Paula Daza

This work proposes a cell–microelectrode model to be used on cell culture assays as an alternative to end-point protocols employed in cell growth and cell biometry applications. The microelectrode model proposed is based on the area overlap between the microelectrode sensor and the living cells as main parameter. This model can be applied to cell size identification, cell count, and their extension to cell growth, motility and dosimetry protocols. A procedure to fit the proposed model to microelectrode electrical performance is presented, enabling the decoding of empirical measurements and its interpretation in terms of number of cells. This fitting procedure depends on three parameters: microelectrode geometry, gap resistance between substrate attached cells and microelectrode and, mainly, on microelectrode area covered by cells. The model has been implemented employing Analog Hardware Descriptions Language (AHDL) to be incorporated also to mixed-mode simulation processes during circuit design flow. Experiments performed with commercial electrodes are described, illustrating a procedure to obtain cell number in real time in both, growth and dosimetry assays, employing an established cell line (AA8). The results are displayed in the form of growth curves (cells were growing during a week), as well as dosimetric response after treatment with MG132, a proteasome inhibitor. The results agree with the expected performances, with errors around 10–20% in the number of cells measured, therefore we think that these results are promising.

The ubiquitin-proteasome system and the autophagy-lysosome pathway are the two main mechanisms for eukaryotic intracellular protein degradation. Proteasome inhibitors are used for the treatment of some types of cancer, whereas autophagy seems to have a dual role in tumor cell survival and death. However, the relationship between both pathways has not been extensively studied in tumor cells. We have investigated both proteolytic systems in the human epithelial breast non-tumor cell line MCF10A and in the human epithelial breast tumor cell line MCF7. In basal condition, tumor cells showed a lower proteasome function but a higher autophagy activity when compared with MCF10A cells. Importantly, proteasome inhibition (PI) leads to different responses in both cell types. Tumor cells showed a dose-dependent glycogen synthase kinase-3 (GSK-3)β inhibition, a huge increase in the expression of the transcription factor CHOP and an active processing of caspase-8. By contrast, MCF10A cells fully activated GSK-3β and showed a lower expression of both CHOP and processed caspase-8. These molecular differences were reflected in a dose-dependent autophagy activation and cell death in tumor cells, while non-tumor cells exhibited the formation of inclusion bodies and a decrease in the cell death rate. Importantly, the behavior of the MCF7 cells can be reproduced in MCF10A cells when GSK-3β and the proteasome were simultaneously inhibited. Under this situation, MCF10A cells strongly activated autophagy, showing minimal inclusion bodies, increased CHOP expression and cell death rate. These findings support GSK-3β signaling as a key mechanism in regulating autophagy activation or inclusion formation in human tumor or non-tumor breast cells, respectively, which may shed new light on breast cancer control.

An alternative approach for cell-culture end-point protocols is proposed herein. This new technique is suitable for real-time remote sensing. It is based on Electrical Cell-substrate Impedance Spectroscopy (ECIS) and employs the Oscillation-Based Test (OBT) method. Simple and straightforward circuit blocks form the basis of the proposed measurement system. Oscillation parameters - frequency and amplitude - constitute the outcome, directly correlated with the culture status. A user can remotely track the evolution of cell cultures in real time over the complete experiment through a web tool continuously displaying the acquired data. Experiments carried out with commercial electrodes and a well-established cell line (AA8) are described, obtaining the cell number in real time from growth assays. The electrodes have been electrically characterized along the design flow in order to predict the system performance and the sensitivity curves. Curves for 1-week cell growth are reported. The obtained experimental results validate the proposed OBT for cell-culture characterization. Furthermore, the proposed electrode model provides a good approximation for the cell number and the time evolution of the studied cultures.

