Ke N.,ACEA Biosciences, Inc. |
Ke N.,Hangzhou High Throughput Drug Screening Center |
Xi B.,ACEA Biosciences, Inc. |
Xi B.,Hangzhou High Throughput Drug Screening Center |
And 16 more authors.
Analytical Chemistry | Year: 2010
Cellular processes such as cell cycle progression, mitosis, apoptosis, and cell migration are characterized by well-defined events that are modulated as a function of time. Measuring these events in the context of time and its perturbation by small molecule compounds and RNAi can provide mechanistic information about cellular pathways being affected. We have used impedance-based time-dependent cell response profiling (TCRP) to measure and characterize cellular responses to antimitotic compounds or siRNAs. Our findings indicate that small molecule perturbation of mitosis leads to unique TCRP. We have further used this unique TCRP signature to screen 119′595 compound library and identified novel antimitotic compounds based on clustering analysis of the TCRPs. Importantly, 113 of the 117 hit compounds in the TCRP antimitotic cluster were confirmed as antimitotic based on independent assays, thus establishing the robust predictive nature of this profiling approach. In addition, potent and novel agents that induce mitotic arrest either by directly interfering with tubulin polymerization or by other mechanisms were identified. The TCRP approach allows for a practical and unbiased phenotypic profiling and screening tool for small molecule and RNAi perturbation of specific cellular pathways and time resolution of the TCRP approach can serve as a complement for other existing multidimensional profiling approaches. © 2010 American Chemical Society.
Guan N.,CAS Shanghai Institute of Materia Medica |
Gao W.,CAS Shanghai Institute of Materia Medica |
He M.,CAS Shanghai Institute of Materia Medica |
Zheng M.,Hangzhou High Throughput Drug Screening Center |
And 4 more authors.
Analytical Biochemistry | Year: 2012
β-Cell injury plays an important role in the development of type 1 and type 2 diabetes. Most of the β-cell bioassays depend on labeling or endpoint assessments that might not capture the true physiology or pathology of the injury process. In the current study, we dynamically detected a broad range of pathological and pharmacological responses to four toxicants (cytokine mixture, free fatty acid mixture, streptozotocin, and hydrogen peroxide) in living β-cells (INS-1E and MIN6) by a label-free, cell-based assay system named xCELLigence, codeveloped by ACEA Biosciences and Roche Diagnostics. Our results suggest that the impedance readout is highly sensitive and provides more information than some of the conventional endpoint cytotoxicity assays for β-cell injury such as the Cell Counting Kit-8 (CCK-8) and morphology measurements. Furthermore, this system was used to evaluate the anti-injury effects of glucagon-like peptide-1 (GLP-1) and its nonpeptidic mimetic Boc5 by monitoring responses to four toxicants in two β-cell lines. This study confirms that the protective property of Boc5 on β-cells is similar to that of GLP-1. © 2012 Elsevier Inc. All rights reserved.
Guan N.,CAS Shanghai Institute of Materia Medica |
Deng J.,CAS Shanghai Institute of Materia Medica |
Li T.,CAS Shanghai Institute of Materia Medica |
Xu X.,CAS Shanghai Institute of Materia Medica |
And 3 more authors.
Molecular BioSystems | Year: 2013
T cells play a critical role in maintaining the normal function of the adaptive immune response, with their dysfunction resulting in a variety of autoimmune and immunodeficiency diseases. Efficient and accurate detection of T cell function is therefore crucial to clinical diagnosis and development of immunomodulators. A variety of in vitro cellular systems are currently employed for analyzing T cell activation, yet all suffer from some combination of low throughput, unnatural conditions and long assay times. Label-free technologies are capable of detecting phenotypic responses to treatments under physiological conditions, thereby potentially accelerating drug discovery by facilitating the use of disease-relevant cell models for functional assessment and clinical diagnosis. The xCELLigence system is an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes in cells, such as those induced during T cell activation. Here we describe the development and validation of a T cell activation assay based upon electrical impedance. Co-activation of Jurkat cells with anti-CD28 and anti-CD3 functional antibodies led to impedance changes that were rapidly and sensitively recorded (within 30 minutes). This phenomenon was also observed in human peripheral blood mononuclear cells. These changes reflect morphological and adhesive alterations correlated with cytoskeletal reorganization as verified by microscopy. They were functionally dependent on canonical T cell signaling pathways, including calcium-mediated signals and Src family kinases because relevant inhibitors impaired T cell activation. Our results provide a convenient approach to measure T cell activation in real-time and to elucidate the underlying mechanisms of action through probing with small molecules. © 2013 The Royal Society of Chemistry.
