Inverted Fluorescence Microscope Research Articles

MicroRNA-155 reduces inflammatory response induced by lipopolysaccharide in alveolar macrophages

To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. (1) The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. (2) Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. (1) After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. (2) Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.

Highly specialized Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of microstructures

AbstractExtremely miniaturized longipedes insects (body length c. 0.3 mm) embedded in two pieces of Cretaceous amber from Myanmar are described and interpreted. Using inverted fluorescence and light microscopy for detailed analysis of microstructures, the inclusions were identified as primary larvae of the beetle family Ripiphoridae, subfamily Ripidiinae. While the structure of thoracic and abdominal segments including appendages corresponds well with the groundplan known in recent members of Ripidiinae, a curved prosternal ridge with prominent spines (each c. 5 μm), the reduced condition of stemmata and antennae and the lack of sharp mandibles are unique features within the entire family, apparently apomorphies of the longipedes larvae. A sinuate prosternal edge with a dense row of spines (prosternoctenidium) might be homologous with ‘head ctenidia’ in some previously described miniaturized conicocephalate larvae, but further investigation is needed. The morphological differences between the head of longipedes larvae and extant Ripidiinae are interpreted as adaptations to different groups of hosts and life strategies. Palaeoethology of the longipedes larvae is briefly discussed. In addition, the systematic placement of conicocephalate larvae from Canadian, Myanmar and Russian Cretaceous ambers, already interpreted by various authors as primary instars within Coleopterida (assigned to either Strepsiptera or to the coleopteran Tenebrionoidea: Ripiphoridae), is discussed.

Mechanical properties and cytotoxicity of hierarchical carbon fiber-reinforced poly (ether-ether-ketone) composites used as implant materials

Weak mechanical properties affect the application of PEEK as an implant. Carbon fiber (CFR) reinforcement provides an excellent solution to improve the mechanical strength of PEEK and to provide perfect matching of elastic modulus between CFR-PEEK composites and human bone. To investigate the effect of carbon fiber content on the mechanical, thermal properties and cytotoxicity of CFR reinforced PEEK composites, a series of CFR-PEEK composites with different carbon fiber content (25 wt%, 30 wt%, 35 wt%, 40 wt%) was prepared in this work. Thermal decomposition behavior and melting temperature were studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), respectively. Subsequently, mechanical properties including bending strength, compressive strength, impact strength and hardness were tested respectively. Afterwards, the fracture morphology of the bending test samples was observed by scanning electron microscopy (SEM). In addition, murine fibroblast L929 cells were adopted for cytotoxicity test by CCK-8 assay in vitro, and the morphology of cells was observed by inverted fluorescence microscope simultaneously, cell compatibility of CFR-PEEK composites was tested.

Astaxanthin protects ARPE-19 cells against oxidative stress injury induced by hydrogen peroxide

The present study aimed to investigate the protective effect and mechanism of action of astaxanthin (AST) on the oxidative stress injury of adult retinal pigment epithelium-19 (ARPE-19) cells induced by hydrogen peroxide (H2O2). ARPE-19 cells were divided into five groups, including control group, H2O2 model group, AST1 group, AST2 group and AST3 group. MTT was used to determine cell viability. Cell morphology was visualized under an inverted fluorescence microscope. TUNEL assay and flow cytometry was performed to detect cell apoptosis. To examine the levels of ROS, SOD and MDA, we used DCFH-DA, WST-1 and TBA assays. The optimal concentration of AST for increasing cell viability was 40 µg/L. Pretreatment with AST alleviated damages in cell morphology induced by H2O2. Pretreatment with AST had the best protective effect on ARPE-19 cells from oxidative stress injury induced by H2O2. AST treatment had protective effect on ARPE-19 cells against apoptosis. Treatment with AST reduced the apoptotic rate of ARPE-19 cells induced by H2O2. Treatment with AST altered the levels of ROS, SOD and MDA in ARPE-19 cells induced by H2O2. The present study demonstrates that AST protects ARPE-19 cells against oxidative stress injury, probably by inhibiting the production of ROS, elevating the activity of SOD and reducing the content of MDA. Of note, preventive delivery of AST (40 µg/L) has the best effect. The present study also provides a theoretical basis for the prevention and treatment of age-related macular degeneration.

