Recognition of healthy and cancerous breast cells: Sensing the differences by dielectric spectroscopy.

PURPOSE: The response of human cells to applied electrical signals depends on the cellular health status, because it is influenced by the composition and structure of the main cellular components. Therefore, electrical impedance-based techniques can be considered as sensitive tools to investigate healthy or disease state at cellular level. The goal of this study is to show that different types of in vitro cellular lines, related to different health status, can be differentiated using impedance spectra analysis. METHODS: Three different types of human breast cell line, corresponding to healthy, cancerous, and metastatic adenocarcinoma cells, were measured by means of electrical impedance spectroscopy. By modeling the investigated cells with proper resistive and capacitive circuital elements, the magnitude of the cell electrical components and spectra of real and imaginary part of dielectric permittivity were obtained. The latter were subsequently examined with a commonly adopted mathematical model, in order to estimate the values of specific dielectric parameters for the three different cellular lines. RESULTS: The relative variation of cellular capacitance with respect to that of the culture medium, estimated at 100 Hz, has a larger value for the two types of cancerous cells with respect to the noncancerous type. Furthermore, the ratio between the real and imaginary part of the dielectric permittivity function has larger values for metastatic cells with respect to the normal and nonmetastatic ones. Therefore, the mentioned relative capacitance allows to discriminate between normal and cancerous cells, whereas the results obtained for the dielectric function can discriminate between metastatic and nonmetastatic cells. CONCLUSIONS: This study can be considered as an exploratory investigation of evaluating in vitro the health status of humans cells using selected electrical impedance parameters as potential markers. The obtained results highlight that a standard cultureware system, provided with interdigitated electrodes and appropriate impedance parameters, that is, cellular capacitance and the ratio between the imaginary and real part of cellular dielectric function, can be used to discriminate between healthy and cancerous breast cell lines, as well as different malignancy degrees.

Raman spectroscopy for the evaluation of the radiobiological sensitivity of normal human breast cells at different time points after irradiation by a clinical proton beam.

Among different radiotherapy techniques, proton irradiation is an established and effective method for treatment of several types of cancer, because less healthy tissue is exposed with respect to conventional radiotherapy by photons/electrons. Recently, proton therapy has been proposed for the treatment of breast cancer. In vitro studies of proton irradiated normal human breast cells can provide information about cellular radioresponse, particularly as far as healthy tissue is concerned. In this paper, a study of the effects at different time points, following proton irradiation at different doses, of human normal MCF10A breast cells is performed by Raman spectroscopy. The aim of this investigation is to detect the unwanted effects of proton treatment and to investigate the possibility of monitoring them and of making an assessment of the cellular sensitivity by means of such a technique. The obtained results seem to indicate a rather significant sensitivity of MCF10A cells to proton irradiation. In fact, even at doses as low as 0.5 Gy, biological effects are clearly detectable in Raman spectra. In particular, ratiometric analysis of the Raman spectra measured from the nucleoplasm compartment showed that DNA/RNA damage increases with time, suggesting that most cells are unable to repair DNA/RNA broken bonds. The results obtained by the Raman spectroscopy analysis exhibit a similar trend with regard to dose to those obtained by commonly used radiobiological assays (i.e. MTT, clonogenic assay, senescence, apoptosis and necrosis). The results of this study strongly suggest the possibility that the Raman technique can be used to identify molecular markers predicting radiation response.

X-ray irradiation effects on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells investigated by Raman micro-spectroscopy.

Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.

Correction: Exposure to 1.8 GHz electromagnetic fields affects morphology, DNA-related Raman spectra and mitochondrial functions in human lympho-monocytes.

[This corrects the article DOI: 10.1371/journal.pone.0192894.].

Exposure to 1.8 GHz electromagnetic fields affects morphology, DNA-related Raman spectra and mitochondrial functions in human lympho-monocytes.

