A Novel RNAi Lethality Rescue Screen to Identify Regulators of Adipogenesis

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an atractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting

Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

© 2009 Paul and Piontkivska; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

What does the structure-function relationship of the HIV-1 Tat protein teach us about developing an AIDS vaccine?

The human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription protein Tat is an important factor in viral pathogenesis. In addition to its function as the key trans-activator of viral transcription, Tat is also secreted by the infected cell and taken up by neighboring cells where it has an effect both on infected and uninfected cells. In this review we will focus on the relationship between the structure of the Tat protein and its function as a secreted factor. To this end we will summarize some of the exogenous functions of Tat that have been implicated in HIV-1 pathogenesis and the impact of structural variations and viral subtype variants of Tat on those functions. Finally, since in some patients the presence of Tat-specific antibodies or CTL frequencies are associated with slow or non-progression to AIDS, we will also discuss the role of Tat as a potential vaccine candidate, the advances made in this field, and the importance of using a Tat protein capable of eliciting a protective or therapeutic immune response to viral challenge