Specialties and Awards

Specialties

Proteomics, Lipidomics, Molecular Biology and Molecular Diagnostics

Awards

2015 KMU Excellent homeroom teacher (校級績優導師)

2016 KMU Outstanding Faculty Research Award (研究績優教師/研究成果績優獎)

2017 Finalist of the Young Investigator Competition of the University of Cologne – Kaohsiung Medical University Joint Symposium

2017 KMU Outstanding Faculty Research Award (研究績優教師/優秀論文獎)

2017 KMU Teaching Excellence Award (106學年度教學優良教師)

Inventions and patents

Inventions and patents

Patent No. I359196:用於檢測伯氏疏螺旋體菌致病性菌株的引子對、檢驗套組以及方法Early detection of Borrelia burgdorferi sensu lato, the PCR primer pair and diagnostic method. (50%)

Patent No. I580782: 去除陰電性低密度脂蛋白之生化反應材料與裝置以及體外處理血液或血漿以去除其中陰電性低密度脂蛋白的方法 (25%)

Patent No. CN106434619A: 生化反应材料与装置以及体外处理血液或血浆的方法(25%)

Patent No. US20170044516A1: Biochemistry reactive material and device for eliminating low-density lipoprotein (LDL) and method for treating blood or plasma ex vivo to eliminate low-density lipoprotein therein (25%)Patent No. US20150148410A1: Identification of specific apolipoprotein epitopes on circulating atherogenic LDL (50%)

