Jinyi Wang

Cancer stem cell labeling using poly(L-lysine)-modified iron oxide nanoparticles

Cell labeling using magnetic nanoparticles is an increasingly used approach in noninvasive behavior tracking, in vitro separation of cancer stem cells (CSCs), and CSC-based research in cancer therapy. However, the impact of magnetic labeling on the biological properties of targeted CSCs, such as self-renewal, proliferation, multi-differentiation, cell cycle, and apoptosis, remains elusive. The present study sought to explore the potential effects on biological behavior when CSCs are labeled with superparamagnetic iron oxide (SPIO) nanoparticles in vitro. The glioblastoma CSCs derived from U251 glioblastoma multiforme were labeled with poly(L-lysine) (PLL)-modified γ-Fe(2)O(3) nanoparticles. The iron uptake of glioblastoma CSCs was confirmed through prussian blue staining, and was further quantified using atomic absorption spectrometry. The cellular viability of the SPIO-labeled glioblastoma CSCs was assessed using a fluorescein diacetate and propidium iodide double-staining protocol. The expressed specific markers and multi-differentiation of SPIO-labeled glioblastoma CSCs were comparatively assessed by immunocytochemistry and semi-quantitative RT-PCR. The effects of magnetic labeling on cell cycle and apoptosis rate of glioblastoma CSCs and their differentiated progenies were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of glioblastoma CSCs and their differentiated progenies were not affected by SPIO labeling compared with their unlabeled counterparts. Moreover, the magnetically labeled CSCs displayed an intact multi-differentiation potential, and could be sub-cultured to form new tumor spheres, which indicates the CSCs capacity for self-renewal. In addition, cell cycle distribution, apoptosis rate of the magnetically labeled glioblastoma CSCs, and their differentiated progenies were not impaired. Therefore, the SPIO-labeled CSCs could be a feasible approach in conducting further functional analysis of targeted CSCs.

An enzymatic immunoassay microfluidics integrated with membrane valves for microsphere retention and reagent mixing

The present study presents a new microfluidic device integrated with pneumatic microvalves and a membrane mixer for enzyme-based immunoassay of acute myocardial infarction (AMI) biomarkers, namely, myoglobin, and heart-type fatty acid binding protein (H-FABP). Superparamagnetic microspheres with carboxyl groups on their surfaces were used as antibody solid carriers. A membrane mixer consisting of four ψ-type membrane valves was assembled under the reaction chamber for on-chip performing microsphere trapping and reagent mixing. The entire immunoassay process, including microsphere capture, reagent input, mixing, and subsequent reaction, was accomplished on the device either automatically or manually. The post-reaction substrate resultant was analyzed using a microplate reader. The results show that the average absorbance value is correlated with the concentration of cardiac markers, in agreement with the results obtained using a conventional microsphere-based immunoassay; this indicated that the proposed on-chip immunoassay protocol could be used to detect both myoglobin and H-FABP. The minimum detectable concentration is 5 ng/mL for myoglobin and 1 ng/mL for H-FABP.

Synthesis of copolymers using dendronized polyethylene glycol and assay of their blood compatibility and antibacterial adhesion activity.

Thrombus formation and microbial invasion are two major complications that impede the widespread application of blood-contacting devices. The development of new materials that have blood compatibility and antibacterial adhesion activity has gained increased attention. In this study, a new class of polymers composed of hydrophilic dendronized polyethylene glycol (PEG) methacrylate and hydrophobic octyne monomethyl ether-glycidyl methacrylate was synthesized via click chemistry and free radical polymerization. Different polymers were synthesized by changing the ratio of the two monomers. The structures of the synthesized polymers were characterized by (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy. Their physical properties such as molecular weight, polydispersity, and glass transition temperature were determined using gel permeation chromatography and differential scanning calorimetry. The synthesized polymers were coated on glass slides to prepare a series of polymeric surfaces. Contact angle measurements and attenuated total reflection Fourier-transform infrared spectroscopy analysis showed that the polymeric surfaces had long-lasting stability. The introduction of the monomer dendronized PEG methacrylate to the polymers greatly improved the hydrophilicity of the polymeric surfaces. The blood compatibility of the synthesized polymers was evaluated by protein (bovine serum albumin and fibrinogen) adsorption and platelet adhesion assays. Their antibacterial adhesion ability was investigated using the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus. The results demonstrated that the amount of adsorbed protein, platelets, and bacteria on the polymeric surfaces decreased with increased content of the hydrophilic monomer dendronized PEG methacrylate in the polymers. However, no obvious difference was observed when such content exceeded 50 mol%. The results suggested that the new kind of polymer could be developed as a promising blood-contact coating material that may have extensive medical applications.

