Correlating nanoscale motion and ATP production in healthy and favism erythrocytes: a real-time nanomotion sensor study.

Introduction: Red blood cells (RBCs) are among the simplest, yet physiologically relevant biological specimens, due to their peculiarities, such as their lack of nucleus and simplified metabolism. Indeed, erythrocytes can be seen as biochemical machines, capable of performing a limited number of metabolic pathways. Along the aging path, the cells' characteristics change as they accumulate oxidative and non-oxidative damages, and their structural and functional properties degrade. Methods: In this work, we have studied RBCs and the activation of their ATP-producing metabolism using a real-time nanomotion sensor. This device allowed time-resolved analyses of the activation of this biochemical pathway, measuring the characteristics and the timing of the response at different points of their aging and the differences observed in favism erythrocytes in terms of the cellular reactivity and resilience to aging. Favism is a genetic defect of erythrocytes, which affects their ability to respond to oxidative stresses but that also determines differences in the metabolic and structural characteristic of the cells. Results: Our work shows that RBCs from favism patients exhibit a different response to the forced activation of the ATP synthesis compared to healthy cells. In particular, the favism cells, compared to healthy erythrocytes, show a greater resilience to the aging-related insults which was in good accord with the collected biochemical data on ATP consumption and reload. Conclusion: This surprisingly higher endurance against cell aging can be addressed to a special mechanism of metabolic regulation that permits lower energy consumption in environmental stress conditions.

Mechano-Transduction Boosts the Aging Effects in Human Erythrocytes Submitted to Mechanical Stimulation.

Erythrocytes' aging and mechano-transduction are fundamental cellular pathways that determine the red blood cells' (RBCs) behavior and function. The aging pattern can be influenced, in morphological, biochemical, and metabolic terms by the environmental conditions. In this paper, we studied the effect of a moderate mechanical stimulation applied through external shaking during the RBCs aging and revealed a strong acceleration of the aging pattern induced by such stimulation. Moreover, we evaluated the behavior of the main cellular effectors and resources in the presence of drugs (diamide) or of specific inhibitors of the mechano-transduction (probenecid, carbenoxolone, and glibenclamide). This approach provided the first evidence of a direct cross-correlation between aging and mechano-transduction and permitted an evaluation of the overall metabolic regulation and of the insurgence of specific morphological features, such as micro-vesicles and roughness alterations. Overall, for the first time the present data provided a schematic to understand the integration of distinct complex patterns in a comprehensive view of the cell and of its interactions with the environment. Mechano-transduction produces structural effects that are correlated with the stimulation and the strength of the environmental stimulation is paramount to effectively activate and trigger the biological cascades initiated by the mechano-sensing.

Surprising Structural and Functional Properties of Favism Erythrocytes Are Linked to Special Metabolic Regulation: A Cell Aging Study.

Favism uniquely arises from a genetic defect of the Glucose-6 Phosphate Dehydrogenase (G6PD) enzyme and results in a severe reduction of erythrocytes' (RBCs) reducing power that impairs the cells' ability to respond to oxidative stresses. After exposure to fava beans or a few other drugs, the patients experience acute hemolytic anemia due to RBCs' lysis both intra and extra-vascularly. In the present paper, we compared selected biochemical, biophysical, and ultra-morphological properties of normal RBCs and cells from favism patients measured along cellular aging. Along the aging path, the cells' characteristics change, and their structural and functional properties degrade for both samples, but with different patterns and effectors that have been characterized in biophysical and biochemical terms. In particular, the analysis revealed distinct metabolic regulation in G6DP-deficient cells that determines important peculiarities in the cell properties during aging. Remarkably, the initial higher fragility and occurrence of structural/morphological alterations of favism cells develop, with longer aging times, into a stronger resistance to external stresses and higher general resilience. This surprisingly higher endurance against cell aging has been related to a special mechanism of metabolic regulation that permits lower energy consumption in environmental stress conditions. Our results provided a direct and coherent link between the RBCs' metabolic regulation and the cell properties that would not have been possible to establish without an investigation performed during aging. The consequences of this new knowledge, in particular, can be discussed in a more general context, such as understanding the role of the present findings in determining the characteristics of the favism pathology as a whole.

Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma.

INTRODUCTION: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established. METHODS: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR. RESULTS: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters. CONCLUSION: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.

A perspective view on the nanomotion detection of living organisms and its features.

The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.

A new tool to determine the cellular metabolic landscape: nanotechnology to the study of Friedreich's ataxia.

Understanding the cell response to oxidative stress in disease is an important but difficult task. Here, we demonstrate the feasibility of using a nanomotion sensor to study the cellular metabolic landscape. This nanosensor permits the non-invasive real-time detection at the single-cell level and offers high sensitivity and time resolution. We optimised the technique to study the effects of frataxin overexpression in a cellular model of Friedreich's ataxia, a neurodegenerative disease caused by partial silencing of the FXN gene. Previous studies had demonstrated that FXN overexpression are as toxic as silencing, thus indicating the importance of a tight regulation of the frataxin levels. We probed the effects of frataxin overexpression in the presence of oxidative stress insults and measured the metabolic response by the nanosensor. We show that the nanosensor provides new detailed information on the metabolic state of the cell as a function of time, that agrees with and complements data obtained by more traditional techniques. We propose that the nanosensor can be used in the future as a new and powerful tool to study directly how drugs modulate the effects of oxidative stress on Friedreich's ataxia patients and, more in general, on other neurodegenerative processes.

Amyloid ß peptide affects erythrocyte morphology: Role of intracellular signaling pathways.

BACKGROUND: Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. OBJECTIVE: Understanding the role of Aß in the cross talk between cell signalling pathways and modulation of the cell structural and biomechanical properties occurring in RBCs during aging. METHODS: The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. RESULTS: We show that treatment with Aß accelerates the occurrence of morphological and biochemical aging markers in human RBC and influences the cell metabolism. Biochemical data demonstrate that contemporaneously to morphological alterations, Aß triggers: (i) metabolic alterations and (ii) a complex signaling pathway involving caspase 3, protein kinase C and nitric oxide derived metabolites. CONCLUSIONS: our study provides a comprehensive picture in which Aß treatment of RBC induces changes in specific cell signalling events and/or metabolic pathways, in turns affecting the membrane-cytoskeleton interaction and the membrane integrity.

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study.

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1-42 (Aß) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aß boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aß-induced effects. Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aß and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites. As a whole, the collected data revealed that, the damaging path induced by Aß in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.

Identification of Oxidative Stress in Red Blood Cells with Nanoscale Chemical Resolution by Infrared Nanospectroscopy.

During their lifespan, Red blood cells (RBC), due to their inability to self-replicate, undergo an ageing degradation phenomenon. This pathway, both in vitro and in vivo, consists of a series of chemical and morphological modifications, which include deviation from the biconcave cellular shape, oxidative stress, membrane peroxidation, lipid content decrease and uncoupling of the membrane-skeleton from the lipid bilayer. Here, we use the capabilities of atomic force microscopy based infrared nanospectroscopy (AFM-IR) to study and correlate, with nanoscale resolution, the morphological and chemical modifications that occur during the natural degradation of RBCs at the subcellular level. By using the tip of an AFM to detect the photothermal expansion of RBCs, it is possible to obtain nearly two orders of magnitude higher spatial resolution IR spectra, and absorbance images than can be obtained on diffraction-limited commercial Fourier-transform Infrared (FT-IR) microscopes. Using this approach, we demonstrate that we can identify localized sites of oxidative stress and membrane peroxidation on individual RBC, before the occurrence of neat morphological changes in the cellular shape.

FC_analysis: a tool for investigating atomic force microscopy maps of force curves.