Publications

Publications

*: corresponding author

1. Chen CH, Ke LY, Chan HC, Chu CS, Lee AS, Lin KD, Lee MY, Hsiao PJ, Chen CH, ShinSJ. Electronegative low-density lipoprotein of patients with metabolic syndrome induces atherogenesis via disruption of STRA6 cascade. Journal of Diabetes Investigation. 2019 (accepted)
2. Yamakado S, Cho H, Inada M, Morikawa M, Jiang YH, Saito K, Nakaishi K, Watabe S, Takagi H, Kaneda M, Nakatsuma A, Ninomiya M, Imachi H, Arai T, Yoshimoto T, Murao K, Chang JH, Chen SM, Shih YC, Zeng MJ , Ke LY, Chen CH, Yoshimura T, Miura T, Ito E*. Urinary Adiponectin as A New Diagnostic Index for Chronic Kidney Disease Due to Diabetic Nephropathy. BMJ Open Diabetes Research & Care 2019;7(2):e000661.
3. Chen CH, Lin KD, Ke LY, Liang CJ, Kuo WC, Lee MY, Lee YL, Hsiao PJ, Hsu CC, Shin SJ. O-GlcNAcylation disrupts STRA6-retinol signals in kidneys of diabetes. Biochim Biophys Acta Gen Subj. Jun 2019;1863(6):1059-1069.
4. Chen FC, Shen KP, Ke LY, Lin HL, Wu CC, Shaw SU*. Flavonoids from Camellia sinensis (L.) O. Kuntze seed ameliorates TNF-a induced insulin resistance in HepG2 cells, Saudi Pharm J. May 2019; 27:4, p.507-516.
5. Law SH†, Chan ML†, Marathe GK, Parveen F, Chen CH, Ke LY*. An Updated Review of Lysophosphatidylcholine Metabolism Beyond the Lands Cycle in Human Diseases. Int. J. Mol. Sci. Mar 2019; 20(5). pii: E1149.
6. Hao CL, Lin HL, Ke LY Yen HW, Shen KP*. Pre‐germinated brown rice extract ameliorates high‐fat diet‐induced metabolic syndrome. Journal of Food Biochemistry 2019;2019:1-e12769.
7. Chu CS†, Chan HC†, Tsai MH, Stancel N, Lee HC, Cheng KH, Tung YC, Chan HCB, Wang CY, Shin SJ, Lai WT, Yang CY, Dixon RA, Chen CH*, Ke LY*. Range of L5 LDL levels in healthy adults and L5’s predictive power in patients with hyperlipidemia or coronary artery disease. Sci Rep. 2018 Aug 8;8(1):11866. doi: 10.1038/s41598-018-30243-w. [IF=4.122; 12/64=18.8 % in MULTIDISCIPLINARY SCIENCES
8. Wang YC†, Lee AS†, Lu LS†, Ke LY, Chen WY, Dong JW, Lu J, Chen ZP, Chu CS, Chan HC, Kuzan TY, Tsai MH, Hsu WL, Dixon RAF, Sawamura T, Chang KC*, Chen CH*. Human electronegative LDL induces mitochondrial dysfunction and premature senescence of vascular cells in vivo. Aging Cell. 2018 Jun 19:e12792. [IF=7.627; 3/53=5.7 % in GERIATRICS & GERONTOLOGY]
9. Lee CH, Chu CS, Tsai HJ, Ke LY, Lee HC, Yeh JL, Chen CH, Wu BN. Xanthine- derived KMUP-1 reverses glucotoxicity-activated Kv channels through the cAMP/PKA signaling pathway in rat pancreatic β cells. Chem Biol Interact. 2018 Jan 5;279:171-176. [IF=3.143; R/C=27/92=29.3% in TOXICOLOGY].
10. Ke LY, Chan HC, Chan HCB, Kalu FCU, Lee HC, Lin IL, Jhuo SJ, Lai WT, Tsao CR, Sawamura T, Dixon RA, Chen CH, Chu CS*, Shin SJ*. Electronegative Low-Density Lipoprotein L5 Induces Adipose Tissue Inflammation Associated With Metabolic Syndrome. The Journal of Clinical Endocrinology & Metabolism 2017 Dec 1;102(12):4615- 4625. [IF=5.455; R/C=20/138=14.5% in ENDOCRINOLOGY & METABOLISM].
11. Akyol S*, Lu J, Akyol O, Akcay F, Armutcu F, Ke LY*, Chen CH. The role of electronegative low-density lipoprotein in cardiovascular diseases and its therapeutic implications. Trends in Cardiovasc Med. 2017 May;27(4):239-246. doi: 10.1016/j.tcm.2016.11.002. Epub 2016 Nov 19. Review. PMID: 28040327. (Featured with editorial comment*). [IF=4.964; R/C=25/126=19.8% in CARDIAC AND CARDIOVASCULAR SYSTEMS].
12. Ke LY, Chang JG, Chang CS, Hsieh LL, Liu TC. Rapid screening for deleted form β-thalassemia by real-time quantitative PCR. J Clin Lab Anal. 2017 Jan;31(1). doi: 10.1002/jcla.22019. Epub 2016 Aug 16.
13. Chu CS, Ke LY, Chan HC, Chan HCB, Chen CC, Cheng KC, Lee HC, Kuo HF, Chang CT, Sheu SH, Chen CH, Lai WT. The Four Statin Benefit Groups Defined by The 2013 ACC/AHA New Cholesterol Guideline are Characterized by Increased Plasma Level of Electronegative LDL. Acta Cardiol Sin. 2016 Nov;32(6):667-675.
14. Chen CH, Ke LY, Chan HC, Lee AS, Lin KD, Chu CS, Lee MY, Hsiao PJ, Hsu C, Chen CH*, Shin SJ*. Electronegative low-density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved. J Lipid Res. 2016 Aug;57(8):1435-46. doi: 10.1194/jlr.M067215. [IF=4.810; R/C=54/286=18.9% in BIOCHEMISTRY MOLECULAR BIOLOGY].
15. Ke LY†, Chan HC†, Chen CC, Lu J, Marathe GK, Chu CS, Chan HC, Wang CY, Tung YC, McIntyre TM, Yen JH*, Chen CH*. Enhanced sphingomyelinase activity in apolipoprotein B100 contributes to the atherogenicity of electronegative LDL. J Med Chem. 2016 Feb 11;59(3):1032-40. [IF=6.259; R/C=3/60=5% in CHEMISTRY, MEDICINAL]
16. Lee HC, Lin HT, Ke LY, Wei C, Hsiao YL, Chu CS, Lai WT, Shin SJ, Chen CH, Sheu SH, Wu BN. VLDL from metabolic syndrome individuals enhanced lipid accumulation in atria with association of susceptibility to atrial fibrillation. Int J Mol Sci. 2016 Jan 20;17(1). [IF=3.226; R/C=54/166=32.5% in CHEMISTRY, MULTIDISCIPLINARY].