Shao X.,Zhejiang University |
Qian Y.,Zhejiang University |
Xu C.,Zhejiang University |
Hong B.,Zhejiang University |
And 6 more authors.
PLoS ONE | Year: 2013
Background: Concanavalin A (ConA)-induced hepatitis is an experimental murine model mirroring the pathology of human autoimmune hepatitis. Aim: To investigate the effects of intrasplenically transplanted fetal hepatocytes (BNL.CL2) transfected with recombinant adenovirus vector expressing the IL-18 binding protein (IL-18BP) and IL-4 fusion protein on ConA-induced hepatitis in mice. Methods: Ad-IL-18BP/IL-4 was used to infect BNL.CL2 cells. IL-4 and IL-18BP fusion protein expression were detected by ELISA and Western blotting. BNL.CL2 cells infected with Ad-IL-18BP/IL-4 were intrasplenically transplanted into mice. After 10 days, mice were injected with ConA (15 mg/kg), and sacrificed 18 hours later. Liver injury was assessed by serum transaminase and liver histology. TNF-α, IL-18, IL-4, IL-10, IL-12p70 and monocyte-chemoattracting protein (MCP)-1 levels in serum and liver homogenates were detected by ELISA. Signaling molecules in liver homogenates were analyzed by Western blotting. Results: Ad-IL-18BP/IL-4 effectively expressed the IL-18BP/IL-4 fusion protein for more than 14 days in BNL.CL12 cells. Treatment of mice with Ad-IL-18BP/IL-4-BNL.CL2 before ConA injection significantly reduced the elevated plasma levels of transaminases compared with ConA control groups. TNF-α, IL-18, IL-12p70 and MCP-1 levels in serum and liver homogenates from mice transplanted with Ad-IL-18BP/IL-4-BNL.CL2 were lower and IL-4 and IL-10 levels were higher than control groups. Phosphorylation levels of NF-κB p65, AKT, p38 and JNK1/2 in liver homogenates were markedly suppressed by Ad-IL-18BP/IL-4. Conclusions: Ad-IL-18BP/IL-4 was effectively transfected into mouse BNL.CL2 cells. Intrasplenic transplantation of Ad-IL-18BP/IL-4-BNL.CL12 cells alleviated the severity of inflammation in ConA-induced experimental hepatitis and provides a useful basis for the targeted gene therapy of liver disease. © 2013 Shao et al.
Xu H.,Capital Medical University |
Xu H.,Anhui Medical University |
Gu N.,Capital Medical University |
Liu Z.-B.,Capital Medical University |
And 5 more authors.
Oncology Letters | Year: 2012
NY-ESO-1 belongs to the cancer testis antigens (CTA) family, and is identified in a variety of tumors. Certain studies have demonstrated that NY-ESO-1 predicts tumor recurrence and treatment response. No reports are currently available regarding the correlation between NY-ESO-1 and the recurrence of hepatocellular carcinoma (HCC) following surgery. The purpose of the present study was to evaluate the association between NY-ESO-1 and relapse of HCC and to explore the possible mechanisms for this correlation. A total of 120 HCC patients were analyzed for the expression of NY-ESO-1 by immunohistochemistry (IHC). A stable NY-ESO-1 over-expressed HepG2 cell line (ESO-HepG2) was established to determine the biological effects of NY-ESO-1 on cell proliferation, cell cycle and migration by using the xCELLigence DP system, flow cytometry and xCELLigence SP system. NY-ESO-1 was positive in 28 of 120 (23.3%) HCC tumor tissues. NY-ESO-1 was not detectable in adjacent normal liver tissues. A close correlation was found between NY-ESO-1 expression and the recurrence of HCC following surgery (P=0.007). Kaplan-Meier analysis showed a shorter recurrence-free survival (RFS) for patients positive for NY-ESO-1 (log-rank test, P=0.003). The Cox regression model demonstrated that NY-ESO-1 expression was a significant independent predictor for the recurrence of HCC following curative surgery (P=0.022). Compared with HepG2 cells, ESO-HepG2 cells have increased migration but not proliferation ability. In conclusion, NY-ESO-1 expression is associated with worse HCC outcome following surgery, and the mechanism for this finding may be that NY-ESO-1 increases tumor cell migration.