Viability of Descemet Membrane Endothelial Keratoplasty Grafts Folded in the Eye Bank

Preloaded, trifolded grafts in Descemet membrane endothelial keratoplasty require transfer of the trifolding process from the corneal transplant surgeon to the eye bank technician. We sought to assess whether trifolding may be safely conducted by an eye bank technician with cell loss comparable to standard peeling and lifting. A total of 10 grafts were stained, peeled, and transferred directly onto a bed of Calcein-AM and Amvisc Plus by an eye bank technician. Five grafts were removed and stained as a scroll, and 5 grafts were trifolded with the endothelium in before transfer. Photographs were acquired with an inverted fluorescence microscope, and image segmentation was performed. A t test was conducted to compare differences in endothelial cell loss across groups. Mean cell loss in the scroll group was 18.5% [95% confidence interval (CI): 15.2%-21.9%] compared with 7.6% of the trifolded group (95% CI: 1.7%-13.5%). A 2-tailed t test indicated decreased cell loss in the trifolded group (P = 0.013). Despite additional manipulation of the graft, trifolding of Descemet membrane and endothelium may be performed by an eye bank technician without significantly increased cell loss relative to graft preparation as a scroll.

PiggyBac Transposon-Mediated Transgenesis in the Pacific Oyster (Crassostrea gigas) - First Time in Mollusks.

As an effective method of transgenesis, the plasmid of PiggyBac transposon containing GFP (PiggyBac) transposon system has been widely used in various organisms but not yet in mollusks. In this work, piggyBac containing green fluorescent protein (GFP) was transferred into the Pacific oyster (Crassostrea gigas) by sperm-mediated gene transfer with or without electroporation. Fluorescent larvae were then observed and isolated under an inverted fluorescence microscope, and insertion of piggyBac was tested by polymerase chain reaction (PCR) using genomic DNA as template. Oyster larvae with green fluorescence were observed after transgenesis with or without electroporation, but electroporation increased the efficiency of sperm-mediated transgenesis. Subsequently, the recombinant piggyBac plasmid containing gGH (piggyBac-gGH) containing GFP and a growth hormone gene from orange-spotted grouper (gGH) was transferred into oysters using sperm mediation with electroporation, and fluorescent larvae were observed and isolated. The insertion of piggyBac-gGH was tested by PCR and genome walking analysis. PCR analysis indicated that piggyBac-gGH was transferred into oyster larvae; genome walking analysis further showed the detailed location where piggyBac-gGH was inserted in the oyster genome. This is the first time that piggyBac transposon-mediated transgenesis has been applied in mollusks.

Effect of rat adipose-derived mesenchymal stem cells on macrophages phenotype in skin wound

Objective To investigate the effect of local injection of rat adipose-derived mesenchymal stem cells (ADSCs)on the phenotype of macrophages in skin wound. Methods The cryopreserved primary SD rat ADSCs were resuscitated and then sub-cultured. ADSCs of the third generation were used in the experiments. Thirty six SD rats were divided into ADSCs group (n=18) and control group(n=18) by random numbers table method. The full thickness skin wounds were established on both sides of the spine. After the model establishment 0.2 ml ADSCs suspension labeled by live cell stain Chloromethylbenzamido derivatives of 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate (CM-Dil) with the concentration of 5×106/ml was subcutaneously annularly injected in the skin wound of SD rats in ADSCs group. The SD rats in control group were given 0.2 ml serum-free Dulbecco modified Eagle medium (DMEM). On 3, 7, and 14 days after injury, six rats were selected from each group to measure the wound area and healing rate. The healed wound tissues were harvested to observe the morphology by HE staining. The expressions of interleukin (IL)-10 were detected by immunohistochemistry staining. The double-positive expressions of CD68 and inducible nitric oxide synthase (iNOS) (M1 type macrophages) and of CD68 and arginase-1 (Arg-1) (M2 type macrophages) were detected by immunofluorescent staining. The distribution of CM-Dil-labeled ADSCs in healed wound tissue 14 days after injury was observed by inverted fluorescence microscope. Results (1) At day 3 after injury, the wound areas in two groups were covered with crust and surrounding redness, and the wound healing rates were slightly different; at day 7 after injury, the wound area of ADSCs group was significantly smaller than that of control group, and the healing speed and rate of ADSCs group was significantly higher than that of control group (P<0.01); at day 14 after injury, the healing rate of ADSCs group was nearly 99% (P<0.01), and the healing skin tissue texture of ADSCs group was better than that of control group. (2) At day 3 after injury, there were a large number of inflammatory cells and disorganized collagen fiber in the wound areas of two groups; at day 7 after injury, the inflammatory cells infiltration reduced in ADSCs group compared with control group, and the collagen fiber arrangement in control group was in disorder; at day 14 after injury, the inflammatory cells in both groups obviously decreased, and ADSCs group had more new vessels and more orderly arrangement of collagen fiber than control group. CM Dil labeled ADSCs were seen in the healing wound tissue in ADSCs group. (3) At day 3 after injury, there was little difference in M1 type macrophage distribution in the two groups; ADSCs group had more M2 type macrophage cells than control group significantly (P<0.01); the expression of IL 10 in ADSCs group was not high, which did not differ from that of control group; at days 7 and 14 after injury, ADSCs group has fewer M1 type macrophage cells, more M2 type macrophage cells, and higher expressions of IL-10 than control group (P<0.01). Conclusion The ADSCs trasplantation can promote the change from M1 type to M2 type macrophages, facilitating wound regeneration and healing. Key words: Mesenchymal stem cells; Macrophages; Wound healing