Diversification of Microfluidic Chip for Applications in Cell-Based Bioanalysis

Abstract Microfluidic chips are a promising platform for the exploration of modern life science. In this review, we outline the functional features of microfluidic chips and recent developments in their applications in cell-based bioanalysis, and highlight the diversification of cell-based microfluidic chips, including animal, plant and microbial cell chips. Future prospects of microfluidic chips in cell-based bioanalysis are also discussed.

Microfluidics-based assay on the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors

A new microfluidic system with four different microchambers (a circle and three equilateral concave polygons) was designed and fabricated using poly(dimethylsiloxane) (PDMS) and the soft lithography method. Using this microfluidic device at six flow rates (5, 10, 20, 30, 40, and 50 μL/h), the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors were investigated. Escherichia coli HB101 pGLO, which could produce a green fluorescent protein induced by l-arabinose, was utilized as the model bacteria. The results demonstrated that bacterial adhesion was significantly related to culture time, microenvironment geometry, and aqueous flow rates. Adhered bacterial density increased with the culture time. Initially, the adhesion occurred at the microchamber sides, and then the entire chamber was gradually covered with increased culture time. Adhesion densities in the side zones were larger than those in the center zones because of the lower shearing force in the side zone. Also, the adhesion densities in the complex chambers were larger than those in the simple chambers. At low flow rates, the orientation of adhered bacteria was random and disorderly. At high flow rates, bacterial orientation became close to the streamline and oriented toward the flow direction. All these results implied that bacterial adhesion tended to occur in complicated aqueous flow areas. The present study provided an on-chip flow system for physiological behavior of biological cells, as well as provided a strategic cue for the prevention of bacterial infection and biofilm formation.

Heat-shock transformation of Escherichia coli in nanolitre droplets formed in a capillary-composited microfluidic device

This work describes an improved method for monodispersed water-in-oil droplet formation and collection using a composite microfluidic device composed of a poly(dimethylsiloxane) (PDMS) microfluidic device and a commercially available quartz capillary. The application of the method to chemical heat-shock (CaCl2-dependent) transformation of Escherichia coli (E. coli) is also presented. With this approach, tunable and uniform different-sized droplets were generated and conveniently collected into a capillary for subsequent experiments. Characterization of droplet size and formation frequency exhibits that droplet behavior is strongly dependent on the ratio (R) of aqueous phase flow rate (Qaq) to oil phase flow rate (Qo). An increase in R induces droplet size and droplet formation frequency increase, which agrees well with a theoretical calculation. To illustrate the application of this droplet-based device in biological fields, as a case study, we also apply this device to the study of heat-shock E. coli transformation. Results demonstrate that plasmid DNA can be effectively transformed into E. coli, and a similar transformation efficiency with the traditional tube-based method can be obtained. This technique provides a new way for droplet generation and easy collection, as well as functional genomics studies by taking advantage of the high throughput of droplet microfluidics.

