated with eight isolates of bacterial biothreat agents (10⁶ CFU ml^?1). The inoculated water samples were incubated at 5, 8 (Francisella tularensis only) or 25°C and assayed for viability by culture and by the presence of metabolic activity as measured by esterase activity (ScanRDI, AES Chemunex). Viability as determined by culture varied from 1 to 30 days, depending upon the organism and the temperature of the water. All organisms were determined viable as measured by esterase activity for the entire 30 days, regardless of the incubation temperature. Conclusion: Francisella tularensis was culturable for at least 21 days if held at 8°C. The remaining nonspore‐forming bacterial biothreat agents were found to be metabolically active for at least 30 days in water held at 5 or 25°C. Significance and Impact of the Study: The data can assist public health officials to determine the safety of drinking water after contamination with a biothreat agent.     

High Temperatures and Net CO2 Uptake, Growth, and Stem Damage for the Hemiepiphytic Cactus Hylocereus undatus1

Hylocereus undatus, which is native to tropical forests experiencing moderate temperatures, would not be expected to tolerate the extremely high temperatures that can be tolerated by cacti native to deserts. Nevertheless, total daily net CO₂ uptake by this hemiepiphytic cactus, which is widely cultivated for its fruits, was optimal at day/night air temperatures of 30/20°C, temperatures that are higher than those optimal for daily net CO₂ uptake by cacti native to arid and semiarid areas. Exposure to 35/25°C for 30 weeks led to lower net CO₂ uptake than at 10 weeks; exposure to 40/30°C led to considerable necrosis visible on the stems at 6 weeks and nearly complete browning of the stems by 19 weeks. Dry mass gain over 31 weeks was greatest for plants at 30/20°C, with root growth being especially noteworthy and root dry mass gain representing an increasing percentage of plant dry mass gain as day/night air temperatures were increased. Viability of chlorenchyma cells, assayed by the uptake of the vital stain neutral red into the central vacuoles, was decreased 50 percent by a one‐hour treatment at 55°C compared with an average of 64°C for 18 species of cacti native to deserts. The lower high‐temperature tolerance for H. undatus reflected its low high‐temperature acclimation of only 1.4°C as growth temperatures were raised by 10°C compared with an average acclimation of 5.3°C for the other 18 species of cacti. Thus, this tropical hemiepiphytic cactus is not adapted to day/night air temperatures above ca 40/30°C, although its net CO₂ uptake is optimal at the relatively high day/night air temperatures of 30/20°C.     

Capillary electrophoresis analysis of isoniazid using luminol-periodate potassium chemiluminescence system

A rapid and simple capillary electrophoresis method coupled with chemiluminescent (CL) detection was proposed for analysis of isoniazid (ISO) based on the enhancement effect of ISO to CL emission of luminol‐periodate potassium reaction. Under the optimal conditions, ISO can be assayed in the range of 7.0 × 10^?7 to 3.0 × 10^?5 g mL^?1 (R² = 0.9990) with a limit of detection of 3.0 × 10^?7 g mL^?1 (signal‐to‐noise ratio of 3). The whole analysis process can be completed within 2.5 min with a theoretical plate number of 6258. The relative standard deviations of the signal intensity and the migration time were 3.1 and 1.4% for a standard sample at 1.0 × 10^?5 g mL^?1 (n = 5), respectively. The presented novel strategy was successfully applied to the determination of ISO in commercial pharmaceutical preparations and spiked human serum samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Studies of Asymmetric Styrene Cyclopropanation with a Rhodium(II) Metallopeptide Catalyst Developed with a High‐Throughput Screen

Dirhodium metallopeptides have been developed as selective catalysts for asymmetric cyclopropanation reactions. A selective ligand sequence has been identified by screening on‐bead metallopeptide libraries in a 96‐well plate format. Efficient ligand synthesis and screening allows a 200‐member library to be created and assayed in less than three weeks. These metallopeptides catalyze efficient cyclopropanation of aryldiazoacetates, providing asymmetric access to cyclopropane products in high diastereoselectivity. Chirality 25:493–497, 2013. © 2013 Wiley Periodicals, Inc.     

