Ren, F. | bioRxiv Science

bioRxiv2018

Absence of GdX/UBL4A protects against inflammatory bowel diseases by regulating NF-κB signaling in DCs and macrophages

Nuclear factor-kappa B (NF-{kappa}B) activation is critical for innate immune responses. Here we report that the UBL4A (Ubiquitin-like protein 4A, also named GdX) enhances dendritic cells (DCs) and macrophages (M{varphi})-mediated innate immune defenses by positively regulating NF-{kappa}B signaling. GdX-deficient mice were resistant to LPS-induced endotoxin shock and DSS-induced colitis. DC- or M{varphi}-specific GdX-deficient mice displayed alleviated mucosal inflammation, and the production of pro-inflammatory cytokines by GdX-deficient DCs and M{varphi} was reduced. Mechanistically, we found that PTPN2 (TC45) and PP2A form a complex with RelA (p65) to mediate its dephosphorylation whereas GdX interrupts the TC45/PP2A/p65 complex formation and restrict p65 dephosphorylation by trapping TC45. Our study provides a mechanism by which NF-{kappa}B signaling is positively regulated by an adaptor protein GdX in DC or M{varphi} to maintain the innate immune response. Targeting GdX could be a strategy to reduce over-activated immune response in inflammatory diseases.

immunology↗

bioRxiv2017

A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

Here we report a novel microdroplet PCR method combined with fluorescence spectrophotometry (MPFS), which allows for qualitative, quantitative and high -throughput detection of multiple DNA targets. In this study, each pair of primers was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA was amplified and labeled with the same fluorophore. After products purification, the DNA products tagged with different fluorophores could be analyzed qualitatively by the fluorescent intensity determination. The relative fluorensence unit was also measured to construct the standard curve and to achieve quantitative analysis. In a reaction, the co -amplified products with different fluorophores could be simultaneously analyzed to achieve high -throughput detection. We used four kinds of GM maize as a model to confirm this theory. The qualitative results revealed high specificity and sensitivity of 0.5% (w / w). The quantitative results revealed that the limit of detection was 103copies and with good repeatability. Moreover, reproducibility assay were further performed using four foodborne pathogenic bacteria. Consequently, the same qualitative, quantitative and high-throughput results were confirmed as the four GM maize.

biochemistry↗