Abstract Diabetes and metabolic syndrome are associated with the typical American high glycemia diet and result in accumulation of high levels of advanced glycation end products (AGEs), particularly upon aging. AGEs form when sugars or their metabolites react with proteins. Associated with a myriad of age‐related diseases, AGEs accumulate in many tissues and are cytotoxic. To date, efforts to limit glycation pharmacologically have failed in human trials. Thus, it is crucial to identify systems that remove AGEs, but such research is scanty. Here, we determined if and how AGEs might be cleared by autophagy. Our in vivo mouse and C . elegans models, in which we altered proteolysis or glycative burden, as well as experiments in five types of cells, revealed more than six criteria indicating that p62‐dependent autophagy is a conserved pathway that plays a critical role in the removal of AGEs. Activation of autophagic removal of AGEs requires p62, and blocking this pathway results in accumulation of AGEs and compromised viability. Deficiency of p62 accelerates accumulation of AGEs in soluble and insoluble fractions. p62 itself is subject to glycative inactivation and accumulates as high mass species. Accumulation of p62 in retinal pigment epithelium is reversed by switching to a lower glycemia diet. Since diminution of glycative damage is associated with reduced risk for age‐related diseases, including age‐related macular degeneration, cardiovascular disease, diabetes, Alzheimer's, and Parkinson's, discovery of methods to limit AGEs or enhance p62‐dependent autophagy offers novel potential therapeutic targets to treat AGEs‐related pathologies.

Autophagy plays a key role in the maintenance of cellular homeostasis, and autophagy deregulation gives rise to severe disorders. Many of the signaling pathways regulating autophagy under stress conditions are still poorly understood. Using a model of proteasome stress in rat hippocampus, we have characterized the functional crosstalk between the ubiquitin proteasome system and the autophagy-lysosome pathway, identifying also age-related modifications in the crosstalk between both proteolytic systems. Under proteasome inhibition, both autophagy activation and resolution were efficiently induced in young but not in aged rats, leading to restoration of protein homeostasis only in young pyramidal neurons. Importantly, proteasome stress inhibited glycogen synthase kinase-3β in young but activated in aged rats. This age-related difference could be because of a dysfunction in the signaling pathway of the insulin growth factor-1 under stress situations. Present data highlight the potential role of glycogen synthase kinase-3β in the coordination of both proteolytic systems under stress situation, representing a key molecular target to sort out this deleterious effect.

DNA end-joining, a process related to illegitimate recombination and capable of rejoining unrelated pairs of DNA ends in the absence of sequence homology, is considered the major pathway of double-strand break (DSB) repair in mammalian cells. Whole cell and nuclear extracts from three human and one mouse cell line were investigated for their capacities to promote nonhomologous DNA end-joining and their relative activities of DNA-PK, a mammalian DNA end-binding protein complex implicated in DSB-repair. The levels of DNA end-joining and the spectra of junctions of the human systems were identical with the ones of a previously described cell-free joining system derived from Xenopus laevis eggs. Due to the presence of potent 3'-5'-exonuclease activities the mouse system displayed decreased levels of DNA end-joining and larger fractions of junctions containing deletions but otherwise the basic mechanisms of junction formation appeared to be identical with the Xenopus system. DNA-PK activity was found to be equally low in the Xenopus and the mouse system but 4- to 6-fold increased in the human systems. Our results suggest that the mechanisms of DNA end-joining may be modulated by the level of exonuclease activities and/or DNA end-protecting factors but are otherwise highly conserved in vertebrate cells.

A field study was carried out in the south of the Iberian Peninsula in an industrial area in the neighbourhood of Huelva city, SW Spain, and in a natural area (Doñana National Park) for comparison, to estimate the genetic risk induced by environmental pollution in wild mice. Genotoxic effects in a sentinel organism, the Algerian mice (Mus spretus) free living in the industrial area were compared with animals of the same species living in the natural protected area. The single cell gel electrophoresis, or Comet assay, was performed as a genotoxicity test in peripheral blood of mice. Our results clearly show that mice free living in the contaminated area bear a high burden of genetic damage as compared with control individuals. The results suggest that the assessing of genotoxicity levels by the Comet assay in wild mice can be used as a valuable test in pollution monitoring and environmental conservation.

Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.

The fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques have been used to determine, respectively, the frequencies of sister chromatid exchanges (SCEs) and stable chromosome aberrations (translocations) induced by different concentrations of BrdU in the Chinese hamster ovary cell mutant EM9 and its parental line AA8. The results indicate that BrdU induced a high frequency of SCEs and translocations in EM9 as compared with AA8, and that the translocation/dicentric ratio was also higher in the mutant cell line than in the parental cell line in both untreated and BrdU-treated cultures. These observations may indicate a possible relationship between the molecular mechanisms involved in the formation of SCEs and translocations.