17. Stancel N, Chen CC, Ke LY, Chu CS, Lu J, Sawamura T, Chen CH*. Interplay between CRP, atherogenic LDL, and LOX-1 and its potential role in the pathogenesis of atherosclerosis. Clin Chem. 2016 Feb;62(2):320-7. PMID: 26607724. [IF=8.008; R/C=1/30=3.3% in MEDICAL LABORATORY TECHNOLOGY]
18. Tung YC, Ke LY, Lu PL, Lin KH, Lee SC, Lin YY, Chou LC, Tsai WC. Comparison of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test v1.0 with v2.0 in HIV-1 viral load quantification. Kaohsiung J Med Sci. 2015 Apr;31(4):188-93.
19. Tung YC, Ke LY, Tsai WC, Lin KH, Tsai SM, Wang CF, Lee HF, Su HJ, Lu PL. The development of a beacon-based real-time PCR for cytomegalovirus and its application in immunocompromised patients. Clin Lab. 2014;60(11):1895-901.
20. Ke LY†, Stancel N†, Bair H, Chen CH*. The underlying chemistry of electronegative LDL’s atherogenicity. Curr Atheroscler Rep. 2014;16:428. doi: 10.1007/s11883-014-0428-y. PMID: 24890631 [IF=2.731; R/C=35/63=55.5% in PERIPHERAL VASCULAR DISEASE]
21. Tung YC, Ke LY, Tsai SM, Lu PL, Tsai WC. High seroprevalence of human herpesvirus 8 infection in patients with systemic lupus erythematosus. Int J Rheum Dis. 2013 Dec;16(6):709-14.
22. Chan HC†, Ke LY†(co-first), Chu CS, Lee AS, Shen MY, Cruz MA, Hsu JF, Cheng KH, Chan HC, Lu J, Lai WT, Sawamura T, Sheu SH, Yen JH*, Chen CH*. Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation. Blood. 2013;122:3632-3641. PMID: 24030386 (Featured with editorial comment*). [IF=13.164; R/C=2/70=2.9% in CARDIAC & CARDIOVASCULAR SYSTEMS]
23. Tung YC, Lu PL, Ke LY, Tsai WC. CMV Infection and Pregnancy. Curr Obstet Gynecol Rep (2012) 1:216-222; 1(4). DOI:10.1007/s13669-012-0028-1.
24. Ke GM, Lin KH, Lu PL, Tung YC, Wang CF, Ke LY, Lee MS, Lin PC, Su HJ, Lin YY, Huang TP, Wang JR, Wang SY, Hsu LC, Chu PY. Molecular epidemiology of Echovirus 30 in Taiwan, 1988-2008. Virus Genes. 2011 Apr;42(2):178-88.
25. Ke LY, Engler DA, Lu J, Matsunami RK, Chan HC, Wang GJ, Yang CY, Chang JG, Chen CH*. Chemical composition–oriented receptor selectivity of L5, a naturally occurring atherogenic low-density lipoprotein. Pure Appl Chem. 2011;83:1731-1740. PMID: 24198440. [IF=2.626; R/C=67/166=40.3% in CHEMISTRY, MULTIDISCIPLINARY]
26. Chan HC, Ke LY, Liu CC, Chang LL, Tsai WC, Liu HW, Yen JH. Increased expression of suppressor of cytokine signaling 1 mRNA in patients with rheumatoid arthritis. Kaohsiung J Med Sci. 2010 Jun;26(6):290-8.
27. Chan HC, Ke LY, Chang LL, Liu CC, Hung YH, Lin CH, Li RN, Tsai WC, Liu HW, Yen JH. Suppressor of cytokine signaling 1 gene expression and polymorphisms in systemic lupus erythematosus. Lupus. 2010 May;19(6):696-702.
28. Ke GM, Yu SW, Ho CH, Chu PY, Ke LY, Lin KH, Tsai YC, Liu HJ, Lin MY. Characterization of newly emerging Newcastle disease viruses isolated during 2002-2008 in Taiwan. Virus Res. 2010 Feb;147(2):247-57.
29. Chu PY, Ke GM, Chang CH, Lin JC, Sun CY, Huang WL, Tsai YC, Ke LY, Lin KH. Molecular epidemiology of coxsackie A type 24 variant in Taiwan, 2000-2007. J Clin Virol. 2009 Aug;45(4):285-91.
30. Chen CH*, Dixon RAF, Ke LY, Willerson JT. Vascular progenitor cells in diabetes mellitus: Roles of Wnt signaling and negatively charged LDL. Circ Res. 2009;104:1038-1040. PMID:19423862. [IF=13.965; R/C=5/126=4.0% in CARDIAC AND CARDIOVASCULAR SYSTEMS].
31. Tung YC, Lin KH, Chiang HC, Ke LY, Chen YH, Ke GM, Chen TC, Chou LC, Lu PL. Molecular epidemiology of dengue virus serotype 2 in the Taiwan 2002 outbreak with envelope gene and nonstructural protein 1 gene analysis. Kaohsiung J Med Sci. 2008 Aug;24(8):398-407.
32. Tung YC, Lin KH, Chang K, Ke LY, Ke GM, Lu PL, Lin CY, Chen YH, Chiang HC. Phylogenetic study of dengue-3 virus in Taiwan with sequence analysis of the core gene. Kaohsiung J Med Sci. 2008 Feb;24(2):55-62.
33. Tsai JJ, Sun HY, Ke LY, Tsai KS, Chang SY, Hsieh SM, Hsiao CF, Yen JH, Hung CC, Chang SC. Higher seroprevalence of Entamoeba histolytica infection is associated with human immunodeficiency virus type 1 infection in Taiwan. Am J Trop Med Hyg. 2006 Jun;74(6):1016-9.
34. Ke GM, Cheng HL, Ke LY, Ji WT, Chulu JL, Liao MH, Chang TJ, Liu HJ. Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus. J Virol Methods. 2006 Apr;133(1):6-13.
35. Hsu MC, Tsai PY, Chen KT, Li LH, Chiang CC, Tsai JJ, Ke LY, Chen HY, Li SY. Genotyping of Chlamydia trachomatis from clinical specimens in Taiwan. J Med Microbiol. 2006 Mar;55(Pt 3):301-8.
36. Lin KH, Hwang KP, Ke GM, Wang CF, Ke LY, Hsu YT, Tung YC, Chu PY, Chen BH, Chen HL, Kao CL, Wang JR, Eng HL, Wang SY, Hsu LC, Chen HY. Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71. J Med Virol. 2006 Feb;78(2):254-62.

