Georgia Moschopoulou

Engineering of the membrane of fibroblast cells with virus-specific antibodies: A novel biosensor tool for virus detection

A novel concept for the assay of viral antigens is described. The methodological approach is based on a membrane-engineering process involving the electroinsertion of virus-specific antibodies in the membranes of fibroblast cells. As a representative example, Vero fibroblasts were engineered with antibodies against Cucumber mosaic virus (CMV) and used for the construction of an ultra-sensitive miniature cell biosensor system. The attachment of a homologous virus triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the bioelectric recognition assay (BERA). No change in the membrane potential was observed upon cell contact with the heterologous cucumber green mottle mosaic virus (CGMMV). Fluorescence microscopy observations showed that attachment of CMV particles to membrane-engineered cells was associated with membrane hyperpolarization and increased [Ca(2+)](cyt). In an additional field-based application, we were able to detect CMV-infected tobacco plants at an essentially 100% level of accuracy.

Effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (Mentha spicata L.).

We investigated the effect of inorganic salt concentration on the in vitro organogenesis of spearmint (Mentha spicata L.) by using Murashige and Skoog culture medium of different strength (full, half- and quarter strength). The highest number of shoots and roots induced per explant (3.5 and 10, respectively), as well as the maximum average shoot length (16 cm) was observed on half strength medium. The maximum leaf number (35 per explant) and average root length (7 cm) were associated with regeneration on full medium. The highest average phenolic content was observed on shoot extracts (7.20 mg/g f.w.) and root extracts (5.93 mg/g f.w.) cultured onto quarter and half strength medium, respectively. The strength of the culture medium was inversely correlated with the antioxidant activity of plant extracts. Therefore, for spearmint organogenesis, half strength MS medium offers a compromise between optimum growth in vitro and antioxidant phenolic accumulation.

Commercially available chemicals as immunizing haptens for the development of a polyclonal antibody recognizing carbendazim and other benzimidazole-type fungicides

Carbendazim is a fungicide widely used for controlling fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops is frequently required to assure compliance with national/European regulations. A polyclonal antibody recognizing carbendazim was developed by using commercially available 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole as immunizing haptens; each of the above derivatives was directly conjugated to the carrier protein keyhole limpet hemocyanin and a mixture of the conjugates was administered to New Zealand white rabbits. Immunochemical functionality of the antisera and the corresponding isolated antibody (whole IgG fraction) was evaluated through titer and displacement curves in an in-house developed ELISA, which employed a 2-mercaptobenzimidazole - functionalized lysine-dendrimer as the immobilized hapten. As shown with ELISA-displacement curves, the above antibody could recognize carbendazim as well as other benzimidazole-type fungicides, i.e. benomyl and thiabendazole, and also intact benzimidazole, while it did not cross-react with the structurally different pesticides carbaryl and imazalil. Considering the rather simple approach which has led to its development and its highly promising immunochemical profile, the new antibody may be exploited in immunoanalytical systems for detecting benzimidazole-type pesticides e.g. in samples of environmental interest. The above antibody is being currently tested as a biorecognition element in the novel FOODSCAN cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay technology.

Pesticide Residue Screening Using a Novel Artificial Neural Network Combined with a Bioelectric Cellular Biosensor

We developed a novel artificial neural network (ANN) system able to detect and classify pesticide residues. The novel ANN is coupled, in a customized way, to a cellular biosensor operation based on the bioelectric recognition assay (BERA) and able to simultaneously assay eight samples in three minutes. The novel system was developed using the data (time series) of the electrophysiological responses of three different cultured cell lines against three different pesticide groups (carbamates, pyrethroids, and organophosphates). Using the novel system, we were able to classify correctly the presence of the investigated pesticide groups with an overall success rate of 83.6%. Considering that only 70,000–80,000 samples are annually tested in Europe with current conventional technologies (an extremely minor fraction of the actual screening needs), the system reported in the present study could contribute to a screening system milestone for the future landscape in food safety control.

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.

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.

Assessment of in vitro dopamine-neuroblastoma cell interactions with a bioelectric biosensor: perspective for a novel in vitro functional assay for dopamine agonist/antagonist activity

Current receptor-binding assays for dopamine do not measure the in vitro whole cellular response against dopamine or potential agonist/antagonist molecules. We herewith report the development of a novel functional assay concept for studying the in vitro interaction of the neurotransmitter dopamine with neural cells bearing dopamine receptors. The concept is based on the ultra-rapid measurement of changes in the electric properties of cultured N2a mouse neuroblastoma cells (corresponding to cumulative changes of the cell membrane potential). A close relationship between cumulative cell membrane potential and dopamine concentration was observed. Membrane depolarization was observed at nanomolar dopamine concentrations, while hyperpolarization was associated with micromolar ones. Treatment with the dopamine D2-receptor antagonist eticlopride resulted to a concentration-dependent membrane depolarization. Treatment with sodium chloride caused considerable weakening of the dopamine-associated hyperpolarization effect. The observed bioelectric response to dopamine was highly inversely correlated with the pattern of dopamine release-uptake balance by N2a cells, as determined with cyclic voltammetry. The bioelectric approach was also used to evaluate the dopaminergic activity of chaste tree (Vitex agnus-castus) extracts. The novel assay concept offers promising perspectives for the development of advanced companion diagnostics system for the high throughput, fast functional characterization of neurotransmitter agonists and antagonists.

Noninvasive Superoxide Monitoring of In Vitro Neuronal Differentiation Using a Cell-Based Biosensor

Membrane-engineered cells bearing superoxide dismutase (SOD) molecules on their surface offer the capability of ultrarapid and ultrasensitive detection of the superoxide anion () through the measurement of changes of their cell membrane potential. We herewith report the application of this technology for the noninvasive determination of superoxide levels during the in vitro differentiation of PC12 cells. We were able to detect changes in accumulation in the culture medium, which were closely associated with the progress of neuronal differentiation.

Increased Antioxidant Activity, Despite Reduced Rosmarinic Acid Accumulation in Methanolic Extracts, of Spearmint (Mentha spicata L.) Plantlets Regenerated In Vitro

One hundred spearmint (Mentha spicata) plantlets were regenerated from apical shoot segments of ten field- grown donor plants. Although the accumulation of rosmarinic acid and total phenolics in vitro was almost half than in vivo, regenerants demonstrated a tenfold-higher hydrogen peroxide compared to the donor plants. This finding may have been associated with the increased activity of hydroxyphenylpyruvate reductase, a key enzyme of the phenolic biosyn- thetic pathway and the increased production of yet unidentified phenolic compounds in vitro. This process of in vitro cul- ture associated with a reduction of rosmarinic acid and total phenolics and with an increase of the antioxidant capacity in- dicated the possible promotion of in vitro-specific biosynthetic pathways.