Ying-Song Wu

The role of translationally controlled tumor protein in tumor growth and metastasis of colon adenocarcinoma cells.

Translationally controlled tumor protein (TCTP) plays a major role in a broad array of biological processes. However, the TCTP-related biological process and interactive proteins still remain poorly characterized. In the present study, we found that knockdown of TCTP inhibited proliferation, migration, and invasion activities of LoVo cells in vitro and in vivo. The whole-cell proteomes were compared by 2D gel electrophoresis before and after knockdown of TCTP. Alterations in 27 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of Ubiquitin-Proteasome System, proteins involved in the cytoskeleton biosynthesis and tumor metastasis were found to be changed upon TCTP removal. These results imply that TCTP might play at least a partial role in colon adenocarcinoma progression.

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was logY=3.65786+0.43863·logX (R=0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4 ng mL(-1). By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.

Heatstroke induces liver injury via IL-1β and HMGB1-induced pyroptosis

BACKGROUND & AIMS Liver injury is a common complication of heat stroke (HS), and often constitutes a direct cause for patient death. The cellular and molecular mechanism underlying HS-induced liver injury remains unclear. Recent evidence indicates that inflammasome plays an important role in mediating sterile inflammation triggered by tissue damage. Using a rat HS model, we identified a novel mechanism by which inflammasome-dependent interleukin-1β (IL-1β) activation and hepatocyte pyroptosis mediate HS-induced liver injury. METHODS To induce HS, rats were subjected to heat exposure. Inhibition of inflammasomes was achieved by RNA silencing and pharmacologic inhibitor prior to heat exposure. Inflammasome assembly, caspase-1 activation, histological changes, as well as serum levels of liver enzymes were measured. RESULTS We demonstrated that the onset of HS activated inflammasome in the liver as evidenced by increased capase-1 activity and the association of inflammasome components NOD-like receptor family pyrin domain containing 3 (Nlrp3) and apoptosis speck-like protein containing a caspase-recruitment domain (ASC); and the activated inflammasome, in turn, induced IL-1β activation and hepatocyte pyroptosis, and subsequent augmented liver injury. HS-induced hepatocyte inflammasome activation seems to be high-mobility group box 1 (HMGB1) dependent. Inhibition of Nlrp3, caspase-1, or HMGB1 prevented HS-induced liver inflammation and ameliorated liver injury. CONCLUSIONS These findings demonstrate an important role of HMGB1 in mediating inflammasome activation in the development of liver injury following HS, and suggest that targeting inflammasome may represent a novel therapeutic strategy to limit cell death and prevent liver failure after HS.

Development of a dual-label time-resolved fluoroimmunoassay for the detection of α-fetoprotein and hepatitis B virus surface antigen

Time-resolved fluorometry of lanthanide chelates is one of the most useful non-isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time-resolved fluoroimmunoassay (TRFIA) to quantify α-fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two-site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co-coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu(3+)- and Sm(3+)-McAbs were added and fluorescence signals of Sm(3+) and Eu(3+) tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP-TRFIA and HBsAg-TRFIA were 1-1000 mIU/L and 0.2-150 µg/L, respectively. Intra- and inter-assay coefficients of variation of AFP-TRFIA were 3.3-4.1% and 5.7-7.2% and for HBsAg-TRFIA were 2.9-3.9% and 4.9-6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu-labeled McAbs were stable for at least one year at -20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use.

Development of an improved time-resolved fluoroimmunoassay for simultaneous quantification of C-peptide and insulin in human serum.

OBJECTIVES Diabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus. DESIGN AND METHODS A highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu(3+) and Sm(3+) chelate labels were used for detection. RESULTS Antibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs. CONCLUSIONS The results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.

PSMA7, A Potential Biomarker of Diseases

Proteasome subunit alpha type 7(PSMA7) is an alpha-type subunit of the 20S proteasome core complex and participates in degrading proteins through ubiquitin-proteasome pathway (UPP) which plays an important role in the regulation of cell proliferation or cell cycle control, transcriptional regulation, immune and stress response, cell differentiation, and apoptosis. Previous studies have demonstrated that PSMA7 can be a target interacting with some important proteins involved in transcription factor regulation, cell cycle transition, viral replication and even tumor initiation and progression, suggesting that PSMA7 could be a potential target for the development of clinical diagnosis and new therapeutic drugs. Here, we review the recent studies on PSMA7 involved in many different cellular processes, ranging from the cell cycle process to antigen processing and tumorigenesis.

A Novel Immunoassay for the Quantization of CYFRA 21–1 in Human Serum

Cytokeratin 19 fragment antigen (CYFRA 21–1) is used to diagnose and monitor neoplasms. However, the main disadvantages of the currently available CYFRA 21–1 assays include heterogenous technology, being time‐consuming, and having low through‐put with low insensitivity. This study investigated the use of amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the quantization of CYFRA 21–1 in human serum.

Simultaneous determination of the cytokeratin 19 fragment and carcinoembryonic antigen in human serum by magnetic nanoparticle-based dual-label time-resolved fluoroimmunoassay

A highly sensitive, rapid and novel simultaneous measurement method for cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum by magnetic nanoparticle-based dual-label time-resolved fluoroimmunoassay was developed. On the basis of a sandwich-type immunoassay format, analytes in the samples were captured by antibodies coated onto the surface of the magnetic beads and sandwiched by other antibodies labeled with europium and samarium chelates. The lower limit of quantitation of the present method for CYFRA 21-1 was 0.77 ng mL−1 and CEA was 0.85 ng mL−1. The coefficient variations of the method were less than 7%, and the recoveries were in the range of 90–110% for serum samples. The concentrations of CYFRA 21-1 and CEA serum samples determined by the present method were compared with those obtained by the chemiluminescence immunoassay. A good correlation was obtained with the correlation coefficients of 0.961 for CYFRA 21-1 and 0.938 for CEA. This novel method demonstrated high sensitivity, wide effective detection range and excellent reproducibility for the simultaneous determination of CYFRA 21-1 and CEA, which can be useful for the early screening and prognosis evaluation of patients with lung cancer.

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum.

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

A direct competitive inhibition time-resolved fluoroimmunoassay for the detection of unconjugated estriol in serum of pregnant women

Unconjugated estriol (uE3) is one of the most important serum markers for prenatal screening. The abnormally low content of uE3 is used as an indicator of fetal DS (Down syndrome) during the second trimester in pregnant women. In the present study, we developed a time-resolved fluoroimmunoassay to detect uE3 by employing microtiter plates with pre-captured primary antibodies. E3-3-CME-BSA (estriol-3-carboxymethyl ether-bovine serum albumin) conjugates served as labels and Eu3+ (europium) as the probe for signal detection. The detection limit of this assay was 0.35 nmol L−1. The within-run and between-run imprecision values for serum control detection were less than 5.0% and 6.0% respectively. The mean recovery was 102.6%. The long-term stability (2–8 °C, 15 months) and thermostability (37 °C, 10 days) were excellent. The uE3 concentrations measured by the present assay in 1168 Chinese maternal serum samples correlated well with those obtained by the chemiluminescence immunoassay assay (r = 0.948). The reference range in normal singleton pregnancies in Southern China was established which provided reference data to adjust the uE3 medians for biochemical screening.