Blood is a fluid connective tissue of human body, where it plays vital functions for the nutrition, defense and well-being of the organism. When circulating in peripheral districts, it is exposed to some physical stresses coming from outside the human body, as electromagnetic fields (EMFs) which can cross the skin. Such fields may interact with biomolecules possibly inducing non thermal-mediated biological effects at the cellular level. In this study, the occurrence of biochemical/biological modifications in human peripheral blood lympho-monocytes exposed in a reverberation chamber for times ranging from 1 to 20 h to EMFs at 1.8 GHz frequency and 200 V/m electric field strength was investigated. Morphological analysis of adherent cells unveiled, in some of these, appearance of an enlarged and deformed shape after EMFs exposure. Raman spectra of the nuclear compartment of cells exposed to EMFs revealed the onset of biochemical modifications, mainly consisting in the reduction of the DNA backbone-linked vibrational modes. Respirometric measurements of mitochondrial activity in intact lympho-monocytes resulted in increase of the resting oxygen consumption rate after 20 h of exposure, which was coupled to a significant increase of the FoF1-ATP synthase-related oxygen consumption. Notably, at lower time-intervals of EMFs exposure (i.e. 5 and 12 h) a large increase of the proton leak-related respiration was observed which, however, recovered at control levels after 20 h exposure. Confocal microscopy analysis of the mitochondrial membrane potential supported the respiratory activities whereas no significant variations in the mitochondrial mass/morphology was observed in EMFs-exposed lympho-monocytes. Finally, altered redox homeostasis was shown in EMFs-exposed lympho-monocytes, which progressed differently in nucleated cellular subsets. This results suggest the occurrence of adaptive mechanisms put in action, likely via redox signaling, to compensate for early impairments of the oxidative phosphorylation system caused by exposure to EMFs. Overall the data presented warn for health safety of people involved in long-term exposure to electromagnetic fields, although further studies are required to pinpoint the leukocyte cellular subset(s) selectively targeted by the EMFs action and the mechanisms by which it is achieved.

Morphology of synthetic DOPA-eumelanin deposited on glass and mica substrates: an atomic force microscopy investigation.

Bright field microscopy and atomic force microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of L-DOPA solution, deposited on glass and mica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates, with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates, several microns long and about 100 nm wide and high, and granular aggregates, ∼50 nm high and 100 nm wide, are found on both substrates, whereas point-like deposits less than 10nm high and less than 50 nm wide are found on mica substrate. Dynamic light scattering measurements and atomic force microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentous structures when deposition occurs, as a consequence of interactions with the substrate surface.

Novel fastidious, partially acid-fast, anaerobic Gram-positive bacillus associated with abscess formation and recovered from multiple medical centers.

We report a novel anaerobe causing abscess in four patients at three hospitals. In the clinical specimen, bacilli were branching, Gram positive, and acid fast. The organism grew slowly and was not identified by 16S rRNA sequencing. Our findings support the description of a new genus and species of the suborder Corynebacterineae.

[Using atomic force microscopy to analyze morphological changes and mechanical properties caused by cellular exposure to low doses of pesticides]. / Utilizzo della microscopia a forza atomica per l'analisi di alterazioni morfologiche e meccaniche cellulari causate da esposizione a basse dosi di pesticidi.

A commercial pesticide is usually composed of active ingredients and formulants. Among the active ingredients, Deltamethrin is a pyrethroid chemical widely used for synthesizing pesticides products which are very effective in damaging the central nervous system of pests. In this work, we analyze, by means of atomic force microscopy (AFM), cellular morphological changes induced by exposure to a Deltamethrin-based commercial pesticide (Decaflow). AFM microscopy, in addition to the well-known characterization of the cellular topography, has the ability to monitor interesting biomechanical parameters of the surface as roughness and elastic modulus. In particular, we exposed normal human keratinocytes for 24 hours at different solutions of Decaflow, well below the threshold of cytotoxicity. The AFM images of exposed cells show alterations of surface cell shape. Moreover exposed cells are characterized by an increase of the value of membrane roughness. The mechanical properties of cells are also modified after Decaflow exposure, as confirmed by a decrease of the elasticity modulus with increasing the concentration of pesticide.

Atomic force microscopy investigation of morphological changes in living keratinocytes treated with HgCl(2) at not cytotoxic doses.

Morphological changes of normal human keratinocyte cells have been monitored by means of atomic force microscopy after the exposure at a mercury solution containing HgCl(2) at 10(-7) M. The measurements have been carried out in contact mode in a thermostated liquid cell, to reproduce a cellular environment similar to the physiologic one. Remarkable alterations of the cellular morphology and volume have been revealed after few minutes from starting the exposure experiment, although the HgCl(2) concentration is several orders of magnitudes lower than the cytotoxic value (10(-4) M). The atomic force microscopy technique results to be a powerful mean to investigate modifications induced in the cell morphology by external chemical agents.

