Mining the effect of the neonicotinoids imidacloprid and clothianidin on the chemical homeostasis and energy equilibrium of primary mouse neural stem/progenitor cells using metabolomics.

The projection of plant protection products' (PPPs) toxicity to non-target organisms at early stages of their development is challenging and demanding. Recent developments in bioanalytics, however, have facilitated the study of fluctuations in the metabolism of biological systems in response to treatments with bioactives and the discovery of corresponding toxicity biomarkers. Neonicotinoids are improved insecticides that target nicotinic acetylocholine receptors (nAChR) in insects which are similar to mammals. Nonetheless, they have sparked controversy due to effects on non-target organisms. Within this context, mammalian cell cultures represent ideal systems for the development of robust models for the dissection of PPPs' toxicity. Thus, we have investigated the toxicity of imidacloprid, clothianidin, and their mixture on primary mouse (Mus musculus) neural stem/progenitor (NSPCs) and mouse neuroblastoma-derived Neuro-2a (N2a) cells, and the undergoing metabolic changes applying metabolomics. Results revealed that NSPCs, which in vitro resemble those that reside in the postnatal and adult central nervous system, are five to seven-fold more sensitive than N2a to the applied insecticides. The energy equilibrium of NSPCs was substantially altered, as it is indicated by fluctuations of metabolites involved in energy production (e.g. glucose, lactate), Krebs cycle intermediates, and fatty acids, which are important components of cell membranes. Such evidence plausibly suggests a switch of cells' energy-producing mechanism to the direct metabolism of glucose to lactate in response to insecticides. The developed pipeline could be further exploited in the discovery of unintended effects of PPPs at early steps of development and for regulatory purposes.

Moderate superoxide production is an early promoter of mitochondrial biogenesis in differentiating N2a neuroblastoma cells.

Reactive oxygen species (ROS) have been widely considered as harmful for cell development and as promoters of cell aging by increasing oxidative stress. However, ROS have an important role in cell signaling and they have been demonstrated to be beneficial by triggering hormetic signals, which could protect the organism from later insults. In the present study, N2a murine neuroblastoma cells were used as a paradigm of cell-specific (neural) differentiation partly mediated by ROS. Differentiation was triggered by the established treatments of serum starvation, forskolin or dibutyryl cyclic AMP. A marked differentiation, expressed as the development of neurites, was detected by fixation and staining with coomassie brilliant blue after 48 h treatment. This was accompanied by an increase in mitochondrial mass detected by mitotracker green staining, an increased expression of the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1-alpha (PGC-1α) and succinate dehydrogenase activity as detected by MTT. In line with these results, an increase in free radicals, specifically superoxide anion, was detected in differentiating cells by flow cytometry. Superoxide scavenging by MnTBAP and MAPK inhibition by PD98059 partially reversed differentiation and mitochondrial biogenesis. In this way, we demonstrated that mitochondrial biogenesis and differentiation are mediated by superoxide and MAPK cues. Our data suggest that differentiation and mitochondrial biogenesis in N2a cells are part of a hormetic response which is triggered by a modest increase of superoxide anion concentration within the mitochondria.

Novel aspects of neuronal differentiation in vitro and monitoring with advanced biosensor tools.

Neuronal differentiation is a very complex and sophisticated cellular process that encompasses the development of mature neurons and their specialization. In this review we will focus on the novel and less well-known aspects of neuronal differentiation. Cell lines, to which some pro-differentiation drugs are added, have been widely used because of their convenience in terms of cost-efficiency, ease of use and reproducibility. After a brief overview of these systems, this review focuses on the new pharmacological aspects of differentiation related to mitochondrial changes and cellular redox homeostasis. A number of different parameters are commonly evaluated to assess neuronal differentiation. These include neurite length, differential gene expression, mitochondrial mass, free radical levels, enzyme induction and others. However, the classical techniques used to detect neuronal differentiation (such as immunochemistry, flow cytometry and gene expression analysis) are time-consuming or dependent on the subjective view of the researcher. On the other hand, emerging novel, miniaturized biosensor technologies have the potential to revolutionize the study of neuronal differentiation, by detecting neuron-derived electrical signals and differentiation markers, such as shape or attachment in a non-invasive and high throughput fashion. These state-of-the-art technologies are being extensively reviewed. Emphasis is given to progress, made in the field of integrated systems (including impedance sensing, microfluidics and associated nanotechnologies), neuronal differentiation in 3-D cultures and the identification of novel agents controlling neuronal cell fate.