Research Plateforms

A. Flowchart for isolating atherogenic lipoproteins

1. Purifications for VLDL, LDL, and HDL: Blood samples to be used for VLDL, LDL and HDL isolation will be obtained from subjects as described in each sub-project. After the initial screening, blood samples will be removed from the subjects with precaution to avoid coagulation and ex vivo oxidation. The plasma will be treated with Complete Protease Inhibitor Cocktail (Roche; Cat. No. 05056489001; 1 tablet/100 mL) to prevent protein degradation. Lipoprotein preparation from the human. Overlay the plasma with 2 mL of Milli-Q water and spin at 20,000 rpm for 2 hours. Remove upper white fraction, chylomicrons, and save the remnant layer which contains VLDL, IDL, LDL, and HDL for a series of isolation steps. To progressively separate VLDL (d=0.93–1.006), IDL (d=1.006–1.019), LDL (1.019 to 1.063 g/dL) and HDL (1.063 to 1.210 g/dL) from one another, the remnant sample will be sequentially adjusted to d=1.006, d=1.019, d=1.063, d=1.210 respectively, by adding potassium bromide and followed by centrifugation at 45,000 rpm for 20 hrs at 4°C. After centrifugation at each isolation step, IDL will be discarded whist VLDL, LDL and HDL will be collected. Isolated VLDL, LDL and HDL samples will be treated with 5 mM EDTA and nitrogen to avoid ex vivo oxidation. Then, VLDL, LDL and HDL samples will be dialyzed against buffer A (20 M, pH8.0, 0.5 M EDTA) for 24 hours (×3 times) to remove excessive potassium bromide, and will be filtrated through a 0.22-µm filter (Sartorius; Minisart®) to sterilize the samples.
2. VLDL, LDL, and HDL subfractions: Approximately 30 mg of VLDL or LDL material will be injected onto a UnoQ12 anion-exchange column (BioRad) by using the ÄKTA fast-protein liquid chromatography (FPLC) pump (GE Healthcare Life Sciences, Pittsburgh, PA). VLDL or LDL will be eluted according to electronegativity by the use of a multistep gradient of buffer B (1 mol/L NaCl in buffer A) at a flow rate of 2 mL/min. In short, samples will be equilibrated with buffer A for 10 mins, following by linearly increase to 15% buffer B in 10 mins (fraction 1), linearly increase to 20% buffer B in 30 mins (fraction 2, 3), keep at 20% buffer B for 10 mins (fraction 4) and linearly increase to100% buffer B in 20 mins (fraction 5). Lastly, the effluents will be monitored at 280 nm.
   Purification of fractionated VLDL, LDL and HDL: Based on the gradient profile, each VLDL/LDL fractions will be pooled. The volume of each subfraction is constant. Dilution of VLDL/LDL during chromatography depends on the injection volume. The respective fractions will be concentrated with Centriprep® filters (YM-30; EMD Millipore Corp., Billerica, MA), be dialyzed against buffer A (20 M, pH8.0, 0.5 M EDTA) for 24 hours (3 days) and be sterilized by passing through 0.22-μm filters (Sartorius; Minisart®). The isolated fractions will be quantified at their protein concentrations by the Lowry method and then be stored at 4°C.
3. Apoptosis Measurements: Endothelial cells, renal cells are used after 3 or 4 passages and maintained in DMEM (Invitrogen) containing 10% FBS. During treatment, FBS reduced to 5% in DMEM. 1×104 cells are seeded in 96-well plate for 24 hours, subconfluent cultures f cultured cells are exposed to PBS (lipoprotein-free, negative control) or graded (25, 50, and 100 μg/mL) LDL subfractions, unfractionated normolipidemic LDL, and for 24 hours. Apoptosis is assessed with visualization by a Zeiss Axiovert 200 fluorescence microscope and filters to capture digital images based on Hoechst 33342, propidium iodide (red), and calcein AM (green) staining of nuclear, apoptotic DNA membrane integrity. Cytoplasmic histone-associated DNA fragmentation is examined by using the Cell Death Detection ELISA Assay (Roche) according to the protocol of the manufacturer.

