Characterization of COVID-19 Vaccine Hesitancy Among Essential Workforce Members of a Large Safety Net Urban Medical Center.

OBJECTIVES: Vaccine hesitancy among essential workers remains a significant public health challenge. We examined psychological constructs of perceived susceptibility, threat, and self-efficacy and their associations with COVID-19 vaccine hesitancy among a racially and ethnically diverse essential workforce population. METHODS: We performed a cross-sectional survey of essential workers from September-December 2020 at a large Los Angeles safety-net medical center as part of a program offering free COVID-19 serology testing. Program participants completed a standardized survey at the time of phlebotomy. Hierarchical logistic regression was utilized to determine factors independently associated with vaccine hesitancy. RESULTS: Among 1327 persons who had serology testing, 1235 (93%) completed the survey. Of these, 958 (78%) were healthcare workers. Based on expressed intent, 22% were vaccine-hesitant 78% were vaccine acceptors. In our multivariate model, vaccine hesitancy was associated with female gender [aOR = 2.09; 95% CI (1.44-3.05)], African American race [aOR = 4.32; (2.16-8.62)], LatinX ethnicity [aOR = 2.47; 95% CI (1.51-4.05)] and history of not/sometimes receiving influenza vaccination [aOR = 4.39; 95% CI (2.98-6.48)]. Compared to nurses, vaccine hesitancy was lower among physicians [aOR = 0.09; 95% CI (0.04-0.23)], non-nursing/non-physician healthcare workers [aOR = 0.55; 95% CI (0.33-0.92)], and non-healthcare care workers [aOR = 0.53; 95% CI (0.36-0.78)]. CONCLUSIONS: Among a racially/ethnically diverse group of safety net medical center essential workers, COVID-19 vaccine hesitancy was associated with racial/ethnic minority groups, employment type, and prior influenza vaccination hesitancy. Interestingly, we found no association with the Health Belief Model construct measures of perceived susceptibility, threat, and self-efficacy. Psychological constructs not assessed may be drivers of vaccine hesitancy in our population.

Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A.

The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

Nephrin regulates lamellipodia formation by assembling a protein complex that includes Ship2, filamin and lamellipodin.

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.

Actin-depolymerizing factor cofilin-1 is necessary in maintaining mature podocyte architecture.

Actin dynamics determines podocyte morphology during development and in response to podocyte injury and might be necessary for maintaining normal podocyte morphology. Because podocyte intercellular junction receptor Nephrin plays a role in regulating actin dynamics, and given the described role of cofilin in actin filament polymerization and severing, we hypothesized that cofilin-1 activity is regulated by Nephrin and is necessary in normal podocyte actin dynamics. Nephrin activation induced cofilin dephosphorylation via intermediaries that include phosphatidylinositol 3-kinase, SSH1, 14-3-3, and LIMK in a cell culture model. This Nephrin-induced cofilin activation required a direct interaction between Nephrin and the p85 subunit of phosphatidylinositol 3-kinase. In a similar fashion, cofilin-1 dephosphorylation was observed in a rat model of podocyte injury at a time when foot process spreading is initially observed. To investigate the necessity of cofilin-1 in the glomerulus, podocyte-specific Cfl1 null mice were generated. Cfl1 null podocytes developed normally. However, these mice developed persistent proteinuria by 3 months of age, although they did not exhibit foot process spreading until 8 months, when the rate of urinary protein excretion became more exaggerated. In a mouse model of podocyte injury, protamine sulfate perfusion of the Cfl1 mutant mouse induced a broadened and flattened foot process morphology that was distinct from that observed following perfusion of control kidneys, and mutant podocytes did not recover normal structure following additional perfusion with heparin sulfate. We conclude that cofilin-1 is necessary for maintenance of normal podocyte architecture and for actin structural changes that occur during induction and recovery from podocyte injury.

Tacrolimus reduces nitric oxide synthase function by binding to FKBP rather than by its calcineurin effect.