Antibacterial activity of naringin derivatives against pathogenic strains

Aims: To study the antimicrobial activity of naringin (NAR), a flavonoid extracted from citrus industry waste, and NAR derivatives [naringenin (NGE), prunin and alkyl prunin esters] against pathogenic bacteria such as L. monocytogenes, E. coli O157:H7 and S. aureus. The relationship between the structure of the chemical compounds and their antagonistic effect was also analysed. Methods and Results: The agar dilution technique and direct contact assaying were applied. NGE, prunin and NAR showed no antimicrobial activity at a concentration of 0·25 mmol l^?1. Similarly, fatty acids with a chain length between C2 and C18 showed no antimicrobial activity at the same concentration. However, prunin‐6″‐O‐acyl esters presented high antibacterial activity, mainly against Gram‐positive strains. This activity increased with increasing chain length (up to 10–12 carbon atoms). Alkyl prunin esters with 10–12 carbon atoms diminished viability of L. monocytogenes by about 3 log orders and S. aureus by 6 log orders after 2 h of contact at 37°C and at a concentration of 0·25 mmol l^?1. The compounds examined were not effective against any of the Gram‐negative strains assayed, even at the highest concentration. Conclusions: Addition of sugars to the aglycone did not enhance its antimicrobial activity. Attachment of a saturated aliphatic chain with 10–12 carbon atoms to the A ring of the flavonoid (or to sugars attached to this ring), seems to be the most promising modification. In conclusion, alkyl prunin esters with a chain length of C10–C12 have promising features as antimicrobial agents because of their high antilisterial and antistaphylococcal activity. Significance and Impact of the Study: This study shows that it is possible to obtain NAR derivatives with important antimicrobial activity, especially against Gram‐positive pathogenic bacteria. It also provides guidelines on the structural modifications in similar molecules to enhance the antimicrobial activity.     

Increased Serum Cholesteryl Ester Transfer Protein in Obese Children

Objective: To determine whether serum cholesteryl ester transfer protein (CETP), which is one of the physiologically active gene products secreted from adipose tissue, is increased and associated with atherogenic lipoprotein profile in obese children. Research Methods and Procedures: Subjects were 42 consecutive outpatient Japanese obese children, 29 boys and 13 girls, ranging in age from 5 to 14 years, and 25 age‐matched non‐obese children, 13 boys and 12 girls, as the control group for measuring CETP mass. Blood was drawn after an overnight fast and, at the same time, and anthropometric measurements including height, body weight, waist girth, hip girth, and triceps and subscapular skinfold thicknesses were taken. Paired samples were obtained from 15 obese children who underwent psychoeducational therapy. Serum CETP mass was assayed by an enzyme‐linked immunosorbent assay. Results: The serum levels of triglyceride, total cholesterol (TC), low‐density lipoprotein cholesterol, TC/high‐density lipoprotein cholesterol (HDLC), apolipoproteins (apo) B, apo B/apo A₁, and insulin in obese children were significantly higher than the respective reference values. Serum CETP level was ～2‐fold higher (98.7 ± 3.6 vs. 50.9 ± 4.0 nM, means ± SEM, p < 0.001) in the obese children than in the controls. In 15 obese children, whose percentage of overweight declined during therapy, CETP levels decreased significantly. CETP level was correlated with HDLC, TC/HDLC, and insulin, and with percentage of overweight when the data of the obese and non‐obese children were combined. Discussion: CETP is increased and associated with the atherogenic lipoprotein profile in obese children.     

Further survey of the occurrence of fusarium toxins in wheat grown in southwest Germany

