Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30-70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins-function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.

OBJECTIVES: Home-based medical care (HBMC) delivers physician or advanced practice provider-led medical services for patients in private residences and domiciliary settings (eg, assisted living facilities, group/boarding homes). We aimed to examine the time trends in HBMC utilization by care settings. DESIGN: Analyses of HBMC utilization at the national and state levels during the years 2012-2019. SETTING AND PARTICIPANTS: With Medicare public use files, we calculated the state-level utilization rate of HBMC among fee-for-service (FFS) Medicare beneficiaries, measured by visits per 1000 FFS enrollees, in private residences and domiciliary settings, both separately and combined. METHODS: We assessed the trend of HBMC utilization over time via linear mixed models with random intercept for state, adjusting for the following state-level markers of HBMC supply and demand: number of HBMC providers, state ranking of total assisted living and residential care capacity, and the proportion of FFS beneficiaries with dementia, dual eligibility for Medicaid, receiving home health services, and Medicare Advantage. RESULTS: Total HBMC visits in the United States increased from 3,911,778 in 2012 to 5,524,939 in 2019. The median (interquartile range) state-level HBMC utilization rate per 1000 FFS population was 67.6 (34.1-151.3) visits overall, 17.3 (7.9-41.9) visits in private residences, and 47.7 (23.1-86.6) visits in domiciliary settings. The annual percentage increase of utilization rates was significant for all care settings in crude models (3%-8%), and remained significant for overall visits and visits in domiciliary settings (2%-4%), but not in private residences. CONCLUSIONS AND IMPLICATIONS: The national-level growth in HBMC from 2012-2019 was largely driven by a growth of HBMC occurring in domiciliary settings. To meet the needs of a growing aging population, future studies should focus efforts on policy and payment issues to address inequities in access to HBMC services for homebound older adults, and examine drivers of HBMC growth at regional and local levels.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.