Integrative Mouse and Human Studies Implicate ANGPT1 and ZBTB7C as Susceptibility Genes to Ischemic Injury

Background and Purpose— The extent of ischemic injury in response to cerebral ischemia is known to be affected by native vasculature. However, the nonvascular and dynamic vascular responses and their genetic basis are not well understood. Methods— We performed a genome-wide association study in 235 mice from 33 inbred strains using the middle cerebral artery occlusion model. Population structure and genetic relatedness were accounted for using the efficient mixed-model association method. Human orthologs to the genes associated with the significant and suggestive single-nucleotide polymorphisms from the mouse strain survey were examined in patients with M1 occlusions admitted with signs and symptoms of acute ischemic stroke. Results— We identified 4 genome-wide significant and suggestive single-nucleotide polymorphisms to be associated with infarct volume in mice (rs3694965, P=2.17×10–7; rs31924033, P=5.61×10–6; rs32249495, P=2.08×10–7; and rs3677406, P=9.56×10–6). rs32249495, which corresponds to angiopoietin-1 (ANGPT1), was also significant in the recessive model in humans, whereas rs1944577, which corresponds to ZBTB7C, was nominally significant in both the additive and dominant genetic models in humans. ZBTB7C was shown to be upregulated in endothelial cells using both in vitro and in vivo models of ischemia. Conclusions— Genetic variations of ANGPT1 and ZBTB7C are associated with increased infarct size in both mice and humans. ZBTB7C may modulate the ischemic response via neuronal apoptosis and dynamic collateralization and, in addition to ANGPT1, may serve as potential novel targets for treatments of cerebral ischemia.

Synthesis of a new chitosan derivative and assay of Escherichia coli adsorption

A new chitosan derivative is prepared using chitosan. Ethyl cholorocarbonate was first introduced to the hydroxyl group of phthaloylchitosan through a nucleophilic reaction. Hydrazine was then added to recover the amino groups of chitosan, and promote cross-linking. The structure of this new chitosan derivative was characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and its physical properties were determined by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The thermal and chemical stabilities of the new derivative were improved compared with those of native chitosan. Assay of Escherichia coli adhesion on a film based on this chitosan derivative showed good adsorption and biofilm formation.

High Signal Intensity in the Dentate Nucleus and Globus Pallidus on Unenhanced T1-Weighted MR Images: Comparison between Gadobutrol and Linear Gadolinium-Based Contrast Agents

This is a retrospective analysis of 59 patients who received only gadobutrol and 60 patients who received only linear gadolinium-based contrast agents. Linear gadolinium-based contrast agents included gadoversetamide, gadobenatedimeglumine, and gadodiamide. T1 signal intensity in the globus pallidus, dentate nucleus, and pons was measured on the precontrast portions of patients first and seventh brain MRIs. The dentate nucleus/pons signal ratio increased in the linear gadolinium-based contrast agent group while no significant increase was seen in the gadobutrol group. The authors conclude that successive doses of gadobutrol do not result in T1 shortening compared with changes seen in linear gadolinium-based contrast agents. BACKGROUND AND PURPOSE: In view of the recent observations that gadolinium deposits in brain tissue after intravenous injection, our aim of this study was to compare signal changes in the globus pallidus and dentate nucleus on unenhanced T1-weighted MR images in patients receiving serial doses of gadobutrol, a macrocyclic gadolinium-based contrast agent, with those seen in patients receiving linear gadolinium-based contrast agents. MATERIALS AND METHODS: This was a retrospective analysis of on-site patients with brain tumors. Fifty-nine patients received only gadobutrol, and 60 patients received only linear gadolinium-based contrast agents. Linear gadolinium-based contrast agents included gadoversetamide, gadobenate dimeglumine, and gadodiamide. T1 signal intensity in the globus pallidus, dentate nucleus, and pons was measured on the precontrast portions of patients first and seventh brain MRIs. Ratios of signal intensity comparing the globus pallidus with the pons (globus pallidus/pons) and dentate nucleus with the pons (dentate nucleus/pons) were calculated. Changes in the above signal intensity ratios were compared within the gadobutrol and linear agent groups, as well as between groups. RESULTS: The dentate nucleus/pons signal ratio increased in the linear gadolinium-based contrast agent group (t = 4.215, P < .001), while no significant increase was seen in the gadobutrol group (t = −1.422, P = .08). The globus pallidus/pons ratios followed similarly, with an increase in the linear gadolinium-based contrast agent group (t = 2.931, P < .0001) and no significant change in those receiving gadobutrol (t = 0.684, P = .25). CONCLUSIONS: Successive doses of gadobutrol do not result in T1 shortening compared with changes seen in linear gadolinium-based contrast agents.