Effect of lentivirus-mediated small interfering RNA on mitogen- and stress-activated protein kinase 1 in spinal cord injury of rats

To investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats. One hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group ( n=35), the rats in SCI group were given SCI according to Allen's method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above ( n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T 10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins. Western blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury ( P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury ( P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment ( P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B ( P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B ( P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B ( P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B ( P<0.05). Lentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.

Preparation of Molecular Stimuli-Responsive Gel Valves That Regulate Microchannels Autonomously

Diagnosis sensors using micro-total analysis systems (m-TAS) have been developed for detecting target biomolecules such as proteins and saccharides because they are signal biomolecules for monitoring body conditions and diseases. Valve systems that regulate the flow of a sample solution in the microchannels are the most important elements in fabricating m-TAS as diagnostic chips. Standard m-TAS uses electromechanical elements fabricated by MEMS technologies. As such electromechanical elements are based on complicated systems, advantages of m-TAS such as simplicity and miniaturization are lost. We have prepared biomolecularly stimuli-responsive hydrogels using biomolecular complexes as dynamic crosslinks that reversibly form or dissociate according to changes in an external environment. For example, biomolecularly stimuli-responsive hydrogels with antigen-antibody complexes or glycopolymer-lectin complexes swelled gradually in response to the concentration of a target biomolecule such as an antigen and glucose. Biomolecularly stimuli-responsive hydrogels that exhibited unique shrinkage in the presence of a target biomolecule were strategically designed by molecular imprinting. Their biomolecularly stimuli-responsive swelling and shrinkage were based on drastic changes in the crosslinking density of the hydrogel networks, induced by the dissociation and formation of biomolecular complexes as dynamic crosslinks in the presence of a target biomolecule. In this study, three types of micron-sized molecular stimuli-responsive hydrogels, bisphenol A (BPA)-, glucose- and concanavalin A (ConA)-responsive micro-hydrogels, were prepared by photopolymerization in a microchannel. We investigated the flow rate of the microchannels with their responsive micro-hydrogels in the absence and presence of the target molecule. ConA-responsive micro-hydrogels were prepared by molecular imprinting as follows. First, glycosyloxyethyl methacrylate as a ligand monomer and ConA as a molecular template were mixed for forming a molecular complex. Acrylamide as a main monomer, N, N’-methylenbisacrylamide as a crosslinker, and 2, 2'-azobis(2-methylpropionamidine) dihydrochloride as a photoinitiator were dissolved in a phosphate buffer solution. The solution was exposed to UV light focused using an inverted fluorescence microscope to polymerize the monomers. A ConA-imprinted micro-hydrogel was prepared near the branch point of the Y-shaped microchannel. To evaluated the ConA-imprinted micro-hydrogel as a self-regulated valve for a microchannel flow control system, the flow rates of the channels were measured while a buffer solution without and with target ConA was pumped into the Y-shape microchannel. After flowing a buffer solution with ConA, the ConA-imprinted micro-hydrogel in the channel shrank in response to ConA, followed by an increase in the flow rate. These results suggest that the ConA-imprinted micro-hydrogel is useful as a self-regulated micro-hydrogel valve for fabricating smart microchannels which can autonomously change the flow rate in response to the target ConA. ConA-responsive micro-hydrogels leads to the fabrication of smart microchannels in which micro-hydrogel valves can open and close autonomously and sensitively. In addition, BPA-responsive and glucose-responsive micro-hyrogels were prepared in a microchannel by photopolymerization using an inverted fluorescence microscope. This paper also demonstrates the autonomous changes in the flow rate in the microchannel with their micro-hydrogels as molecularly stimuli-responsive valves.