BACKGROUND: The collection and analysis of Atomic Force Microscopy force curves is a well-established procedure to obtain high-resolution information of non-topographic data from any kind of sample, including biological specimens. In particular, these analyses are commonly employed to study elasticity, stiffness or adhesion properties of the samples. Furthermore, the collection of several force curves over an extended area of the specimens allows reconstructing maps, called force volume maps, of the spatial distribution of the mechanical properties. Coupling these maps with the conventional high-resolution topographic reconstruction of the sample's surface, provides a deeper insight on the sample composition from the structural and nanomechanical point of view. RESULTS: In this paper we present the open source software package FC_analysis that automatically analyses single force curves or entire force volume maps to yield the corresponding elasticity and deformability images. The principal characteristic of the FC_analysis is a large adaptability to the various experimental setups and to different analysis methodologies. For instance, the user can provide custom values for the detector sensitivity, scanner-z sensitivity, cantilever's elastic constant and map's acquisition modality and can choose between different analysis methodologies. Furthermore, the software allows the optimization of the fitting parameters and gives direct control on each step of the analysis procedure. Notably, to overcome a limitation common to many other analysis programs, FC_analysis can be applied to a rectangular portion of the image, allowing the analysis of inhomogeneous samples. Finally, the software allows reconstructing a Young's modulus map at different penetration depths, enabling the use of modern investigation tools such as the force tomography. CONCLUSIONS: The FC_analysis software aims to become a useful tool for the analysis of force curves maps collected using custom or commercial Atomic Force Microscopes, and is especially useful in those cases for which the producer doesn't release a dedicated software.

Methods for Atomic Force Microscopy of Biological and Living Specimens.

Two main precautions must be taken into account to obtain high-resolution morphological and nanomechanical characterization of biological specimens with an atomic force microscope: the tip-sample interaction and the sample-substrate adhesion. In this chapter we discuss the necessary steps for a correct preparation of three types of biological samples: erythrocytes, bacteria, and osteoblasts. The main goal is to deliver reproducible protocols to produce good cellular adhesion and minimizing the morphological alterations of the specimens.

Modelling the pathogenesis of Myotonic Dystrophy type 1 cardiac phenotype through human iPSC-derived cardiomyocytes.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease, autosomal dominant, caused by a CTG repeat expansion in DMPK gene. We assessed the appropriateness of patient-specific induced pluripotent stem cell-derived cardiomyocytes (CMs) as a model to recapitulate some aspects of the pathogenetic mechanism involving cardiac manifestations in DM1 patients. Once obtained in vitro, CMs have been characterized for their morphology and their functionality. CMs DM1 show intranuclear foci and transcript markers abnormally spliced respect to WT ones, as well as several irregularities in nuclear morphology, probably caused by an unbalanced lamin A/C ratio. Electrophysiological characterization evidences an abnormal profile only in CMs DM1 such that the administration of antiarrythmic drugs to these cells highlights even more the functional defect linked to the disease. Finally, Atomic Force Measurements reveal differences in the biomechanical behaviour of CMs DM1, in terms of frequencies and synchronicity of the beats. Altogether the complex phenotype described in this work, strongly reproduces some aspects of the human DM1 cardiac phenotype. Therefore, the present study provides an in vitro model suggesting novel insights into the mechanisms leading to the development of arrhythmogenesis and dilatative cardiomyopathy to consider when approaching to DM1 patients, especially for the risk assessment of sudden cardiac death (SCD). These data could be also useful in identifying novel biomarkers effective in clinical settings and patient-tailored therapies.

Behind Resveratrol Stabilization by Carboxymethylated (1,3/1,6)-ß-d-Glucan: Does the Polyphenol Play a Role in Polymer Structural Organization?

Resveratrol stability in solution can be improved by combining the polyphenol with carboxymethylated (1,3/1,6)-ß-d-glucan (CM-glucan), a carbohydrate polymer widely used in the food and pharmaceutical industries. The present work was undertaken to elucidate the mechanism behind this stabilizing effect. The supramolecular structural, physico-chemical and morphological features of the CM-glucan/resveratrol complex have been studied under different physical and chemical stimuli by means of spectroscopic techniques, microscopy and physical methods such as UV-Visible spectroscopy (UV-Vis), spectrofluorimetry, Circular Dichroism (CD), Infrared spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). Our experimental data indicate that CM-glucan conformational organized architecture in aqueous solution is enhanced in the presence of resveratrol, suggesting that the polyphenol is able to confer a high degree of order to the polymer by a probable cooperative structural organization that results in a long term stabilization for the polyphenol.

MicroRNA-155 in serum-derived extracellular vesicles as a potential biomarker for hematologic malignancies - a short report.

