Fluctuation Spectroscopy Analysis of Glucose Capped Gold Nanoparticles.

In this work, we report the synthesis and biophysical studies carried out on a new kind of biocompatible and very stable gold nanoparticle (GNP) stabilized with glucose through a PEG linker (AuNP-PEG-Glu). The synthetic path was optimized to obtain nanoparticles of controlled sizes. ζ-potential and dynamic light scattering measurements allowed assessment of the nanodimension, dispersity, surface charge, and stability of our GNPs. Confocal microscopy demonstrated qualitatively that glucose molecules are successfully bonded to GNP surfaces. For our study, we selected nanoparticles with diameter in a range that maximizes the internalization efficiency in cells (40 nm). A detailed investigation about the biophysical proprieties of AuNP-PEG-Glu was carried out by means of fluorescence correlation spectroscopy (FCS) and orbital tracking techniques. This work gives new insights about the uptake mechanism of gold nanoparticles capped with glucose molecules.

Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells.

BACKGROUND: Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease. METHODS: We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging. RESULTS: Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional. CONCLUSIONS: We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells. GENERAL SIGNIFICANCE: The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinson's disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Measuring protein-membrane interaction through radial fluorescence correlation in 2 dimensions.

The cell membrane has a fundamental role in the cell life cycle but there's still much to be learned about its heterogeneous structure, regulation, and protein interaction. Additionally, the protein-membrane interaction is often overlooked when studying specific protein dynamics. In this work, we present a new tool for a better understanding of protein dynamics and membrane function using live cells and fast non-invasive techniques without the need for individual particle tracking. To this end, we used the 2D-pair correlation function (2D-pCF) to study protein interactions across cellular membranes. We performed numerical simulations and confocal experiments using a GAP-mEGFP fusion construct known to interact with the plasmatic membrane. Our results demonstrate that based on a quantitative correlation analysis as the 2D pair correlation of the signal intensities, is possible to characterize protein-membrane interactions in live systems and real-time. Combining experimental and numerical results this work presents a new powerful approach to the study of the dynamic protein-membrane interaction.

Experiences of parents and caregivers in pediatric intensive care units: A qualitative study.

BACKGROUND: In pediatric intensive care units (PICUs), parents and healthcare professionals attend to children who verbally and non-verbally express their pain and suffering, fears, anxieties, desires, and wishes in complex intensive care situations. What can we learn from these experiences to improve the way we can take care of and support children? OBJECTIVES: The main objective of this clinical ethics study was to focus on the experience stories of parents during their child's hospitalization in a PICU, to analyze their discourse, and to propose an ethical perspective. METHOD: The current research collects the experience reports of parents during their child's hospitalization in a PICU and those of the caregivers who treated them. A total of 17 semi-directive interviews were conducted in the PICU of the Nantes University Hospital from November 2017 to June 2019. Each interview lasted around 1 h. The main results of our study are analyzed and informed by the four ethical principles of T. Beauchamp and J. Childress: autonomy, beneficence, non-maleficence, and justice. RESULTS: The interviews highlighted the difficulties encountered by parents during the hospitalization of their children, such as the distance between their home and the hospital, the technicality of PICU environment, and the difficulty in finding their place as parents. For medical and paramedical teams, their main concerns are undoubtedly to improve the coherence and continuity of their stand toward parents, to promote parental autonomy, and to remain fully aware of the profound existential changes that the child's illness brings about for parents: It is the caregivers' duty to take this into account and to respect the parents' rhythm as much as possible. CONCLUSIONS: The main disagreements between healthcare teams and parents, where they exist, are communication problems that are easily controlled, for the most part, by caregivers.

Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation.

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.

The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging.

Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.

Comprehensive correlation analysis for super-resolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector.

The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.

An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Modular Polymer Biosensors by Solvent Immersion Imprint Lithography.

We recently demonstrated Solvent Immersion Imprint Lithography (SIIL), a rapid benchtop microsystem prototyping technique, including polymer functionalization, imprinting and bonding. Here, we focus on the realization of planar polymer sensors using SIIL through simple solvent immersion without imprinting. We describe SIIL's impregnation characteristics, including an inherent mechanism that not only achieves practical doping concentrations, but their unexpected 2-fold enhancement compared to the immersion solution. Subsequently, we developed and characterized optical sensors for detecting molecular O2. To this end, a substantially high dynamic range is reported, including its control through the immersion duration, a manifestation of SIIL's modularity. Overall, SIIL exhibits the potential of improving the operating characteristics of polymer sensors, while significantly accelerating their prototyping, as it requires a few seconds of processing and no need for substrates or dedicated instrumentation. These are critical for O2 sensing as probed by way of example here, as well as any polymer permeable reactant.

Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions.

The effect of cholesterol on microheterogeneity of liposomes obtained from saturated and unsaturated phospholipids was studied by measuring the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Data obtained by frequency domain fluorometry have been analyzed either by discrete exponential or continuous lifetime distribution approaches. In egg phosphatidylcholine liposomes, the addition of cholesterol increases the lifetime value or the centre of the lifetime distribution. At high cholesterol concentration, good fits are obtained using a monomodal distribution analysis or single exponential component. At low cholesterol concentration an additional short component of low fractional intensity must be included to obtain a good fit. In dipalmitoylphosphatidylcholine, the addition of cholesterol decreases the long lifetime component centre value both in the gel and in the liquid-crystalline state. The DPH lifetime value is sensitive to the dielectric constant of the probe microenvironment, and cholesterol has been shown to modify water penetration in the bilayer. Using this information our data indicate that cholesterol affects the polarity of the microenvironment in liposomes of unsaturated phosphatidylcholine and saturated phosphatidylcholine in different ways. Although the major conclusions of this paper are obtained using changes of the distribution centre upon cholesterol addition, there are also preliminary indications that the lifetime distribution width decreases as cholesterol is added. We have interpreted this observation as being due to the homogenizing effect of cholesterol.

