The hen's egg test for micronucleus induction (HET-MN): Novel analyses with a series of well-characterized substances support the further evaluation of the test system

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint “formation of micronuclei” with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics—including those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay.We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC).The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87‰ ± 0.87; average ± s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05 mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4‰ ± 6.8 (average ± s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.     

Parasitoids of Monochamus galloprovincialis (Coleoptera,Cerambycidae), vector of the pine wood nematode,with identification key for the Palaearctic region

The parasitoid complex associated with Monochamus galloprovincialis (Olivier), vector of the pine wood nematode, is discussed. Four species of the family Braconidae and one Ichneumonidae were found associated with Monochamus galloprovincialis in Portugal, namely Atanycolus denigrator (Linnaeus), Atanycolus ivanowi (Kokujev), Cyanopterus flavator (Fabricius), Doryctes striatellus (Nees) (Braconidae), and Xorides depressus (Holmgren) (Ichneumonidae). Atanycolus ivanowi, Atanycolus denigrator, Doryctes striatellus and Xorides depressus are new species for Portugal fauna, and Monochamus galloprovincialis is recorded as a new host of Xorides depressus. A key for determination of the ichneumonoid parasitoids of the pine sawyer is provided for the Palaearctic fauna.     

Application of high‐throughput mini‐bioreactor system for systematic scale‐down modeling,process characterization,and control strategy development

High‐throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high‐throughput mini‐bioreactor system viz. the Advanced Microscale Bioreactor (ambr15^TM), to perform process characterization in less than a month and develop an input control strategy. As a pre‐requisite to process characterization, a scale‐down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO₂ profiles as well as several other process and product quality parameters. The scale‐down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15^TM system is capable of replacing the bench scale bioreactor system for routine process development and process characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1623–1632, 2015     

Application of a two-stage temperature control strategy for enhanced glutathione production in the batch fermentation by Candida utilis

In batch culture for glutathione production with Candida utilis, a higher temperature (30 °C) was required to hasten cell growth while a lower temperature (26 °C) was needed to increase the production of glutathione. A two-stage temperature control strategy was used to enhance both the yield and the productivity of glutathione. As a result, glutathione production was increased by 5% and 23% of that at 26 °C and 30 °C, respectively, and the intracellular glutathione content reached 2.5% (w/w).     

Nematophagous fungus <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis> (Thom) Samson is also a biological agent for control of greenhouse insects and mite pests

Pathogenicity of nematophagous fungus Paecilomyces lilacinus (Thom) Samson in control of the most destructive greenhouse pests such as: greenhouse whitefly, Trialeurodes vaporariorum, glasshouse red spider mite, Tetranychus urticae, the cotton aphid, Aphis gossypii and western flower thrips, Frankliniella occidentalis was examined in laboratory and pot experiments. The fungus showed the greatest efficacy in controlling winged and wingless forms of the cotton aphid. The cotton aphid’s population was almost totally eliminated. In controlling the greenhouse whitefly, P. lilacinus was most successful when applied against nymphal growth stages (L₃-L₄). Control of the western flower thrips was most efficient against prepupal and pupal stages when the fungus was applied as a water spore suspension to the soil. When the fungus was applied at temperatures below 10 °C, it was able to reduce a glasshouse red spider mite population by 60%.     

An <Emphasis Type="Italic">in situ</Emphasis> method for the investigation of vertical distributions of zooplankton in lakes: test of a two-compartment enclosure

Two-section enclosures were designed for the investigation of the effect of various physicochemical and biological factors on vertical distribution of zooplankton in situ. The framework of the enclosure was a cylindrical polyethylene column without any partitions inside, in which the isolation of animals in different sections after in situ exposure was achieved by pinching the flexible central part of the column. Enclosures were tested at the brackish stratified meromictic Lake Shira (Russia, Khakasia). The absence of fish and carnivorous zooplankton in the lake suggests that the vertical distribution of zooplankton is mainly determined by physicochemical gradients in the water column. Experiments and field observations demonstrated that all age and size groups of Arctodiaptomus salinus and Brachionus plicatilis strongly avoided surface layers during the daylight. The escape of zooplankton from the anoxic hypolimnion was less active. Statistically significant avoidance was observed only for copepodites C4–C5 and females of A. salinus. The relatively simple construction of the columns and easy handling during the experiment were the factors that favoured the use of this device to perform in situ basic tests of the effect of different factors on the vertical distribution of zooplankton.     

Auxin concentration control of the average DNA content in cells of in vitro cultures: a theoretical model and comparison to experimental data for <Emphasis Type="Italic">Allium cepa</Emphasis> and <Emphasis Type="Italic">Allium sativum</Emphasis>

A theoretical model explaining the average DNA content in cells of in vitro cultures as a function of concentration of auxin 2,4-D in medium is provided. The model assumes influence of auxin on the functioning of the main G1/S and G2/M control points of the cell cycle. Theoretical results are compared to experimental data obtained from the callus cultures of Allium sativum and Allium cepa conducted during 18 months. Adaptation of the model to explanation of the shape of the dose–response curves is suggested and successfully verified for the example of Nicotiana tabacum cultures.     

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA–protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model.