Exploiting endocytosis for transfection of mRNA for cytoplasmatic delivery using cationic gold nanoparticles.

Introduction: Gene therapy holds promise to cure various diseases at the fundamental level. For that, efficient carriers are needed for successful gene delivery. Synthetic 'non-viral' vectors, as cationic polymers, are quickly gaining popularity as efficient vectors for transmitting genes. However, they suffer from high toxicity associated with the permeation and poration of the cell membrane. This toxic aspect can be eliminated by nanoconjugation. Still, results suggest that optimising the oligonucleotide complexation, ultimately determined by the size and charge of the nanovector, is not the only barrier to efficient gene delivery. Methods: We herein develop a comprehensive nanovector catalogue comprising different sizes of Au NPs functionalized with two different cationic molecules and further loaded with mRNA for its delivery inside the cell. Results and Discussion: Tested nanovectors showed safe and sustained transfection efficiencies over 7 days, where 50 nm Au NPs displayed the highest transfection rates. Remarkably, protein expression was increased when nanovector transfection was performed combined with chloroquine. Cytotoxicity and risk assessment demonstrated that nanovectors are safe, ascribed to lesser cellular damage due to their internalization and delivery via endocytosis. Obtained results may pave the way to design advanced and efficient gene therapies for safely transferring oligonucleotides.

Rapid identification of in vitro cell toxicity using an electrochemical membrane screening platform.

This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.

Detection and estimating the blood accumulation volume of brain hemorrhage in a human anatomical skull using a RF single coil.

OBJECTIVE: An experimental study for testing a simple robust algorithm on data derived from an electromagnetic radiation device that can detect small changes in the tissue/fluid ratio in a realistic head configuration. METHODS: Changes in the scattering parameters (S21) of an inductive coil resulting from injections of chicken blood in the 0-18 ml range into calf brain tissue in a human anatomical skull were measured over a 100-1,000 MHz frequency range. RESULTS: An algorithm that combines amplitude and phase results was found to detect changes in the tissue/fluid ratio with 90% accuracy. An algorithm that estimated the injected blood volume was found to have a 1-4 ml average error. This demonstrates the possibility of the inductive coil-based device to possess a practical ability to detect a change in the tissue/fluid ratio in the head. SIGNIFICANCE: This study is an important step towards the goal of building an inexpensive and safe device that can detect an early brain hemorrhagic stroke.

Multifrequency Analysis of Single Inductive Coil Measurements Across a Gel Phantom Simulation of Internal Bleeding in the Brain.

The present study is part of an ongoing effort to develop a simple diagnostic technology for detecting internal bleeding in the brain, which can be used in lieu or in support of medical imaging and thereby reduce the cost of diagnostics in general, and in particular, would make diagnostics accessible to economically disadvantaged populations. The study deals with a single coil inductive device to be used for detecting cerebral hemorrhage. It presents a first-order experimental study that examines the predictions of our recently published theoretical study. The experimental model employs a homogeneous cylindrical phantom in which internal head bleeding was simulated by way of a fluid inclusion. We measured the changes in amplitude and phase across the coil with a network vector analyzer as a function of frequency (100-1,000 MHz), volume of blood simulating fluid, and the site of the fluid injection. We have developed a new mathematical model to statistically analyze the complex data produced in this experiment. We determined that the resolution for the fluid volume increase following fluid injection is strongly dependent on frequency as well as the location of liquid accumulation. The experimental data obtained in this study supports the predictions of our previous theoretical study, and the statistical analysis shows that the simple single coil device is sensitive enough to detect changes due to fluid volume alteration of two milliliters. Bioelectromagnetics. 2020;41:21-33 © 2019 Bioelectromagnetics Society.

Functional, inflammatory and interstitial impairment due to artificial stone dust ultrafine particles exposure.