Hypertension develops in many patients receiving the immunosuppressive drug tacrolimus (FK506). One possible mechanism for hypertension is a reduction in vasodilatory nitric oxide. We found that tacrolimus and a calcineurin autoinhibitory peptide significantly decreased vascular calcineurin activity; however, only tacrolimus altered intracellular calcium release in mouse aortic endothelial cells. In mouse aortas, incubation with tacrolimus increased protein kinase C activity and basal endothelial nitric oxide synthase phosphorylation at threonine 495 but reduced basal and agonist-induced endothelial nitric oxide synthase phosphorylation at serine 1177, a mechanism known to inhibit synthase activity. While this decreased nitric oxide production and endothelial function, the calcineurin autoinhibitory peptide had no such effects. Inhibition of ryanodine receptor opening or protein kinase C blocked the effects of tacrolimus. Since it is known that the FK506 binding protein (FKBP12/12.6) interacts with the ryanodine receptor to regulate calcium release, we propose this as the mechanism by which tacrolimus alters intracellular calcium and endothelial nitric oxide synthase rather than by its effect on calcineurin. Our study shows that prevention of the tacrolimus-induced intracellular calcium leak may attenuate endothelial dysfunction and the consequent hypertension.

Uncoupled endothelial nitric oxide synthase and oxidative stress in a rat model of pregnancy-induced hypertension.

BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.

Removal of FKBP12/12.6 from endothelial ryanodine receptors leads to an intracellular calcium leak and endothelial dysfunction.

OBJECTIVES: FK506 Binding Protein 12 and its related isoform 12.6 (FKBP12/12.6) stabilize a closed state of intracellular Ca2+ release channels (ryanodine receptors [RyRs]), and in myocytes removal of FKBP12/12.6 from RyRs alters intracellular Ca2+ levels. The immunosuppressive drugs rapamycin and FK506 bind and displace FKBP12/12.6 from RyRs, and can also cause endothelial dysfunction and hypertension. We tested whether rapamycin and FK506 cause an intracellular Ca2+ leak in endothelial cells and whether this affects endothelial function and blood pressure regulation. METHODS AND RESULTS: Rapamycin or FK506 concentration-dependently caused a Ca2+ leak in isolated endothelial cells, decreased aortic NO production and endothelium-dependent dilation, and increased systolic blood pressure in control mice. Rapamycin or FK506 at 10 micromol/L abolished aortic NO production and endothelium-dependent dilation. Similar results were obtained in isolated endothelial cells and aortas from FKBP12.6-/- mice after displacement of FKBP12 with 1 micromol/L rapamycin or FK506. In hypertensive FKBP12.6-/- mice, systolic blood pressures were further elevated after treatment with either rapamycin or FK506. Blockade of the Ca2+ leak with ryanodine normalized NO production and endothelium-dependent dilation. CONCLUSIONS: Complete removal of FKBP12 and 12.6 from endothelial RyRs induces an intracellular Ca2+ leak which may contribute to the pathogenesis of endothelial dysfunction and hypertension caused by rapamycin or FK506.

FK506 binding protein 12/12.6 depletion increases endothelial nitric oxide synthase threonine 495 phosphorylation and blood pressure.

Chronic treatment with the immunosuppressive drug rapamycin leads to hypertension; however, the mechanisms are unknown. Rapamycin binds FK506 binding protein 12 and its related isoform 12.6 (FKBP12/12.6) and displaces them from intracellular Ca2+ release channels (ryanodine receptors) eliciting a Ca2+ leak from the endoplasmic/sarcoplasmic reticulum. We tested whether this Ca2+ leak promotes conventional protein kinase C-mediated endothelial NO synthase phosphorylation at Thr495, which reduces production of the vasodilator NO. Rapamycin treatment of control mice for 7 days, as well as genetic deletion of FKBP12.6, increased systolic arterial pressure significantly compared with controls. Untreated aortas from FKBP12.6-/- mice and in vitro rapamycin-treated control aortas had similarly decreased endothelium-dependent relaxation responses and NO production and increased endothelial NO synthase Thr495 phosphorylation and protein kinase C activity. Inhibition of either conventional protein kinase C or ryanodine receptor restored endothelial NO synthase Thr495 phosphorylation and endothelial function to control levels. Rapamycin induced a small increase in basal intracellular Ca2+ levels in isolated endothelial cells, and rapamycin or FKBP12.6 gene deletion decreased acetylcholine-induced intracellular Ca2+ release, all of which were reversed by ryanodine. These data demonstrate that displacement of FKBP12/12.6 from ryanodine receptors induces an endothelial intracellular Ca2+ leak and increases conventional protein kinase C-mediated endothelial NO synthase Thr495 phosphorylation leading to decreased NO production and endothelial dysfunction. This molecular mechanism may, in part, explain rapamycin-induced hypertension.

