Gasdermins are main effectors of pyroptosis, an inflammatory form of cell death. Released by proteolysis, the N-terminal gasdermin domain assembles large oligomers to punch lytic pores into the cell membrane. While the endpoint of this reaction, the fully formed pore, has been well characterized, the assembly and pore-forming mechanisms remain largely unknown. To resolve these mechanisms, we characterize mouse gasdermin-A3 by high-resolution time-lapse atomic force microscopy. We find that gasdermin-A3 oligomers assemble on the membrane surface where they remain attached and mobile. Once inserted into the membrane gasdermin-A3 grows variable oligomeric stoichiometries and shapes, each able to open transmembrane pores. Molecular dynamics simulations resolve how the membrane-inserted amphiphilic β-hairpins and the structurally adapting hydrophilic head domains stabilize variable oligomeric conformations and open the pore. The results show that without a vertical collapse gasdermin pore formation propagates along a set of multiple parallel but connected reaction pathways to ensure a robust cellular response.
Standard dead-end sample filtration is used to improve sample purity, but is limited as particle build-up fouls the filter, leading to reduced recovery. The fouling layer can be periodically cleared with backflush algorithms applied through a customized fluidic actuator using variable duty cycles, significantly improving particulate recovery percentage. We show a Pulse Width Modulation (PWM) process can periodically backflush the filter membrane to repeatedly interrupt cake formation and reintegrate the fouling layer into the sample, improving net permeate flux per unit volume of sample by partially restoring filter flux capacity. PWM flow for 2.19 um (targeted) and 7.32 um (untargeted) polystyrene microbeads produced 18-fold higher permeate concentration, higher recovery up to 68.5%, and an 8-fold enrichment increase, compared to a uniform flow. As the duty cycle approaches 50%, the recovery percentage monotonically increases after a critical threshold. Further, we developed and validated a mathematical model to determine that fast, small-volume backflush pulses near 50% duty cycle yield higher recovery by decreasing fouling associated with the cake layer. Optimized PWM flow was then used to purify custom particles for immune activation, achieving 3-fold higher recovery percentage and providing a new route to improve purification yields for diagnostic and cellular applications. Dead-end filtration using patterned microsieves, fiber meshwork, and membranes of various materials is a standard technique to isolate desired particles of various sizes and is often used in clinical and laboratory settings for therapeutic and diagnostic applications 1-4. Both biological and physical suspensions can be filtered to yield high purity and enrichment at a high throughput. Dead-end filters are especially susceptible to fouling, however, which leads to lower recovery percentage and yield as a direct result 2,5,6. Because a decreasing yield negatively impacts therapeutic quality 7-9 , clinical and industrial therapeutic manufacturing will frequently change or increase the surface area of the dead-end filter 10 , or switch to crossflow filtration modalities 11,12 , which further decreases the throughput and increases processing time. Membrane fouling is caused by pore blocking followed by cake layer formation, resulting in an exponential decay with time in the flux of permeating particulate 13-19. Membrane fouling also affects crossflow systems and, in this case, numerous studies were conducted to disrupt cake formation and reintegrate particulates into the bulk flow feed stream 11,12,20-25. For example, crossflow filtration can disrupt caking by implementing an oscillatory flow with a sinusoidal flow velocity or a pulsatile flow, consisting of a steady flow with oscillations superimposed 20. These studies examined the effects of numerous waveforms, including variations of sinusoids, saw tooth, and square waves 12,20,23. Oscillatory and pulsatile techniques showed improved clearance of the crossflow membranes, leading to an incre...
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