Browsing by Author "Chen, Wen Liang"

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An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate
(2007-01-15)
Yueh, Sunny C. H.
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Lai, Yi An
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Chen, Wen Liang
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Hsu, Hsiao Han
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Mao, Simon J. T.
Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we had isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp beta-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we have developed a novel affinity column procedure using an mAb prepared against a-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl and 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then prewashed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HC1, pH 6.8. As a result, the isolated Hp was devoid of apoA-1 and was able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1, or 2-2 was greater than 95% with an yield greater than 50%. The procedure described here is significantly improved in time consumption, recovery, and purity. The rationale, design, and optimization for each step are described in detail. (c) 2006 Elsevier B.V. All rights reserved.
beta-Lactoglobulin Influences Human Immunity and Promotes Cell Proliferation
(2016-01-01)
Tai, Chun San
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Chen, Yi Yun
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Chen, Wen Liang
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生物科技學系
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分子醫學與生物工程研究所
beta-Lactoglobulin (LG) is suspected to enhance ormodulate humanimmune responses. Moreover, LGis also hypothesized to increase human cell proliferation. However, these potential functions of LG have not been directly or thoroughly addressed. In this study, we demonstrated that LG is a potent stimulator of cell proliferation using a hybridoma cell (a splenocyte fused with amyeloma cell) model. LG's ability to promote cell proliferation was lost when the protein is denatured. To further investigate the influence of LG's conformation on cell proliferation, we chemically modified LG by either carboxymethylation (CM) or acetylation and observed significantly reduced cell proliferation when the protein structure was altered. Furthermore, we proved that LG enhances cell proliferation via receptor-mediated membrane IgM receptor. These data indicated that nondenatured LG is the major component in milk that modulates cell proliferation. Collectively, our study showed that LG plays a key role in enhancing immune responses by promoting cell proliferation through IgM receptor.
Gold nanoparticles regulate the blimp1/pax5 pathway and enhance antibody secretion in B-cells
(2014-03-28)
Lee, Chia-Hui
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Syu, Shih-Han
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Chen, Yu-Shiun
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Hussain, Saber M.
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Onischuk, Andrei Aleksandrovich
Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 h after treatment but returned to control levels 24 h after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and western blot experiments indicates that GNP-treatment upregulated B-lymphocyte-induced maturation protein 1 (blimp1) and downregulated paired box 5 (pax5). Immunostaining for blimp1 and pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity, the immune-stimulatory activity of nanoparticles could play an important role in human health and could have an environmental impact.
Haptoglobin expression correlates with tumor differentiation and five-year overall survival rate in hepatocellular carcinoma
(2017-02-03)
Tai, Chun-San
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Lin, Yan-Ren
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Teng, Tsung-Han
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Lin, Ping-Yi
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Tu, Siang-Jyun
Elevated serum haptoglobin (Hp) is identified as a prognostic marker in multiple types of solid tumors, which is correlated with poor prognosis. HCC is one of the major causes of cancer deaths in worldwide, which remains poor prognosis and is clinically urgent for discovering early diagnostic markers. However, except for serum Hp, the correlation of tumor Hp expression with hepatocellular carcinoma (HCC) progression is still unclear. In this study, we evaluated and identified the tissue Hp expression as a prognostic marker to predict the survival rate of HCC patients. To evaluate the prognostic value of Hp expression for HCC, two cohorts were enrolled in our study, including total 130 matched pair tissue sections (both adjacent non-tumorous and tumor tissue derived from same patient) of HCC patients from Changhua Christian Hospital (CCH) and total 316 RNA-seq data with clinical information of HCC patients from The Cancer Genome Atlas (TCGA) database. In contrast to other types of cancers, HCC tumor tissues have lower Hp protein expression in CCH cohort and have lower Hp mRNA expression in TCGA cohort as compared with adjacent non-tumorous tissues (p < 0.001). Moreover, lower Hp expression is significantly correlated with different stages of HCC cancer differentiation in CCH cohort (one-way ANOVA, p < 0.001). Most importantly, lower Hp expression is highly correlated with poor five-year overall survival rate in TCGA cohort (p < 0.01). Based on our data, we conclude that tissue Hp expression positively correlates with better HCC tumor differentiation and increased five-year overall survival rate of HCC patients. The results indicated that tissue Hp is potentially a prognostic marker for HCC patients. Our findings may further provide a new insight of effective treatments along with biopsy diagnosis of HCC patients.
