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Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein
(2015-11-01)
Tang, Yin-Liang
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Chiu, Chien-Yu
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Lin, Chun-Yu
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Huang, Chung-Hao
;
Chen, Yen-Hsu
Dengue virus (DENV) infection is currently at pandemic levels, with populations in tropical and subtropical regions at greatest risk of infection. Early diagnosis and management remain the cornerstone for good clinical outcomes, thus efficient and accurate diagnostic technology in the early stage of the disease is urgently needed. Serotype-specific monoclonal antibodies (mAbs) against the DENV1 nonstructural protein 1 (NS1), DA12-4, DA13-2, and DA15-3, which were recently generated using the hybridoma technique, are suitable for use in diagnostic platforms. Immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis further confirmed the serotype specificity of these three monoclonal antibodies. The ELISA-based diagnostic platform was established using the combination of two highly sensitive mAbs (DA15-3 and DB20-6). The same combination was also used for the flow cytometry-based diagnostic platform. We report here the detection limits of flow cytometry-based and ELISA-based diagnostic platforms using these mAbs to be 0.1 and 1 ng/mL, respectively. The collected clinical patient serum samples were also assayed by these two serotyping diagnostic platforms. The sensitivity and specificity for detecting NS1 protein of DENV1 are 90% and 96%, respectively. The accuracy of our platform for testing clinical samples is more advanced than that of the two commercial NS1 diagnostic platforms. In conclusion, our platforms are suitable for the early detection of NS1 protein in DENV1 infected patients.
A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model
(2017-06-23)
Tu, Shih-Hsin
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Hsieh, Yi-Chen
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Huang, Li-Chi
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Lin, Chun-Yu
;
Hsu, Kai-Wen
Background: As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models. Methods: In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D-imaging on orthotropic-developed breast-tumor-bearing mice. Results: In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x10(11) cell numbers with great correlation (R-2 = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R-2 > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0.001), whereas IVIS Spectrum-CT 3D-visualization showed that blood of mice with lung metastasis contained more than twice the CTC numbers than ordinary tumor-bearing mice. We demonstrated a positive correlation between lung metastasis status and CTC numbers in peripheral mouse blood. Conclusion: Collectively, the techniques developed for this study resulted in the integration of CTC assessments into preclinical models both in vivo and ex vivo, which will facilitate translational targeted therapy in clinical practice.
Reconstructing Genome-Wide Protein-Protein Interaction Networks Using Multiple Strategies with Homologous Mapping
(2015-01-20)
Lo, Yu-Shu
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Huang, Sing-Han
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Luo, Yong-Chun
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Lin, Chun-Yu
;
Yang, Jinn-Moon
Background One of the crucial steps toward understanding the biological functions of a cellular system is to investigate protein-protein interaction (PPI) networks. As an increasing number of reliable PPIs become available, there is a growing need for discovering PPIs to reconstruct PPI networks of interesting organisms. Some interolog-based methods and homologous PPI families have been proposed for predicting PPIs from the known PPIs of source organisms. Results Here, we propose a multiple-strategy scoring method to identify reliable PPIs for reconstructing the mouse PPI network from two well-known organisms: human and fly. We firstly identified the PPI candidates of target organisms based on homologous PPIs, sharing significant sequence similarities (joint E-value <= 1 x 10(-40)), from source organisms using generalized interolog mapping. These PPI candidates were evaluated by our multiple-strategy scoring method, combining sequence similarities, normalized ranks, and conservation scores across multiple organisms. According to 106,825 PPI candidates in yeast derived from human and fly, our scoring method can achieve high prediction accuracy and outperform generalized interolog mapping. Experiment results show that our multiple-strategy score can avoid the influence of the protein family size and length to significantly improve PPI prediction accuracy and reflect the biological functions. In addition, the top-ranked and conserved PPIs are often orthologous/ essential interactions and share the functional similarity. Based on these reliable predicted PPIs, we reconstructed a comprehensive mouse PPI network, which is a scale-free network and can reflect the biological functions and high connectivity of 292 KEGG modules, including 216 pathways and 76 structural complexes. Conclusions Experimental results show that our scoring method can improve the predicting accuracy based on the normalized rank and evolutionary conservation from multiple organisms. Our predicted PPIs share similar biological processes and cellular components, and the reconstructed genome-wide PPI network can reflect network topology and modularity. We believe that our method is useful for inferring reliable PPIs and reconstructing a comprehensive PPI network of an interesting organism.
