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Three protocols of accelerated startup and shutdown (SU/SD) test were investigated: startup and shutdown with air supply and soak to both anode and cathode (air-SU/SD), hydrogen protected startup and shutdown (H 2 -SU/SD), and hydrogen protected startup and shutdown with a load (H 2 -SU/SD with a load). The performance losses, electrochemical surface area (ECSA) reduction, and catalyst layer degradation were characterized and compared for these SU/SD protocols. Air-SU/SD protocol showed much more severe performance loss and catalyst layer degradation than hydrogen protected ones, which confirmed the benefits of hydrogen protection. The temperature effect on air-SU/SD was significant in a broad range from 20 °C to 70 °C, with low temperature greatly reducing the degradation. The mechanism of H 2 protection and load drawn in alleviating carbon corrosion was explained based on reactions and charge conservation during SU/SD. This paper provides comprehensive test data and failure analysis to quantify the benefits of H 2 protection and load drawn and to facilitate future enhancement of system strategies on SU/SD durability. 

Abstract Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.