B. Flowchart for studying proteomics and Lipidomics

1. 2-Dimensional electrophoresis: Protein contents of VLDL/LDL/HDL subfractions will be analyzed by 2-dimensional electrophoresis. Twice delipidated (1:1 EtAc+EtOH, 0.3 mL/30 μg LDL protein) VLDL/LDL/HDL particles will be centrifuged for 30 min (14000 rpm, 4°C). After removal of the solution, the lipoprotein pellet will be resuspended in 30 μL H2O. Resuspended samples will be incubated in ZOOM IPGRunner Cassette with Strip, 1X ZOOM 2D Protein Solubilizer 1, 1X Protease Inhibitor Cocktail, 20 mM DTT, and 3.5 mM Tris base at pH 7.4 for 2 hours. Two-dimensional-PAGE (isoelectrofocusing, equilibrating, performing) will be performed by ZOOM IPGRunner, ZOOM Equilibration Tray, and XCell SureLock Mini-Cel according to the user manual. The 4–20% 2-dimensional gels will be stained with SYPRO Ruby Protein Gel Stain (Ex/Em: 280, 450/610 nm).
2. LC/MSE analysis for protein composition: Proteins in each VLDL/LDL/HDL subfractions will be quantified by data-independent parallel-fragmentation mass spectrometry (LC/MSE), a Waters Xevo G2 Q-TOF mass spectrometer equipped with a nano-electrospray ionization (ESI) interface (Waters Corporation, MA, USA) and ProgenesisTM QI-P software (Nonlinear Dynamics, USA). Total proteins from HDL will be digested with trypsin, and be separated on a M-class ultra-performance liquid chromatography (UPLC; Waters Corporation, MA, USA). A 50 fmol-on-column tryptic digest of yeast alcohol dehydrogenase will be used as the internal standard. Peptide elution will be executed through a 75 μm × 25 cm BEH C-18 column under gradient conditions at a flow rate of 300 nL/min over 70 min at 35°C. The mobile phase will be composed of acetonitrile as the organic modifier and formic acid (0.1% v/v) for molecule protonation. Parallel ion fragmentation will be programmed to switch between low (4 eV) and high (15–45 eV) energies in the collision cell, and data will be collected from 50 to 2000 m/z utilizing glu-fibrinopeptide B (Sigma Aldrich, m/z 785.8426) as the separate data channel lock mass calibrant. Data will be processed with ProgenesisTM QI-P for relative quantification. In addition, the ProteinLynx Global Server v2.5 (Waters) will be further used to search for possible modifications. Deisotoped results will be searched for protein association from the UniProt (www.uniprot.org) human protein database.
3. LC/MSE analysis for lipid composition: Total lipids from each subfraction of HDL will be quantified by LC/MSE and ProgenesisTM QI software. Different from a proteomic approach, an ACQUITY UPLC I-Class System equipped with CSHTM 1.7 um, 2.1 mm × 10 cm C-18 column and a flow rate of 400 uL/min over 18 min at 55°C will be used for lipidomic studies. The mobile phase A will be composed of 10mM NH4HCO2 in ACN/H2O (60/40) and 0.1% formic acid (0.1% v/v), mobile phase B will be composed of 10mM NH4HCO2 in IPA/ACN (90/10) and 0.1% formic acid (0.1% v/v) for molecule protonation. Mass spectrometry will be performed on Xevo G2 Q-Tof (Waters) instrument equipped with a low flow electrospray ionization probe (Waters) interface and operated in the data-independent collection mode (MSE). Parallel ion fragmentation will be programmed to switch between low (4 eV) and high (35–55 eV) energies in the collision cell, and data will be collected from 200 to 1600 m/z utilizing leucine (Sigma Aldrich, m/z 556.2771) as the separate data channel lock mass calibrant. Data will be processed with MarkerLynx (Waters) and ProgenesisTM QI software.

Recent Research

•  1. An Updated Review of Lysophosphatidylcholine Metabolism Beyond the Lands Cycle in Human Diseases. Int. J. Mol. Sci. 2019 Mar 6;20(5). pii: E1149.
• 
• Lysophosphatidylcholine (LPC) is increasingly recognized as a key marker/factor positively associated with cardiovascular and neurodegenerative diseases. However, findings from recent clinical lipidomic studies of LPC have been controversial. A key issue is the complexity of the enzymatic cascade involved in LPC metabolism. Here, we address the coordination of these enzymes and the derangement that may disrupt LPC homeostasis, leading to metabolic disorders. LPC is mainly derived from the turnover of phosphatidylcholine (PC) in the circulation by phospholipase A₂ (PLA₂). In the presence of Acyl-CoA, lysophosphatidylcholine acyltransferase (LPCAT) converts LPC to PC, which rapidly gets recycled by the Lands cycle. However, overexpression or enhanced activity of PLA₂ increases the LPC content in modified low-density lipoprotein (LDL) and oxidized LDL, which play significant roles in the development of atherosclerotic plaques and endothelial dysfunction. The intracellular enzyme LPCAT cannot directly remove LPC from circulation. Hydrolysis of LPC by autotaxin, an enzyme with lysophospholipase D activity, generates lysophosphatidic acid, which is highly associated with cancers. Although enzymes with lysophospholipase A₁ activity could theoretically degrade LPC into harmless metabolites, they have not been found in the circulation. In conclusion, understanding enzyme kinetics and LPC metabolism may help identify novel therapeutic targets in LPC-associated diseases.

• 2. Range of L5 LDL levels in healthy adults and L5’s predictive power in patients with hyperlipidemia or coronary artery disease. Sci Rep. 2018 Aug 8;8(1):11866.
• 
• Electronegative L5 low-density lipoprotein (LDL) level may be a useful biomarker for predicting cardiovascular disease. We determined the range of plasma L5 levels in healthy adults (n = 35) and examined the power of L5 levels to differentiate patients with coronary artery disease (CAD; n = 40) or patients with hyperlipidemia (HLP) without evidence of CAD (n = 35) from healthy adults. The percent L5 in total LDL (L5%) was quantified by using fast-protein liquid chromatography with an anion-exchange column. Receiver operating characteristic curve analysis was performed to determine cut-off values for L5 levels. The mean L5% and plasma concentration of L5 (ie, [L5]) were significantly higher in patients with HLP or CAD than in healthy adults (P < 0.001). The ranges of L5% and [L5] in healthy adults were determined to be <1.6% and 1.6%, the odds ratio was 9.636 for HLP or CAD. In individuals with [L5] >1.7 mg/dL, the odds ratio was 17.684 for HLP or CAD. The power of L5% or [L5] to differentiate patients with HLP or CAD from healthy adults was superior to that of the LDL/high-density lipoprotein ratio. The ranges of L5% and [L5] in healthy adults determined here may be clinically useful in preventing and treating cardiovascular disease.

• 3. Electronegative Low-Density Lipoprotein L5 Induces Adipose Tissue Inflammation Associated With Metabolic Syndrome. The Journal of Clinical Endocrinology & Metabolism 2017 Dec 1;102(12):4615- 4625.
• 
• Electronegative low-density lipoprotein (LDL) L5 is a naturally occurring, atherogenic entity found at elevated levels in the plasma of patients with metabolic syndrome (MetS) in the absence of elevated plasma LDL levels. Objective: To investigate the role of L5 in the mechanism of adipose tissue inflammation associated with MetS. Patients/Setting: Plasma LDL isolated from patients with MetS (n = 29) and controls (n = 29) with similar plasma LDL levels was separated into five subfractions, L1 to L5, with increasing electronegativity. Design: We examined the invivo effects of L5 on adipose tissue in mice and the in vitro effects of L5 on adipocytokine signaling and monocytes. Results: Tail-vein injection of human L5 but not L1 into C57BL/6 mice induced the accumulation of F4/80+ and CD11c+ M1 macrophages. The effects of L5 were attenuated in mice deficient for L5's receptor, lectin-like oxidized LDL receptor 1 (LOX-1). L5 but not L1 induced human adipocytes to release inflammatory adipocytokines. Incubating human THP-1 monocytes with LDL-free culture media from L5-treated adipocytes enhanced the migration of monocytes by 300-fold (P < 0.001 vs L1-treated adipocyte media)-effects that were attenuated by LOX-1 neutralizing antibody. Migrated cells were positive for mature macrophage marker PM-2K, indicating the transformation of monocytes into macrophages. The infiltration of M1 macrophages in adipose tissue was also observed in a previously established hamster model of endogenously elevated L5. Conclusions: L5 induces adipose inflammation through LOX-1 by promoting macrophage maturation and infiltration into adipose tissue. Elevated plasma L5 levels may be a novel etiology of adipose tissue inflammation in patients with MetS.

• 4. The role of electronegative low-density lipoprotein in cardiovascular diseases and its therapeutic implications. Trends in Cardiovasc Med. 2017 May;27(4):239-246.
• 
• Cardiovascular disease (CVD) is a health problem of great concern to both the public and medical authorities. Low-density lipoprotein (LDL) has been reported to play an important role in both the development and progression of CVD, but studies are underway to determine how LDL exerts its effects. In recent years, it has been found that LDL has several subfractions, each of which affects endothelial function differently; L5, the most electronegative fraction, has been shown to be unique in that it induces an atherogenic response. This review examines the current knowledge concerning the relationships between L5 and CVD and highlights the role of L5 in the pathophysiology of CVD, especially with regards to atherosclerosis.

• 5.The Four Statin Benefit Groups Defined by The 2013 ACC/AHA New Cholesterol Guideline are Characterized by Increased Plasma Level of Electronegative LDL. Acta Cardiol Sin. 2016 Nov;32(6):667-675.
• 
• BACKGROUND: Significantly higher cytotoxic and thrombogenic human electronegative low-density lipoprotein (LDL), or L5, has been found in patients with stable coronary artery disease and acute coronary syndrome. We hypothesized that the statin-benefit groups (SBGs) defined by the new cholesterol guideline were of higher electronegative L5. METHODS: In total, 62 hyperlipidemia patients (mean age 59.4 ± 10.5, M/F 40/22) were retrospectively divided into SBGs (n = 44) and N-SBGs (n = 18). The levels of complete basic lipid panel, biochemical profile and electronegative L5 of each individual were obtained before and after rosuvastatin 10 mg/day for 3 months. RESULTS: After 3 months' statin therapy, significant reduction of total cholesterol, LDL-C and triglyceride were demonstrated (all p-values < 0.05), with 38.4% LDL-C reduction. The percentage of L5 was significantly reduced by 40.9% (from 4.4% to 2.6%) after statin therapy (p = 0.001). Regarding absolute L5 concentration, derived from L5% multiplied by LDL-C, there was approximate 63.8% reduction (from 6.3 mg/dL to 2.3 mg/dL) of absolute L5 (p < 0.001) after statin treatment. Notably, while plasma LDL-C levels were similar between SBGs and N-SBGs (152.8 ± 48.6 vs. 146.9 ± 35.0 mg/dL), the SBGs had significantly elevated L5% (5.2 ± 7.4% vs. 2.6 ± 1.9%, p = 0.031) and higher absolute L5 concentration (7.4 ± 10.4 vs. 3.7 ± 3.1 mg/dL, p = 0.036). Linear regression showed the significantly positive correlation between the plasma L5 concentration and the 10-year cardiovascular risk by pooled cohort equation (r = 0.297, p < 0.05). CONCLUSIONS: The four SBGs defined by the 2013 ACC/AHA new cholesterol guideline tend to have increased atherogenic electronegative L5. Statin therapy can effectively reduce the electronegative L5 of these four major SBGs.

