Yu-Chang Tyan

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.

Properties of osteoconductive biomaterials: Calcium phosphate cement with different ratios of platelet-rich plasma as identifiers

This study aims to evaluate further the performance of a platelet-rich plasma (PRP) additive incorporated with calcium phosphate bone cement (CPC) in vitro to prove its efficiency as bone graft substitutes and its compatibility to be incorporated into the CPC with other techniques in clinical restoration in vivo. The growth factor release ability and the osteogenic evaluation of PRP, CPC, and PRP/CPC testing groups with 5, 10, and 15 wt.% PRP were compared in vitro. Four groups were measured using non-decalcified staining methods in vivo, which include the testing group of 10 wt.% PRP/CPC selected from the evaluation in vitro, by using both the autograft with rabbit trabecular and CPC-only as comparison groups and the group without grafting material as the control sample. The results obtained through specimen immersion show that growth factor release and alkaline phosphatase activities after osteoprogenitor cell culture had a significantly better effect on 10 and 15 wt.% PRP/CPC than on the other groups in vitro. Analysis results suggest that PRP was still retained in the CPC matrix even after 32 days of immersion. The results in vivo show that the histology of the autograft bone and the control group without grafting material exhibited fibrous connective and adipose tissues, which obviously filled the created cavity even at nine weeks after the operation. Osteoregeneration was more successful in the PRP-additive group, which accumulated bone remodeling than in the other groups. In conclusion, CPC could be a potential carrier with adequate PRP additives that bear a therapeutic potential for enhanced bone tissue regeneration.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate‐washed membrane standard

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate‐washing step enriches for integral and lipid‐anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.

Deriving fast setting properties of tetracalcium phosphate/ dicalcium phosphate anhydrous bone cement with nanocrystallites on the reactant surfaces

OBJECTIVE This study attempts to reveal how nanocrystallites on the ceramic surfaces of non-dispersive calcium phosphate cement (nd-CPC) participate in setting processes as compared with conventional CPC (c-CPC). METHODS The compositions and morphologies of CPC during the early setting reactions were studied with X-ray diffraction and a scanning transmission electron microscope equipped with an energy dispersive spectroscopy system. The pH values and dispersive properties of CPC during the early setting reactions were investigated as well as the compressive strength of nd-CPC after 24h of immersion with varying liquid to powder ratios. RESULTS The mechanical strength of nd-CPC was approximately 60MPa after a 24h immersion in simulate body solution with a P/L ratio between 3.3 and 4.2g/mL. The nanocrystallites on the particle surfaces of nd-CPC were shown to grow rapidly and provided interlocking sites that allowed rapid development of the apatite phase in the cement, and were also shown to be non-dispersive in solution as determined by an injection test of c-CPC. CONCLUSIONS The interlocking particles produced by whisker growth on the ceramic particles or new crystallites formed between the ceramic particles caused the cement to be non-dispersive in solution. The particles of reactants with nanocrystallites on surfaces also gave this cement the ability to be shaped easily as a paste during an operation or to be injected into a cavity.

Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.

The Contribution of Antibiotic Resistance Mechanisms in Clinical Burkholderia cepacia Complex Isolates: An Emphasis on Efflux Pump Activity

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.

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.

A comparative proteomics analysis of peritoneal dialysate before and after the occurrence of peritonitis episode by mass spectrometry

BACKGROUND Peritoneal dialysis (PD) is one of the therapeutic options for the end-stage renal disease (ESRD) patients. The peritoneal membrane is immersed in a high glucose concentration of the peritoneal dialysate, which may cause structural and functional damage. Peritonitis is one of the major complications of PD, which will accelerate the damage of peritoneal membrane by increasing the peritoneal permeability and decrease the ultrafiltration efficiency. It will cause the peritoneal membrane dysfunction and the patient may have fluid overload related complications or hemodialysis. METHODS To enhance our understanding of peritoneal dialysate, the peritoneal dialysate proteins were identified by 2-dimensional gel electrophoresis (2DE) combined with reverse phase nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) followed by peptide fragmentation pattern. RESULTS We performed 2DE on the 12 peritoneal dialysate before/after peritonitis, and more than 350 spots were detected. Among these protein spots, 136 spots of the 2DE were excised, in-gel digested and identified by nano-HPLC-ESI-MS/MS. A total of 41 proteins were identified with high levels of confidence. Ten of these were significantly differentially expressed between the peritoneal dialysate samples before/after peritonitis. CONCLUSION The present study was designed to establish optimal techniques to develop a proteomic map of peritoneal dialysate proteins. These proteins may not be new biomarkers; however, they may indicate a situation for possible drug treatment and can be the predictors of peritonitis for the validation study in the further.

Urinary protein profiling by liquid chromatography/tandem mass spectrometry: ADAM28 is overexpressed in bladder transitional cell carcinoma

Bladder cancer is the most common urological cancer with higher incidence rate in the endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to utilize the proteomic approach to establish urinary protein patterns of bladder cancer. The experimental results showed that most patients with bladder cancer had proteinuria or albuminuria. The urine arsenic concentrations of bladder cancer patients in BFD areas were significantly higher than those patients from non-BFD areas. In the proteomic analysis, the urinary proteome was identified by nano-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (nano-HPLC/ESI-MS/MS) followed by peptide fragmentation pattern analysis. We categorized 2782 unique proteins of which 89 proteins were identified with at least three unique matching peptide sequences. Among these 89 proteins, thirteen of them were not found in the control group and may represent proteins specific for bladder cancer. In this study, three proteins, SPINK5, ADAM28 and PTP1, were also confirmed by Western blotting and showed significant differential expression compared with the control group. ADAM28 may be used as a possible biomarker of bladder cancer.