Fluorescence lifetime distributions of DNA-4',6-diamidino-2-phenylindole complex.

Time-resolved fluorescence of 4',6-diamidino-2-phenylindole (DAPI) complexes show that for a homogeneous polymer (polyd(AT) or polyd(A).polyd(T)) at high P/D (phosphate/dye) ratio, a single exponential component adequately describes the fluorescence decay. For the AT polymers at low P/D ratio or for native DNA, the decay cannot be described by a single-exponential term. A continuous distribution of lifetime values of Gaussian shape gives a good fit to the decay data. We propose that the lifetime distribution method for the analysis of the fluorescence decay of DNA-DAPI complexes provides a useful method of characterizing the microheterogeneity of site binding.

Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry.

The fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) in K562 cell membranes has been studied using multifrequency phase and modulation fluorimetry. The DPH decay data collected at various modulation frequencies were analysed by assuming either a model of discrete exponential components or a model of continuous lifetime distribution. The fits showed smaller values of the reduced chi square using the model of continuous lifetime distribution. The K562 cell membranes dynamics were investigated during the cell differentiation along the erythroid pathway. By using the continuous lifetime distribution method for the analysis of the DPH decay, marked variations were observed during the four initial days of the erythroid differentiation. Namely, the width of the DPH lifetime distribution increased by a factor of about two, while the center value of the distribution remained constant. By using the discrete exponential components model for the analysis of the DPH decay no variations were observed during the K562 differentiation.

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins.

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene reveal the effect of cholesterol on the microheterogeneity of erythrocyte membrane.

The fluorescence decay of 1,6 diphenyl-1,3,5-hexatriene (DPH) has been used to characterize aspects of the erythrocyte membrane structure related to the microheterogeneity of the lipid bilayer. The DPH decay has been studied using frequency domain fluorometry and the data analyzed either by a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values. The main intensity fraction was associated with a lifetime value centered at about 11 ns in the erythrocyte membrane, but a short component of very low fractional intensity had to be considered to obtain a good fit to the data. The lifetime value of the long component was insensitive to temperature, while the width of the distribution decreased with increasing temperature. In multilamellar liposomes prepared from phospholipids extracted from the erythrocytes, the long lifetime component showed a temperature dependence. The depletion of 27% of the cholesterol in the erythrocyte membrane induced a broadening of the distribution, suggesting a homogenizing effect of cholesterol. This effect has also been detected in egg phosphatidylcholine at a very low cholesterol/phospholipid molar ratio. The role of cholesterol on membrane heterogeneity is discussed in relation to the effect of cholesterol on water penetration.

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids.

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.

Infrared stretching frequencies of CO in carbomonoxyhemoglobin from trout.

The infrared spectra of the carbomonoxy derivatives of the hemoglobin components I and IV from trout have been measured in the CO stretching frequency region using a high resolution infrared spectrometer. The CO stretching frequency of Hb I CO is very close to that of carbomonoxy human hemoglobin and is pH-independent. In contrast, the CO stretching frequency of Hb IV CO is higher and shows a small but significant pH dependence in the range 6.2-7.8. These results point to a decreased strength of the iron-CO bond in Hb IV CO at low pH, in agreement with the conclusions drawn from the reported difference spectra of Hb IV CO as a function of pH.

Chlorophyll a fluorescence lifetime distributions in open and closed photosystem II reaction center preparations.

We have measured the decay of chlorophyll a fluorescence at 4 degrees C under anaerobic conditions in stabilized photosystem II reaction center complex isolated from spinach, using multifrequency (2-400 MHz) cross-correlation phase fluorometry. Examination of our data shows that although the fluorescence decay of open reaction centers (i.e., when both the electron donor P-680 and the electron acceptor pheophytin are capable of engaging in charge separation) can be analyzed as a multiexponential decay, another representation of the data is obtained when the decay is analyzed using a continuous distribution of lifetimes. Our results on the open reaction center differ from the two lifetime components of 25 ps and 35 ns published by Mimuro et al. (Biochim. Biophys. Acta 933 (1988) 478-486) for the D1-D2-cytochrome b-559 complex, obtained for F682 at 4 degrees C by a time-resolved photon-counting spectrofluorometer. When the reaction centers are closed by pretreatment with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of reduced pheophytin, a dramatic decrease in the contribution of the slow lifetime component(s) is observed. These results suggest that the slow distribution lifetime component(s) in the 5-20 ns range originate(s) in the back reaction of the charge separated state. On the other hand, the fast lifetime component(s) in the picosecond range may be only partially related to the charge separation, since no dramatic change is observed upon closure of the reaction center. Perhaps, this component is related, in part, to the excitation energy migration among the various chromophores in the reaction center preparations.

Surface properties of cholesterol-containing membranes detected by Prodan fluorescence.

The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase model for cholesterol-containing membranes and show that the presence of cholesterol, in addition to modification to the phase state of the hydrophobic portion of the bilayer, strongly affects the packing and the polarity of the membrane hydrophobic-hydrophilic interface.

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.