• 6. Enhanced sphingomyelinase activity in apolipoprotein B100 contributes to the atherogenicity of electronegative LDL. J Med Chem. 2016 Feb 11;59(3):1032-40. 

Sphingomyelinase (SMase) catalyzes the degradation of sphingomyelin to ceramide. In patients with metabolic syndrome or diabetes, circulating plasma ceramide levels are significantly higher than in normal individuals. Our data indicate that electronegative low-density lipoprotein (LDL) shows SMase activity, which leads to increased ceramide levels that can produce pro-inflammatory effects and susceptibility to aggregation. According to sequence alignment and protein structure predictions, the putative catalytic site of SMase activity is in the α2 region of apoB-100. To identify specific post-translational modifications of apoB100 near the catalytic region, we performed data-independent, parallel-fragmentation liquid chromatography/mass spectrometry (LC/MSE), followed by data analysis with ProteinLynx GlobalServer v2.4. Results showed that the serine of apoB100 in electronegative LDL was highly O-glycosylated, including S1732, S1959, S2378, S2408 and S2429. These findings may support the changing of α-helix/β-pleated sheets ratio in protein structure analysis. Further study is necessary to confirm the activation of SMase activity by electronegative LDL.

7. The Underlying Chemistry of Electronegative LDL's Atherogenicity. (First author). Curr Atheroscler Rep. 2014 Aug;16(8):428) 

Electronegative low-density lipoprotein (LDL) found in human plasma is highly atherogenic and is elevated in individuals with increased cardiovascular risk. In this review, we summarize the available data regarding the elevation of electronegative LDL in the plasma of patients with various diseases. In addition, we discuss the harmful effects and underlying mechanisms of electronegative LDL in various cell types. We also highlight the known biochemical properties of electronegative LDL that may contribute to its atherogenic functions, including its lipid and protein composition, enzymatic activities, and structural features. Given the increasing recognition of electronegative LDL as a potential biomarker and therapeutic target for the prevention of cardiovascular disease, key future goals include the development of a standard methodology for the detection of electronegative LDL that can be used in a large-scale population survey and the identification and testing of strategies for eliminating electronegative LDL from the blood.

8. Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation. (Blood. 2013;122:3632-3641). 

We provide evidence that plasma levels of L5—the most electronegative and atherogenic subfraction of low-density lipoprotein—are drastically elevated in patients with ST-elevation myocardial infarction (STEMI). Furthermore, we show that when L5 isolated from the plasma of STEMI patients was injected into the tail vein of mice, it induced platelet activation and shortened the tail-bleeding time. These results indicate that L5 is prothrombotic, which was further demonstrated by the ability of L5 to increase tissue factor and P-selectin expression on endothelial cells, enhance platelet aggregation, and promote platelet–endothelial cell interactions. We also showed that L5 enhanced ADP-induced platelet activation through the PAFR- and LOX-1–mediated PKCα signaling pathway. Our findings strongly suggest that L5 may be a key factor in promoting acute thrombosis that leads to STEMI.

9. Chemical composition-oriented receptor selectivity of L5, a naturally occurring atherogenic low-density lipoprotein. (Pure Appl Chem. 2011;83(9). 

Analysis with SDS-PAGE and 2-dimensional electrophoresis showed that the protein framework of L1 was composed mainly of apolipoprotein (apo) B100, with an isoelectric point (pI) of 6.620. There was a progressively increased association of additional proteins, including apoE (pI 5.5), apoAI (pI 5.4), apoCIII (pI 5.1), and apo(a) (pI 5.5), from L1 to L5. LC/MSE was used to quantify protein distribution in all subfractions. On the basis of weight percentages, L1 contained 99% apoB-100 and trace amounts of other proteins. In contrast, L5 contained 60% apoB100 and substantially increased amounts of apo(a), apoE, apoAI, and apoCIII. The compositional characteristics contribute to L5's electronegativity, rendering it unrecognizable by LDLR. Thus, the chemical composition-oriented receptor selectivity hinders normal metabolism of L5, enhancing its atherogenicity through LOX-1.