PURPOSE: The use of extracellular vesicles (EVs) from body fluids as "liquid biopsies" is emerging as a promising approach for the diagnosis, prognosis and therapeutic monitoring of cancer patients. MicroRNA-155 (miR155), a non-coding transcript of the B-cell integration cluster (BIC) gene, has been reported to play a critical role in the pathogenesis of several types of hematologic malignancies (HMs) in which high miR155 levels have been found. At yet, however, the EV miR155 level and its putative clinical relevance in sera of HM patients have not been reported. METHODS: EVs from sera of representative patients with eight different HMs and healthy subjects (controls) were isolated using differential centrifugation. The identity and quality of the EVs were verified by atomic force and transmission electron microscopy. The EV miR155 levels were measured by quantitative RT-PCR. The sensitivity, specificity and area under the curve (AUC) of differences in EV miR155 levels were determined using ROC curve analyses. RESULTS: We found that the EV miR155 levels were significantly higher in chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and Waldenström's macroglobulinemia (WM) cases compared to controls. Conversely, we found that the EV miR155 levels were significantly lower in myelodysplastic syndrome (MDS) and multiple myeloma (MM) cases. No differences were found in follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) or Hodgkin's Lymphoma (HL) cases compared to controls. EV miR155 ROC curve analyses revealed significantly different patterns in CLL and AML cases compared to controls, and in AML cases compared to MDS cases (p = 0.004, p = 0.01 and p = 0.04, respectively). In addition, we found that high EV miR155 levels correlated with high white blood cell counts in AML patients. CONCLUSION: Our data indicate that EV miR155 may serve as an attractive new, non-invasive diagnostic biomarker in human hematologic malignancies.

Vascular dysfunction-associated with Alzheimer's disease.

Our attention is focused on the study of a new model based on the red blood cell (RBC) and on its interaction with amyloid beta peptide 1-42 (Aß). RBC are highly deformable to assist blood flow in the microcirculation. For this reasons RBC abnormalities could contribute to Alzheimer's disease (AD) by obstructing oxygen delivery to brain, causing hypoxia. In our work, considering that RBC membrane contains, among blood elements, higher acetylcholinesterase (AChE) levels, we can assume that in blood occurs a mechanism similar to the one which occurs at the neuronal level leading to an increase of Aß toxicity mediated by its binding with AChE, located on the RBC external face. Furthermore, since mechanical properties of RBC membrane are regulated by a number of molecular components of signalling and/or regulatory pathways, of these, particular interest has been addressed toward Nitric Oxide (NO) metabolism, due to its dependence to AChE.

Induced movements of giant vesicles by millimeter wave radiation.

Our previous study of interaction between low intensity radiation at 53.37GHz and cell-size system - such as giant vesicles - indicated that a vectorial movement of vesicles was induced. This effect among others, i.e. elongation, induced diffusion of fluorescent dye di-8-ANEPPS, and increased attractions between vesicles was attributed to the action of the field on charged and dipolar residues located at the membrane-water interface. In an attempt to improve the understanding on how millimeter wave radiation (MMW) can induce this movement we report here a real time evaluation of changes induced on the movement of giant vesicles. Direct optical observations of vesicles subjected to irradiation enabled the monitoring in real time of the response of vesicles. Changes of the direction of vesicle movement are demonstrated, which occur only during irradiation with a "switch on" of the effect. This MMW-induced effect was observed at a larger extent on giant vesicles prepared with negatively charged phospholipids. The monitoring of induced-by-irradiation temperature variation and numerical dosimetry indicate that the observed effects in vesicle movement cannot be attributed to local heating.

Structure and function in native and pathological erythrocytes: a quantitative view from the nanoscale.

