Hypoxia and inflammation induce synergistic transcriptome turnover in macrophages.

Macrophages are effector immune cells that experience substantial changes to oxygenation when transiting through tissues, especially when entering tumors or infected wounds. How hypoxia alters gene expression and macrophage effector function at the post-transcriptional level remains poorly understood. Here, we use TimeLapse-seq to measure how inflammatory activation modifies the hypoxic response in primary macrophages. Nucleoside recoding sequencing allows the derivation of steady-state transcript levels, degradation rates, and transcriptional synthesis rates from the same dataset. We find that hypoxia produces distinct responses from resting and inflammatory macrophages. Hypoxia induces destabilization of mRNA transcripts, though inflammatory macrophages substantially increase mRNA degradation compared to resting macrophages. Increased RNA turnover results in the upregulation of ribosomal protein genes and downregulation of extracellular matrix components in inflammatory macrophages. Pathways regulated by mRNA decay in vitro are differentially regulated in tumor-associated macrophages implying that mixed stimuli could induce post-transcriptional regulation of macrophage function in solid tumors.

An integrated platform for high-throughput nanoscopy.

Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, implemented within the PYthon-Microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions.