BACKGROUND: With the commercialization of genetically modified organisms (GMOs) in the market, laboratories have undergone a significantly increased workload. A universal analytical approach was designed to achieve cost-efficient and high-throughput GMOs screening with high specificity and accuracy. The approach provides accurate qualification of authorized and unauthorized GMOs.
OBJECTIVE: This article describes the assessment of this analytical approach developed to detect the majority of commercialized GMOs over the world.
METHODS: Seven elements and three events were detected by qPCR in a single laboratory to detect 59 commercialized GMOs. Certificated reference materials and food/feed samples from the Chinese market were also evaluated for the specificity, conformity, and robustness of this approach and were challenged in the interlaboratory study.
RESULTS: The results showed that elements and events selected can best detect GMO presence with good specificity and sensitivity. The results showed a concordance between 97.5 and 99.56% and the variance between 0.65 and 12.88%, which is in line with the minimum requirement of analytical methods of GMO testing.
CONCLUSIONS: The approach validated here can be used to manipulate GMO presence in food and feed and showed the capacity to manipulate GMO trace in the trade and domestic agriculture market in China.
CONCLUSIONS: A universal analytical approach used to track GMO presence was evaluated for its specificity, sensitivity, and robustness.

The ability of Saccharomyces cerevisiae to reconstitute viability after strong peroxide-induced oxidative stress during liquid holding (LH) in non-nutrient medium has been compared with that of Ustilago maydis. It was found that like U. maydis, S. cerevisiae was capable of reconstituting viability through multiplication of the survivors. However, differences were observed in the pattern of their response: (i) the reconstitution of viability was slower in S. cerevisiae; (ii) before the viability was progressively increasing the treated samples of this fungus reproducibly passed through a phase of additional decrease of the surviving fraction and (iii) the final yields of viable cells attained in S. cerevisiae were below those achieved by U. maydis. The reason for the relative superiority of U. maydis is twofold: (1) early initiated and faster degradation and leakage of the intracellular biomolecules and (2) greater ability of U. maydis cells to recycle damaged and released intracellular compounds. Conceptually similar studies extended to another oxidative-stress-inducing condition, namely desiccation, indicated that the marked differences between these fungi in their patterns of the post-stress regrowth, cellular leakage and macromolecule decomposition are reproduced during LH of desiccated cells. The concordance of the findings obtained upon these two approaches was also corroborated by an analysis of the post-desiccation LH response of U. maydis mutants (adr1, did4, kel1 and tbp1) that were previously identified as defective in post-peroxide LH restitution of viability. We discuss the findings in terms of their possible relevance to the mechanisms of the ecological and evolutionary adaptation of free-living microorganisms to fluctuating and severely inhospitable environments.

Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.

BACKGROUND: There are still no data on the attitudes and acceptance of genetic modification (GM) food in European developing countries, such as the Western Balkan countries. The aim of the study was to assess the knowledge, attitudes, and acceptance of GM but also to shed light on the multifactorial process leading to acceptance of genetic modifications among Western Balkan students of life sciences.
METHODS: In this cross-sectional study, the final study population sample was composed of 1251 university students. The instrument for data collection was a questionnaire consisting of 49 items composed of 5 sections taken from the literature. Attitudes toward GM were analyzed by using Q-mode factor analysis and principal component analysis was run for the assessment of perception of personal health risks. The acceptability of GM was analyzed in binary probit models assessing the acceptability of GM products in different areas of application with Q models, sociodemographic variables, perception of personal health risks factors, respondents\' knowledge about biotechnology, gender, and age as explanatory variables.
RESULTS: This study demonstrated that students of life sciences supported the implementation of GM in industry and medicine production but not in food production. Their acceptance was most influenced by 3 out of 5 attitude models that were identified (p < 0.0001). Regarding the perception of personal health risks, the factor \"credence risks\" was seen as a negative predictor of acceptance of GM in industry and food production (p < 0.05). The main knowledge predictor of rejecting GM was misconception, whereas real knowledge had no impact (p < 0.0001).
CONCLUSIONS: The AGREE study provided the first rough picture of the knowledge, attitudes, and acceptance of GM in this area. Given the target population, it could be expected that the general population\'s acceptance of all observed elements, especially knowledge, would be lower.

The transgalactosilating ability of beta-galactosidase preparations obtained from Kluyveromyces fragilis, Penicillium canescens, and Escherichia coli was investigated. In samples of hydrolysates of 20% lactose solutions without or with a 2% addition of glucose, the galactose or fructose obtained with the use of the investigated preparations and the content of saccharides was determined with the HPLC method. Under experimental conditions, the highest quantity (56.0-64.1%) of galactooligosaccharides was synthesised by beta-galactosidase obtained from E. coli, while the lowest number (11.25-25.2%) was obtained by beta-galactosidase obtained from P. canescens. Beta-galactosidase from E. coli also synthesised considerable amounts of lactulose.

Natural and modified nucleoside-5\'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

Direct detection of biological transformations of single living cells in vivo has been performed by the advanced combination of local topographic imaging by Atomic Force Microscopy (AFM) and label-free sub-surface chemical characterization using new μ-Tip-Enhanced Raman Spectroscopy (μ-TERS). The enhancing mechanism for μ-TERS tips with micrometre range radius differs significantly to that of the conventional tapered structures terminated by a sharp apex and conditioned by the effects of propagating instead of localizing surface plasmon resonance phenomena. Sub-wavelength light confinement in the form of a nonradiative evanescent wave near the tip surface with penetration depth in the sub-micrometre range opens the way for monitoring of subsurface processes near or within the cell wall, inaccessible by other methods. The efficiency of the approach has been demonstrated by the analysis of the cell envelope of genetically modified (by glucose dehydrogenase (GDH) gene bearing Kluyveromyces lactis toxin signal sequence) yeast cells enriched by GDH protein. The presence of trans-membrane fragments in GDH together with the tendency to form active dimers and tetramers causes the accumulation of the proteins within the periplasmic space. These results demonstrate that the advanced combination of AFM imaging and subsurface chemical characterization by the novel μ-TERS technique provides a new analytical tool for the investigation of single living cells in vivo.

Oncolytic virotherapy is a promising novel therapy for glioblastoma that needs to be optimized before introduced to clinic. The targeting of conditionally replicating adenoviruses (CRAds) can be improved by relying on the tumor-tropic properties of neural stem cells (NSCs). Here, we report the characterization of an FDA approved NSC, HB1.F3-CD, as a cell carrier for CRAd-S-pk7, a glioma-tropic oncolytic adenovirus. We show that NSCs replicate and release infectious CRAd-S-pk7 progeny capable of lysing glioma cell lines. Moreover, ex-vivo-loaded NSCs, injected intracranially in nude mice bearing human glioma xenografts (i) retained their tumor tropism, (ii) continued to replicate CRAd-S-pk7 for more than a week after reaching the tumor site and (iii) successfully handed off CRAd-S-pk7 to glioma cells in vivo. Delivery via carrier cells reduced non-specific adenovirus distribution in the mouse brain. Moreover, we assessed biodistribution of loaded NSCs after intracranial injection in animal models semi-permissive to adenovirus replication, the Syrian hamster and cotton rat. NSCs did not migrate to distant organs and high levels of CRAd-S-pk7 DNA were observed only in the injected hemisphere. In conclusion, this optimized carrier system, with high efficiency of adenovirus delivery and minimal systemic toxicity, poses considerable advantages for anti-glioma oncolytic virotherapy.

Ecological risk analysis (ERA) is a structured evaluation of threats to species, natural communities, and ecosystem processes from pollutants and toxicants and more complicated living stressors such as invasive species, genetically modified organisms, and biological control agents. Such analyses are typically conducted by a narrowly-focused group of scientific experts using technical information. We evaluate whether the inclusion of more diverse experts and practitioners in ERA improved the ecological knowledge base about South African biodiversity and the potential impacts of genetically modified (GM) crops. We conducted two participatory ERA workshops in South Africa, analyzing potential impacts of GM maize on biodiversity. The first workshop involved only four biological scientists, who were joined by 18 diverse scientists and practitioners in the second, and we compared the ERA process and results between the two using descriptive statistics and semi-structured interview responses. The addition of diverse experts and practitioners led to a more comprehensive understanding of biological composition of the agro-ecosystem and a more ecologically relevant set of hazards, but impeded hazard prioritization and the generation of precise risk assessment values. Results suggest that diverse participation can improve the scoping or problem formulation of the ERA, by generating an ecologically robust set of information on which to base the subsequent, more technical risk assessment. The participatory ERA process also increased the transparency of the ERA by exposing the logic and rationale for decisions made at each step.

A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMM Φ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC (50) 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC (50) 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC (50) of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group PERI to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity IN VITRO against the clinically relevant intracellular amastigote with an IC (50) of 1.1 µM. With IC (50) values mostly in the range of 1-3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a crucial role in the host resistance against intracellular pathogens, excluding activation of microbicidal mechanisms in macrophages. The mode of action apparently depended on the substitution pattern, associated with the electrophilicity of the naphthoquinone or the efficiency of redox cycling. Conspicuously, members oxygenated in the quinone ring proved to be leishmanicidal when coincubated with glutathione, while the majority of the remaining compounds lost activity.