OBJECTIVE: Artificial stone dust (ASD) contains high levels of ultrafine particles (UFP <1 µm) which penetrate deeply into the lungs. This study aimed to demonstrate the direct effect of UFP in the lungs of ASD-exposed workers on functional inflammatory and imaging parameters. METHODS: 68 workers with up to 20 years of ASD exposure at the workplace were recruited from small enterprises throughout the country and compared with 48 non-exposed individuals. Pulmonary function test (PFT), CT, induced sputum (IS) and cytokine analyses were performed by conventional methods. The CT scans were evaluated for features indicative of silicosis in three zones of each lung. UFP were quantitated by the NanoSight LM20 system (NanoSight, Salisbury) using the Nanoparticle Tracking Analysis. Interleukin (IL)-6, IL-8 and tumour necrosis factor alpha (TNF-α) levels were measured by Luminex (R&D Systems). RESULTS: Thirty-four patients had CT scores between 0 and 42, and 29 of them were diagnosed with silicosis. Content of the UFP retrieved from IS supernatants correlated negatively with the PFT results (total lung capacity r=-0.347, p=0.011; forced expiratory volume in 1 s r=-0.299, p=0.046; diffusion lung carbon monoxide in a single breath r=-0.425, p=0.004) and with the CT score (r=0.378, p=0.023), and with the inflammatory cytokines IL-8 (r=0.336, p=0.024), IL-6 (r=0.294, p=0.065) and TNF-α (r=0.409, p=0.007). Raw material of ASD was left to sedimentate in water for <15 min, and 50% of the floating particles were UFP. A cut-off of 8×106 UFP/mL in IS samples had a sensitivity of 77% to predict pulmonary disease. CONCLUSIONS: This is the first demonstration of an association between UFP-related decreased PFT results, worsening of CT findings and elevation of inflammatory cytokines, which may be attributed to high-dose inhalation of UFP of ASD at the workplace.

Non-Contact Monitoring of Temporal Volume Changes of a Hematoma in the Head by a Single Inductive Coil: A Numerical Study.

OBJECTIVE: This numerical study was designed to evaluate the feasibility of using an inductive coil for monitoring the changes in the volume of a hematoma in the head in situ and to compare the inductive coil performance to that of a spiral antenna based on the radar principle. METHODS: Numerical analysis was used to solve the complete set of Maxwell's equations in full three-dimensional anatomical model of a head and brain with data on clinical occurrence of hematomas from the clinical literature, for frequencies of 100 MHz, 500 MHz, and 1 GHz. RESULTS: 1) The analysis shows that the spiral radar antenna provides a better resolution when the antenna can be placed exactly facing the center of the volume of blood. Under any other circumstance, the inductive coil has a better resolution at both 500 MHz and 1 GHz. 2) The induction coil is more sensitive to rotation artifacts than the spiral antenna. 3) Single frequency measurements do not provide conclusive results. CONCLUSION: The inductive coil has the ability to monitor small changes in the volume of a hematoma in the head. However, multifrequency measurements are required for correct diagnostic. SIGNIFICANCE: This study provides a new, low-cost alternative to the conventional medical imaging for monitoring the hematoma increase.

Radar based technology for non-contact monitoring of accumulation of blood in the head: A numerical study.

BACKGROUND: This theoretical study examines the use of radar to continuously monitor "accumulation of blood in the head" (ACBH) non-invasively and from a distance, after the location of a hematoma or hemorrhage in the brain was initially identified with conventional medical imaging. Current clinical practice is to monitor ABCH with multiple, subsequent, conventional medical imaging. The radar technology introduced in this study could provide a lower cost and safe alternative to multiple conventional medical imaging monitoring for ACBH. MATERIALS AND METHODS: The goal of this study is to evaluate the feasibility of using radar to monitor changes in blood volume in the brain through a numerical simulation of ACBH monitoring from remote, with a directional spiral slot antennae, in 3-D models of the brain. The focus of this study is on evaluating the effect of frequencies on the antennae reading. To that end we performed the calculations for frequencies of 100 MHz, 500 MHz and 1 GHz. RESULTS AND DISCUSSION: The analysis shows that the ACBH can be monitored with radar and the monitoring resolution improves with an increase in frequency, in the range studied. However, it also appears that when typical clinical dimensions of hematoma and hemorrhage are used, the variable ratio of blood volume radius and radar wavelength can bring the measurements into the Mie and Rayleigh regions of the radar cross section. In these regions there is an oscillatory change in signal with blood volume size. For some frequencies there is an increase in signal with an increase in volume while in others there is a decrease. CONCLUSIONS: While radar can be used to monitor ACBH non-invasively and from a distance, the observed Mie region dependent oscillatory relation between blood volume size and wavelength requires further investigation. Classifiers, multifrequency algorithms or ultra-wide band radar are possible solutions that should be explored in the future.