Electrical pulse stimulation has been used to enhance the differentiation or proliferation of neuronal progenitor cells in tissue engineering and cancer treatment. Therefore, a comprehensive investigation of the effects caused by its parameters is crucial for improvements in those fields. We propose a study of pulse parameters, to allow the control of N2a cell line fate and behavior. We have focused on designing an experimental setup that allows for the knowledge and control over the environment and the stimulation signals applied. To map the effects of the stimulation on N2a cells, their morphology and the cellular and molecular reactions induced by the pulse stimulation have been analyzed. Immunofluorescence, rt-PCR and western blot analysis have been carried out for this purpose, as well as cell counting. Our results show that low-amplitude electrical pulse stimulation promotes proliferation of N2a cells, whilst amplitudes in the range 250 mV/mm-500 mV/mm induce differentiation. Amplitudes higher than 750 mV/mm produce cell damage at low frequencies. For high frequencies, large amplitudes are needed to cause cell death. An inverse relation has been found between cell density and pulse-induced neuronal differentiation. The best condition for neuronal differentiation was found to be 500 mV/mm at 100 Hz. These findings have been confirmed by up-regulation of the Neurod1 gene. Our preliminary study of the molecular effects of electrical pulse stimulation on N2a offers premonitory clues of the PI3K/Akt/GSK-3β pathway implications on the neuronal differentiation process through ES. In general, we have successfully mapped the sensitivity of N2a cells to electrical pulse stimulation parameters.

Plants are used as representative reference biota for the biological assessment of environmental risks such as ionizing radiation due to their immobility. This study proposed a faster, more economical, and more effective method than conventional cytogenetic methods for the biological dosimetry of ionizing radiation in plants (phytodosimetry). We compared various dose-response curves for the radiation-induced DNA damage response (DDR) in Arabidopsis thaliana after relatively “low-dose” gamma irradiation (3, 6, 12, 24, and 48 Gy) below tens of Gy using comet (or single-cell gel electrophoresis), gamma-H2AX, and transcriptomic assays of seven DDR genes (AGO2, BRCA1, GRG, PARP1, RAD17, RAD51, and RPA1E) using quantitative real time PCR. The DDR signal from the comet assay was saturated at 6 Gy, while the gamma-H2AX signal increased up to 48 Gy, following a linear-quadratic dose-response model. The transcriptional changes in the seven DDR genes were fitted to linear or supra-linear quadratic equations with significant dose-dependency. The dose-dependent transcriptional changes were maintained similarly until 24 h after irradiation. The integrated transcriptional dose-response model of AGO2, BRCA1, GRG, and PARP1 was very similar to that of gamma-H2AX, while the transcriptional changes in the BRCA1, GRG, and PARP1 DDR genes revealed significant dependency on the dose-rate, ecotype, and radiation dose. These results suggest that the transcriptome-based dose-response model fitted to a quadratic equation could be used practically for phytodosimetry instead of conventional cytogenetic models, such as the comet and gamma-H2AX assays. The effects of dose-rate and ecotype on the transcriptional changes of DDR genes should also be considered to improve the transcriptome-based phytodosimetry model.

This paper proposes a new yet efficient method allowing a significant improvement in the on-line analysis of biological cell growing and evolution. The procedure is based on an empirical-mathematical approach for calibration and fitting of any cell-electrode electrical model. It is valid and can be extrapolated for any type of cellular line used in electrical cell-substrate impedance spectroscopy (ECIS) tests. Parameters of the bioimpedance model, acquired from ECIS experiments, vary for each cell line, which makes obtaining results difficult and-to some extent-renders them inaccurate. We propose a fitting method based on the cell line initial characterization, and carry out subsequent experiments with the same line to approach the percentage of well filling and the cell density (or cell number in the well). To perform our calibration technique, the so-called oscillation-based test (OBT) approach is employed for each cell density. Calibration results are validated by performing other experiments with different concentrations on the same cell line with the same measurement technique. Accordingly, a bioimpedance electrical model of each cell line is determined, which is valid for any further experiment and leading to a more precise electrical model of the electrode-cell system. Furthermore, the model parameters calculated can be also used by any other measurement techniques. Promising experimental outcomes for three different cell-lines have been achieved, supporting the usefulness of this technique.