The red blood cells (RBCs) are among the most simple and less expensive cells to purify; for this reason and for their physiological relevance, they have been extensively studied with a variety of techniques. The picture that results is that these cells have several peculiarities including extreme mechanical performances, relatively simple architecture, biological relevance and predictable behavior that make them a perfect laboratory of testing for novel techniques, methodologies and ideas. These include the re-evaluation of old concepts, such as the relationship between structure and function (which is one of the guideline of this report) but considered at the cellular level. The studies reported on this paper, indeed, exploit the full potential of an high resolution quantitative microscopy such as the atomic force microscopy (AFM) to investigate different aspect of the erythrocytes' life, death and interaction with the environment. Indeed, the erythrocytes have a special relationship with the environment that is able to deeply influence their morphology as consequence of alteration of their biochemical or biophysical status. In this context the conditions under which the erythrocytes can be considered as biochemically programmable systems have been investigated by analyzing different environmentally induced alteration of the cell's morphology and comparing the results with naturally occurring pathological morphologies. This class of studies takes great advantage by the additional consideration of the nanomechanical properties of the cells. These latter are particularly important for the cell functionality and are shown to be of practical usefulness to discriminate and partition environmental effects charging different cellular structure (e.g. membrane or membrane-skeleton). Moreover, the development of novel morphological parameter can be important to push the level of investigation on the RBCs' status towards the molecular level. In particular, we describe the introduction and use of the plasma membrane roughness as a morphometric parameter of simple derivation from the AFM images and that results sensitive to the structural integrity of the cells' membrane-skeleton. This offer a remarkable opportunity to investigate the relationship between structure and function in normal and pathological cells by using a morphometric parameter that probes the cell surface at the nanoscale level. At last, a complex but physio-pathologically important phenomenon such as the erythrocytes aging was considered. To properly analyze the many variation that the cells experience during the whole aging path we used all the parameters that the AFM can provides: quantitative imaging, analysis of the membrane roughness and local measure of the nanomechanical properties analyzed together with biochemical parameter such as the ATP content. The picture that emerged is that the aging path is triggered by the ATP intracellular concentration that influence the membrane-skeleton structure and the support exerted on the plasma membrane. The consequences of the membrane-skeleton involvement can be monitored by AFM and showed the occurrence of peculiar morphologies and morphological defects that appear in the very place where the membrane-skeleton contact with the membrane became loose. As a whole, the collected data enable to describe the entire phenomenon as a sequence of morphological intermediates following one another along the aging path.

The how, when, and why of the aging signals appearing on the human erythrocyte membrane: an atomic force microscopy study of surface roughness.

We recently developed an atomic force microscopy-based protocol to use the roughness of the plasma membrane of erythrocytes (red blood cells, RBCs) as a morphological parameter, independently from the cell shape, to investigate the membrane-skeleton integrity in healthy and pathological cells. Here we apply the method to investigate a complex physiological phenomenon, the RBCs aging, that plays a major role in the regulation of the RBCs' turnover. The aging, monitored morphologically and biochemically, has been accelerated and modulated by preventing oxidative stresses as well as the effects of proteases and divalent cations, and by artificially consuming the intracellular adenosine triphosphate. The collected data evidence that the progression of aging causes a drastic decrease of the measured roughness that is diagnostic of a progressive, adenosine triphosphate-dependent alteration of the membrane-skeleton properties. Finally, the degree of reversibility of such effects has been investigated as a function of aging time, enabling the detection of irreversible transformation in the RBCs' structure and metabolism.

Erythrocyte death in vitro induced by starvation in the absence of Ca(2+).

Human erythrocytes (RBCs), stored at 4 degrees C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R(rms)) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4 days of storage at 4 degrees C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4-5 days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties - as measured by R(rms) - are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (> or =90%) depleted in an irreversible way of the energetic stores.

The response of giant phospholipid vesicles to millimeter waves radiation.

Due to the increasing interest in millimeter waves (MMW) applications in medicine and telecommunications, the investigation of their potential biological effects is of utmost importance. Here we report results of the study of interaction between low-intensity radiation at 53.37 GHz and giant vesicles. Direct optical observations of vesicles subjected to irradiation enabled the monitoring in real time of the response of vesicles. Physical changes of vesicles, i.e. elongation, induced diffusion of fluorescent dye di-8-ANEPPS, and increased attractions between vesicles are demonstrated. These effects are reversible and occur only during irradiation with a "switch on" of the effect requiring a short time. Since the average temperature change was very small the effects could not be attributed to thermal mechanisms. We assume that the interaction of MMW with lipid membrane leads to changes at the membrane-water interface, where charged and dipolar residues are located.