Mechanistic Insight into Electrode Processes by Operando Visualization of Interfacial pH Using Fluorescent Nanosensors.

Operando visualization of interfacial pH is crucial, yet challenging in electrochemical processes. Herein, we report the fabrication and utilization of ratiometric, fluorescent pH-sensitive nanosensors for operando quantification of fast-dynamic, interfacial pH changes in electrochemical processes and environments where unprotected fluorescent dyes would be degraded. Spatio-temporal pH changes were detected using an electrochemically coupled laser scanning confocal microscope (EC-LSCM) during the electrocoagulation treatment of model and field samples of oil-sands-produced water. Operando visualization of interfacial pH provided new insights into the electrode processes, including ion speciation, electrode fouling, and Faradaic efficiency. We provide compelling evidence that formed metal complexes precipitate at the edge of the pH boundary layer and that there is a strong coupling between the thickness of the interfacial pH layer and the electrode fouling. Furthermore, these findings provide a powerful pathway for optimizing the operating conditions, minimizing electrode passivation, and enhancing the efficiency of electrochemical processes, e.g., electrocoagulation, flow batteries, capacitive deionization, and electrolyzes.

[Three Different Digestion Methods to Separate Neonatal Rat Cardiomyocytes].

OBJECTIVES: To investigate the efficacy of three different digestion methods for the separation of neonatal rat cardiomyocytes. METHODS: We employed three different digestion methods to separate neonatal rat cardiomyocytes. Group A was 0.08% trypsin digestion alone, group B was 0.08% trypsin+0.08% type 2 collagenase mixed digestion, group C was 0.08% trypsin and 0.08% type 2 collagenase isolated digestion. The number of cells and cell viability after differential adhesion were recorded. The purity of cardiomyocytes was evaluated by immunofluorescence staining. The cell vitality was assessed by the detection of mitochondrial membrane potential with JC-1 staining, and the ratio of red to green fluorescence intensity by laser scanning confocal microscope. RESULTS: There was nostatistically significant difference in the number of cells between three groups (P >0.05).The rate of cell viability in group C was significantly higher than that in group A (P <0.01). No statistically significant difference was found in the purity of cardiomyocytes between three groups (P >0.05). The ratio of red to green fluorescence intensity in group A, B and C were 0.928±0.078, 0.943±0.099 and 1.160±0.089, respectively; the ratio in group C was significantly higher than that in group A and group B (P <0.01). CONCLUSION: The cell isolation method with 0.08% trypsin and 0.08% type 2 collagenase isolated digestion could be served as conventional method to separate neonatal rat cardiomyocytes.

Intercellular transfer of P-glycoprotein from the drug resistant human bladder cancer cell line BIU-87 does not require cell-to-cell contact.

PURPOSE: The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. MATERIALS AND METHODS: Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. RESULTS: The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. CONCLUSIONS: Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading.

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca2+ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca2+ overload, L-type Ca2+ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca2+ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca2+ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32 h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca2+, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca2+ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca2+ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

Fluorescence Microscopy in Adeno-Associated Virus Research.

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.

Modified Pseudo-Schiff Propidium Iodide for Staining the Shoot Apical Meristem in Arabidopsis.

Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.

Enhanced rheological and tribological properties of nanoenhanced greases by tuning interparticle contacts.

HYPOTHESIS: Despite the wide spectrum of available nanoparticles, their utilization in lubricant and grease formulations remains challenging. To enhance their performance, an improved link between the interparticle contacts, brittleness of the resulting particle network, time-dependent rheology and tribology is required. EXPERIMENTS: We systematically changed interparticle contacts and examined their effect on the colloidal stability, microstructure, rheological and tribological behavior of model greases by investigating four types of nanoclays: montmorillonite (Cloisite Na+), oleic-acid functionalized Cloisite Na+ (OA-Cloisite Na+), organomodified montmorillonite (C20A) and oleic-acid functionalized C20A (C20A-OA). FINDINGS: We observed a range of behaviors, starting from the lack of colloidal stability in greases derived with Cloisite Na+ and OA-Cloisite Na+ to semi-solid type systems with C20A and C20A-OA. Consistent with previous studies, the rheological and tribological properties of C20A systems scale with nanoclay loadings. Surprisingly, the functionalized C20A-OA system exhibited a delayed transition towards hydrodynamic lubrication, and enhanced lubrication properties, both of which were largely independent of nanoclay loadings. Coupled microstructural investigation and time-dependent rheology reveal that this behavior is governed by increasing repulsive forces, decreasing inter-particle friction between C20A-OA nanoparticles, and faster reorganization of the C20A-OA nanoparticle network under shear. Increased interparticle repulsion enables C20A-OA nanoclays to pass each other under shear and align in direction of shear, which reduces the overall viscosity, while the presence of OA on nanoclays decreases inter-particle friction and particle-steel surface friction.

