Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM.

The ß-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by "slow" and "fast" diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.

Experiences of Black Adults Evaluated in a Locked Psychiatric Emergency Unit: A Qualitative Study.

OBJECTIVE: Evidence shows that Black individuals have higher rates of coercive emergency psychiatric interventions than other racialized groups, yet no studies have elevated the voices of Black patients undergoing emergency psychiatric evaluation. This qualitative study sought to explore the experiences of Black individuals who had been evaluated in a locked psychiatric emergency unit (PEU). METHODS: Electronic health records were used to identify and recruit adult patients (ages ≥18 years) who self-identified as Black and who had undergone evaluation in a locked PEU at a large academic medical center. In total, 11 semistructured, one-on-one interviews were conducted by telephone, exploring experiences during psychiatric evaluation. Transcripts were analyzed with thematic analysis. RESULTS: Participants shared experiences of criminalization, stigma, and vulnerability before and during their evaluation. Although participants described insight into their desire and need for treatment and identified helpful aspects of the care they received, they noted a mismatch between their expectations of treatment and the treatment received. CONCLUSIONS: This study reveals six major patient-identified themes that supplement a growing body of quantitative evidence demonstrating that racialized minority groups endure disproportionate rates of coercive interventions during emergency psychiatric evaluation. Interdisciplinary systemic changes are urgently needed to address structural barriers to equitable psychiatric care.