Pei-Yu Chu

Molecular epidemiology of enterovirus 71 in Taiwan.

Summary. Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10−3vs. 1.4 × 1010−3) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance.

Activity-dependent neuroprotector homeobox protein: A candidate protein identified in serum as diagnostic biomarker for Alzheimer's disease

Alzheimers disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.

Characterization of newly emerging Newcastle disease viruses isolated during 2002–2008 in Taiwan

To elucidate the epidemiological relationships between ND outbreaks and genetic lineages, a portion of the F gene (535 bp) and the full-length HN gene (1922 bp) of recent Taiwanese NDVs isolated in 2002-2008 was amplified by reverse transcription (RT)-polymerase chain reaction (PCR) and sequenced. Only a portion of above amplified PCR products of the F and HN genes (374 and 1713 bp) and their deduced amino acid residues were compared with the other 60 NDVs retrieved from GenBank. Most (29/30) of the recent Taiwanese isolates were clustered in subgenotype VIIe while only one isolate was classified as subgenotype VIIc. All the 29 isolates of subgenotype VIIe were further subclassified and termed provisionally as sub-subgenotypes VIIe2 (13 isolates), VIIe3 (5 isolates), and VIIe4 (11 isolates). The sub-subgenotype VIIe2 isolates possessing the motif (112)R-R-Q-K-R-F(117) and amino acid residue substitutions at positions 23 (L to F) and 90 (T to A) were collected during 2002-2005. The sub-subgenotype VIIe3 isolates possessing the motif (112)R-R-K-K-R-F(117) and amino acid residue substitutions at positions 74 (E to G) and 75 (A to G) within epitopes and 114 (Q to K) within cleavage site of F protein were collected during 2003-2006. The sub-subgenotype VIIe4 isolates possessing the motif (112)R-R-Q-K-R-F(117) and amino acid residue substitutions at positions 23 (L to F), 26 (I to T), and 90 (T to A) were collected during 2007-2008. All the NDV isolates in this study exhibited a high intra-cerebral pathogenicity index (ICPI), they were all classified as velogenic type of NDVs. The sub-subgenotype VIIe2 and VIIe4 viruses are now dominant and have been implicated in most of the recent ND outbreaks in Taiwan. Phylogenetic analysis of these isolates revealed that they may have evolved from previously reported local strains (VIIe1). This finding is essential for improving the disease control strategies and development of vaccines for ND.

Characterization of ADAM28 as a biomarker of bladder transitional cell carcinomas by urinary proteome analysis

Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer.

Proteomic Profiling of Neuroblastoma Cells Adhesion on Hyaluronic Acid-Based Surface for Neural Tissue Engineering

The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA) biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM) in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.

Molecular epidemiology of Coxsackievirus B3.

Molecular epidemiological characteristics are needed to understand the impact of Coxsackievirus B3 (CV-B3) infection, since no CV-B3 genotyping literature is available. Twenty-nine CV-B3 Taiwan strains obtained from 1992 to 2005 were analyzed. A phylogenetic tree was constructed based on the 290 nucleotide sequence of the VP1 gene of Taiwan isolates and in 91 other CV-B3 GenBank sequences. Five genotypes (GI-GV) were depicted. The GI, GII, and GIII were dominant in America and Europe, whereas GIV and GV were prevalent in Asia. In Taiwan, a transient outbreak of GIV occurred in 2000, while GV has been the main genotype circulating since 1992. Patient age ranged from 0.1 to 81 months (median, 4.3 months). The male:female ratio was 1.9:1. More than 60% (17/29) of cases involved children younger than 1 year. Half of them contracted respiratory tract infection (12/24). Nine of the 24 (37.5%) cases with available medical records had central nervous system (CNS) involvement. Eight of the nine patients were younger than 3 months. The CV-B3 has evolved and circulated for the past 60 years. Although the nucleotide sequence of the VP1 is highly variably, amino acids were relatively conserved within the same genotype of CV-B3. CNS infections were not associated with a specific strain or genotype. The CV-B3 poses a significant health threat to children younger than 1 year, especially those younger than 3 months old.

Transmission and Demographic Dynamics of Coxsackievirus B1

The infectious activity of coxsackievirus B1 (CV-B1) in Taiwan was high from 2008 to 2010, following an alarming increase in severe neonate disease in the United States (US). To examine the relationship between CV-B1 strains isolated in Taiwan and those from other parts of the world, we performed a phylodynamic study using VP1 and partial 3Dpol (414 nt) sequences from 22 strains of CV-B1 isolated in Taiwan (1989–2010) and compared them to sequences from strains isolated worldwide. Phylogenetic trees were constructed by neighbor-joining, maximum likelihood, and Bayesian Monte Carlo Markov Chain methods. Four genotypes (GI–IV) in the VP1 region of CV-B1 and three genotypes (GA–C) in the 3Dpol region of enterovirus B were identified and had high support values. The phylogenetic analysis indicates that the GI and GIII strains in VP1 were geographically distributed in Taiwan (1993–1994) and in India (2007–2009). On the other hand, the GII and GIV strains appear to have a wider spatiotemporal distribution and ladder-like topology A stair-like phylogeny was observed in the VP1 region indicating that the phylogeny of the virus may be affected by different selection pressures in the specified regions. Further, most of the GI and GII strains in the VP1 tree were clustered together in GA in the 3D tree, while the GIV strains diverged into GB and GC. Taken together, these data provide important insights into the population dynamics of CV-B1 and indicate that incongruencies in specific gene regions may contribute to spatiotemporal patterns of epidemicity for this virus.

Protein Profiling of Human Nonpigmented Ciliary Epithelium Cell Secretome: The Differentiation Factors Characterization for Retinal Ganglion Cell line

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

Molecular Epidemiology and Phylogenetic Analysis of Human Adenovirus Caused an Outbreak in Taiwan during 2011.

An outbreak of adenovirus has been surveyed in Taiwan in 2011. To better understand the evolution and epidemiology of adenovirus in Taiwan, full-length sequence of hexon and fiber coapsid protein was analyzed using series of phylogenetic and dynamic evolution tools. Six different serotypes were identified in this outbreak and the species B was predominant (HAdV-3, 71.50%; HAdV-7, 15.46%). The most frequent diagnosis was acute tonsillitis (54.59%) and bronchitis (47.83%). Phylogenetic analysis revealed that hexon protein gene sequences were highly conserved for HAdV-3 and HAdV-7 circulation in Taiwan. However, comparison of restriction fragment length polymorphism (RFLP) analysis and phylogenetic trees of fiber gene in HAdV-7 clearly indicated that the predominant genotype in Taiwan has shifted from 7b to 7d. Several positive selection sites were observed in hexon protein. The estimated nucleotide substitution rates of hexon protein of HAdV-3 and HAdV-7 were 0.234×10-3 substitutions/site/year (95% HPD: 0.387~0.095×10-3) and 1.107×10-3 (95% HPD: 0. 541~1.604) respectively; those of the fiber protein of HAdV-3 and HAdV-7 were 1.085×10-3 (95% HPD: 1.767~0.486) and 0.132×10-3 (95% HPD: 0.283~0.014) respectively. Phylodynamic analysis by Bayesian skyline plot (BSP) suggested that using individual gene to evaluate the effective population size might possibly cause miscalculation. In summary, the virus evolution is ongoing, and continuous surveillance of this virus evolution will contribute to the control of the epidemic.

Mass Spectrometry in Clinical Diagnosis: A Preliminary Application in Tumor Cellular Proteomics for Biomarker Discovery

Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells, which cause high mortality rates. The purpose of this study is to characterize the proteins secreted from the HepG2, MCF-7 and HT-29 cells, which may relate to cell differentiation and tumor metastasis. In the proteomic analysis, the secretome proteins were identified by reverse phase nano-high performance liquid chromatography/electrospray ionization tandem mass spectrometry (RP-nano-UPLC-ESI-MS/MS) followed by peptide fragmentation pattern analysis. Moreover, identifications of tumor cell protein expressions by proteomic approaches were indicated that several proteins may activate or enhance the PI3K/AKT/mTOR pathway. The PI3K/AKT/mTOR pathway has been shown to be related to the cancer cell metastasis and proliferation, and the ubiquitin C (UBC) may serve as potential protein diagnostic biomarker to be examined in further investigations.