Extracellular vesicle-loaded hydrogels for tissue repair and regeneration.

Extracellular vesicles (EVs) are a collective term for nanoscale or microscale vesicles secreted by cells that play important biological roles. Mesenchymal stem cells are a class of cells with the potential for self-healing and multidirectional differentiation. In recent years, numerous studies have shown that EVs, especially those secreted by mesenchymal stem cells, can promote the repair and regeneration of various tissues and, thus, have significant potential in regenerative medicine. However, due to the rapid clearance capacity of the circulatory system, EVs are barely able to act persistently at specific sites for repair of target tissues. Hydrogels have good biocompatibility and loose and porous structural properties that allow them to serve as EV carriers, thereby prolonging the retention in certain specific areas and slowing the release of EVs. When EVs are needed to function at specific sites, the EV-loaded hydrogels can stand as an excellent approach. In this review, we first introduce the sources, roles, and extraction and characterization methods of EVs and describe their current application status. We then review the different types of hydrogels and discuss factors influencing their abilities to carry and release EVs. We summarize several strategies for loading EVs into hydrogels and characterizing EV-loaded hydrogels. Furthermore, we discuss application strategies for EV-loaded hydrogels and review their specific applications in tissue regeneration and repair. This article concludes with a summary of the current state of research on EV-loaded hydrogels and an outlook on future research directions, which we hope will provide promising ideas for researchers.

Mechanism of Morphology Development in HDGEBA/PAMS Hybrid Thermosets: Monte Carlo Simulation and LSCM Study.

Reactive combinations of aliphatic epoxy resins and functional polysiloxanes form a class of hybrid thermosetting materials with properties that may come from both the organic and the inorganic phases. The two typically immiscible phases form a suspension whose morphology, composition, and thermal properties vary with curing time. The aim of this research was to elucidate the mechanism by which morphology changed with time and to simulate it through Metropolis-Monte Carlo. The selected system was hydrogenated epoxy (HDGEBA) and a synthetic polyaminosiloxane (PAMS). It was studied by DSC, FTnIR, gel point, viscometry, and in-situ laser scanning confocal microscopy. A mechanism for morphology generation was proposed and simulated, exploring a wide range of values of the "a priori" relevant variables. The essential features were captured by simulations with a reasonable agreement with experimental data. However, the complete process was more complex than the geometrical approach of the simulation. The main deviations that were found and qualitatively explained are: (i) the induction period on the rate of coalescence, and (ii) PAMS-rich domain average size increases faster than predictions.

Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans.

Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).

Multicolor Laser Scanning Confocal Immunofluorescence Microscopy of DNA Damage Response Biomarkers.

DNA damage through endogenous and environmental toxicants is a constant threat to both a human's ability to pass on intact genetic information to its offspring as well as in somatic cells for its own survival. To counter these threats posed by DNA damage, cells have evolved a series of highly choreographed mechanisms-collectively defined as the DNA-damage response (DDR)-to sense DNA lesions, signal their presence, and mediate their repair. Thus, regular DDR signaling cascades are vital to prevent the initiation and progression of many human diseases including cancer. Consequently, quantitative assessment of DNA damage and response became an important biomarker for assessment of human health and disease risk in biomonitoring studies. However, most quantitative DNA damage biomarker techniques require dissolution of the nuclear architecture and hence loss of spatial information. Laser scanning confocal immunofluorescence microscopy (LSCIM) of three-dimensionally preserved nuclei can be, quantitative and maintain the spatial information. Here we describe the experimental protocols to quantify individual key events of the DDR cascade in three-dimensionally preserved nuclei by LSCIM with high resolution, using the simultaneous detection of Rad50 as well as phosphorylated H2AX and ATM and in somatic and germ cells as an example.