The pesticide malathion reduces survival and growth in developing zebrafish.

Malathion is an organophosphorous pesticide widely used to control mosquitoes in urban areas and pests, such as boll weevils, in agricultural areas. Zebrafish, Danio rerio, are model organisms for developmental toxicology research because they are readily available, produce large numbers of clear embryos, and are sensitive to environmental changes. The nonlethal effects of malathion on developing zebrafish embryos, however, previously have not been analyzed quantitatively. We exposed zebrafish embryos to sublethal malathion concentrations to determine malathion's effects on a developing vertebrate. Zebrafish exposed to 0.5, 1.0, or 1.5 mg/L of malathion consistently elicited more rapid hatching from the chorion than zebrafish exposed to 2.0-, 2.5-, or 3.0-mg/L malathion concentrations. In addition, exposure to 2.0, 2.5, or 3.0 mg/L of malathion resulted in significantly shorter body length and eye diameters, indicating that malathion had teratogenic effects on zebrafish embryos. Malathion's action as an acetylcholinesterase inhibitor and the toxicity of the metabolites of malathion may be responsible for malathion's teratogenic effects on fish development.

Effects of prey type on specific dynamic action, growth, and mass conversion efficiencies in the horned frog, Ceratophrys cranwelli.

To be most energetically profitable, predators should ingest prey with the maximal nutritional benefit while minimizing the cost of processing. Therefore, when determining the quality of prey items, both the cost of processing and nutritional content must be considered. Specific dynamic action (SDA), the increase in metabolic rate associated with feeding in animals, is a significant processing cost that represents the total cost of digestion and assimilation of nutrients from prey. We examined the effects of an invertebrate diet (earthworms) and a vertebrate diet (newborn mice) on mass conversion efficiencies, growth, and SDA in the Chacoan horned frog, Ceratophrys cranwelli. We found the earthworm diet to be significantly lower in lipid, protein, and energy content when compared to the diet of newborn mice. Growth and mass conversion efficiencies were significantly higher in frogs fed newborn mice. However, mean SDA did not differ between frogs fed the two diets, a finding that contradicts many studies that indicate SDA increases with the protein content of the meal. Together, our results indicate that future studies evaluating the effect of meal type on bioenergetics of herpetofauna are warranted and may provide significant insight into the underlying factors driving SDA.

Dietary soy protein and isoflavones have no significant effect on bone and a potentially negative effect on the uterus of sexually mature intact Sprague-Dawley female rats.

OBJECTIVE: To evaluate the effect of dietary soy protein and isoflavones on bone and the reproductive tract in premenopausal rats. DESIGN: Three-month-old intact Sprague-Dawley female rats (N = 50) were fed diets containing casein, soy protein, or casein with isoflavone extract for 12 weeks. The amount of casein, soy protein, and extract (per kilogram diet) in each group was: (1) 200 g casein (control); (2) 100 g casein plus 100 g soy protein (low soy); (3) 200 g soy protein (high soy); 4) 200 g casein plus 17.2g extract (low extract); and (5) 200 g casein plus 34.4 g extract (high extract). Diet consumption, body weight, uterine wet weight, urinary deoxypyridinoline concentration, and bone mineral density of the femur and lumbar vertebrae were measured. Femur rigidity was evaluated by histomorphometry. The uterus and vagina were studied histologically. RESULTS: Rats in all treatment groups had lower body weights and lower deoxypyridinoline concentrations compared with controls, but none of the differences was statistically significant. There was no significant difference in femur and lumbar bone mineral density, uterine wet weights, or histomorphometry between the control and treatment groups. Histologically, uteri and vaginae were normal in all groups except that 1 of 10 rats in the high-soy group and 2 of 10 rats in the high-extract group showed extensive squamous metaplasia in the uterine gland. CONCLUSION: These results suggest that dietary isolated soy protein and isoflavones have no effect on bone and the vagina during premenopausal period, but may have an adverse effect on the uterus.