Multiplexed patterning of hybrid lipid membrane and protein arrays for cell signaling study.

The supported lipid bilayer (SLB) is a powerful tool for studying dynamic cell-environment interactions and has been widely used for biosensing applications. Using a reusable microfluidic chip, we present here a strategy to fabricate highly multiplexed SLB and protein arrays for cell signaling research. This approach allows for the rapid patterning of hundreds of highly reproducible and size-tunable SLB arrays with distinct lipid composition and mobility. Using fluorescence microscopy and fluorescence correlation spectroscopy, the lipid mobility is found to play a central role for patterning this membrane assay. Adding protein rings as diffusion barriers extends the accessible mobility range and maintains long-term stability of the hybrid array. Subsequent protein functionalizations on the SLB could be conducted using standard conjugation methods. The utility of the hybrid array for cell signaling experiments is demonstrated by studying the immune NF-κB signaling, whose activity is triggered by the binding of the membrane receptor, toll-like-receptor 4 (TLR 4), to its ligand, lipopolysaccharide (LPS), that is functionalized on the SLB. The patterned array allows cells to adhere and spread on areas without LPS before migrating to interact with membrane-bound LPS to initiate NF-κB activation. Overall, the strategy offers an efficient route to rapidly generate easily controllable and multiplexed molecular arrays that can serve as versatile platforms for biosensing and cell signaling research.

Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling.

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

The role of transthoracic ultrasonography in predicting the outcome of community-acquired pneumonia in hospitalized children.

CONCLUSION: TUS findings of fluid bronchogram, multifocal involvement, and pleural effusion were associated with adverse outcomes, including longer hospital stay, ICU admission, and tube thoracotomy in hospitalized CAP children. Therefore, TUS is a novel tool for prognostic stratifications of CAP in hospitalized children.