Electrical stimulation is a novel tool to promote the differentiation and proliferation of precursor cells. In this work we have studied the effects of direct current (DC) electrical stimulation on neuroblastoma (N2a) and osteoblast (MC3T3) cell lines as a model for nervous and bone tissue regeneration, respectively. We have developed the electronics and encapsulation of a proposed stimulation system and designed a setup and protocol to stimulate cell cultures.Cell cultures were subjected to several assays to assess the effects of electrical stimulation on them. N2a cells were analyzed using microscope images and an inmunofluorescence assay, differentiated cells were counted and neurites were measured. MC3T3 cells were subjected to an AlamarBlue assay for viability, ALP activity was measured, and a real time PCR was carried out.Our results show that electrically stimulated cells had more tendency to differentiate in both cell lines when compared to non-stimulated cultures, paired with a promotion of neurite growth and polarization in N2a cells and an increase in proliferation in MC3T3 cell line.These results prove the effectiveness of electrical stimulation as a tool for tissue engineering and regenerative medicine, both for neural and bone injuries. Bone progenitor cells submitted to electrical stimulation have a higher tendency to differentiate and proliferate, filling the gaps present in injuries. On the other hand, neuronal progenitor cells differentiate, and their neurites can be polarized to follow the electric field applied.

Many biological parameters and processes can be sensed and monitored using their impedance as marker (Grimmes, 2008), (Beach. 2005), (Yúfera, 2005), (Radke, 2004), with the advantage that it is a non-invasive, relatively cheap technique.Cell growth, cell activity, changes in cell composition, shapes or cell location are only some examples of processes which can be detected by microelectrode-cell impedance sensors (Huang, 2004) (Borkholder, 1998).The electrical impedance of a biological sample reflects actual physical properties of the tissue.In frequency dependent analyses, the  -dispersion ranging from kilohertzs to hundreds of megahertzs (Schwan, 1957) is mainly affected by the shape of the cells, the structure of the cell membranes, and the amount of intra and extra cellular solution.Electrical bio-impedance can be used to assess the properties of biological materials (Ackmann, 1993) involved in processes such as cancer development (Giaever, 1991), (Blady, 1996), (Aberg, 2004); because the cells of healthy tissues and cancer are different in shape, size and orientation, abnormal cells can be detected using their impedance as a marker.Among Impedance Spectroscopy (IS) techniques, Electrical Cell-substrate Impedance Spectroscopy (ECIS) (Giaever, 1986), based on two-electrode setups, allows the measurement of cell-culture impedances and makes it possible to determine the biological condition (material, internal activity, motility and size) of a cell type and its relationship with the environment; for example, the transfer flow through the cell membrane (Wang, 2010).One of the main drawbacks of the ECIS technique is the need to use efficient models to decode the electrical results obtained.To efficiently manage bio-impedance data, reliable electrical models of the full system comprising electrodes, medium and cells are required.Several studies have been carried out in this field (Giaever, 1991), (Huang, 2004), (Borkholder, 1998), (Joye, 2008), (Olmo, 2010), some of them employing Finite Element simulation (FEM) for impedance model extraction.These models are the key for matching electrical simulations to real system performances and hence for correctly decoding the results obtained in experiments.

The present study was performed to elucidate the extent of damage and the ability of lung epithelial cells to recover or to undergo apoptosis after in vitro treatment with the volatile anaesthetic halothane. The results obtained from the comet assay clearly show that halothane, applied at 3.0 mM concentration, causes DNA and cell damage. Cells exhibited nuclear fragmentation and budding early after treatment and these events gradually increased during the next few days. The presence of a large number of mini-comets after single cell gel electrophoresis was found to represent apoptotic bodies with fragmented DNA. Our results demonstrate apoptosis-like changes after in vitro exposure of A549 cells to the volatile anaesthetic halothane. The majority of the affected cells did not recover and were directed to cell death.

The cell concentration measurement on a culture assay using bioimpedance is a very useful tool, but it is complex to translate impedance to cell concentration values. The purpose of this work is to find a method to obtain in real time the cell concentration values for a given cell-culture assay using an oscillator as the measurement circuit. From a basic cell-electrode model, enhanced models of the cell culture immersed in a saline solution (culture medium) can be derived. These models can be used in a fitting routine to real time estimation of the cell concentration in a cell culture, using the oscillation frequency and amplitude delivered by measurement circuits proposed by the authors. Based on real experimental data (frequency and amplitude of oscillations) obtained by connecting the cell culture to an oscillator as a load, the fitting routine is simulated, and real time data of cell concentration is achieved. These results are compared with concentration data found by traditional optical methods for counting. In addition, the error obtained is divided and analyzed in two parts: in the first part of the experiment (when the few cells are adapting to the culture medium) and the second part of the experiment (when the cells grow exponentially until they completely cover the well).Low error values are obtained in the growth phase of the cell culture (the relevant phase), therefore the results obtained are considered to be promising, proving that the fitting routine is valid, and that the cell concentration can be measured in real time using an oscillator.