[Production cycles and risk agents in the agri-food sector]. / I cicli produttivi ed i relativi fattori di rischio nel settore agroalimentare.

Italian Food Processing Section includes primary sector (agriculture, fishing and farming), secondary industry and service sector (trade, transport and marketing). The whole process is structured in a sequence of production structures, which constitute a die. The most important food processing dies are fruit and vegetable, cereals, wine, oil, bovine, swine and avian breeding, fishing, milk and cheese. Every die presents very different production cycles, jobs and working professionals. Considering the heterogeneity of food processing dies, all occupational risk factors, such as chemical, biological, physical, ergonomic, psychosocial and injuries risks, are very frequent in such working activities. In a pilot study carried out in the province of Foggia (Apulia, Southern Italy) we showed that the major perceived risk factors where dust and physical overload in the cereal-pasta processing die, and noise and awkward postures in olive-oil die. However, perceived risk factors are biased by low risk perception due to poor information about health occupational hazards, this representing an important health safety problem. For this reason, the activity of preventive authorities at various levels is highly recommended.

[Promoter effect induced by HgCl2 by studying the intercellular communication]. / Effetto promoter indoitto da HgCl2 attraverso lo studio della comunicazione intercellulare.

This work aims at assessing at molecular level the effect caused by the HgCl9 intercellular communication inhibition at non-cytotoxic doses. On the basis of our previous experiences, we exposed the human keratinocytes (HUKE) at 10 nM of HgCl2 for 24 hours Next, we estimated: a) the protein expression of connexines Cx43, Cx32 and Cx26 by western blotting; b) the amount of mRNA corresponding to the three connexines by semi-quantitative RT-PCR; and c) the production of reactive oxygen species in HgCl2 treated cells using a specific probe, i.e. DCF in confocal microscopy. Our study demonstrated a higher expression of the transcripts for Cx26, Cx32, Cx43, and a higher amount of proteins Cx43, Cx32 and Cx26, compared to the negative controls. Furthermore, we studied the effect of HgCl2 on the ROS production in keratinocytes, by the analysis in confocal microscopy carried out with the DCF, fit for marking the oxygen free radicals. In HgCl2 treated keratinocytes we obtained an increase of the ROS production compared to controls; and further the mitochondrions resulted the place of ROS production. The results of this study suggest that non-cytotoxic HgCl2 concentrations, might cause an unbalancing of the redox cellular state (ROS increased level), and we can assume that the activation of a redox signalling involves the inactivation of gap junctions.

Salivary markers of work stress in an emergency team of urban police (1 degree step).

INTRODUCTION: Stress is usually defined as the experience of negative events or the perceptions of distress and negative affect that are associated with the inability to cope with them. The parameter most suitable for large-scale field studies is the determination of endocrine activity by measurement of salivary cortisol. The aim of the present study is to identify the presence of sources of stress in an emergency team of urban police and to objective such stress using the PSS (Professional Stress Scale) test and bioumoral markers as salivary cortisol and interleukin 1 /f (IL-IB). MATERIALS AND METHODS: We studied 30 policemen who belonged to an emergency team. Salivary samples were collected at the start and at the end of the work-shift. As control we used the same subjects during the holiday. T test was performed to evaluate the differences between the means, the Chi Square's Test was performed to determine the statistically significant association between PSS subscales and salivary cortisol and ILl-B concentrations. RESULTS: Thirty policeman were evaluated, their mean age was 44,5 years, their mean work experience was 17,1 years. The PSS test indicated high scores in three subscales, they were work load, organizational structure and processes and lack of resources. SALIVARY CORTISOL: The mean concentration at the start of work-shift was higher than at the end of shift-work (p<0,05). T test indicated a statistically significant difference between mean cortisol concentrations at the same hour during the work (start and end) and during the holiday (P<0,05). A statistically significant negative association was noted between the PERC1 and PSS subscale called "work load" (p<0,05). SALIVARY IL-1B: the mean concentration of ILl-B at the start of the work-shift resulted higher than at the end, such reduction was statistically significant (P<0.05). We verified a positive association between the subclass of PSS Test called "conflict with other professionals" and salivary IL-1B concentration at the start of shift-work (p<0,05). DISCUSSION. Several precedent studies agree with our results. Our study has suggested a work related stress in urban police employed in an emergency team. We might conclude that salivary IL-1B and cortisol are useful markers of stress. We think that our findings, surely preliminary, have be corroborated by the study of vegetative parameters (heart rate, heart rate variability) that is still current. It might be useful to evaluate again the cortisol and IL-1B variations after some structure organizational modifications and after training that will teach the workers coping strategies. We might conclude that the stress discovered in this study is not hazardous for the health, if the workers have adequate holidays.

