Study on Single-molecule Biophysics and Biochemistry in Dilute Liquids and Live Cells without Immobilization or Significant Hydrodynamic Flow: The Thermodynamic Single-molecule Demon.

Since mathematics provides a way to answer questions about the thermodynamic jitter in a clear, rational manner, with evidence to support it, mathematics is the reliable method necessary to get the best information on the movement of a single molecule / a single particle at the molecular scale in dilute liquids and live cells without immobilization or hydrodynamic flow. The Brownian movement (normal diffusive systems) and generally the thermodynamic jitter (anomalous diffusive systems) are ultimately the direct or indirect cause of every measurement signal at the molecular scale in diffraction limited and unlimited optical systems in dilute liquids and live cells without immobilization or hydrodynamic flow. For example, emitted photons are the epiphenomenon of the underlying process of thermodynamic jitter of single molecules / single particles at the molecular scale. The key question is: How far apart do two molecules / two particles have to be in the time domain so that the required degree of separation between the two individual molecules / the two individual particles can be quantified at the molecular scale in order to distinguish them as separate entities without immobilization or hydrodynamic flow? The Földes-Papp's limits of the singlemolecule time resolution in dilute liquids and live cells without immobilization or hydrodynamic flow are the exact answers. The diffusive process is complicated and not minimalist. A minimalist model has a third possibility, it may be right but irrelevant.

Visualization of subdiffusive sites in a live single cell.

We measured anomalous diffusion in human prostate cancer cells which were transfected with the Alexa633 fluorescent RNA probe and co-transfected with enhanced green fluorescent protein-labeled argonaute2 protein by laser scanning microscopy. The image analysis arose from diffusion based on a "two-level system". A trap was an interaction site where the diffusive motion was slowed down. Anomalous subdiffusive spreading occurred at cellular traps. The cellular traps were not immobile. We showed how the novel analysis method of imaging data resulted in new information about the number of traps in the crowded and heterogeneous environment of a single human prostate cancer cell. The imaging data were consistent with and explained by our modern ideas of anomalous diffusion of mixed origins in live cells. Our original research presented in this study is significant as we obtained a complex diffusion mechanism in live single cells.

Personalised Healthcare: The DiMA Clinical Model.

Large-scale application of Personalized Medicine requires a multi-disciplinal environment allowing synergic cooperation among different competences. Strict collaboration between medical and medical sciences, as informatics, ethics, politics, are needed to face the challenges of one-sized healthcare. In spite of the increasing interest, how this system can be globally realized remains tentative. Yet, a relatively small, Personalised Healthcare Service is providing a proof-of principle organizational model to guide implementation into clinical practice.

A Retrospective Study on Advanced Maternal Age and Assisted Reproductive Techniques, Medico-Legal Advice, "Food for Thought".

BACKGROUND: Pregnancy in advanced reproductive age is nowadays part of the social and welfare scenario. The effects and assessment of the risks and complications in women over the age of 43 must still be more specifically defined. The aim of this study is to compare the outcomes between spontaneous pregnancies with those induced by assisted reproductive technology (ART) in women ≥ 43 years. METHODS: This retrospective observational study enrolled 114 women with an age of ≥ 43 divided as follows: 74 with spontaneous pregnancies and 40 with ART-induced pregnancy. For statistical analysis, a t-test was used to compare the parameters analyzed for quantitative variables and χ2 was used for qualitative variables. A p-value ≤ 0.05 was considered statistically significant. Statistical Analysis was performed using the program SPSS 16.0 for Windows. RESULTS: The statistically significant differences between IVF and spontaneous pregnancy groups were respectively: gestational hypertension (30% vs 6.8%), preeclampsia (17.5% vs 2.7%), preterm delivery (47.5% vs 13.5%), IUGR (17.5% vs 4.1%), caesarian section (95% vs 70.3%), length of recovery (8.6±7.2 vs 5.9±3) and mean birth weight (2641± 695 g vs 3207±496 g). CONCLUSION: Women in advanced reproductive age (≥ 43 years) who undergo assisted fertilization procedures are at a higher risk of complications compared to women of the same age with spontaneous pregnancies.

Individual macromolecule motion in a crowded living cell.

The application of the general model of the law of mass action itself, of the achievement of dynamic, statistical equilibria, has led to great successes in describing the theory of single-molecule biophysics and biochemistry based on individually and freely diffusing molecules in dilute liquid and crowded living cells. For example, anomalous diffusion is a general phenomenon in living cells. There is solid evidence for analyzing fluorescence correlation and dual color fluorescence crosscorrelation spectroscopy data (FCS and dual color FCCS) in cellular applications by equations based on anomalous subdiffusion. Using equations based on normal diffusion causes artifacts of the fitted biological system response parameters and of the interpretations of the FCS and dual color FCCS data in the crowded environment of living cells. Equations based on normal diffusion are not valid in living cells. The original article embraces the status of the experimental situation and touches obstacles that still hinder the applications of single molecules in the cellular environment.

Amniotic fluid embolism: what level of scientific evidence can be drawn? A systematic review.

Amniotic fluid embolism (AFE) is a rare and severe obstetric emergency and a significant cause of maternal mortality in developed countries and its incidence varies according to different studies. Presently, advances in the understanding of this pathology continue to be slowed down for the absence of generally accepted diagnostic criteria, the clinical analogies of this entity to other types of acute dangerous maternal illnesses and the presence of a wide range of disease severity. The aim of this review has been to evaluate the incidence of AFE, the role of possible risk factors, the clinical presentation (signs and symptoms) and outcome. Secondly the authors reviewed the management of these very difficult patients, including treatments and interventions in order to extrapolate sharable recommendations for the management of these complicated patients.

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

Measurements of single molecules in solution and live cells over longer observation times than those currently possible: the meaningful time.

Monitoring translational diffusion of single molecules in solution or in a living cell, particularly DNA and proteins brings valuable information unperturbed by interaction with an artificial surface. The article derives theoretical relationships for time intervals during which just one molecule in the effective probe region can be studied, the time we call meaningful time. This time is greater than the transit time of the molecule through the detection volume, as a single molecule will likely reenter the detection volume several times during measurement. From the infinitely stretched molecular Poisson distribution of single molecules or particles, we select the contribution of the selfsame molecule or particle by applying rules for choosing appropriate statistics for the single-molecule trajectories. The results point to a useful and sensitive predictive power of the derived relationships. The meaningful time relationships are the criteria to check the experimental single molecule data measured under conditions of normal and anomalous Brownian diffusion of the molecules of interest. At femtomolar bulk concentration, it would be possible to observe an individual molecule over a second time interval or longer during which biological processes - and not conformational biophysical changes - are just starting.

Detection of hyaluronidase activity using fluorescein labeled hyaluronic acid and Fluorescence Correlation Spectroscopy.

The over-expression of hyaluronidase has been observed in many types of cancer, suggesting that it may have utility for diagnosis. Here we present a technique for the detection of hyaluronidase using Fluorescence Correlation Spectroscopy (FCS). Hyaluronan macromolecules (HAs) have been heavily labeled with fluorescein amine resulting in strong self-quenching. In the presence of hyaluronidase, HA is cleaved into smaller, fluorescein-labeled fragments and the self-quenching is released. Such cleavage is manifested by the increased average diffusion rate of the HA fragments, increased concentration of individual, fluorescent HA fragments, and increased intensity. All three of these properties are monitored simultaneously throughout FCS measurements, both as a function of time and hyaluronidase concentration. The method we present provides a sensitive measure of hyaluronidase activity and requires extremely small amounts of the HA substrate.

Fluorescence molecule counting for single-molecule studies in crowded environment of living cells without and with broken ergodicity.

We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N <1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ T(m) between the measurement time T and the meaningful time T(m), which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

Single actomyosin motor interactions in skeletal muscle.

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:10(5). Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~9s during muscle contraction. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Anomalous behavior in length distributions of 3D random Brownian walks and measured photon count rates within observation volumes of single-molecule trajectories in fluorescence fluctuation microscopy.

Based on classical mean-field approximation using the diffusion equation for ergodic normal motion of single 24-nm and 100-nm nanospheres, we simulated and measured molecule number counting in fluorescence fluctuation microscopy. The 3D-measurement set included a single molecule, or an ensemble average of single molecules, an observation volume DeltaV and a local environment, e.g. aqueous solution. For the molecule number N << 1 per DeltaV, there was only one molecule at a time inside DeltaV or no molecule. The mean rate k of re-entries defined by k = N/tau(dif) was independent of the geometry of DeltaV but depended on the size of DeltaV and the diffusive properties tau(dif). The length distribution l of single-molecule trajectories inside DeltaV and the measured photon count rates I obeyed power laws with anomalous exponent kappa =-1.32 approximately -4/3.

Defining features and exploring chemical modifications to manipulate RNAa activity.

RNA interference (RNAi) is an evolutionary conserved mechanism by which small double-stranded RNA (dsRNA)--termed small interfering RNA (siRNA)--inhibit translation or degrade complementary mRNA sequences. Identifying features and enzymatic components of the RNAi pathway have led to the design of highly-effective siRNA molecules for laboratory and therapeutic application. RNA activation (RNAa) is a newly discovered mechanism of gene induction also triggered by dsRNAs termed small activating RNA (saRNA). It offers similar benefits as RNA interference (RNAi), while representing a new method of gene overexpression. In the present study, we identify features of RNAa and explore chemical modifications to saRNAs that improve the applicability of RNAa. We evaluate the rate of RNAa activity in order to define an optimal window of gene induction, while comparing the kinetic differences between RNAa and RNAi. We identify Ago2 as a conserved enzymatic component of both RNAa and RNAi implicating that saRNA may tolerate modification based on Ago2 function. As such, we define chemical modifications to saRNAs that manipulate RNAa activity, as well as exploit their effects to design saRNAs with enhanced medicinal properties. These findings reveal functional features of RNAa that may be utilized to augment saRNA function for mechanistic studies or the development of RNAa-based drugs.

Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent alpha also determines the resolution of differences in diffusion times between two components in addition to photophysical parameters well-known for normal motion in dilute solution. The resolution limit between two different kinds of single molecule species is also analyzed under translational anomalous motion with broken ergodicity. We apply our theoretical predictions of diffusion times and lower limits for the time resolution of two components to fluorescence images in human prostate cancer cells transfected with GFP-Ago2 and GFP-Ago1. In order to mimic heterogeneous behavior in crowded environments of living cells, we need to introduce so-called continuous time random walks (CTRW). CTRWs were originally performed on regular lattice. This purely stochastic molecule behavior leads to subdiffusive motion with broken ergodicity in our simulations. For the first time, we are able to quantitatively differentiate between anomalous motion without broken ergodicity and anomalous motion with broken ergodicity in time-dependent fluorescence microscopy data sets of living cells. Since the experimental conditions to measure a selfsame molecule over an extended period of time, at which biology is taken place, in living cells or even in dilute solution are very restrictive, we need to perform the time average over a subpopulation of different single molecules of the same kind. For time averages over subpopulations of single molecules, the temporal auto- and crosscorrelation functions are first found. Knowing the crowding parameter alpha for the cell type and cellular compartment type, respectively, the heterogeneous parameter gamma can be obtained from the measurements in the presence of the interacting reaction partner, e.g. ligand, with the same alpha value. The product alpha x gamma = gamma is not a simple fitting parameter in the temporal auto- and two-color crosscorrelation functions because it is related to the proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in cellular systems.We have already derived an analytical solution gamma for in the special case of gamma = 3/2. In the case of two-color crosscorrelation or/and two-color fluorescence imaging (co-localization experiments), the second component is also a two-color species gr, for example a different molecular complex with an additional ligand. Here, we first show that plausible biological mechanisms from FCS/ FCCS and fluorescence imaging in living cells are highly questionable without proper quantitative physical models of subdiffusive motion and temporal randomness. At best, such quantitative FCS/ FCCS and fluorescence imaging data are difficult to interpret under crowding and heterogeneous conditions. It is challenging to translate proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in living cells and their cellular compartments like the nucleus into biological models of the cell biological process under study testable by single-molecule approaches. Otherwise, quantitative FCS/FCCS and fluorescence imaging measurements in living cells are not well described and cannot be interpreted in a meaningful way.

Confocal fluctuation spectroscopy and imaging.

Currently, work with subnanomolar concentrations is routine while femtomolar and even single-molecule studies are possible with some efforts getting high on single-molecule biophysics and biochemistry. Methodological breakthroughs, such as reducing the background light contribution in single-molecule studies, which has plagued many studies of molecular fluorescence in dilute solution, and particularly in live cells, have recently described by us. We first demonstrated how optimized time-gating of the fluorescence signal, together with time-correlated, single-photon counting, can be used to substantially boost the experimental signal-to-noise ratio about 140-fold, making it possible to measure analyte concentrations that are as low as 15 pM. By detection of femtomolar bulk concentrations, confocal microsopy has the potential to address the observation of one and the same molecule in dilute solution without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than currently available. We present relevant physics. The equations are derived using Einstein's approach showing how it fits with Fick's law and the autocorrelation function. An improved technology is being developed at ISS for femtomolar microscopy. The general concepts and provided experimental examples should help to compare our approach to those used in conventional confocal microscopy.

Polarized fluorescent nanospheres.

Fluorescent beads (nanoparticles, nanospheres) are commonly used in fluorescence spectroscopy and microscopy. Due to the random distribution of dye and high dye to nanoparticle ratio, the fluorescence polarization observed from the beads is low. Therefore beads are not used for polarization study. We demonstrate that photoselective bleaching creates beads with highly polarized fluorescence. First, the beads were immobilized in a PVA polymer. Second, the beads-doped PVA film was exposed to the illumination within the dye absorption band. A progressive decrease of absorption was observed. Next, photophysical properties of photobleached and not bleached films dissolved in water were compared.

Myocardial expression of TNF-alpha, IL-1beta, IL-6, IL-8, IL-10 and MCP-1 after a single MDMA dose administered in a rat model.

Indirect effects of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and metabolites on the cardiac cells are well-known, the mechanism(s) underlying direct MDMA-induced cardiotoxicity remaining to be clarified. To better understand the immuno-inflammatory phenomena accompanying the cardiac alterations during MDMA administration, we conducted a study in an in vivo animal model to evaluate the cellular morphological alterations related to the biological response between MDMA administration and inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, 8, 10, and monocyte chemotactic protein-1). A total of 25 male rats were used. The effects were evaluated at 6, 16 and 24 hours after a single dose MDMA administered (20 mg/kg i.p.). We found high levels of the cardioinhibitory cytokines in rat heart after 3 and 6 hs from MDMA administration. Strongest reaction was observed at 24 hs for TNF-alpha, IL-1beta, IL-6, 8, 10 and for MCP-1. Furthermore, we still determined the presence of MDMA and MDA in the plasma of rats treated with MDMA intra-peritoneal single injection; it was present as early at 6 hs and still present 24 hs after treatment. Western blot analysis in cardiac samples demonstrated the IL-1beta and IL-6 reactions in rats died spontaneously at fourth hour. The rise of the selective cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium to the cardiac dysfunction resulting from MDMA injection.

Reducing background contributions in fluorescence fluctuation time-traces for single-molecule measurements in solution.

We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

Trafficking of mature miRNA-122 into the nucleus of live liver cells.

The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

Fluorescence fluctuation spectroscopic approaches to the study of a single molecule diffusing in solution and a live cell without systemic drift or convection: a theoretical study.

Reentries of a single molecule in the confocal, femtoliter-sized probe region (about 10(-16) L and less) are significant because during measurement times they give rise to fluctuation phenomena such as molecule number fluctuations at the single-molecule level in solution without immobilization or hydrodynamic focusing. These fluctuations are the fundamental physical process on which, for example, fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy are based. The reentries of just one molecule in the confocal probe region are theoretically examined in this original article using a hidden, continuous-time Markov model. The system is not set up to have systemic drift or convection. It is found that the reentries obey certain conditions and analytical expressions for the reentry probabilities are obtained first. In particular, the time constant of the mean value and the variance of the reentry probabilities are obtained. The fractions of non-meaningful reentries and meaningful reentries are found for these experimental situations. Therewith, the concentration dependence of the meaningful time that one can study bimolecular reactions of the selfsame molecule in the confocal probe region is derived for the first time. The meaningful time in the probe volume is proportional to the diffusion time of the selfsame molecule and related inversely to the size of the given confocal probe volume. For small molecules, i.e. small diffusion times at a given size of the confocal probe region, one needs lower concentrations of molecules of the same kind in the bulk phase, whereas large molecules can be studied at higher concentrations. The selfsame molecule scenario is compared with the molecular scenario that a second molecule enters the probe volume at random as a function of the meaningful time. The analytical solutions of the physical reentry model (mechanism) hold for the one-, two- (membrane), or three- (solution, live cell) dimensional Brownian motion.