The present study was carried out in order to analyze how persistent the lesions in DNA are which elicit sister-chromatid exchanges (SCEs), induced by three different chemical agents, mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) and ethyl methanesulfonate (EMS), in proliferating human lymphocytes. Cells were exposed to the mutagens for 1 h just before starting bromodeoxyuridine substitution and SCEs were examined in third-cycle metaphases showing three-way-differential staining, by means of our previously standardized method. The results show that, in spite of the fact that these three compounds have different modes of action, the lesions induced by all of them seem to be capable of persisting in DNA and eliciting SCEs for at least three successive cell cycles.

Camptothecin (CPT) and actinomicyn-induced strand-breaks, repair and apoptosis in unstimulated human blood cells were studied using the DNA comet assay, and electrophoresis of low molecular weight DNA extracts. On the one hand, incubation of G0 leukocytes for 1 h with CPT induced DNA strand-breaks that were observed using the single cell gel electrophoresis technique. On the other hand, internucleosomal DNA fragments were not observed, suggesting that apoptosis had not occurred. DNA-strand-breaks caused by CPT were repaired 24 h after treatment; the migration of DNA fragments was assessed by a reduction in the number of comets. These data strongly suggest that the unexpected clastogenic effect of this topoisomerase I inhibitor is not due to the collision of the cleavage complex with the replication fork, since replication does not occur in G0. In our opinion, this effect could be due instead to the topoisomerase I enzyme being able to bind DNA in the absence of replication, probably in a way that is not strictly related to the progression of the cell cycle. Thus, CPT does not provoke apoptosis in quiescent leukocytes.

Radiosensitivity and repair of DNA damage induced by ionizing radiation and restriction enzymes were investigated in three human epithelial cell lines: two tumorigenic squamous carcinoma cell lines (SCC-4 and SCC-25), and a non-tumorigenic epidermal keratinocyte cell line (RHEK-1). Sensitivity to ionizing radiation was determined using a clonogenic cell survival assay, which showed SCC-4 to be more radiosensitive than SCC-25 and RHEK-1, which in turn displayed about equal sensitivity. Using DNA precipitation under alkaline conditions for the analysis of induction and repair of DNA single-strand breaks (ssb), an increased level of ssb induction was found for SCC-4 while the efficiency of ssb repair was about equal in all three cell lines. Using pulsed-field gel electrophoresis (PFGE) for the measurement of induction and repair of DNA double-strand breaks (dsb), no consistent differences were detected between the three cell lines. A plasmid reconstitution assay was used to determine the capacity to rejoin restriction enzyme-induced dsb in whole-cell extracts prepared from the three cell lines. In these experiments, dsb rejoining was shown to be significantly reduced in the most radiosensitive SCC-4 cell line while it was about equal in RHEK-1 and SCC-25. The results indicate that plasmid reconstitution in cell-free extracts is a sufficiently sensitive assay to detect differences in repair capacity among tumour cell lines of different radiosensitivity which remain undetectable by DNA precipitation and PFGE.

Our main aim was to establish the efficiency of the single cell electrophoresis technique for differentiating between drugs that bind DNA and those that do not. The alkaline comet assay was used to test the responses of human leukocytes (quiescent cells) to damage induced by reportedly genotoxic and reportedly cytotoxic agents. Incubation of G0 leukocytes for 1 h with the genotoxic agents camptothecin and actinomycin C provoked DNA migration, observed as comet figures. On the other hand, when cells were treated with the cytotoxic agents cordycepin, fluorodeoxyuridine and puromycin, the leukocyte nuclei were indistinguishable from those of untreated cells. In addition, we have developed a rapid method using non-proliferating cells that requires neither culture nor lymphocyte isolation. This method promises to be useful as a rapid in vitro screening assay.