Improving the reproducibility, accuracy, and stability of an electrochemical biosensor platform for point-of-care use
(2020-05-01)
Chen, Lung-Chieh
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Wang, Erick
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Tai, Chun-San
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Chiu, Yuan-Chen
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Li, Chang-Wei
Electrochemical biosensors possess numerous desirable qualities for target detection, such as portability and ease of use, and are often considered for point-of-care (POC) development. Label-free affinity electrochemical biosensors constructed with semiconductor manufacturing technology (SMT)-produced electrodes and a streptavidin biomediator currently display the highest reproducibility, accuracy, and stability in modern biosensors. However, such biosensors still do not meet POC guidelines regarding these three characteristics. The purpose of this research was to resolve the limitations in reproducibility and accuracy caused by problems with production of the biosensors, with the aim of developing a platform capable of producing devices that exceed POC standards. SMT production settings were optimized and bioreceptor immobilization was improved through the use of a unique linker, producing a biosensor with exceptional reproducibility, impressive accuracy, and high stability. Importantly, the three characteristics of the sensors produced using the proposed platform all meet POC standards set by the Clinical and Laboratory Standards Institute (CLSI). This suggests possible approval of the biosensors for POC development. Furthermore, the detection range of the platform was demonstrated by constructing biosensors capable of detecting common POC targets, including circulating tumor cells (CTCs), DNA/RNA, and curcumin, and the devices were optimized for POC use. Overall, the platform developed in this study shows high potential for production of POC biosensors.
Nanochip-Induced Epithelial-to-Mesenchymal Transition: Impact of Physical Microenvironment on Cancer Metastasis
(2018-04-11)
Dhawan, Udesh
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Sue, Ming-Wen
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Lan, Kuan-Chun
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Buddhakosai, Waradee
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Pao Hui Huang
Epithelial-to-mesenchymal transition (EMT) is a highly orchestrated process motivated by the nature of physical and chemical compositions of the tumor microenvironment (TME). The role of the physical framework of the TME in guiding cells toward EMT is poorly understood. To investigate this, breast cancer MDA-MB-231 and MCF-7 cells were cultured on nanochips comprising tantalum oxide nanodots ranging in diameter from 10 to 200 nm, fabricated through electrochemical approach and collectively referred to as artificial microenvironments. The 100 and 200 nm nanochips induced the cells to adopt an elongated or spindle-shaped morphology. The key EMT genes, E-cadherin, N-cadherin, and vimentin, displayed the spatial control exhibited by the artificial microenvironments. The E-cadherin gene expression was attenuated, whereas those of N-cadherin and vimentin were amplified by 100 and 200 rim nanochips, indicating the induction of EMT. Transcription factors, snail and twist, were identified for modulating the EMT genes in the cells on these artificial microenvironments. Localization of EMT proteins observed through immunostaining indicated the loss of cell cell junctions on 100 and 200 rim nanochips, confirming the EMT induction. Thus, by utilizing an in vitro approach, we demonstrate how the physical framework of the TME may possibly trigger or assist in inducing EMT in vivo. Applications in the fields of drug discovery, biomedical engineering, and cancer research are expected.
Nanochips of Tantalum Oxide Nanodots as artificial-microenvironments for monitoring Ovarian cancer progressiveness
(2016-08-18)
Dhawan, Udesh
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Wang, Ssu-Meng
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Chu, Ying Hao
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Huang, Guewha S.
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Lin, Yan Ren
Nanotopography modulates cell characteristics and cell behavior. Nanotopological cues can be exploited to investigate the in-vivo modulation of cell characteristics by the cellular microenvironment. However, the studies explaining the modulation of tumor cell characteristics and identifying the transition step in cancer progressiveness are scarce. Here, we engineered nanochips comprising of Tantalum oxide nanodot arrays of 10, 50, 100 and 200 nm as artificial microenvironments to study the modulation of cancer cell behavior. Clinical samples of different types of Ovarian cancer at different stages were obtained, primary cultures were established and then seeded on different nanochips. Immunofluorescence (IF) was performed to compare the morphologies and cell characteristics. Indices corresponding to cell characteristics were defined. A statistical comparison of the cell characteristics in response to the nanochips was performed. The cells displayed differential growth parameters. Morphology, Viability, focal adhesions, microfilament bundles and cell area were modulated by the nanochips which can be used as a measure to study the cancer progressiveness. The ease of fabrication of nanochips ensures mass-production. The ability of the nanochips to act as artificial microenvironments and modulate cell behavior may lead to further prospects in the markerless monitoring of the progressiveness and ultimately, improving the prognosis of Ovarian cancer.
Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi
(2017-01-01)
Liu, Yu-Chen
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Chen, Wen Liang
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Kung, Wei-Hsiang
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Huang, Hsien-Da
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生物科技學系
Background: Emerging evidence indicates that plant miRNAs can present within human circulating system through dietary intake and regulate human gene expression. Hence we deduced that comestible plants miRNAs can be identified in the public available small RNA sequencing data sets. Results: In this study, we identified abundant plant miRNAs sequences from 410 human plasma small RNA sequencing data sets. One particular plant miRNA miR2910, conserved in fruits and vegetables, was found to present in high relative amount in the plasma samples. This miRNA, with same 6mer and 7mer-A1 target seed sequences as hsa-miR-4259 and hsa-miR-4715-5p, was predicted to target human JAK-STAT signaling pathway gene SPRY4 and transcription regulation genes. Conclusions: Through analysis of public available plasma small RNA sequencing data, we found the supporting evidence for the plant miRNAs cross kingdom RNAi within human circulating system.
Spatial Control of Cell-Nanosurface Interactions by Tantalum Oxide Nanodots for Improved Implant Geometry
(2016-06-30)
Dhawan, Udesh
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Pan, Hsu An
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Lee, Chia Hui
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Chu, Ying Hao
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Huang, Guewha Steven
Nanotopological cues can be exploited to understand the nature of interactions between cells and their microenvironment to generate superior implant geometries. Nanosurface parameters which modulate the cell behavior and characteristics such as focal adhesions, cell morphology are not clearly understood. Here, we studied the role of different nanotopographic dimensions in modulating the cell behavior, characteristics and ultimately the cell fate and accordingly, a methodology to improve implant surface geometry is proposed. Tantalum oxide nanodots of 50, 100nm dot diameter with an inter-dot spacing of 20, 70nm and heights 40, 100nm respectively, were engineered on Silicon substrates. MG63 cells were cultured for 72 hours and the modulation in morphology, focal adhesions, cell extensible area, cell viability, transcription factors and genes responsible for bone protein secretion as a function of the nanodot diameter, inter-dot distance and nanodot height were evaluated. Nanodots of 50nm diameter with a 20nm inter-dot spacing and 40nm height enhanced cell spreading area by 40%, promoted cell viability by 70% and upregulated transcription factors and genes twice as much, as compared to the 100nm nanodots with 70nm inter-dot spacing and 100nm height. Favorable interactions between cells and all dimensions of 50nm nanodot diameter were observed, determined with Scanning electron microscopy and Immunofluorescence staining. Nanodot height played a vital role in controlling the cell fate. Dimensions of nanodot features which triggered a transition in cell characteristics or behavior was also defined through statistical analysis. The findings of this study provide insights in the parameters of nanotopographic features which can vitally control the cell fate and should therefore be taken into account when designing implant geometries.
The Spatiotemporal Control of Osteoblast Cell Growth, Behavior, and Function Dictated by Nanostructured Stainless Steel Artificial Microenvironments
(2017-02-06)
Dhawan, Udesh
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Pan, Hsu-An
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Shie, Meng-Je
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Chu, Ying Hao
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Huang, Guewha S.
The successful application of a nanostructured biomaterial as an implant is strongly determined by the nanotopography size triggering the ideal cell response. Here, nanoporous topography on 304L stainless steel substrates was engineered to identify the nanotopography size causing a transition in the cellular characteristics, and accordingly, the design of nanostructured stainless steel surface as orthopedic implants is proposed. A variety of nanopore diameters ranging from 100 to 220 nm were fabricated by one-step electrolysis process and collectively referred to as artificial microenvironments. Control over the nanopore diameter was achieved by varying bias voltage. MG63 osteoblasts were cultured on the nanoporous surfaces for different days. Immunofluorescence (IF) and scanning electron microscopy (SEM) were performed to compare the modulation in cell morphologies and characteristics. Osteoblasts displayed differential growth parameters and distinct transition in cell behavior after nanopore reached a certain diameter. Nanopores with 100-nm diameter promoted cell growth, focal adhesions, cell area, viability, vinculin-stained area, calcium mineralization, and alkaline phosphatase activity. The ability of these nanoporous substrates to differentially modulate the cell behavior and assist in identifying the transition step will be beneficial to biomedical engineers to develop superior implant geometries, triggering an ideal cell response at the cell-nanotopography interface.