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Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery ofStentor coeruleus(SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery onStentoras our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.

When granular materials, colloidal suspensions, and even animals and crowds exit through a narrow outlet, clogs can form spontaneously when multiple particles or entities attempt to exit simultaneously, thereby obstructing the outlet and ultimately halting the flow. Counterintuitively, the presence of an obstacle upstream of the outlet has been found to suppress clog formation. For soft particles such as emulsion drops, clogging has not been observed in the fast flow limit due to their deformability and vanishing interparticle friction. Instead, they pinch off each other and undergo break up when multiple drops attempt to exit simultaneously. Similar to how an obstacle reduces clogging in a rigid particle system, we hypothesize and demonstrate that an obstacle could suppress break up in the two-dimensional hopper flow of a microfluidic crystal consisting of dense emulsion drops by preventing the simultaneous exit of multiple drops. A regime map plotting the fraction of drops that undergo break up in a channel with different obstacle sizes and locations delineates the geometrical constraints necessary for effective break up suppression. When optimally placed, the obstacle induced an unexpected ordering of the drops, causing them to alternate and exit the outlet one at a time. Droplet break up is suppressed drastically by almost three orders of magnitude compared to when the obstacle is absent. This result can provide a simple, passive strategy to prevent droplet break up and can find use in improving the robustness and integrity of droplet microfluidic biochemical assays as well as in extrusion-based three-dimensional printing of emulsion or foam-based materials.

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.