Enzymatic time-temperature indicator with cysteine-loaded chitosan microspheres/silver nanoparticles.

A time-temperature indicator (TTI) based on acid-base reaction was developed by applying a new pH dye composed of cysteine-loaded chitosan (Cys-CS) microspheres and silver nanoparticles (AgNPs). It was hypothesized that cysteine released by the disintegration of Cys-CS microspheres at a critical pH would cause AgNPs to aggregate, leading to color change. Cys-CS microspheres were produced as water-in-oil (paraffin oil, MCT oil, soybean oil) emulsions according to the KOH addition method. An enzymatic TTI was made using glucose oxidase, glucose, and catalase. Only paraffin oil produced Cys-CS microspheres (average diameter, 335 ± 100 µm), whereas the others did not, probably due to saponification with KOH. FTIR analysis confirmed that cysteine was encapsulated in the microspheres. The microspheres disintegrated at pH 6.18 in a titration test. The TTI pH gradually decreased and showed a sudden color change at pH 6.10, which was similar to the critical pH of microsphere disintegration.

Polycaprolactone/Anthocyanin-Based Electrospun Volatile Amines Gas Indicator with Improved Visibility by Varying Bi-Solvent Ratio: A Case of Intelligent Packaging of Mackerel.

Electrospun nanofibers have been applied as a new technology for gas indicators in food intelligent packaging. A poly(ε-caprolactone) (PCL)/red cabbage anthocyanin (RCA)-based nanofiber volatile amines gas indicator was developed by applying a bi-solvent of acetic acid (AA) and formic acid (FA) in electrospinning. The visibility of color change was improved from pink to blue, compared to blue to yellow-green, when using a single solvent of AA. The solutes of PCL (12.5, 15, 17.5, and 20%) and RCA (10, 20, 30, and 40%) and the solvents of AA/FA (9:1, 7:3, 5:5, 3:7, and 1:9) were applied in electrospinning under the condition of 12.5 cm, 1.0 mL/h, and 20 kV. The optimal microstructure with the thinnest fiber diameter and constant arrangement without forming NF beads appeared under the 7:3 FA/AA, 15% PCL, and 20% RCA condition. The indicator changed from pink to blue with the values of total color change (ΔE) of 10, 14, and 18 when exposed to the saturated gas of ammonia solutions of 8, 80, and 800 mM, respectively. The indicator was stable and unchanged in color for 28 days when exposed to light at room temperature. In the application to mackerel packaging, the built-in indicator changed from pink to purple regardless of storage temperature when the spoilage point was reached.

First-in-Human Studies of [18F] Fluorohydroxyphenethylguanidines.

BACKGROUND: Disease-induced damage to cardiac autonomic nerve populations is associated with an increased risk of sudden cardiac death. The extent of cardiac sympathetic denervation, assessed using planar scintigraphy or positron emission tomography, has been shown to predict the risk of arrhythmic events in heart failure patients staged for implantable cardioverter defibrillator therapy. The goal of this study was to perform first-in-human evaluations of 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine, 2 new positron emission tomography radiotracers developed for quantifying regional cardiac sympathetic nerve density. METHODS AND RESULTS: Cardiac positron emission tomography studies with 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine were performed in normal subjects (n=4 each) to assess their imaging properties and organ kinetics. Patlak graphical analysis of their myocardial kinetics was evaluated as a technique for generating nerve density metrics. Whole-body biodistribution studies (n=4 each) were acquired and used to calculate human radiation dosimetry estimates. Patlak analysis proved to be an effective approach for quantifying regional nerve density. Using 960 left ventricular volumes of interest, across-subject Patlak slopes averaged 0.107±0.010 mL/min per gram for 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 0.116±0.010 mL/min per gram for 3-[18F]fluoro-para-hydroxyphenethylguanidine. Tracer uptake was highest in heart, liver, kidneys, and salivary glands. Urinary excretion was the main elimination pathway. CONCLUSIONS: 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine each produce high-quality positron emission tomography images of the distribution of sympathetic nerves in human heart. Patlak analysis provides reproducible measurements of regional cardiac sympathetic nerve density at high spatial resolution. Further studies of these tracers in heart failure patients will be performed to identify the best agent for clinical development. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT02385877.

Constriction model of actomyosin ring for cytokinesis by fission yeast using a two-state sliding filament mechanism.