Lipopolysaccharide-Induced Nitric Oxide, Prostaglandin E2, and Cytokine Production of Mouse and Human Macrophages Are Suppressed by Pheophytin-b
(2017-12-01)
Lin, Chun-Yu
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Wang, Wen-Hung
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Chen, Shin-Huei
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Chang, Yu-Wei
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Hung, Ling-Chien
Sepsis is an overwhelming systemic response to infection that frequently results in tissue damage, organ failure, and even death. Nitric oxide (NO), prostaglandin E2 (PGE2), and cytokine overproduction are thought to be associated with the immunostimulatory cascade in sepsis. In the present study, we analyzed the anti-inflammatory efficacy of the pheophytin-b on both RAW264.7 murine macrophage and purified human CD14(+) monocytes stimulated with lipopolysaccharide (LPS) and elucidated the mechanisms by analyzing the cell signaling pathways known to be activated in sepsis. Pheophytin-b suppressed the overexpression of NO, PGE2, and cytokines in LPS-stimulated macrophages without inducing cytotoxicity. It also reduced NOS2 and COX-2 mRNA and protein levels. The inhibitory effects on NO, PGE2, and cytokine overproduction arose from the suppression of STAT-1 and PI3K/Akt pathways; no changes in NF-kappa B, MAPK, and AP-1 signaling were detected. Thus, pheophytin-b may represent a potential candidate to beneficially modulate the inflammatory response in sepsis.
Module organization and variance in protein-protein interaction networks
(2015-03-23)
Lin, Chun-Yu
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Lee, Tsai-Ling
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Chiu, Yi-Yuan
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Lin, Yi-Wei
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Lo, Yu-Shu
A module is a group of closely related proteins that act in concert to perform specific biological functions through protein-protein interactions (PPIs) that occur in time and space. However, the underlying module organization and variance remain unclear. In this study, we collected module templates to infer respective module families, including 58,041 homologous modules in 1,678 species, and PPI families using searches of complete genomic database. We then derived PPI evolution scores and interface evolution scores to describe the module elements, including core and ring components. Functions of core components were highly correlated with those of essential genes. In comparison with ring components, core proteins/PPIs were conserved across multiple species. Subsequently, protein/module variance of PPI networks confirmed that core components form dynamic network hubs and play key roles in various biological functions. Based on the analyses of gene essentiality, module variance, and gene co-expression, we summarize the observations of module organization and variance as follows: 1) a module consists of core and ring components; 2) core components perform major biological functions and collaborate with ring components to execute certain functions in some cases; 3) core components are more conserved and essential during organizational changes in different biological states or conditions.
