Molecular mechanisms underlying the BIRC6-mediated regulation of apoptosis and autophagy.

Procaspase 9 is the initiator caspase for apoptosis, but how its levels and activities are maintained remains unclear. The gigantic Inhibitor-of-Apoptosis Protein BIRC6/BRUCE/Apollon inhibits both apoptosis and autophagy by promoting ubiquitylation of proapoptotic factors and the key autophagic protein LC3, respectively. Here we show that BIRC6 forms an anti-parallel U-shaped dimer with multiple previously unannotated domains, including a ubiquitin-like domain, and the proapoptotic factor Smac/DIABLO binds BIRC6 in the central cavity. Notably, Smac outcompetes the effector caspase 3 and the pro-apoptotic protease HtrA2, but not procaspase 9, for binding BIRC6 in cells. BIRC6 also binds LC3 through its LC3-interacting region, probably following dimer disruption of this BIRC6 region. Mutation at LC3 ubiquitylation site promotes autophagy and autophagic degradation of BIRC6. Moreover, induction of autophagy promotes autophagic degradation of BIRC6 and caspase 9, but not of other effector caspases. These results are important to understand how the balance between apoptosis and autophagy is regulated under pathophysiological conditions.

S-S-PEG-COOH Self-Assembled Monolayer on Gold Surface Enabled a Combined Assay for Serological EBV Antibody Isotypes.

PURPOSE: Epstein-Barr virus (EBV) is a ubiquitous human gamma herpes virus that infects human epithelial cells and B lymphocytes. It would be potentially valuable to develop novel combined assays to benefit screening for large panels of samples of EBV infectious diseases. EXPERIMENTAL DESIGN: A simple antigen-probed biochip that is modified with S-S-PEG-COOH and is used as a label-free high-throughput screening method for a combined detection of EBV capsid antigen IgM antibody, capsid antigen IgG antibody, and nuclear antigen IgG antibody. RESULTS: This protein biochip has similar feasibility, sensitivity, and specificity in comparison with Liaison chemiluminescent immunoassay (CLIA). Detection limit of the EBV antibodies by the biochip is almost identical to that by CLIA-L (2.91 U mL-1 vs 3.00 U mL-1 for EBNA-1 IgG, 8 U mL-1 vs10 U mL-1 for EBV-VCA IgG, and 3.5 U mL-1 vs 10 U mL-1 for EBV-VCA IgM). Tests of the three serological antibodies against EBV by the biochip are consistent with the CLIA-L method in 274 clinical sera, respectively. Finally, the combined biochip is successfully utilized for diagnostic identification of EBV infection in 14 patients with infectious mononucleosis (IM) and 25 patients with systemic lupus erythematosus SLE, as well as additional 10 known real-time PCR positive patients. CONCLUSIONS AND CLINICAL RELEVANCE: This biochip format will enable concurrent detection of antibodies against EBV infection and confirm infection status of EBV. It will be a versatile tool for large-scale epidemiological screening in view of its miniaturization and high throughput.

A biochip-based combined immunoassay for detection of serological status of Borrelia burgdorferi in Lyme borreliosis.

BACKGROUND: Dithiobis (succinimidyl undecanoate) modified gold surface biochip were used as a combined immunoassay platform for concurrently detecting immune responses to Borrelia burgdorferi (B. burgdorferi) sensu lato antigens, flagellin, outer surface protein C, variable major protein-like sequence proteins, and 3 VlsE protein IR6 peptides. The peptides represented intrinsic Borrelia genospecies: B. burgdorferi sensu stricto, B. garinii, and B. afzelii, respectively. METHODS: Fourier transform infrared spectroscopy was utilized to validate the surface chemical characteristics on the modified gold surface. RESULTS: The limits in detection of IgG antibody on the biochips were as little as 0.39µg/ml for anti-VlsE and 0.78µg/ml for anti-flagellin and anti-OspC, respectively. Samples from 56 neuroborreliosis (NB) patients and 114 healthy individuals were analyzed by the combined biochip. We found that the seroprevalences of IgM or IgG antibody against the 6 antigens were contributed to increased overall sensitivity by the multiplex immunobiochip assay. Serum combined positive rates of the 6 antigens in the patients were 92.86% for IgM antibody and 91.07% for IgG antibody. Part of the patients bore antibody responses against the 3 VlsE IR6 variant peptides, indicating that Lyme borreliosis would attribute to consequence of multiple infections by one or more Borrelia burgdorferi strains. Concurrent assessment for both IgM and IgG antibodies against the protein antigens and B. burgdorferi IR6 peptides in the sera of NB patients was beneficial from the biochip format, enabling detection of expanded serologic infection status and therapy strategy-making more efficiently. CONCLUSIONS: The combined biochip-based immunoassay, as a potential substitution of ELISA, provided a promising approach to extend the detection spectrum of infectious antibodies against a panel of Borrelia antigens.

Development of a novel protein biochip enabling validation of immunological assays and detection of serum IgG and IgM antibodies against Treponema pallidum pathogens in the patients with syphilis.

In this study, we developed a novel protein biochip methodology that was characterized by dithiobis (succinimidyl undecanoate) (DSU) and specialized for detection of serum IgG and IgM antibodies against Treponema pallidum pathogens in the patients with syphilis, respectively. The biochips were validated by a dimension of atomic force microscope (AFM). The visualized detection limit of IgG antibody on the biochip was 0.39µg/ml. Finally, 286 serum samples from the patients with syphilis were simultaneously tested on the rTpN15-17-47 coated biochips. The results were evaluated in comparison with the assays of T. pallidum particle agglutination (TPPA) and the toluidine red unheated serum test (TRUST). The result demonstrated that the relative positive rate in the 286 patients by biochip was 99.0%, similar to that by TPPA (97.9%, P>0.05) and higher than that by TRUST, (76.2%, P<0.01). The detection specificities were 100% for the biochip and the TPPA and 97.0% for the TRUST. Thus, the protein biochip would provide a useful platform not only for enabling concurrent detection of the infectious antibodies directed against T. pallidum on a larger scale, but also for monitoring therapy modality of the disease.