Studies on neuronal differentiation and signalling processes with a novel impedimetric biosensor.

The differentiation of neural cells is an important process during the development of the central nervous system. Studies on the mechanisms of the differentiation process is of special importance, e.g. in the field of regenerative medicine. In this contribution the cellular differentiation of gel matrix embedded neuronal cells was studied. The three-dimensional organization of neuronal cells represents a new cellular model system closer to the physiology than conventional two-dimensional cell cultures. Neuro2a (N2a) neuroblastoma cells were immobilized in different gel matrices and the grade of differentiation was compared. Furthermore, the impact of the cell number and selected differentiation factors were analyzed. Experimental results revealed that gel matrices based on collagen-laminin mixtures in contact with serum free medium enable neural differentiation. Therefore, collagen-laminin gels appear as a suitable three-dimensional model for drug screening in developmental neurobiology. Following optimization of the immobilization process, a novel impedimetric sensor and electrical impedance spectroscopy technique was applied to on-line monitor the differentiation process by means of changes in the dielectric and conductive properties. Experimental results showed an increase in the impedance magnitude that can be mainly attributed to differentiating cells accompanied by an increase in the specific resistivity of the bare gel mixture.

Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: a comparative study.

In order to investigate the effect of cell immobilization in calcium alginate gels on cell physiology, we immobilized Vero or N2a neuroblastoma cells in gels shaped either as spherical beads or as thin membrane layers. Throughout a culture period of 4 weeks cell viability, RNA and cytoplasmic calcium concentration and glutathione accumulation were assayed by fluorescence microscopy after provision of an appropriate dye. Non-elaborate culture conditions were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Vero cell proliferation was observed only in spherical beads, while N2a cell proliferation was observed in both configurations until the third week of culture. Increased [Ca2+]cyt could be associated with cell proliferation only when cells were immobilized in spherical beads, while a considerable decrease in the biosynthesis of reduced glutathione and RNA was observed in cells immobilized in thin membrane layers. The observed effects of the shape of the immobilization matrix may be due to differences in external mass transfer resistance. Therefore, depending on cell type, cell proliferation could have been promoted by either increased (Vero) or decreased (N2a) nutrient and oxygen flow to immobilized cells. The results of the present study could contribute to an improvement of immobilized cell sensor storability.

Development of a novel, multi-analyte biosensor system for assaying cell division: identification of cell proliferation/death precursor events.

A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.

Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments.

The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell-virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.

In vitro germination, protocorm formation and plantlet development of mature versus immature seeds from several Ophrys species (Orchidaceae).

We investigated the effect of genotype, seed maturity and culture medium on the in vitro germination and development of protocorms and plantlets from seeds of 13 different Ophrys species (O. apifera, O. attica, O. cornuta, O. delfinensis, O. ferrum-equinum, O. lutea, O. mammosa, O. speculum, O. spruneri, O. umbilicata, O. argolica, O. irricolor and O. tenthredinifera) collected in Greece, some of which are endemic to this country. Mature seeds (10 months after collection) and immature seeds (2 months after anthesis) were cultured in a coconut milk-enriched or a pineapple-enriched medium (CEM or PEM, respectively). The highest percentage of callogenesis (96%) was observed in immature seeds of O. delphinensis in the CEM, while the highest percentage of protocorm formation (52%) was observed in mature seeds of O. spuneri in the CEM. Protocorm formation was significantly lower in immature seeds than in mature seeds in both culture media. Eventually almost all of the transferred protocorms developed to plantlets, which later formed minitubers. PEM appeared to be the most suitable for the development of minitubers from plantlets. All of the factors investigated--as well as their interactions--significantly affected callogenesis and protocorm formation. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several under-utilized Ophrys species.

Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots.

An efficient transformation system for the medicinal plant Pueraria phaseoloides was established by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants or via callus 10 days after inoculation with the bacteria. The highest frequency of explant transformation by A. rhizogenes ATCC15834 was about 70% after infection for 30 days. Hairy roots could grow rapidly on solid, growth regulator-free Murashige and Skoog medium and had characteristics of transformed roots such as fast growth and high lateral branching. Paper electrophoresis revealed that bacteria-free hairy roots of P. phaseoloides could synthesize agropine and mannopine. The polymerase chain reaction amplification of rooting locus genes showed that left-hand transferred DNA of the root inducing plasmid of A. rhizogenes was inserted into the genome of transformed P. phaseoloides hairy roots. The content of puerarin in hairy roots reached a level of 1.190 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants.

Developing a scale-up system for the micropropagation of cucumber (Cucumis sativus L.): the effect of growth retardants, liquid culture and vessel size.

We investigated the effect of the growth retardant flurprimidol, the phase of the culture medium (solid versus liquid) and the size of the liquid culture vessel (250-ml flask versus 2.5-l airlift bioreactor) on the micropropagation of cucumber (Cucumis sativus L.) from nodal explants. Flurprimidol at concentrations of 0.1-2 mg l(-1) caused considerable growth retardation but increased, albeit slightly, the number of branches and buds and stimulated (solid medium) or reduced (liquid medium) the accumulation of NO(3)-and PO(4)(3-). Flurprimidol had varying effects on the accumulation of soluble sugars and antioxidant compounds. Bioreactor-derived plants showed an increased fresh weight and size but a decreased content of macronutrients, solid sugars, ascorbic acid and free antioxidant phenolics. The majority (95%) of the plants were successfully acclimatized after being graft on squash. The perspective for an efficient, commercial-level use of bioreactors in combination with growth retardants of this commercially important vegetable species is discussed.

The application of the bioelectric recognition assay for the detection of human and plant viruses: definition of operational parameters.

The bioelectric recognition assay (BERA) is a novel biosensory method based on a unique combination of a group of cells, their immobilization in a matrix that preserves their physiological functions and the expression of the cell interaction with viruses as a change in electrical properties. A BERA sensor consists of an electroconductive, tube-like probe containing components of immobilized cells in a gel matrix. Cells are selected to specifically interact with the virus under detection. In this way, when a positive sample is added to the probe, a characteristic, 'signature-like' change in electrical potential occurs upon contact between the virus and the gel matrix. In the present study, we demonstrate that BERA can be used for the detection of viruses in humans (hepatitis C virus) and plants (tobacco and cucumber viruses) in a remarkably specific, rapid (1-2 min), reproducible and cost-efficient fashion. The sensitivity of the virus detection with BERA (0.1 ng) is equal or even better than with advanced immunological, cytological and molecular techniques, such as the reverse transcription polymerase chain reaction. Moreover, a good storability of the sensors can be achieved without affecting their performance. The potential use of portable BERA biosensors in medicine, for mass screening purposes, as well as for the detection of biological warfare agents without prior knowledge of a specific receptor-molecule interaction is discussed.

Bioelectric recognition assay (BERA).

A novel biosensory method has been developed for the determination of various chemical and biological molecules by assessing their electrophysiological interactions with a group of cells and cell components immobilized in a gel matrix that preserves their 'physiological' functions. The method was applied for the detection of: (i) hepatitis C virus in human blood samples; (ii) plant viruses; and (iii) a herbicide (glyphosate) in aqueous solutions. It was able to rapidly (assay time 3-5 min) and specifically detect the molecules in question at a concentration lower than 100 pg/ml, among other compounds f similar structure. The potential use of BERA biosensors for a rapid and cost-efficient molecule determination without prior knowledge of a specific receptor-molecule interaction is discussed.