A smart sensor system for cell culture real-time supervision is proposed, allowing for a significant reduction in human effort applied to this type of assay. The approach converts the cell culture under test into a suitable “biological” oscillator. The system enables the remote acquisition and management of the “biological” oscillation signals through a secure web interface. The indirectly observed biological properties are cell growth and cell number, which are straightforwardly related to the measured bio-oscillation signal parameters, i.e., frequency and amplitude. The sensor extracts the information without complex circuitry for acquisition and measurement, taking advantage of the microcontroller features. A discrete prototype for sensing and remote monitoring is presented along with the experimental results obtained from the performed measurements, achieving the expected performance and outcomes.

The present study was carried out in Chinese hamster ovary cells in order to determine whether lesions induced by three different mutagens, namely 4-nitroquinoline-1-oxide (4-NQO), Mitomycin C (MMC) and Ethyl methanesulfonate (EMS), can persist for more than one cell generation leading to sister chromatid exchanges (SCE) or, alternatively, they are efficiently repaired during the next replicative period after treatment. In order to accurately score the number of SCEs arising during the first (S1), second (S2) and third (S3) DNA synthetic periods, third-cycle (M3) metaphases showing three-way differential (TWD) staining were analyzed. Our results show that, even though the three compounds tested were efficient in increasing the yield of SCE, the frequency of SCE was more dramatically increased after MMC treatment. Differences were also observed among the three mutagens with regard to the persistence of the lesions leading to SCE throughout successive cell generations. EMS-induced lesions appeared as more persistent than those induced by MMC. However, most of the damage induced by the UV mimetic agent 4-NQO seems to be efficiently repaired after the first round of DNA replication following treatment with the drug.

This paper proposes a cell-microelectrode model for cell biometry applications, based on the area overlap as main parameter. The model can be applied to cell size identification, cell count, and their extension to cell growth and dosimetry protocols. Experiments performed with comercial electrodes are presented, illustrating a procedure to obtain cell number in both growth and dosimetry processes. Results obtained for the AA8 cell line are promising.

Obtaining cell concentration measurements from a culture assay by using bioimpedance is a very useful method that can be used to translate impedances to cell concentration values. The purpose of this study was to find a method to obtain the cell concentration values of a given cell culture assay in real time by using an oscillator as the measurement circuit. From a basic cell-electrode model, enhanced models of a cell culture immersed in a saline solution (culture medium) were derived. These models were used as part of a fitting routine to estimate the cell concentration in a cell culture in real time by using the oscillation frequency and amplitude delivered by the measurement circuits proposed by previous authors. Using real experimental data (the frequency and amplitude of oscillations) that were obtained by connecting the cell culture to an oscillator as the load, the fitting routine was simulated, and real-time data of the cell concentration were obtained. These results were compared to concentration data that were obtained by using traditional optical methods for counting. In addition, the error that we obtained was divided and analyzed in two parts: the first part of the experiment (when the few cells were adapting to the culture medium) and the second part of the experiment (when the cells exponentially grew until they completely covered the well). Low error values were obtained during the growth phase of the cell culture (the relevant phase); therefore, the results obtained were considered promising and show that the fitting routine is valid and that the cell concentration can be measured in real time by using an oscillator.

This work describes Electrical Stimulations (ES) assays on stem cells.The neuroblastoma (N2A) cell linage was submitted to several electrical fields to enable and enhance its differentiation toward neurons.Both Direct Current (DC) and Alternated Current (AC) time dependent electric field protocols were applied to N2A cell culture under differentiation conditions, obtaining different responses.Control and electrically excited samples' number of differentiated cells and neurite lengths were measure after differentiation.Results showed that DC fields have a strong influence on N2A differentiation since the percentage of differentiated cells and the neurites lengths were the highest.In addition, a significant alignment of neurites measured with the applied electrical field has been detected, which demonstrates the high sensitivity of differentiation processes to electrical field polarity.

An approach based on the synchronization of CHO cells after a first cell cycle incorporating a relatively low amount of bromodeoxyuridine (BrdUrd) into DNA, followed by mutagenic treatment and subsequent culture for second and third generations of BrdUrd incorporation for the scoring of sister chromatid exchanges (SCEs) per cell cycle in three-way differentially (TWD) stained chromosomes, has been used to investigate the possible cancellation of SCEs. Cancellation is expected to occur if two mutagen-induced SCEs occur at exactly the same site in subsequent rounds of replication. Lesions in DNA seem to persist and are able to induce SCE throughout two cell cycles after treatment with the three mutagens tested--mitomycin C (MMC), ethyl methanesulfonate (EMS) and ultraviolet (UV) light--though this latter agent was shown as only moderately persistent. Our results seem to indicate that SCEs induced by these mutagens do not take place at the same locus in successive cell generations, as assessed by a lack of SCE cancellation.