A total of 53, 54, 57, 52 and 60 wheat samples for feed use were collected randomly after the 1989, 1990, 1991, 1992 and 1993 crops, respectively, from farms in an area of southwest Germany. Deoxynivalenol (DON), 3‐ and 15‐acetyldeoxynivalenol (3‐, 15‐ADON), nivalenol (NIV), HT‐2 toxin (HT‐2), T‐2 toxin (T‐2), diacetoxyscirpenol (DAS), and fusarenon‐X (FUS‐X) were determined by gas chromatography, combined with mass selective detection (GC‐MS), zearalenone (ZEA), α‐ and β‐zearalenol (α‐ß‐ZOL) were determined by HPLC. DON was the major toxin, with incidences at 77 to 93% and mean contents at 167 to 735 μg/kg. In contrast, incidences of ZEA, 3‐ADON, NIV, HT‐2, and T‐2 were at 13 to 37%, 10 to 44%, 15 to 67%, 0 to 11%, and 0 to 12%, respectively, with mean contents in positive samples between 2 and 73 μg/kg, except for 948 μg/kg 3‐ADON in samples from 1993. 15‐ADON and FUS‐X were assayed in samples from 1991, 1992 and 1993. 15‐ADON was found in 0 to 11% of samples at mean levels ≤ 17 μ/kg, DAS, α‐ and β‐ZOL, and FUS‐X were not detected in any sample. Over the years, incidences and levels of toxins remained constant, decreased or increased, with most differences between years being slight and insignificant. The risk for livestock due to DON, HT‐2 and ZEA was estimated based on maximum tolerated levels recommended for these toxins in some countries.     

PRELIMINARY EVALUATION OF THE BIOREMEDIATION POTENTIAL OF PORPHYRA: PHOTOSYNTHETIC PRODUCTION BY BLADES AND CONCHOCELIS

Given their rapid growth and nutrient assimilation rates, Porphyra spp. are good candidates for bioremediation. The production potential of two northeast U.S. Porphyra species currently in culture (P. purpurea and P. umbilicalis) was evaluated by measuring rates of photosynthesis (as O₂ evolution) of samples grown at 20° C. Gametophytes of P. umbilicalis photosynthesized at rates that were 80% higher than those of P. purpurea over 5–20° C at both sub‐saturating and saturating irradiances (37 and 289 μmol photons m^?2 s^?1). Porphyra umbilicalis was both more efficient at low irradiances (higher alpha) and had a higher P_max than did P. purpurea (23.0 vs. 15.6 μmol O₂ g^?1 DW min^?1), suggesting that P. umbilicalis is a better choice for mass culture where self‐shading may be severe. The photosynthesis‐irradiance relationship for the Conchocelis stage of P. purpurea was also examined. Tufts of filaments, grown at 10, 15, and 20° C, were assayed at growth temperatures at irradiances ranging from 0–315 μmol photons m^?2 s^?1. Tufts were slightly more productive at 15° than at 10° C, but only ca. 4–6% as productive as gametophytes. Maximum rates of net photosynthesis were reduced by 66–74% in tufts grown at 20° C (only about 2% of gametophytes). The Conchocelis stage, however, need not limit mariculture operations; once Conchocelis cultures are established, they can be maintained over the long‐term as ready sources of spores for net seeding.     

Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos

Abstract. The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV‐1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field‐collected Culicoides were fed on sheep blood containing 7.5 log₁₀TCID₅₀/mL of BTV‐1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK‐21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30°C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge ≥ 3 log₁₀ TCID₅₀) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4–10 post‐infection (dpi), at 15–30°C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0–13.7% and 0–46.8%. In both species the highest IR was recorded at 25°C and the highest TP at 30°C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25°C to 8 dpi at 15°C. In C. imicola it ranged from 4 dpi at 30°C to 10 dpi at 23.5°C; no individuals with TP were detected at 15°C. There was no evidence of virus replication in flies held at 10°C. When, at various points of incubation, individual flies were transferred from 10°C to 23.5°C and then assayed 4–10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaço was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV‐1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa.     

Effects of different nitrogen compounds and temperatures on the germination ofAvena sterilis spp.macrocarpa Mo.

The effect of several nitrogen compounds on the germination of the first seed of each spikelet ofAvena sterilis spp.macrocarpa Mo. was studied on Petri dishes placed in germination chamber. The compounds assayed were ammonium sulphate, ammonium nitrate, urea and calcium nitrate, generally used as fertilizer for cereal crops, as well as a control supplied with water only The concentration of the assayed compounds were 100, 1000, 2500 and 5000 ppm either at a constant temperature of 15 ° C or at alternated temperatures 5°C and 15 °C, for 16 and 8 h, respectively