Shared IgG Infection Signatures vs. Hemorrhage-Restricted IgA Clusters in Human Dengue: A Phenotype of Differential Class-Switch via TGF beta 1
(2017-12-04)
Huang, Chung-Hao
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Chang, Ya-Hui
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Lin, Chun-Yu
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Wang, Wen-Hung
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Kuan, Hui-Chung
Phenotypic manifestations of infectious diseases are closely related to individual immune responses. Methods to extract information from patients' own immune reactions would be of great use for both diagnosis and treatment. Dengue fever is one of the diseases that clinical aggravations could occur paradoxically after humoral immunity appears. This property makes dengue fever an excellent disease model to explore. A principal component analyses (PCAs)-based framework derived from a prior vaccination study was developed. The framework was verified by successful demonstrations of known IgG signatures from a Mexico Dengue data set. Afterward the pipeline was tested upon de novo IgG and IgA libraries of Dengue patients from southern Taiwan. We discovered four infection signatures within IgG repertoires, two of which were identical to previous reports. However, it was IgA but not IgG that could differentiate hemorrhagic from non-hemorrhagic patients. IgA repertoires were found more diversified among bleeders, from whom seven signature clusters were characterized. The expressions of transforming growth factor beta 1 (TGF beta 1) and accordingly mediated class-switch activity of IgA were distinct only among the PCA-segregated bleeding group. In sum, intercontinental sharing of IgG signatures in dengue fever was demonstrated via a unified working flow. Differential regulation of IgA class-switch with associated diversity expansion plus existences of hemorrhage-restricted clusters were shown. The ability of the framework to find common IgG signatures would implicate applications to infections even from unknown pathogens. The clusters within IgA repertoires could offer perspectives to other IgA-related bleeding disorders such as Henoch-Schonlein purpura or IgA nephropathy. Substantiated grounds for IgA-specific effector function via TGF beta 1-mediated class-switch would be a new factor to consider for infectious diseases.
Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model
(2017-11-03)
Huang, Li-Chi
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Lin, Ching-Ling
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Qiu, Jia-Zheng
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Lin, Chun-Yu
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Hsu, Kai-Wen
Triple-negative breast cancer (TNBC) subtype is associated with poor prognosis and a high risk of recurrence-related death in women. Despite the aggressiveness of TNBCs, targeted TNBC therapy is not yet available in the clinic. To overcome this challenge, we generated highly metastatic TNBC cells (LM) derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to identify targetable molecules for attenuating the progression of TNBC metastasis. Gene analysis of primary tumor (P), first-round (1LM) and second-round (2LM) metastasized lung cells revealed that mesenchymal-related genes were significantly expressed in LM cells, especially in 2LM cells. Interestingly, alpha 9-nAChR gene expression was also dramatically induced in LM cells, confirming our previous finding that alpha 9-nAChR plays important roles in receptor-mediated carcinogenic signals in human breast cancer development. Using alpha 9-nAChR as a biomarker, we transfected 2LM cells with CRISPR/Cas9 lentivirus targeting the alpha 9-nAChR genomic region (2LM-alpha 9-nAChR-null), showing that mesenchymal markers and the migration and invasion abilities of 2LM cells were significantly attenuated in 2LM-alpha 9-nAChR-null cells both in vitro and in vivo. In addition, the high efficiency of editing the alpha 9-nAChR gene using a CRISPR/Cas9 lentivirus was demonstrated by gene sequencing, genomic indel frequency and protein expression analyses. Collectively, these results confirmed those of our previous study that advanced-stage breast tumors are associated with substantially higher levels of alpha 9-nAChR gene expression, indicating that alpha 9-nAChR expression is essential for mediating TNBC metastasis during cancer development and may potentially act as a biomarker for targeted therapy in clinical investigations.
Serum Galectin-9 and Galectin-3-Binding Protein in Acute Dengue Virus Infection
(2016-06-01)
Liu, Kuan-Ting
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Liu, Yao-Hua
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Chen, Yen-Hsu
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Lin, Chun-Yu
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Huang, Chung-Hao
Dengue fever is a serious threat for public health and induces various inflammatory cytokines and mediators, including galectins and glycoproteins. Diverse immune responses and immunological pathways are induced in different phases of dengue fever progression. However, the status of serum galectins and glycoproteins is not fully determined. The aim of this study was to investigate the serum concentration and potential interaction of soluble galectin-1, galectin-3, galectin-9, galectin-3 binding protein (galectin-3BP), glycoprotein 130 (gp130), and E-, L-, and P-selectin in patients with dengue fever in acute febrile phase. In this study, 317 febrile patients (187 dengue patients, 150 non-dengue patients that included 48 patients with bacterial infection and 102 patients with other febrile illness) who presented to the emergency department and 20 healthy controls were enrolled. Our results showed the levels of galectin-9 and galectin-3BP were significantly higher in dengue patients than those in healthy controls. Lower serum levels of galectin-1, galectin-3, and E-, L-, and P-selectin in dengue patients were detected compared to bacteria-infected patients, but not to healthy controls. In addition, strong correlation between galectin-9 and galectin-3BP was observed in dengue patients. In summary, our study suggested galectin-9 and galectin-3BP might be critical inflammatory mediators in acute dengue virus infection.