Background: Salmonella Typhi and Paratyphi infections were hyperendemic in Santiago, Chile from 1977-1991. In 1980, bile cultures from 1000 cholecystectomies yielded S. Typhi in 3·8% and S. Paratyphi in 3·5%. Typhoid transmission plummeted following prohibitions against irrigating crops with untreated sewage after a 1991 cholera outbreak. Typhoid is currently rare in Santiago.Methods: Two groups of Santiago residents awaiting cholecystectomy in four hospitals during June 2017-June 2019, age ≥55 years (adults exposed during typhoid era) and 18-34 years (non-exposed), were enrolled after informed consent. Bile and pulverized gallstone specimens were tested by qPCR and cultured (bile only) to detect typhoidal Salmonella.Findings: 1208 subjects age ≥55 and 1030 age 18-34 years provided specimens. S. Typhi was detected by qPCR in bile or pulverized gallstones of 3/1208 (0·25%) older subjects. S. Paratyphi B was cultured from bile of a 70-year-old. High anti-Vi titers (≥30 mcg/mL) were detected in 2 older patients (111·3 mcg/mL; 46·3 mcg/mL). Neither typhoidal Salmonella nor high Vi titers were detected in younger patients.Interpretation: S. Typhi (N=3) and S. Paratyphi B (N=1) in 4 older but no younger patients coincide with their S. Typhi exposure. Over their lifespan, older subjects’ receipt of antibiotics for urinary tract and other infections may have eliminated Salmonella from gallbladders of chronic typhoid carriers who were infected during the hyperendemic era. Alternatively, Typhi detected by qPCR from pulverized gallstones may represent decades-old DNA of non-viable bacteria. Low carrier prevalence among cholecystectomized young adults coincides with the current rarity of typhoid in Santiago.Funding Information: This work was supported by the Bill & Melinda Gates Foundation [OPP1161058].Declaration of Interests: MML and SMT report grants from the Bill & Melinda Gates Foundation during the conduct of the study. MML, SMT, and RS have patents issued for: a ‘Broad spectrum vaccine against typhoidal and nontyphoidal Salmonella disease’ (U.S. Patent 9,011,871); and ‘Compositions and Methods for Producing Bacterial Conjugate Vaccines’ (U.S. Patent 10,716,839). MML also has a patent issued for: 'Attenuated Salmonella Enterica Serovar Paratyphi and Uses Thereof' (U.S. Patent 8,137,930). All other authors report no potential conflicts.Ethics Approval Statement: Protocols were approved by University of Maryland Baltimore’s Institutional Review Board (HP00073809). Pontificia Universidad Católica de Chile Ethics Committee oversaw ethical approval for the four study sites. Written informed consent was obtained from volunteers prior to enrollment.

The possible modulation by T4 DNA ligase of the DNA double-strand breaks produced by restriction endonucleases in living mammalian cells was studied. A clear decrease in the frequency of chromosomal aberrations was observed when T4 DNA ligase was included in the electroporation treatment along with restriction enzymes inducing either cohesive- or blunt-ends in DNA. The relative proportions of exchange-type aberrations were similar for both kinds of treatments (with and without ligase), which seems to suggest an error-free ligation by T4 DNA ligase.

The biomedical engineering must frequently develop sensor designs by including information from performance of bio-samples (cell cultures or tissues), technical specifications of transducers, and constrains from electronic circuits. A computer program for real-time cell culture monitoring system design is developed; analyzing, modelling and integrating into the program design flow the electrodes, cell culture and test circuit's influences. The computer tool, first, generates an equivalent electric circuit model for the cell-electrode bio-systems based on the area covered by cells, which also considers the cell culture dynamics. Second, proposes an Oscillation Based Test (OBT) parameterized circuit, for Electrical Cell-Substrate Sensing (ECIS) measurements of the cell culture system bioimpedance. Third, simulates electrically the full system to define the best system parameter values for the sensor. Reported experimental results are based on commercial gold electrodes and the AA8 cell line. Characteristics of the cell lines, as time-division or cell size, are incorporated into the program design flow, showing that for a given assay, the optimal OBT circuit parameters can be selected with the help of the computer tool. The electrical simulations of the full system demonstrate that the can be correctly predicted the output frequency and amplitude ranges of the voltage response, obtaining accurate results when cell culture approaches to confluence phase. It is proposed a computer program for system design of biosensors applied to monitoring cell culture dynamics. The program allows obtaining confident system information by electrical stimulation. All system components (electrodes, cell culture and test circuits) are properly modelled. The employed procedure can be applied to any other 2D electrode layout or alternative circuit technique for ECIS test. Finally, deep insight information on cell size, number, and time-division can be extracted from the comparison with real cell culture assays in the future.