Study on Crude Flavonoids Extraction from Longan Leaves and its anti Human Lung Adenocareinoma Cell Line A549

The crude extracts of flavonoids were obtained from longan leaves via ultrasonic and purified with macroporous resin of AB-8. Taking rutin as the standard, the amount of flavonoids in crude extracts was measured by ultraviolet spectrophotometer. The concentration of flavonoids after extraction and purification was 0.775 μg/mL. Total flavonoids in the extractum accounted for 77.5%. Total flavonoids accounted for 77.49% of the dry weight of the longan leaves. MTT assay was applied to test the A549 cell activities treated by a series of concentrations of flavonoids (50, 100, 200, 400 and 800 μg/mL) for 24h, 48h and 72h, respectively. The morphological changes of A549 cells treated by 100, 200 and 400μg/mL flavonoids for 48h were observed by inverted microscope and fluorescence microscope, respectively. The results showed that the total flavonoids of longan leaves obviously inhibit the proliferation of A549 cells, with cell shrinkage, nuclear fragmentation and apoptosis occurred.

Cytotoxicity Assessment of a Poly(methyl methacrylate) Synthesized for the Direct Fabrication of Bone Tissues

ABSTRACT A cytotoxicity study is performed on a poly(methyl methacrylate) polymer (PMMA) to be used for the fabrication of bone tissue by Rapid Prototyping (RP). The solution polymerization is conducted in a pilot plant reactor using more appropriated reagents in consideration of the medical application. Moreover, the polymer is efficiently handled to avoid the side effect of the monomer, reducing the concentration of this specie to 287,731 µg MMA/kg PMMA. The cytotoxicity of the polymer is determined through growth monitoring, adherence and morphology of L-929 cells. Additionally, MTT and LIVE/DEAD tests are performed. The results showed continuous and progressive growth of the cells on the surface of the specimens. Moreover, the material did not influence on the viability of mesenchymal cells and inverted fluorescence microscopy images showed a polyanionic dye calcein well retained in the cells in contact with the PMMA as well as the negative control after 72 hours. Thus, the polymer was efficiently synthesized and handled for the expected demands.

Cytotoxic Activities of Total Saponins from Plena Clematis on Human Tumor Cell Lines In Vitro.

To investigate the anti-proliferative effects of saponins prepared from Plena Clematis (PC) cultured in Fujian Province, China on 4 human tumor cell lines and its possible anti-tumor mechanism. The growth inhibition assays of saponins on human esophageal squamous carcinoma cell line (EC9706), human hepatoma cell line (HepG-2), human oral cancer cell line (KB) and human gastric cancer cell line (BGC-823) were evaluated in vitro by thiazolyl blue (MTT) method. The inhibitory effects on EC9706 treated with different concentrations of saponins (15.62, 31.25, 62.50, 125, 250 and 500 μg/mL) were performed in vitro by MTT method. The morphology and nuclear staining with acridine orange/ethidium bromide of EC9706 cells treated with saponins were illustrated under an inverted phase fluorescence microscope. The apoptotic effects of saponins were further evaluated by annexin-V/propidium iodide dual staining experiment to examine the occurrence of phosphatidylserine externalization onto the cell surface by a flflow cytometer. MTT assay showed that the saponins could inhibit the proliferation of 4 tumor cell lines. Among them, the maximum inhibition rate of 73.1% was detected in EC9706 cells at the saponins concentration of 250 μg/mL for 24 h. Further investigation indicated that the saponins induced EC9706 cells apoposis. The EC9706 cells presented apoptotic characteristics when treated with saponins, including that the morphologies of EC9706 cells were appeared round-shaped with higher refraction, and the cell nuclear stained orange with EB after 250 μg/mL saponins exposure. The flow cytometry analysis results showed that the induction of cell cycle arrest in apoptotic system may participate in the anti-proliferative activity of saponins on EC9706 cells. The saponins from PC exhibited significant cytotoxicity against human EC9706, KB, BGC-823, and HepG-2 cells and might be beneficial to development of ethnic pharmaceutical plant for potential anti-tumor drugs.

Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10μs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45kV/cm and 100 pulses at 1kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

A novel and easy-to-use approach for assessment of chimeric antigen receptor (CAR) modified immune cell cytotoxicity

Abstract Cancer immunotherapies have shown promising results for treating various cancers. One such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), thus combining tumor antigen specificity with immune cell activation in a single receptor. Currently, assessment of this CAR-modified immune cell cytotoxicity is limited by conventional tools (e.g., standard Chromium-51 release assay), which are time-consuming, costly, and labor-intensive. In this study, we developed a novel, easy-to-use, and non-radioactive approach for assessment of CAR-modified immune cell cytotoxicity on the basis of luciferase bioluminescent signal. After a 4- hour incubation of CAR-modified immune cells in a 96-Well optical-bottom microplate, which was pre-seeded with targeted cell stablely expressed eGFP -firefly luciferase fusion gene (GFP-FFluc), the chemical bioluminescent signal of GFP-FFluc was quantified by a fluorescent microplate reader. The positive correlation between FFLuc signal and number of target cells was observed. The dose-dependent specific lysis is comparable with Chromium-51 release assays. The cytotoxicity activity measured by this approach can be further quantified under a common inverted fluorescence microscope to evaluate the morphology and dynamics of CAR-modified immune cells. In conclusion, this newly developed approach provides the scientific community with a powerful tool to study the CAR-modified immune cell cytotoxicity.

Activation of the SphK1/ERK/p-ERK pathway promotes autophagy in colon cancer cells.

Sphingosine kinase 1 (SphK1) is a master kinase that catalyzes the synthesis of sphingosine 1 phosphate and participates in the regulation of cell proliferation and autophagy. The present study aimed to assess the effects of the activation of the SphK1/extracellular signal-regulated kinase (ERK)/phosphorylated (p-)ERK pathway in the regulation of autophagy in colon cancer (HT-29) cells. Inverted fluorescence microscopy was used to detect the expression of green fluorescent protein (GFP) in the SphK1-overexpressing HT-29 cells [SphK1(+)-HT-29] and the negative control HT-29 cells (NC-HT-29). Western blotting was used to detect the protein expression levels of SphK1, ERK1/2, p-ERK1/2, as well as those of the autophagy-associated markers LC3A, ATG5, and ULK1. Protein localization and expression of the LC3A antibody were detected by immunofluorescence. The results demonstrated that GFP was similarly expressed in SphK1(+)-HT-29 and NC-HT-29 cells. However, significantly increased SphK1 mRNA and protein expression levels were detected in SphK1(+)-HT-29 cells compared with in NC-HT-29 cells, which resulted in upregulated ERK/p-ERK. Furthermore, the protein expression levels of the three autophagy-associated markers increased. LC3A protein was localized in the cytoplasm of SphK1(+)-HT-29 cells, indicating autophagy. In summary, the findings of the present study suggested that activation of the SphK1/ERK/p-ERK pathway promotes autophagy in colon cancer HT-29 cells.

Glass-based microchip with finger electrodes for the formation, collection, and biointeractions of liposomes

A concept using a glass-based microchip with finger electrodes, under a two-step voltage-controlled manner, to accomplish the formation and collection of liposomes is presented. By the actions of electro-osmotic and electrolytic bubbles in low and high applied voltages, respectively, the liposomes were first formed in an electrolyte reservoir; then, they were successfully moved to a collected reservoir, which is helpful for subsequent on-chip processing. This study evaluated the distribution of an electric field for different couple-finger distances using finite-element analysis software. The simulation result shows that the electric field density increases rapidly with the decrease of finger distance, with a quadratic relationship. Experiment results show that, when the applied AC voltage is 1 Vp-p, at a driving frequency of 10 Hz, the liposomes are first formed near the boundary of the electrolyte reservoir, and when the voltage is higher, the mass production of liposomes is achieved. When the voltage is higher than 4 Vp-p, the electrolytic bubbles grow cyclically to separate the liposomes from the electrolyte reservoir and therefore collect in a specific reservoir. Liposomes encapsulate doxorubicin, and their drug release to the lung cancer cell line H1299 was also implemented to verify their biointeraction characteristics. These experimental results were all identified using an inverted fluorescence microscope.