CompNanoTox2015: novel perspectives from a European conference on computational nanotoxicology on predictive nanotoxicology.

A first European Conference on Computational Nanotoxicology, CompNanoTox, was held in November 2015 in Benahavís, Spain with the objectives to disseminate and integrate results from the European modeling and database projects (NanoPUZZLES, ModENPTox, PreNanoTox, MembraneNanoPart, MODERN, eNanoMapper and EU COST TD1204 MODENA) as well as to create synergies within the European NanoSafety Cluster. This conference was supported by the COST Action TD1204 MODENA on developing computational methods for toxicological risk assessment of engineered nanoparticles and provided a unique opportunity for cross fertilization among complementary disciplines. The efforts to develop and validate computational models crucially depend on high quality experimental data and relevant assays which will be the basis to identify relevant descriptors. The ambitious overarching goal of this conference was to promote predictive nanotoxicology, which can only be achieved by a close collaboration between the computational scientists (e.g. database experts, modeling experts for structure, (eco) toxicological effects, performance and interaction of nanomaterials) and experimentalists from different areas (in particular toxicologists, biologists, chemists and material scientists, among others). The main outcome and new perspectives of this conference are summarized here.

Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation.

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label-free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive-index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive-index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label-free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label-free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

Artificial stone dust-induced functional and inflammatory abnormalities in exposed workers monitored quantitatively by biometrics.

The manufacture of kitchen and bath countertops in Israel is based mainly on artificial stone that contains 93% silica as natural quartz, and ∼3500 workers are involved in cutting and processing it. Artificial stone produces high concentrations of silica dust. Exposure to crystalline silica may cause silicosis, an irreversible lung disease. Our aim was to screen exposed workers by quantitative biometric monitoring of functional and inflammatory parameters. 68 exposed artificial stone workers were compared to 48 nonexposed individuals (controls). Exposed workers filled in questionnaires, and all participants underwent pulmonary function tests and induced sputum analyses. Silica was quantitated by a Niton XL3 X-ray fluorescence spectrometer. Pulmonary function test results of exposed workers were significantly lower and induced sputa showed significantly higher neutrophilic inflammation compared to controls; both processes were slowed down by the use of protective measures in the workplace. Particle size distribution in induced sputum samples of exposed workers was similar to that of artificial stone dust, which contained aluminium, zirconium and titanium in addition to silica. In conclusion, the quantitation of biometric parameters is useful for monitoring workers exposed to artificial stone in order to avoid deterioration over time.

Detection and controlled depletion of cancer cells using photothermal phase microscopy.

We present a dual-modality technique based on wide-field photothermal (PT) interferometric phase imaging and simultaneous PT ablation to selectively deplete specific cell populations labelled by plasmonic nanoparticles. This combined technique utilizes the plasmonic reaction of gold nanoparticles under optical excitation to produce PT imaging contrast by inducing local phase changes when the excitation power is weak, or ablation of selected cells when increasing the excitation power. Controlling the entire process is carried out by dynamic quantitative phase imaging of all cells (labelled and unlabelled). We demonstrate our ability to detect and specifically ablate in vitro cancer cells over-expressing epidermal growth factor receptors (EGFRs), labelled with plasmonic nanoparticles, in the presence of either EGFR under-expressing cancer cells or white blood cells. The latter demonstration establishes an initial model for depletion of circulating tumour cells in blood. The proposed system is able to image in wide field the label-free quantitative phase profile together with the PT phase profile of the sample, and provides the ability of both detection and selective cell ablation in a controlled environment. Quantitative phase imaging with molecular specificity and specific cell depletion. (a) Label-free quantitative phase profiles of mixed population of EGFR(+) /EGFR(-) cancer cells. (b) When weak modulated PT excitation is applied, selective phase contrast is generated in the modulation frequency only for the EGFR(+) cancer cells labelled with plasmonic nanoparticles. (c) When stronger modulated PT excitation is applied, selective ablation of the EGFR(+) cancer cells labelled with plasmonic nanoparticles occurs. White scalebars represent 10 µm upon sample.