It is proposed the use of a cell-microelectrode model for cell biometry applications. The model can be applied to cell size identification, cell count, and extended to cell growth and dosimetry protocols, by identifing the total area covered by cells. The work develops Analog Hardware Description Language (AHDL) codes for cell-microelectrode systems, considering the geometrical and technological parameters, enabling the possibility to simulate biological complex systems with convencional mixed-mode simulators as SpectreHDL. Experimental results are presented to calculate the cell number in growth and dosimetry experiments using AA8 cell line.

In this paper the application of a cell- microelectrode model to cell biometry experiments is proposed, using the cell-electrode area overlap as main parameter. The model can be applied to cell size identification, cell count, and their extension to cell growth and dosimetry protocols. Experimental results using AA8 cell line are presented, obtaining promising results.

Abstract Background: Electrical stimulation is a novel tool to promote the differentiation and proliferation of precursor cells. In this work we have studied the effects of direct current (DC) electrical stimulation on neuroblastoma (N2a) and osteoblast (MC3T3) cell lines as a model for nervous and bone tissue regeneration, respectively. We have developed the electronics and encapsulation of a proposed stimulation system and designed a setup and protocol to stimulate cell cultures. Methods: Cell cultures were subjected to several assays to assess the effects of electrical stimulation on them. N2a cells were analyzed using microscope images and an inmunofluorescence assay, differentiated cells were counted and neurites were measured. MC3T3 cells were subjected to an AlamarBlue assay for viability, ALP activity was measured, and a real time PCR was carried out. Results: Our results show that electrically stimulated cells had more tendency to differentiate in both cell lines when compared to non-stimulated cultures, paired with a promotion of neurite growth and polarization in N2a cells and an increase in proliferation in MC3T3 cell line. Conclusions: These results prove the effectiveness of electrical stimulation as a tool for tissue engineering and regenerative medicine, both for neural and bone injuries. Bone progenitor cells submitted to electrical stimulation have a higher tendency to differentiate and proliferate, filling the gaps present in injuries. On the other hand, neuronal progenitor cells differentiate, and their neurites can be polarized to follow the electric field applied.

The cytochrome P-450-dependent O-dealkylation of all alkoxyresorufins was used to study the effect of cumene hydroperoxide on cytochrome P-450 IIB1 and IA1 in microsomal and reconstituted systems. In liver microsomal systems from respectively phenobarbital and 3-methylcholanthrene pretreated male Wistar rats, cytochrome P-450 IIB1-dependent pentoxyresorufin-O-dealkylation appeared to be more sensitive to cumene hydroperoxide treatment than cytochrome P-450 IA1-dependent ethoxyresorufin-O-dealkylation. This phenomenon was also observed when the cumene hydroperoxide sensitivity of P-450 IIB1 and IA1 was studied in an isosafrole pretreated rat liver microsomal system. The decrease in alkoxy-O-dealkylating activities appeared to proceed by destruction of the cytochrome P-450 component of the enzyme system. Purification and reconstitution of the enzyme system components in a system in which the isolated proteins were not incorporated into a membrane resulted in the disappearance of the difference in sensitivity between the two P-450 enzymes. However, in a reconstituted system with membrane incorporated proteins, again cytochrome P-450 IIB1 expressed a higher sensitivity towards cumene hydroperoxide than cytochrome P-450 IA1. From this it was concluded tha the differential cumene hydroperoxide sensitivity of cytochrome P-450 IIB1 and IA1 is not caused by an intrinsic difference in their sensitivity but by a differential effect of membrane incorporated on their cumene hydropeoxide sensitivity.