Transport of Flavanolic Monomers and Procyanidin Dimer A2 across Human Adenocarcinoma Stomach Cells (MKN-28).

It has been proven that A-type procyanidins, containing an additional ether bond, compared to B-type procyanidins are also bioavailable in vitro and in vivo. However, their bioavailability and absorption in the gastrointestinal tract remain uncertain. In this study, a model of the human adenocarcinoma stomach cell line (MKN-28) was established to explore the cellular transport of flavanolic monomers and procyanidin dimer A2, which were isolated from the litchi pericarp extract. After the integrity and permeability of the cell monolayer were ensured by measurement of the transepithelial electrical resistance and the apparent permeability coefficient for Lucifer yellow, the transportation of procyanidins A2 and B2, (-)-epicatechin (EC), and (+)-catechin (CC) was studied at pH 3.0, 5.0, or 7.0 in the apical side, with compound concentrations of 0.05 and 0.1 mg/mL based on the cytotoxicity test. High-performance liquid chromatography and liquid chromatography-mass spectrometry analyses indicated that EC, CC, and A2 were transported in the MKN-28 cell line from 30 to 180 min, while B2 showed no transport. The maximal transport efficiencies of EC, CC, and A2 were 23 ± 0.81, 13.16 ± 1.53, and 16.41 ± 1.36%, respectively, existing at 120, 180, and 120 min of transportation. Laser scanning confocal microscopy analysis presented the dynamic transmission of EC, in accordance with the result of concentration determination, suggesting that the A-type procyanidins are possibly absorbed through the stomach barrier, which is pH- and time-dependent.

Study on Bainitic Transformation by Dilatometer and In Situ LSCM.

This study investigates the bainitic transformation kinetics of carbide-free bainitic steel with Si + Al and carbide-bearing bainitic steel without Si + Al, as well as the phase transformation and microstructure through in situ high-temperature laser scanning confocal microscopy. Results show that bainitic ferrite plates preferentially nucleate at the grain boundary. New plates nucleate on previously formed ones, including two dimensions which appear on a plane where a three-dimensional space of bainitic ferrite forms. Nucleation on the formed bainitic ferrite is faster than that at the grain boundary in some grains. The bainitic ferrite growth at the austenite grain boundary is longer and has a faster transformation rate. The bainitic ferrite growth on the formed bainitic ferrite plate is shorter and has a slower transformation rate. The location and number of nucleation sites influence the thickness of the bainitic ferrite. The higher the number of plates preferentially nucleating at the original austenite grain boundary, the greater the thickness of the bainitic ferrite.

Application of laser scanning confocal microscopy in the soft tissue exquisite structure for 3D scan.

Three-dimensional (3D) printing is a new developing technology for printing individualized materials swiftly and precisely in the field of biological medicine (especially tissue-engineered materials). Prior to printing, it is necessary to scan the structure of the natural biological tissue, then construct the 3D printing digital model through optimizing the scanned data. By searching the literatures, magazines at home and abroad, this article reviewed the current status, main processes and matters needing attention of confocal laser scanning microscope (LSCM) in the application of soft tissue fine structure 3D scanning, empathizing the significance of LSCM in this field.

Choosing the right microscope to image mitosis in zebrafish embryos: A practical guide.

Tissue growth and organismal development require orchestrated cell proliferation. To understand how cell division guides development, it is important to explore mitosis at the tissue-wide, cellular, and subcellular scale. At the tissue level this includes determining a tissue's mitotic index, at the cellular level the tracing of cell lineages, and at the subcellular level the characterization of intracellular components. These different tasks can be addressed by different imaging approaches (e.g., laser-scanning confocal, spinning disk confocal, and light-sheet fluorescence microscopy). Here, we summarize three protocols for exploring different facets of mitosis in developing zebrafish embryos. Zebrafish embryos are transparent and their rapid external development greatly facilitates the study of cellular processes and developmental dynamics using microscopy. A critical step in all imaging studies of mitosis in development is to choose the most suitable microscope for each scientific question. This choice is important in order to ensure a balance between the required temporal and spatial resolution and minimal phototoxicity that could otherwise perturb the process of interest. The use of different microscopy techniques, best suited for the purpose of each experiment, thus permits to generate a comprehensive and unbiased view on how mitosis influences development.