10. Vascular progenitor cells in diabetes mellitus: roles of Wnt signaling and negatively charged low-density lipoprotein. (Chen CH, Dixon RA, Ke LY, Willerson JT. Circ Res. 2009 May 8;104(9):1038) 

Expression of miR-8 potently antagonizes Wnt signaling, both by inhibiting Wnt-Fz interaction on the plasma membrane and by reducing TCF protein intracellularly. Additionally, miR-15a and miR-16-1 can also antagonize Wnt signaling, and reduction of these microRNAs promotes growth of prostate cancer cells. Thus, the role of Wnt signaling in EC or vascular PC activity is not totally clear at this time, and uncontrolled stimulation of this pathway may invite dangerous consequences. These considerations, including how L5 may counteract the CD133+ PC and Wnt interactions, are schematically summarized in the Figure.

Postgraduate Students

Ph.D. Students

Farzana Parveen

Vineet Kumar Mishra

Shi Hui Law

Master Students

Keng-Chih Yueh

Bo-Xuan Yao

Liu-Fang Wang

Yong Hong Tan

2019 Lab Activities

20190312 Professor Etsuro Ito from Waseda University visited Research Center of Graduate Institute of Animal Vaccine, National Ping-Tung University Science and Technology.

20190129 Medical Laboratory Science and Biotechnology invited Günter Schwarz from Cologne University, Germany, Gopal K. Marathe from Mysore University, India visited at KMU for International Symposium.

20190129 Chuan-Fa Chang.

20190129 Long-Sheng Lu

20190128 Meeting with Professor Günter Schwarz from Cologne University, Germany.

2018 Lab Activities

20181227 Medical Laboratory Science and Biotechnology Associate professor Liang-Yin Ke invited Etsuro Ito from Waseda University visited at KMU for collaborative teaching. Classes were for bachelor, master and Ph. D students: “Development of ultrasensitive ELISA for early diagnosis” and “Biostatistics”

20181119 Dr. Daniel Bender from Institute of Biochemistry, University of Cologne, Germany gave a talk on “Sulfite oxidase in human health and disease”.

20181027 At 2018 Mass Spectrometry for Clinical Diagnosis Conference, NSYSU, Kaohsiung, with Professor Markus R WENK from National University of Singapore.

20181025 At China Medical University.

20181012 Shi Hui Law won the Best Poster Presentation Award in the international conference- Baltic Conference Series Fall 2018, Stockholm, held by International Association of Advanced Materials (IAAM).

20181011 Scientific presentation in the international conference- Baltic Conference Series Fall 2018, Stockholm, held by International Association of Advanced Materials (IAAM).

20180907 University of Cologne, Germany (with Professor Schwarz)

20180723 Dissertation Defense (with Associate Professor An-Sheng Lee from Mackay Medical College)

20180708-0713 Summer Workshop

Professor Chu-Huang (Mendel) Chen

Professor Gopal K. Marathe

Professor Ming-Shi Shiao

Professor Gopal K. Marathe

Professor Chuan-Fa Chang （張權發）from National Cheng Kung University

Associate Professor Liang-Yin (Maurice) Ke

20180706 Students' presentation to professor Gopal K. Marathe from Mysore University, India.

20180119-0209 University of Cologne, Germany

2017 Lab Activities

20171108-15 American Heart Association 2017 Scientific Secessions

20171103 【專題演講】Precision Diagnosis with Ultrasensitive ELISA and Surface-enhanced Roman Spectroscopy

20171012 【專題演講】「An Omics Avenue to Precision Medicine」

20170807 【專題演講】「Biology of a Lectin-Like Receptor」

20170706 【專題演講】「A Look into the Secrets of Aging and Metabolic Disorders from a Metabolomic Window」邀請蕭明邀請蕭明熙教授、Fionn Quinlan專題演講

20170616 【專題演講】「Genetic, structural and functional studies of cardiac gap junctions 」研討會邀請 Western University London Professor Donglin Bai專題演講「Connexin Molecules in Heart Rhythm Synchronization」 

20170602 White Coat Ceremony

20170518 Invite Prof. Tatsuya Sawamura from Shinshu University, Japan

20170516-0518 Invite Prof. Kazushi Motomura from Nagasaki University, Japan

20170512 香港中文學者張志強教授、黃永德助理教授、王家驥專員及13名學生至高醫醫技系進行交流訪問。

20170325 「我的一日實驗室」活動

2016 Lab Activities

20161017 Dinner party with Dr. Günter Schwarz, (University of Cologne, Köln)

20160628 Dinner party with Rena and Chihiro, two students from the School of Medicine, Shinshu University 

20160624 Students' Party at PERFUME DANCE CAFE

20160607-0609 The 2016 Alzheimer's disease congress

20160309-0311 IBMS-KMU Joint Symposium

20160304 KMU-Waters Symposium on Metabolomics and Aging 

20160129 CLB Happy-New-Year Party 

2015 Lab Activities

20151216 KMU-Waters Symposium

20151109 American Heart Association Scientific Sessions in Orlando, FL, November 7–11, 2015.

20151028-1030 Kazushi MOTOMURA Collaborative teaching on "Biotechnology"

20150825 Mamiko Koshiba, Ph.D. from Saitama Medical University 

20150518 Lee Jun C. Wong's talk on NGS

20150323 International Symposium: New Era of Lipid and Glycomedicine Research 

20141225 X'mas Party