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New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-μl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.

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BackgroundThe periplasmic High Temperature Requirement protein A (HtrA) plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA) in chlamydial pathogenesis is not clear.ResultsThe cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor.ConclusionSince it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and timeresolved fluorescence resonance energy transfer (TR-FRET). Compounds (~42,000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists. (Journal of Biomolecular Screening 2003:381-392)

Allergic asthma and obesity are major public health problems in the world. Recent Meta-analysis studies implicated a positive relationship between serum leptin, which is elevated in obese individuals, and the risk of asthma. However, it is not well understood how obesity-associated elevation of leptin increases the risk of asthma. In the current study, we have found that leptin induces the unfolded protein response factor XBP1s in an mTOR- and MAPK-dependent manner in pro-allergic TH2 cells; in vivo, mice fed with high fat diet had increased serum leptin as observed in human obese population and exacerbated asthmatic symptoms, associated with increased XBP1s expression in splenic CD4+ T cells. XBP1s is required for leptin-mediated pro-allergic TH2 cell survival and cytokine production. Our results reveal a previously unappreciated insight that obesity-associated hyperleptinemia contributes to enhanced pro-allergic lymphocyte responses through induction of XBP1s, leading to exacerbation of allergic asthma.