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli.

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force - laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.

Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium.

Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts.

Monitoring the architecture of anionic κ-carrageenan/cationic glycine betaine amide surfactant assemblies by dilution: A multiscale approach.

The interaction between glycine betaine-based cationic surfactant and algal polysaccharide κ-carrageenan was studied by investigating the dilution effect of the surfactant/polymer assemblies driven by electrostatic interactions. Two aqueous solutions of cationic surfactant and κ-carrageenan at two molar ratios (3.5 and 0.8) diluted with factors of 5 and 10 times, were tested by various analytical methods including a multiscale observation by Transmission Electron Microscopy (TEM) and Laser Scanning Confocal Microscopy (LSCM) to understand the solution behavior of surfactant and oppositely charged polymer at both nano- and micrometer scale. Raman spectroscopy as well as confocal Raman imaging were applied to give Supplementary information about the surfactant/polysaccharide interactions and the distribution of assemblies. These analyses confirmed the formation of singular hybrid surfactant/polymer nano-, microobjects and they revealed the influence of dilution on the nanostructures. These results give an insight of the mechanism of the dilution effect on surfactant/polymer assemblies that could be valuable in pharmaceutical formulations, food and cosmetics fields.

Chrysin-organogermanium (IV) complex induced Colo205 cell apoptosis-associated mitochondrial function and anti-angiogenesis.

Colorectal cancer, a kind of malignant cancer, has more than 1 million new patients and results in 0.5 million deaths every year globally based on the estimation of Globocan in 2008. One of the most important issues against colon cancer is tumor metastasis. Anti-angiogenesis, a form of targeted therapy uses drugs or other substances to prevent the new blood vessel formation, which is critical for tumor metastasis. In our previous studies, we have demonstrated a simple method to synthesize Chry-Ge complex through the reaction between chrysin and triphenylgermanium bromide. In this work, we investigated the mechanism of Chry-Ge induced Colo205 cell apoptosis. We found that Chry-Ge could induce apoptosis in Colo205 cells in mitochondrial-dependent pathway, cause the reorganization of cytoskeleton and induce the damage of nucleus in Colo205 cells. Besides, Chry-Ge was also found to induce membrane ultrastructural changes in Colo205 cells by AFM. Further, we found that Chry-Ge can inhibit tube formation of human umbilical vascular endothelial cell in vitro. Chry-Ge was also tested in vivo in the chicken chorioallantoic membrane (CAM) assay and found to inhibit bFGF-treated CAMs development. These results suggested that Chry-Ge could induce Colo205 cell apoptosis by mitochondrial pathway and anti-angiogenesis, highlighting the use of organic germanium agents for the treatment of colorectal cancer.

In vitro cytotoxicity of gold nanorods in A549 cells.

Gold nanoparticles, which have unique physicochemical characteristics, are being used for an increasingly wide range of applications in biomedical research. In this study, gold nanorods (width of 25 nm, length of 52 nm) were found to be internalized by A549 cells and were primarily localized in the lysosomes and membranous vesicles. The integrity of the membranes of A549 cells exposed to gold nanorods for 4h was damaged, as indicated by laser scanning confocal microscopy (LSCM). Increased lactate dehydrogenase (LDH) leakage and decreased cell viability further indicated the concentration-dependent cytotoxicity of the gold nanorods to the A549 cells. Reactive oxygen species (ROS) production was induced in the A549 cells by the gold nanorods, and this effect was positively correlated with the concentration of the gold nanorods. The results of this study indicated that exposure to gold nanorods caused dose-dependent cytotoxicity in A549 cells and that oxidative stress may be the main factor causing cytotoxicity.