Optimal nano-descriptors as translators of eclectic data into prediction of the cell membrane damage by means of nano metal-oxides.

Systematization of knowledge on nanomaterials has become a necessity with the fast growth of applications of these species. Building up predictive models that describe properties (both beneficial and hazardous) of nanomaterials is vital for computational sciences. Classic quantitative structure-property/activity relationships (QSPR/QSAR) are not suitable for investigating nanomaterials because of the complexity of their molecular architecture. However, some characteristics such as size, concentration, and exposure time can influence endpoints (beneficial or hazardous) related to nanoparticles and they can therefore be involved in building a model. Application of the optimal descriptors calculated with the so-called correlation weights of various concentrations and different exposure times are suggested in order to build up a predictive model for cell membrane damage caused by a series of nano metal-oxides. The numerical data on correlation weights are calculated by the Monte Carlo method. The obtained results are in good agreement with the experimental data.

The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis.

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10=1.1, proceeding through a prompt (<5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10=1.44, accompanied by activation energy Ea=4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10=2.2 and Ea=7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence.

Activation of local and systemic anti-tumor immune responses by ablation of solid tumors with intratumoral electrochemical or alpha radiation treatments.

Cancer, the most devastating chronic disease affecting humankind, is treated primarily by surgery, chemotherapy, and radiation therapy. Surgery and radiotherapy are mainly used for debulking the primary tumor, while chemotherapy is the most efficient anti-metastatic treatment. To control better metastatic cancer, the host immune system should be stimulated. Yet, successful specific stimulation of the immune system against tumors was seldom achieved even in antigenic tumors. Our working hypothesis is that aggressive in situ tumor ablation can release tumor antigens and danger signals, which will enhance anti-tumor T cell responses resulting in the destruction of residual malignant cells in primary tumors and distant metastases. We developed two efficient in situ ablation treatments for solid cancer, which can be used to destroy the primary tumors and stimulate anti-tumor immune responses. The first treatment, electrochemical ablation, is applied through intratumoral electrodes, which deliver unipolar-pulsed electric currents. The second treatment, diffusing alpha-emitters radiation therapy (DaRT), is based on intratumoral (224)Ra-loaded wire(s) that release by recoil its daughter atoms. These short-lived alpha-emitting atoms spread in the tumor and spray it with lethal alpha particles. It was confirmed that these treatments effectively destroy various malignant animal and human primary solid tumors. As a consequence of such tumor ablation, tumor-derived antigenic material was released and provoked systemic T cell-dependent anti-tumor immunological reactions. These reactions conferred protection against a secondary tumor challenge and destroyed remaining malignant cells in the primary tumor as well as in distant metastases. Such anti-tumor immune responses could be further amplified by the immune adjuvant, CpG. Electrochemical ablation or DaRT together with chemotherapy and immunostimulatory agents can serve as treatment protocols for solid metastatic tumors and can be applied instead of or in combination with surgery.

Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).

One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.

Proton-induced endocytosis is dependent on cell membrane fluidity, lipid-phase order and the membrane resting potential.

Recently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4-36°C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones.

Predictive toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods of knowledge discovery from data.