We developed a model describing the structure and contractile mechanism of the actomyosin ring in fission yeast, Schizosaccharomyces pombe. The proposed ring includes actin, myosin, and α-actinin, and is organized into a structure similar to that of muscle sarcomeres. This structure justifies the use of the sliding-filament mechanism developed by Huxley and Hill, but it is probably less organized relative to that of muscle sarcomeres. Ring contraction tension was generated via the same fundamental mechanism used to generate muscle tension, but some physicochemical parameters were adjusted to be consistent with the proposed ring structure. Simulations allowed an estimate of ring constriction tension that reproduced the observed ring constriction velocity using a physiologically possible, self-consistent set of parameters. Proposed molecular-level properties responsible for the thousand-fold slower constriction velocity of the ring relative to that of muscle sarcomeres include fewer myosin molecules involved, a less organized contractile configuration, a low α-actinin concentration, and a high resistance membrane tension. Ring constriction velocity is demonstrated as an exponential function of time despite a near linear appearance. We proposed a hypothesis to explain why excess myosin heads inhibit constriction velocity rather than enhance it. The model revealed how myosin concentration and elastic resistance tension are balanced during cytokinesis in S. pombe.

Radiotracers for cardiac sympathetic innervation: transport kinetics and binding affinities for the human norepinephrine transporter.

INTRODUCTION: Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [(11)C]-(-)-meta-hydroxyephedrine, [(11)C]-(-)-epinephrine, and a series of [(11)C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [(3)H]-labeled biogenic amines were also determined. METHODS: Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [(3)H]mazindol provided estimates of binding affinities (KI) for NET. RESULTS: Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for 'tracer concentrations' of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r(2)=0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. CONCLUSION: The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density.

A computational study of ion conductance in the KcsA K(+) channel using a Nernst-Planck model with explicit resident ions.

The biophysical mechanisms underlying the relationship between the structure and function of the KcsA K(+) channel are described. Because of the conciseness of electrodiffusion theory and the computational advantages of a continuum approach, the Nernst-Planck (NP) type models, such as the Goldman-Hodgkin-Katz and Poisson-NP (PNP) models, have been used to describe currents in ion channels. However, the standard PNP (SPNP) model is known to be inapplicable to narrow ion channels because it cannot handle discrete ion properties. To overcome this weakness, the explicit resident ions NP (ERINP) model was formulated, which applies a local explicit model where the continuum model fails. Then, the effects of the ERI Coulomb potential, the ERI induced potential, and the ERI dielectric constant for ion conductance were tested in the ERINP model. The current-voltage (I-V) and current-concentration (I-C) relationships determined in the ERINP model provided biologically significant information that the traditional continuum model could not, explicitly taking into account the effects of resident ions inside the KcsA K(+) channel. In addition, a mathematical analysis of the K(+) ion dynamics established a tight structure-function system with a shallow well, a deep well, and two K(+) ions resident in the selectivity filter. Furthermore, the ERINP model not only reproduced the experimental results with a realistic set of parameters, but it also reduced CPU costs.

Radiolabeled phenethylguanidines: novel imaging agents for cardiac sympathetic neurons and adrenergic tumors.

The norepinephrine transporter (NET) substrates [123I]-m-iodobenzylguanidine (MIBG) and [11C]-m-hydroxyephedrine (HED) are used as markers of cardiac sympathetic neurons and adrenergic tumors (pheochromocytoma, neuroblastoma). However, their rapid NET transport rates limit their ability to provide accurate measurements of cardiac nerve density. [11C]Phenethylguanidine ([11C]1a) and 12 analogues ([11C]1b-m) were synthesized and evaluated as radiotracers with improved kinetics for quantifying cardiac nerve density. In isolated rat hearts, neuronal uptake rates of [11C]1a-m ranged from 0.24 to 1.96 mL min-1 (g wet wt)-1, and six compounds had extremely long neuronal retention times (clearance T1/2 > 20 h) due to efficient vesicular storage. Positron emission tomography (PET) studies in nonhuman primates with [11C]1e, N-[11C]guanyl-m-octopamine, which has a slow NET transport rate, showed improved myocardial kinetics compared to HED. Compound [11C]1c, [11C]-p-hydroxyphenethylguanidine, which has a rapid NET transport rate, avidly accumulated into rat pheochromocytoma xenograft tumors in mice. These encouraging findings demonstrate that radiolabeled phenethylguanidines deserve further investigation as radiotracers of cardiac sympathetic innervation and adrenergic tumors.