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.

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.

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.

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.

A simple, safe and effective citrate anticoagulation protocol for the genius dialysis system in acute renal failure.

BACKGROUND: The Genius dialysis system is a close loop dialysis batch system increasingly used as an intermittent hemodialysis device in intensive care units. The aim of this study was to test the safety and feasibility of a regional citrate anticoagulation protocol with respect to acid-base and electrolyte disarrangements in critically ill patients with acute renal failure. A standard heparin anticoagulation protocol served as control. METHODS AND RESULTS: In a cross-over study design, 27 acute renal failure patients were allocated to a citrate- and heparin-anticoagulated dialysis sessions (4-6 h). For citrate anticoagulation, a 4% sodium-citrate solution was infused into the arterial line of the extracorporeal circuit. A low calcium dialysate (1 mmol/l) was used for all dialysis sessions. Citrate dosing was adjusted according to the post-filter ionized calcium concentration (targeted values 0.5-0.7 mmol/l). There was no routine calcium substitution. Heparin anticoagulation was started with a heparin-loading dose followed by an individual, patient-adjusted continuous heparin infusion. Electrolyte disarrangements, namely hypernatremia, hypo- and hypercalcemia did not occur in either group. Although the highest bicarbonate levels were achieved during citrate anticoagulation (p = 0.021 versus heparin) the acid base values remained equilibrated in both groups. Filter longevity was excellent and the targeted dialysis time was achieved in all but 1 patient. Citrate anticoagulation was well tolerated with respect to cardiovascular hemodynamics. CONCLUSIONS: Citrate anticoagulation can be safely and effectively performed during intermittent Genius dialysis. Calcium supplementation is not routinely required.

Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics.

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

Renal replacement therapy with high-cutoff hemofilters: Impact of convection and diffusion on cytokine clearances and protein status.

BACKGROUND: High-cutoff hemofilters are characterized by an increased effective pore size designed to facilitate the elimination of inflammatory mediators in sepsis. This study compares diffusive versus convective high-cutoff renal replacement therapy (RRT) in terms of cytokine clearance rates and effects on plasma protein levels. METHODS: Twenty-four patients with sepsis-induced acute renal failure were studied. A polyflux hemofilter with a cutoff point of approximately 60 kd was used for RRT. Patients were randomly allocated to either continuous venovenous hemofiltration (CVVH) with an ultrafiltration rate of 1 L/h (group 1) or 2.5 L/h (group 2) or continuous venovenous hemodialysis (CVVHD) with a dialysate flow rate of 1 L/h (group 3) or 2.5 L/h (group 4). Interleukin-1 (IL-1) receptor antagonist (IL-1ra), IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), and plasma proteins were measured daily. RESULTS: CVVH achieved significantly greater IL-1ra clearance compared with CVVHD (P = 0.0003). No difference was found for IL-6 (P = 0.935). Increasing ultrafiltration volume or dialysate flow led to a highly significant increase in IL-1ra and IL-6 clearance rates (P < 0.00001). Peak clearances were 46 mL/min for IL-1ra and 51 mL/min for IL-6. TNF-alpha clearance was poor for both RRT modalities. A significant decline in plasma IL-1ra and IL-6 clearance was observed in patients with high baseline levels. Protein and albumin losses were greatest during the 2.5-L/h hemofiltration mode. CONCLUSION: High-cutoff RRT is a novel strategy to clear cytokines more effectively. Convection has an advantage over diffusion in the clearance capacity of IL-1ra, but is associated with greater plasma protein losses.

Intermittent high-permeability hemofiltration modulates inflammatory response in septic patients with multiorgan failure.

BACKGROUND/AIM: Continuous venovenous hemofiltration with high-permeability hemofilters is a novel approach in the adjuvant therapy of septic patients. High-permeability hemofilters are characterized by an increased pore size which facilitates the filtration of inflammatory mediators. The present study examines whether intermittent high-permeability hemofiltration has an immunomodulatory effect on polymorphonuclear leukocytes and mononuclear cells. METHODS: Twenty-eight septic patients with acute renal failure were randomly allocated to either receive intermittent high-permeability or conventional hemofiltration. Intermittent high-permeability hemofiltration consisted of a daily 12-hour course of high-permeability hemofiltration alternated by conventional hemofiltration. For high-permeability hemofiltration, a newly developed high-flux polyamide membrane (P2SH) with a nominal cutoff point of 60 kD was used. For conventional hemofiltration a high-flux polyamide hemofilter (Polyflux 11S, cutoff point 30 kD) was used. RESULTS: The polymorphonuclear leukocyte phagocytosis activity before starting hemofiltration was almost double the rate of healthy controls in both groups (p < 0.001). The phagocytosis rate decreased significantly during the course of intermittent high-permeability hemofiltration (p < 0.05), whereas the values remained unchanged in the conventional hemofiltration group. Incubation of high-permeability filtrates with blood from healthy donors resulted in a significant induction of phagocytosis (p < 0.001), whereas conventional filtrates had no phagocytosis-stimulating effects. In addition, incubation of healthy-donor mononuclear cells with high-permeability but not conventional filtrates resulted in a significant tumor necrosis factor alpha release (p < 0.001). CONCLUSIONS: Intermittent high-permeability hemofiltration is a novel extracorporeal elimination modality which exhibits immunomodulatory effects on leukocytes, attenuating polymorphonuclear neutrophil phagocytosis. Further studies are necessary to elucidate whether these effects translate in a clinical improvement in patients suffering from sepsis.