The impact of comorbidity on survival after hemorrhagic stroke among dialysis patients: a nationwide population-based study
(2014-11-27)
Lin, Chun-Yu
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Chien, Chih-Chiang
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Chen, Hung-An
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Su, Fu-Mei
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Wang, Jhi-Joung
Background: This study was aimed at determining the outcome and examining the association between comorbidities and mortality after intracerebral hemorrhage in chronic dialysis patients. Methods: We used the Taiwan National Health Insurance Research Database and enrolled patients who underwent maintenance dialysis between 2000 and 2007. Annual incidence of intracerebral hemorrhage in patients receiving dialysis from 2000 to 2007 was determined. To identify predictors of hemorrhagic stroke, we used logistic regression model to estimate the relative ratio of factors for intracerebral hemorrhage in the most recent cohort (2007). The cumulative survival rate and comorbid conditions associated with mortality after intracerebral hemorrhage among all dialysis patients between 2000 and 2007 was calculated using the Kaplan-Meier method and Cox regression analysis. Results: We identified 57,261 patients on maintenance dialysis in the cohort of 2007, and 340 patients had history of intracerebral hemorrhage among them. Hypertension was the most common comorbidity of dialysis patients. The incidence rate of intracerebral hemorrhage among dialysis patients was about 0.6%. Adjusted logistic regression model showed that male gender, middle age (45-64 years), hypertension, and previous history of stroke were the independent predictors for the occurrence of intracerebral hemorrhage among chronic dialysis patients. 1,939 dialysis patients with development of intracerebral hemorrhage in the analysis period from 2000 to 2007 were identified. In-hospital mortality was high (36.15%) following intracerebral hemorrhage. They were followed up after intracerebral hemorrhage for a mean time of 41.56 months. Adjusted Cox regression analyses demonstrated that the factors independently associated with mortality after intracerebral hemorrhage among dialysis patients included diabetes mellitus, malignancy and a history of prior stroke. Conclusions: Dialysis patients who have history of prior stroke, diabetes and malignancy have worse survival than patients without these comorbidities. Attention must focus on providing optimal medical care after hemorrhagic stroke for these target groups to reduce mortality.
Graphene oxide dot-based microstructures via dispersion and support of bovine serum albumin
(2016-10-01)
Chang, Hsin-Yu
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Lin, Chun-Yu
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Chang, Chia-Yuan
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Teng, Hsisheng
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Campagnola, Paul J.
Three-dimensional graphene oxide-based microstructures have great potential for use in microelectronic and biomedical applications and can be fabricated by using two-photon excitation photochemical processes. In such excitation processes, rose Bengal is the preferred candidate as the photoactivator in two-photon crosslinking; nevertheless, it induces graphene oxide self-aggregation. Herein, bovine serum albumin (BSA) is adopted to prevent graphene oxide dot (GOD) aggregation even at very high GOD fabrication-solution concentrations, yet still supports GOD microstructures. In this manner, three-dimensional freeform GOD microstructures with different GOD concentrations were developed. Furthermore, the biocomparability of the GOD microstructure with the help of added biomaterials such as BSA was verified under living cell incubation. (C) 2016 Optical Society of America

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