Role of tumour necrosis factor alpha and interleukin 1 beta in promoter effect induced by mercury in human keratinocytes.

In the progress of a carcinogenetic process, the promoter effect was seen as the final event able to determine uncontrolled proliferation. The promoter effect begins with an inhibition of gap junctional intercellular communication. Previously we observed an inhibitory effect of Mercury (HgCl2) on Gap Junction Intercellular Communication of Human Keratinocytes in culture. Here we evaluate the effect of Mercury on gap junctional intercellular communication, on cytokines intracellular concentrations and on cytokines secretion of Human Keratinocytes. In particular, we report a reduction of the intracellular concentrations and secretions of Tumour Necrosis alpha and Interleukin 1 beta. It is known that the inhibitory effect on Gap Junctional Intercellular Communication is correlated with a promoter effect induced by carcinogens. In this paper we discuss the relationship between the inhibition of gap junctional intercellular communication and cytokine production, and whether these effects are related in an xenobiotic carcinogenesis process. Our considerations could be seen as too adventurous, but they may set the stage for an open discussion of our results according to the literature. An intriguing relationship appears to develop when comparing the effects of proinflammatory mediators on GJIC. Although highly speculative, a review of the current literature would suggest that the GJIC inhibition induced by mercury might be the beginning of the promoter effect, but the role induced by cytokines on initiated cells to stimulate its proliferation remains to be determined We think that the reduction of TNF-alpha, and in part IL-1beta, induced by mercury might favour the cancer. We hypothesise that the reduction of cytokines and inhibition of the gap junction intercellular communication are correlated and they may play a role in the xenobiotic carcinogenesis process.

[Assessment of heart rate variability (HRV) as a stress index in an emergency team of urban police]. / Valutazione della variabilità della frequenza cardiaca (HRV) come indice di stress in addetti al pronto intervento nella polizia urbana.

Stress is usually defined as the experience of negative events or the perceptions of distress and negative affect that are associated with the inability to cope with them. The parameter most suitable for large-scale field studies is the determination of endocrine activity by measurement of salivary cortisol. The aim of the present study is to identify the presence of sources of stress in an emergency team of urban police and to objective such stress using the PSS (Professional Stress Scale) test and bioumoral markers as salivary cortisol and interleukin 1 beta (IL1-B). Moreover it will be determined the Heart Rate Variability (HRV) that is able to mirror the sympathetic-parasympathetic balance. We studied 30 policemen who belonged to an emergency team. Thirty policemen made cardiologic exams as ECG, Heart Eco-doppler and Holter Ecg during the work and during the holiday. Particularly we studied HRV using frequency-domain based HRV measures. The results were evidenced as the differences of the spectral power. As control we used the same subjects during the holiday. T test was performed to evaluate the differences between the means, the Chi Square's Test was performed to determine the statistically significant association between PSS subscales, HRV, salivary cortisol and IL1-B concentrations. The study of HRV demonstrated a statistically significant association between HRV parameters during the holiday, salivary cortisol concentrations and PSS test. IL1-B, instead, was not associated with HRV parameters. Several precedent studies agree with our results. Our study has suggested a work related stress in urban police employed in an emergency team. We might conclude that salivary IL1-B and cortisol are useful markers of stress. The study of HRV parameters gave evidence that LF/HF during the holiday is a useful marker of work stress, because it correlated with cortisol difference during the work. Conclusively we can believe that the HRV parameters evaluated during the work are useful as confront, instead the HRV parameters during the holiday are surest indexes of work stress. Probably the effect of stress on the heart aren't present during the work because the work experience reduces these effects, they appear during the holiday when the imagination could make the conflicts or the problems more complex than they are. It might be useful to repeat this study after some structure organizational modifications and after training that will teach the workers coping strategies. If we consider only the bioumoral and PSS results, we might conclude that the stress discovered in this study is not hazardous for the health when the workers have adequate holidays, but if we evaluate the HRV parameters, we have believe that, for a heart health, it's necessary either to increment the holidays or, it can appear paradoxical, to work again.

Evaluation of the Alexon-trend ProSpecT Campylobacter microplate assay.

We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection.