TMAO, a seafood-derived molecule, produces diuresis and reduces mortality in heart failure rats.

Trimethylamine-oxide (TMAO) is present in seafood which is considered to be beneficial for health. Deep-water animals accumulate TMAO to protect proteins, such as lactate dehydrogenase (LDH), against hydrostatic pressure stress (HPS). We hypothesized that TMAO exerts beneficial effects on the circulatory system and protects cardiac LDH exposed to HPS produced by the contracting heart. Male, Sprague-Dawley and Spontaneously-Hypertensive-Heart-Failure (SHHF) rats were treated orally with either water (control) or TMAO. In vitro, LDH with or without TMAO was exposed to HPS and was evaluated using fluorescence correlation spectroscopy. TMAO-treated rats showed higher diuresis and natriuresis, lower arterial pressure and plasma NT-proBNP. Survival in SHHF-control was 66% vs 100% in SHHF-TMAO. In vitro, exposure of LDH to HPS with or without TMAO did not affect protein structure. In conclusion, TMAO reduced mortality in SHHF, which was associated with diuretic, natriuretic and hypotensive effects. HPS and TMAO did not affect LDH protein structure.

Heart failure is a common cause of death in industrialized countries with aging populations. Japan, however, has lower rates of heart failure and fewer deaths linked to this disease than the United States or Europe, despite having the highest proportion of elderly people in the world. Dietary differences between these regions may explain the lower rate of heart failure in Japan. The Japanese diet is rich in seafood, which contains nutrients that promote heart health, such as omega-3 fatty acids. Seafood also contains other compounds, including trimethylamine oxide (TMAO). Fish that live in deep waters undergo high pressures, which can damage their proteins, but TMAO seems to protect the proteins from harm. In humans, eating seafood increases TMAO levels in the blood and urine, but it is unclear what effects this has on heart health. Increased levels of TMAO in the blood are associated with cardiovascular diseases, but scientists are not sure whether TMAO itself harms the heart. A toxic byproduct of gut bacteria called TMA is converted in TMAO in the body, so it is possible that TMA rather than TMAO is to blame. To assess the effects of dietary TMAO on heart failure, Gawrys-Kopczynska et al. fed the compound to healthy rats and rats with heart failure for one year. TMAO had no effects on the healthy rats. Of the rats with heart failure that were fed TMAO, all of them survived the year, while one third of rats with heart failure that were not fed TMAO died. TMAO-treated rats with heart failure had lower blood pressure and urinated more than untreated rats with the condition. The experiments suggest that dietary TMAO may mimic the effects of heart failure treatments, which remove excess water and salt and lower pressure on the heart. More studies are needed to confirm whether TMAO has this same effect on humans.

Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population.

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.

Kinetics and equilibrium constants of oligonucleotides at low concentrations. Hybridization and melting study.

Although DNA hybridization/melting is one of the most important biochemical reactions, the non-trivial kinetics of the process is not yet fully understood. In this work, we use Förster resonance energy transfer (FRET) to investigate the influence of temperature, ionic strength, and oligonucleotide length on the kinetic and equilibrium constants of DNA oligonucleotide binding and dissociation. We show that at low reagent concentrations and ionic strength, the time needed to establish equilibrium between single and double strand forms may be of the order of days, even for simple oligonucleotides of a length of 20 base pairs or less. We also identify and discuss the possible artifacts related to fluorescence-based experiments conducted in extremely dilute solutions. The results should prove useful for the judicious design of technologies based on DNA-matching, including sensors, DNA multiplication, sequencing, and gene manipulation.

Fluorescence correlation spectroscopy for multiple-site equilibrium binding: a case of doxorubicin-DNA interaction.

Quantitative description of the interaction between doxorubicin (DOX), a broadly used anticancer drug, and DNA is the key to understand the action mechanism and side effects of its clinical use. However, the reported equilibrium constants of DOX-DNA interaction obtained using a range of different analytical methods vary even by several orders of magnitude. Herein, we propose a novel application of a single-molecule technique - fluorescence correlation spectroscopy (FCS) - to probe the interaction between DOX and two types of DNA (pUC19 and calf thymus DNA), taking advantage of intrinsic self-fluorescence of DOX. We provide an analytical formula for autocorrelation analysis to determine the equilibrium constant of DOX-DNA complex-formation, where binding of multiple DOX molecules to a DNA chain is included in the reaction-diffusion model. Our FCS-based method not only quantitatively revealed the values of equilibrium constant, but also implied that the stability of DOX-DNA complex is related to the types of base pair rather than the length or structure of the DNA. This work opens a promising pathway toward quantitative determination of molecular interactions in complex systems such as living cells or organisms at single-molecule level.

Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level.

Equilibrium and rate constants are key descriptors of complex-formation processes in a variety of chemical and biological reactions. However, these parameters are difficult to quantify, especially in the locally confined, heterogeneous, and dynamically changing living matter. Herein, we address this challenge by combining stimulated emission depletion (STED) nanoscopy with fluorescence correlation spectroscopy (FCS). STED reduces the length-scale of observation to tens of nanometres (2D)/attoliters (3D) and the time-scale to microseconds, with direct, gradual control. This allows one to distinguish diffusional and binding processes of complex-formation, even at reaction rates higher by an order of magnitude than in confocal FCS. We provide analytical autocorrelation formulas for probes undergoing diffusion-reaction processes under STED condition. We support the theoretical analysis of experimental STED-FCS data on a model system of dye-micelle, where we retrieve the equilibrium and rates constants. Our work paves a promising way toward quantitative characterization of molecular interactions in vivo.

Apparent Anomalous Diffusion in the Cytoplasm of Human Cells: The Effect of Probes' Polydispersity.

This work, based on in vivo and in vitro measurements, as well as in silico simulations, provides a consistent analysis of diffusion of polydisperse nanoparticles in the cytoplasm of living cells. Using the example of fluorescence correlation spectroscopy (FCS), we show the effect of polydispersity of probes on the experimental results. Although individual probes undergo normal diffusion, in the ensemble of probes, an effective broadening of the distribution of diffusion times occurs-similar to anomalous diffusion. We introduced fluorescently labeled dextrans into the cytoplasm of HeLa cells and found that cytoplasmic hydrodynamic drag, exponentially dependent on probe size, extraordinarily broadens the distribution of diffusion times across the focal volume. As a result, the in vivo FCS data were effectively fitted with the anomalous subdiffusion model while for a monodisperse probe the normal diffusion model was most suitable. Diffusion time obtained from the anomalous diffusion model corresponds to a probe whose size is determined by the weight-average molecular weight of the polymer. The apparent anomaly exponent decreases with increasing polydispersity of the probes. Our results and methodology can be applied in intracellular studies of the mobility of nanoparticles, polymers, or oligomerizing proteins.

Motion of Molecular Probes and Viscosity Scaling in Polyelectrolyte Solutions at Physiological Ionic Strength.

We investigate transport properties of model polyelectrolyte systems at physiological ionic strength (0.154 M). Covering a broad range of flow length scales-from diffusion of molecular probes to macroscopic viscous flow-we establish a single, continuous function describing the scale dependent viscosity of high-salt polyelectrolyte solutions. The data are consistent with the model developed previously for electrically neutral polymers in a good solvent. The presented approach merges the power-law scaling concepts of de Gennes with the idea of exponential length scale dependence of effective viscosity in complex liquids. The result is a simple and applicable description of transport properties of high-salt polyelectrolyte solutions at all length scales, valid for motion of single molecules as well as macroscopic flow of the complex liquid.

Small Crowders Slow Down Kinesin-1 Stepping by Hindering Motor Domain Diffusion.

The dimeric motor protein kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of kinesin-1: small crowders stop the motor at a viscosity of 5 mPa·s-corresponding to a hydrodynamic load in the sub-fN (~10^{-4} pN) range-whereas large crowders have no impact even at viscosities above 100 mPa·s. This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor determining kinesin's functionality. Our results emphasize the role of diffusion in the kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.

Motion of nanoprobes in complex liquids within the framework of the length-scale dependent viscosity model.

This paper deals with the recent phenomenological model of the motion of nanoscopic objects (colloidal particles, proteins, nanoparticles, molecules) in complex liquids. We analysed motion in polymer, micellar, colloidal and protein solutions and the cytoplasm of living cells using the length-scale dependent viscosity model. Viscosity monotonically approaches macroscopic viscosity as the size of the object increases and thus gives a single, coherent picture of motion at the nano and macro scale. The model includes interparticle interactions (solvent-solute), temperature and the internal structure of a complex liquid. The depletion layer ubiquitously occurring in complex liquids is also incorporated into the model. We also discuss the biological aspects of crowding in terms of the length-scale dependent viscosity model.

Tracking structural transitions of bovine serum albumin in surfactant solutions by fluorescence correlation spectroscopy and fluorescence lifetime analysis.

The structural dynamics of proteins is crucial to their biological functions. A precise and convenient method to determine the structural changes of a protein is still urgently needed. Herein, we employ fluorescence correlation spectroscopy (FCS) to track the structural transition of bovine serum albumin (BSA) in low concentrated cationic (cetyltrimethylammonium chloride, CTAC), anionic (sodium dodecyl sulfate, SDS), and nonionic (pentaethylene glycol monododecyl ether, C12E5 and octaethylene glycol monododecyl ether, C12E8) surfactant solutions. BSA is labelled with the fluorescence dye called ATTO-488 (ATTO-BSA) to obtain steady fluorescence signals for measurements. We find that the diffusion coefficient of BSA decreases abruptly with the surfactant concentration in ionic surfactant solutions at concentrations below the critical micelle concentration (CMC), while it is constant in nonionic surfactant solutions. According to the Stokes-Sutherland-Einstein equation, the hydrodynamic radius of BSA in ionic surfactant solutions amounts to ∼6.5 nm, which is 1.7 times larger than in pure water or in nonionic surfactant solutions (3.9 nm). The interaction between BSA and ionic surfactant monomers is believed to cause the structural transition of BSA. We confirm this proposal by observing a sudden shift of the fluorescence lifetime of ATTO-BSA, from 2.3 ns to ∼3.0 ns, in ionic surfactant solutions at the concentration below CMC. No change in the fluorescence lifetime is detected in nonionic surfactant solutions. Moreover, by using FCS we are also able to identify whether the structural change of protein results from its self-aggregation or unfolding.

A depletion layer in polymer solutions at an interface oscillating at the subnano- to submicrometer scale.

The mobility of segments of the polymer mesh in a solution determines the dynamic response of the depletion layer (DL) to mechanical stimuli. This phenomenon can be used to vastly decrease the local viscosity experienced by any device performing periodic motion at the nano- and microscale in complex liquids. We refined the vibrating quartz tuning fork (QTF) method to probe the viscosity of model aqueous solutions of polyethylene glycol, covering a broad range of molecular weights (3 kDa to 1 MDa) and QTF oscillation amplitudes (50 pm to 100 nm). For semidilute solutions of PEGs of high molecular weight, we found a drop of local viscosity, up to two orders of magnitude below the bulk value. We propose a simple explanation based on the motion of the depletion layer, strongly supported by rheometry and dynamic light scattering results. We show that it is possible to directly probe the viscosity of the DL and increase its thickness far above the equilibrium value. The key role is played by the rate of relaxation of the entangled system. The relevance of this paradigm ranges from the basic research on dynamics of entangled systems to design of energy-efficient nanomachines operating in a crowded environment.

Length-scale dependent transport properties of colloidal and protein solutions for prediction of crystal nucleation rates.

We propose a scaling equation describing transport properties (diffusion and viscosity) in the solutions of colloidal particles. We apply the equation to 23 different systems including colloids and proteins differing in size (range of diameters: 4 nm to 1 µm), and volume fractions (10(-3)-0.56). In solutions under study colloids/proteins interact via steric, hydrodynamic, van der Waals and/or electrostatic interactions. We implement contribution of those interactions into the scaling law. Finally we use our scaling law together with the literature values of the barrier for nucleation to predict crystal nucleation rates of hard-sphere like colloids. The resulting crystal nucleation rates agree with existing experimental data.

Activation energy for mobility of dyes and proteins in polymer solutions: from diffusion of single particles to macroscale flow.