Influence on invasion and migration of hepatocellular carcinoma tumor endothelial cells by up-regulation of microRNA-3178

Objective To explore the effect of invasion and migration of hepatocellular carcinoma (HCC) endothelial cells (TECs) affected by overexpression of microRNA-3178 (miR-3178) through the transfection of miR-3178 mimic. Methods Real-time polymerase chain reaction (Real-time PCR) was used to identify differential expression of miR-3178 in normal hepatic sinusoidal endothelial cells (HSECs ) and HCC TECs. Furthermore, HCC TECs were divided into 3 groups: control (CON) group, miRNA-3178 up-regulation (Mimics, up-regulation of miR-3178 expression was achieved using miR-3178 mimics transfected into HCC TECs) group and negative control (NC, negative control sequence was transfected into HCC TECs) group. RT-PCR was used to detect expression of miR-3178 in HCC TECs before and after transfection. Transfection efficiency was observed by using an inverted fluorescence microscope. HCC TECs invasionand migration were measured by matrigel invasion and transwell migration assay. EGR3 protein expression of HCC TECs were identified by Western blotting analysis. EGR3 mRNA expression of HCC TECs were identified by RT-PCR analysis. Results The results of RT-PCR showed that miR-3178 was significantly down-regulated in HCC TECs compared to HSECs (P<0.05), and expression of miR-3178 was significantly increased after the transcienttransfection (P<0.05). The transfection efficiency in HCC TECs was morethan 90%. Number of migrated and invaded cells and in miR-3178 group was significantly less than those in other groups. Target gene prediction software showed EGR3 was a possible candidate target. Transfection of miR-3178 mimic significantly decreased the mRNA and protein expression levels of EGR3. Conclusion MiR-3178 was downregulated in HCC TECs and overexpression of miR-3178 can specifically inhibit migration and invasion of HCC TECsin vitro through inhibiting EGR3 expression, thus, miR-3178 might be a critical targeted therapy strategy for HCC. Key words: Hepatocellular carcinoma; Tumor vascular endothelial cells; MicroRNA-3178; Invasion; Migration

Cytoprotective effects of diosmetin against hydrogen peroxide-induced L02 cell oxidative damage via activation of the Nrf2-ARE signaling pathway.

Oxidative stress is considered a crucial mediator in the pathogenesis of various liver diseases. The flavone diosmetin has been reported to exhibit antioxidant activities; however, the hepatoprotective effects of diosmetin against oxidative stress, and the underlying molecular mechanisms, remain unknown. The present study aimed to investigate the potential hepatoprotective effects of diosmetin on hydrogen peroxide (H2O2)‑induced oxidative damage in L02 cells and attempted to evaluate the role of the nuclear factor erythroid 2‑related factor 2 (Nrf2)/antioxidant response element pathway in this process. L02 cells were divided into groups: Control (DMSO, diosmetin), H2O2, Trolox or tertiary butylhydroquinone and diosmetin (different doses). Protective effects in L02 cells were determined by CCK‑8, cell apoptosis and lactate dehydrogenase leakage assays. Flow cytometry and inverted fluorescence microscope were used to measure the intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Protein expression levels were of Nrf2, heme oxygenase‑1 (HO‑1) and NAD(P)H quinone oxidoreductase‑1 (NQO1) were determined by western blotting and mRNA levels were determined by reverse transcription‑quantitative polymerase chain reaction. The results revealed that H2O2 induced notable injury to L02 cells, as demonstrated by decreased cell viability, increased lactate dehydrogenase release, apoptotic rate and intracellular ROS production, and by the loss of MMP. Conversely, diosmetin (20‑40µM) significantly reversed the damaging effects of H2O2, which indicated that diosmetin may exhibit potent hepatoprotective potential against H2O2‑induced oxidative damage. Furthermore, pretreatment with diosmetin elevated mRNA and protein expression levels of Nrf2, HO‑1 and NQO1. The present study is the first, to the best of our knowledge, to demonstrate that activation of the Nrf2/NQO1‑HO‑1 signaling pathway maybe involved in the cytoprotective effects of diosmetin against oxidative stress. Therefore, diosmetin may be considered a promising therapeutic agent for the treatment of various liver diseases associated with oxidative stress.

Construction of tissue engineered cartilage based on acellular cartilage extracellular matrix oriented scaffold and chondrocytes

To observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage. Chondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining). After chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining. ACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.