BACKGROUND: Cobalt-ferrite nanoparticles (Co-Fe NPs) are attractive for nanotechnology-based therapies. Thus, exploring their effect on viability of seven different cell lines representing different organs of the human body is highly important. METHODS: The toxicological effects of Co-Fe NPs were studied by in-vitro exposure of A549 and NCIH441 cell-lines (lung), precision-cut lung slices from rat, HepG2 cell-line (liver), MDCK cell-line (kidney), Caco-2 TC7 cell-line (intestine), TK6 (lymphoblasts) and primary mouse dendritic-cells. Toxicity was examined following exposure to Co-Fe NPs in the concentration range of 0.05 -1.2 mM for 24 and 72 h, using Alamar blue, MTT and neutral red assays. Changes in oxidative stress were determined by a dichlorodihydrofluorescein diacetate based assay. Data analysis and predictive modeling of the obtained data sets were executed by employing methods of Knowledge Discovery from Data with emphasis on a decision tree model (J48). RESULTS: Different dose-response curves of cell viability were obtained for each of the seven cell lines upon exposure to Co-Fe NPs. Increase of oxidative stress was induced by Co-Fe NPs and found to be dependent on the cell type. A high linear correlation (R2=0.97) was found between the toxicity of Co-Fe NPs and the extent of ROS generation following their exposure to Co-Fe NPs. The algorithm we applied to model the observed toxicity belongs to a type of supervised classifier. The decision tree model yielded the following order with decrease of the ranking parameter: NP concentrations (as the most influencing parameter), cell type (possessing the following hierarchy of cell sensitivity towards viability decrease: TK6 > Lung slices > NCIH441 > Caco-2 = MDCK > A549 > HepG2 = Dendritic) and time of exposure, where the highest-ranking parameter (NP concentration) provides the highest information gain with respect to toxicity. The validity of the chosen decision tree model J48 was established by yielding a higher accuracy than that of the well-known "naive bayes" classifier. CONCLUSIONS: The observed correlation between the oxidative stress, caused by the presence of the Co-Fe NPs, with the hierarchy of sensitivity of the different cell types towards toxicity, suggests that oxidative stress is one possible mechanism for the toxicity of Co-Fe NPs.

Low electric fields induce ligand-independent activation of EGF receptor and ERK via electrochemical elevation of H(+) and ROS concentrations.

Physiological electric fields are involved in many biological processes and known to elicit their effects during long exposures ranging from a few hours to days. Following exposure to electric fields of physiological amplitude, epidermal growth factor receptor (EGFR) was demonstrated to be redistributed and upregulated with further intracellular signaling such as the MAPK signaling cascade. In our study we demonstrated EGFR activation and signaling induced by short train of pulsed low electric field (LEF) (10V/cm, pulse-width 180µs, 500Hz, 2min) in serum-free medium, following 24-hour starvation, and in the absence of exogenous EGF ligand, suggesting a ligand-independent pathway for EGFR activation. This ligandless activation was further confirmed by using neutralizing antibodies (LA1) that block the EGFR ligand-binding site. EGFR activation was found to be EGFR kinase dependent, yet with no dimerization following exposure to LEF. ERK activation was found to be mainly a result of EGFR downstream signaling though it partially occurred via EGFR-independent way. We demonstrate that reactive oxygen species and especially decrease in pH generated during exposure to LEF are involved in EGFR ligandless activation. We propose a possible mechanism for the LEF-induced EGFR ligand-independent activation and show activation of other receptor tyrosine kinases following exposure to LEF.

Actin-cytoskeleton rearrangement modulates proton-induced uptake.

Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process.

Enhancing pigmentation via activation of A3 adenosine receptors in B16 melanoma cells and in human skin explants.

A3 adenosine receptor, A3AR, belongs to the Gi proteins coupled receptors. Activation of A3AR by its agonist, IB-MECA, decreases cAMP and was expected to reduce melanin level. Unexpectedly, B16 melanoma cells exposed to IB-MECA increased melanin levels in a dose-dependent manner. Human skin explants exposed to IB-MECA showed an increase in DOPA positive cells and in melanin deposition in keratinocytes. The agonist induced AKT phosphorylation, leading to a rapid translocation of the transcription factor MiTF towards the nucleus. DOPA oxidase activity and melanin levels induced by IB-MECA were further enhanced by PD98509, an inhibitor ERK signalling pathway. Our study shows that IB-MECA decreases cAMP while inducing melanogenesis. The proposed mechanism involves activation of PI3K/AKT signalling pathway by ß/γ subunits of the G protein coupled to A3AR. The increase in melanin level in human skin explants suggests that IB-MECA may be a potential candidate to the treatment of hypopigmentation of skin.