Pulmonary endothelial cells express a store-operated calcium entry current ( I), which contributes to inter-endothelial cell gap formation. I is regulated by a heterocomplex of proteins that includes the immunophilin FKBP51. FKBP51 inhibits I by mechanisms that are not fully understood. In pulmonary artery endothelial cells (PAECs) we have shown that FKBP51 increases microtubule polymerization, an event that is critical for I inhibition by FKBP51. In neurons, FKBP51 promotes microtubule stability through facilitation of tau dephosphorylation. However, FKBP51 does not possess phosphatase activity. Protein phosphatase 5 (PP5C/PPP5C) can dephosphorylate tau, and similar to FKBP51, PP5C possesses tetratricopeptide repeats (TPR) that mediate interaction with heat shock protein-90 (HSP90) chaperone/scaffolding complexes. We therefore tested whether PP5C contributes to FKBP51-mediated inhibition of I. Both siRNA-mediated suppression of PP5C expression in PAECs and genetic disruption of PP5C in HEK293 cells attenuate FKBP51-mediated inhibition of I. Reintroduction of catalytically competent, but not catalytically inactive PP5C, restored FKBP51-mediated inhibition of I. PAEC cell fractionation studies identified both PP5C and the ISOC heterocomplex in the same membrane fractions. Further, PP5C co-precipitates with TRPC4, an essential subunit of ISOC channel. Finally, to determine if PP5C is required for FKBP51-mediated inhibition of calcium entry-induced inter-endothelial cell gap formation, we measured gap area by wide-field microscopy and performed biotin gap quantification assay and electric cell-substrate impedance sensing (ECIS®). Collectively, the data presented indicate that suppression of PP5C expression negates the protective effect of FKBP51. These observations identify PP5C as a novel member of the ISOC heterocomplex that is required for FKBP51-mediated inhibition of I.

Pulmonary artery endothelial cells (PAECs) express a cation current, ISOC (store-operated calcium entry current), which when activated permits calcium entry leading to inter-endothelial cell gap formation. The large molecular weight immunophilin FKBP51 inhibits ISOC but not other calcium entry pathways in PAECs. However, it is unknown whether FKBP51-mediated inhibition of ISOC is sufficient to protect the endothelial barrier from calcium entry-induced disruption. The major objective of this study was to determine whether FKBP51-mediated inhibition of ISOC leads to decreased calcium entry-induced inter-endothelial gap formation and thus preservation of the endothelial barrier. Here, we measured the effects of thapsigargin-induced ISOC on the endothelial barrier in control and FKBP51 overexpressing PAECs. FKBP51 overexpression decreased actin stress fiber and inter-endothelial cell gap formation in addition to attenuating the decrease in resistance observed with control cells using electric cell-substrate impedance sensing. Finally, the thapsigargin-induced increase in dextran flux was abolished in FKBP51 overexpressing PAECs. We then measured endothelial permeability in perfused lungs of FKBP51 knockout (FKBP51–/–) mice and observed increased calcium entry-induced permeability compared to wild-type mice. To begin to dissect the mechanism underlying the FKBP51-mediated inhibition of ISOC, a second goal of this study was to determine the role of the microtubule network. We observed that FKBP51 overexpressing PAECs exhibited increased microtubule polymerization that is critical for inhibition of ISOC by FKBP51. Overall, we have identified FKBP51 as a novel regulator of endothelial barrier integrity, and these findings are significant as they reveal a protective mechanism for endothelium against calcium entry-induced disruption.

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter‐endothelial cell gaps and barrier disruption. The immunophilin FK506‐binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC. Free PPP5C assumes an autoinhibitory state, which has low “basal” catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium‐dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C‐FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C‐dependent manner and by FKBP51 in a S100A6‐dependent manner. Further, calcium entry‐induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C‐FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry‐induced disruption.

We study the fully nonlinear, nonlocal dynamics of two-dimensional vesicles in a time-dependent, incompressible viscous flow at finite temperature. We focus on a transient instability that can be observed when the direction of applied flow is suddenly reversed, which induces compressive forces on the vesicle interface, and small-scale interface perturbations known as wrinkles develop. These wrinkles are driven by regions of negative elastic tension on the membrane. Using a stochastic immersed boundary method with a biophysically motivated choice of thermal fluctuations, we investigate the wrinkling dynamics numerically. Different from deterministic wrinkling dynamics, thermal fluctuations lead to symmetry-breaking wrinkling patterns by exciting higher order modes. This leads to more rapid and more realistic wrinkling dynamics. Our results are in excellent agreement with the experimental data by Kantsler et al. We compare the nonlinear simulation results with perturbation theory, modified to account for thermal fluctuations. The strength of the applied flow strongly influences the most unstable wavelength characterizing the wrinkles, and there are significant differences between the results from perturbation theory and the fully nonlinear simulations, which suggests that the perturbation theory misses important nonlinear interactions. Strikingly, we find that thermal fluctuations actually have the ability to attenuate variability of the characteristic wavelength of wrinkling by exciting a wider range of modes than the deterministic case, which makes the evolution less constrained and enables the most unstable wavelength to emerge more readily. We further find that thermal noise helps prevent the vesicle from rotating if it is misaligned with the direction of the applied extensional flow.

To understand county-level variation in case fatality rates of COVID-19, a statewide analysis of COVID-19 incidence and fatality data was performed, using publicly available incidence and case fatality rate data of COVID-19 for all 67 Alabama counties and mapped with health disparities at the county level. A specific adaptation of the Shewhart p-chart, called a funnel chart, was used to compare case fatality rates. Important differences in case fatality rates across the counties did not appear to be reflective of differences in testing or incidence rates. Instead, a higher prevalence of comorbidities and vulnerabilities was observed in high fatality rate counties, while showing no differences in access to acute care. Funnel charts reliably identify counties with unexpected high and low COVID-19 case fatality rates. Social determinants of health are strongly associated with such differences. These data may assist in public health decisions including vaccination strategies, especially in southern states with similar demographics. KEY WORDS: case fatality rate, COVID-19, funnel chart, social determinants of healthU nderstanding variation in case fatality rates (CFRs) in patients with COVID-19 across different geographical regions may have profound implications for prevention and control, and could assist health care leaders and legislators in identifying areas requiring further attention. The reported incidence and CFRs of disease, however, are affected by many factors, including availability of testing. 1 There are also variations in reporting time of death to the National Center for Health Statistics across the