Homocysteine induces calcium overload in neonatal rat atrial cells through activation of sodium current and CaMKⅡδ

Objective: To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). Methods: NRICs were assigned to 9 groups after culture for 3 days: (1) control group; (2) Hcy group (0, 50, 100, 200, 500 μmol/L for 48 hours); (3) antioxidant group (NAC, 10 μmol/L for 24 hours); (4) Hcy+NAC group (500 μmol/L Hcy for 48 hours, then treated with 10 μmol/L NAC for 24 hours); (5) calcium/calmodulin dependent protein kinase Ⅱδ (CaMKⅡδ) inhibitor group (KN-93, 3 μmol/L KN-93 for 5 hours); (6) specific sodium current inhibitor group (ELE, 1 μmol/L ELE for 5 hours); (7) Hcy+KN-93 group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 for 5 hours); (8) Hcy+ELE group (500 μmol/L Hcy for 48 hours, then treated with 1 μmol/L ELE for 5 hours; (9) Hcy+KN-93+ELE group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 and 1 μmol/L ELE for 5 hours). Moreover, NRICs were also treated with CaMKⅡδ-siRNA lentivirus, and Nav1.5-siRNA lentivirus, negative lentivirus carrier containing green fluorescent protein (GFP) for 24 hours. The MOI values of the three groups were 10. Infection efficiency of lentivirus was determined by observing the percentage of GFP fluorescence under inverted fluorescence microscope after transfection for 24 hours, and cultured regularly with simultaneous Puro screening, then cells were grouped as Hcy+CaMKⅡδ-siRNA group, Hcy+Nav1.5-siRNA group and Hcy+negative group. The concentration of Ca(2+) in NRICs ([Ca(2+)]i) of various groups was detected through Fluo-4/AM fluorescence probe, then 2', 7'- two chlorofluorescein diacetate (DCFH-DA) was used as a probe to detect reactive oxygen species (ROS) in NRICs by flow cytometry. The malondialdehyde (MDA) was detected by the activity of superoxide dismutase (SOD) and xanthine oxidase was detected by thiobarbituric acid colorimetry. The protein and mRNA expression level of CaMKⅡδ and Nav1.5 in NRICs were detected by Western blot and quantitative real-time PCR. Results: (1) ROS, MDA and SOD were similar between NAC group and control group, ROS and MDA were significantly increased, while SOD was significantly reduced in Hcy group in a concentration-dependent manner. (2) [Ca(2+)]i: The level of [Ca(2+)]i was (155.57+7.25), (187.43+13.07), (248.98+27.22) and (307.36+15.09) nmol/L in 50, 100, 200 and 500 μmol/L Hcy groups, which was significantly higher than that in the control group ((123.18+7.24) nmol/L, P<0.01). In addition, the level of [Ca(2+)]i in Hcy+NAC group ((222.87+23.71)nmol/L) was significantly lower than that in Hcy 500 μmol/L group ((305.15+39.45) nmol/L, P<0.05), while [Ca(2+)]i level was similar between NAC group and the control group. (3) The protein expression of CaMKⅡδ and Nav1.5 was significantly upregulated in Hcy groups than in the control group. The protein expression level of CaMKⅡδ-Thr287 was significantly lower in NAC group than in Hcy 500 μmol/L group (P<0.01), however, there was no significant difference on the protein expression levels of CaMKⅡδ-Thr287 and Nav1.5 between NAC group and control group (all P>0.05). (4) The protein expression levels of CaMKⅡδ-Thr287 and the concentration of [Ca(2+)]i were significantly lower in Hcy+KN-93 group and Hcy+KN-93+ELE group than in Hcy 500 μmol/L group (P<0.05). [Ca(2+)]i concentration was significantly lower in Hcy+KN-93 group, Hcy+ELE group and KN-93+ELE+Hcy group than in Hcy 500 μmol/L group (P<0.05). (5) The mRNA and protein expression levels of CaMKⅡδ and Nav1.5 in each group infected with lentivirus: the GFP expression was ideal post lentivirus transfection for 24 hours (up to 90%), which was significantly lower in the CaMKⅡδ-siRNA group and Nav1.5-siRNA group than in the negative infection group (all P<0.05), which was similar between negative infection group and control group (P>0.05). Moreover, the mRNA and protein expression levels of CaMKⅡδ and CaMKⅡδ-Thr287 was significantly lower in Hcy+Nav1.5-siRNA group than in Hcy+negative infection group (P<0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group (P>0.05). Conclusions: Hcy can induce calcium overload in NRICs by increasing oxidative stress, upregulating the sodium channel protein, and activating the late sodium current and phosphorylating CaMKⅡδ.