We measure the activation energy Ea for the diffusion of molecular probes (dyes and proteins of radii from 0.52 to 6.9 nm) and for macroscopic flow in a model complex liquid-aqueous solutions of polyethylene glycol. We cover a broad range of polymer molecular weights, concentrations, and temperatures. Fluorescence correlation spectroscopy and rheometry experiments reveal a relationship between the excess of the activation energy in polymer solutions over the one in pure solvent ΔEa and simple parameters describing the structure of the system: probe radius, polymer hydrodynamic radius, and correlation length. ΔEa varies by more than an order of magnitude in the investigated systems (in the range of ca. 1-15 kJ/mol) and for probes significantly larger than the polymer hydrodynamic radius approaches the value measured for macroscopic flow. We develop an explicit formula describing the smooth transition of ΔEa from the diffusion of molecular probes to macroscopic flow. This formula is a reference for the quantitative analysis of specific interactions of moving nano-objects with their environment as well as active transport. For instance, the power developed by a molecular motor moving at constant velocity u is proportional to u2exp(Ea/RT).

Anomalous effect of flow rate on the electrochemical behavior at a liquid|liquid interface under microfluidic conditions.

We have investigated the oxidation of ferrocene at a flowing organic solvent|aqueous electrolyte|solid electrode junction in a microfluidic setup using cyclic voltammetry and fluorescent laser scanning confocal microscopy. At low flow rates the oxidation current decreases with increasing flow, contrary to the Levich equation, but at higher flow rates the current increases linearly with the cube root of the flow rate. This behavior is explained using a simple model postulating a smallest effective width of the three-phase junction, which after fitting to the data comes to be ca. 20 µm. The fluorescence microscopy reveals mixing of the two phases close to the PDMS cover, but the liquid|liquid junction is stable close to the glass support. This study shows the importance of the solid|liquid|liquid junctions for the behavior of multiphase systems under microfluidic conditions.

Spontaneous self-assembly of partially fluorinated bolaamphiphiles into ordered layered structures.

We developed a simple method for preparation of well-defined films of X-, T- and anchor-shaped bolaamphiphiles. The compounds were judiciously chosen to investigate the influence of the general molecular structure on the self-assembly properties. Precisely calculated (on the basis of Langmuir π(A) isotherms) volumes of chloroform solutions of the compounds of known concentrations were spread (drop-casted) directly onto the surface of water or silicon wafer. During the solvent evaporation, regular thin films were spontaneously formed. With use of the drop-casting (DC) method, films of thickness of up to three molecular layers could be obtained. X-ray reflectivity (XRR) measurements gave insight into arrangement of the molecules within the films. Different models of molecular organisation in the films were confronted with the experimental results. Advanced fitting procedures allowed for precise determination of the structure of the films. Comparison of films of different compounds obtained using different methods (Langmuir-Blodgett, DC) allowed for a deeper insight into the process of self-assembly, providing guidelines for designing functional molecules spontaneously forming thin, regular films. The proposed DC procedure is a novel alternative to broadly used self-assembled monolayers (SAMs). Unlike SAMs, our method does not require specific molecule-surface interactions and allows formation of films thicker than a monolayer.

Formation of net-like patterns of gold nanoparticles in liquid crystal matrix at the air-water interface.

Controlled patterning and formation of nanostructures on surfaces based on self-assembly is a promising area in the field of "bottom-up" nanomaterial engineering. We report formation of net-like structures of gold nanoparticles (Au NPs) in a matrix of liquid crystalline amphiphile 4'-n-octyl-4-cyanobiphenyl at the air-water interface. After initial compression to at least 18 mN m(-1), decompression of a Langmuir film of a mixture containing both components results in formation of net-like structures. The average size of a unit cell of the net is easily adjustable by changing the surface pressure during the decompression of the film. The net-like patterns of different, desired average unit cell areas were transferred onto solid substrates (Langmuir-Blodgett method) and investigated with scanning electron microscopy and X-ray reflectivity (XRR). Uniform coverage over large areas was proved. XRR data revealed lifting of the Au NPs from the surface during the formation of the film. A molecular mechanism of formation of the net-like structures is discussed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11051-012-0826-4) contains supplementary material, which is available to authorized users.