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Purpose: Most patients are escorted to exam rooms (escorted rooming) although patients directing themselves to their exam room (self-rooming) saves patient and staff time while increasing patient satisfaction. This study assesses patient and staff perceptions after pragmatic implementation of self-rooming., Methods: In October-December 2020, we surveyed patients and staff in 25 primary care clinics after our institution expanded self-rooming from 4 specially built clinics during the COVID-19 pandemic. Semi-structured surveys asked about rooming process used, rooming process preferred, and perceptions of self-rooming compared with escorted rooming., Results: Most patients (n = 1,561) preferred self-rooming (86%), especially among patients aged <65 years and in family medicine clinics. Few patients felt less welcomed (10.6%), less cared about (6.8%), more isolated (15.6%), more lost/confused (7.6%), or more frustrated (3.2%) with self-rooming compared with escorted rooming. Early-adopter clinics that implemented self-rooming ≤2016 had even lower rates of patients feeling more isolated, lost/confused, or frustrated with self-rooming compared with escorted rooming.Over one-half of staff (n = 241; 180 clinical, 61 nonclinical) preferred self-rooming (59%) and thought most patients liked self-rooming (65.8%), especially among clinical staff and in early adopter clinics (≤2016). Few staff reported worse waiting times for patients (12.4%), medical assistants (MAs) (15.9%), and clinicians (16.4%) or worse crowding in waiting areas (1.7%) and hallways (10.1%). Unlike patient-reported confusion (7.6%), most staff thought self-rooming led to more patient confusion (63.8%), except in early-adopter clinics (44.4%)., Conclusions: Self-rooming is a patient-centered innovation that is also acceptable to staff. We demonstrated that pragmatic implementation is feasible across primary care without expensive technology or specially designed buildings., (© 2023 Annals of Family Medicine, Inc.)

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization. Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

Dense-core granules (DCGs) are organelles found in neuroendocrine cells and neurons that house, transport, and release a number of important peptides and proteins. In neurons, DCG cargo can include the secreted neuromodulatory proteins tissue plasminogen activator (tPA) and/or brain-derived neurotrophic factor (BDNF), which play a key role in modulating synaptic efficacy in the hippocampus. This function has spurred interest in DCGs that localize to synaptic contacts between hippocampal neurons, and several studies recently have established that DCGs localize to, and undergo regulated exocytosis from, postsynaptic sites. To complement this work, we have studied presynaptically localized DCGs in hippocampal neurons, which are much more poorly understood than their postsynaptic analogs. Moreover, to enhance relevance, we visualized DCGs via fluorescence labeling of exogenous and endogenous tPA and BDNF. Using single-particle tracking, we determined trajectories of more than 150 presynaptically localized DCGs. These trajectories reveal that mobility of DCGs in presynaptic boutons is highly hindered and that storage is long-lived. We also computed mean-squared displacement curves, which can be used to elucidate mechanisms of transport. Over shorter time windows, most curves are linear, demonstrating that DCG transport in boutons is driven predominantly by diffusion. The remaining curves plateau with time, consistent with motion constrained by a submicron-sized corral. These results have relevance to recent models of presynaptic organization and to recent hypotheses about DCG cargo function. The results also provide estimates for transit times to the presynaptic plasma membrane that are consistent with measured times for onset of neurotrophin release from synaptically localized DCGs., (Copyright © 2011 Wiley Periodicals, Inc.)

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy.

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning-related synaptic plasticity and potentiating N-methyl-D-aspartate (NMDA) receptor-dependent signaling. These findings suggest that tPA may localize to, and undergo activity-dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization-induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense-core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca(+2) concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse-pinch-linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA-type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory.

Dense-core granules (DCGs) are organelles found in specialized secretory cells, including neuroendocrine cells and neurons. Neuronal DCGs facilitate many critical processes, including the transport and secretion of proteins involved in learning, and yet their transport and exocytosis are poorly understood. We have used wide-field and total internal reflection fluorescence microscopy, in conjunction with transport theory, to visualize the transport and exocytosis of DCGs containing a tissue plasminogen activator-green fluorescent protein hybrid in cell bodies, neurites, and growth cones of developing hippocampal neurons and to quantify the roles that diffusion, directed motion, and immobility play in these processes. Our results demonstrate that shorter-ranged transport of DCGs near sites of exocytosis in hippocampal neurons and neuroendocrine cells differs markedly. Specifically, the immobile fraction of DCGs within growth cones and near the plasma membrane of hippocampal neurons is small and relatively unaltered by actin disruption, unlike in neuroendocrine cells. Moreover, transport of DCGs in these domains of hippocampal neurons is unusually heterogeneous, being significantly rapid and directed as well as slow and diffusive. Our results also demonstrate that exocytosis is preceded by substantial movement and heterogeneous transport; this movement may facilitate delivery of DCG cargo in hippocampal neurons, given the relatively low abundance of neuronal DCGs. In addition, the extensive mobility of DCGs in hippocampal neurons argues strongly against the hypothesis that cortical actin is a major barrier to membrane-proximal DCGs in these cells. Instead, our results suggest that extended release of DCG cargo from hippocampal neurons arises from heterogeneity in DCG mobility.

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.

To define the contribution of exoenzyme S to the pathogenesis of infections with Pseudomonas aeruginosa, we have compared the ability of an exoenzyme S-deficient mutant, 388 exs1::Tn1, and that of its exoenzyme S-producing parent to colonize and disseminate in burned mice infected with this organism. Both the exoenzyme S-deficient mutant and the parent strain proliferated in burned skin, but only the parent strain was able to effectively disseminate to blood and other tissues. The reduced ability of the mutant to disseminate was not due to alterations in serum sensitivity, lipopolysaccharide composition, or motility. The exoenzyme S-deficient mutant was able to disseminate in the presence of the exoenzyme S-producing parent. Antibody to purified exoenzyme S was able to greatly reduce dissemination of the exoenzyme S-producing parent strain but did not prevent colonization in the burned skin. These data suggest that exoenzyme S does not contribute to the initial colonization but does contribute to the establishment of disseminated infection.

Legionella pneumophila was susceptible to the antimicrobial action of oxygen metabolites generated by both the myeloperoxidase-H(2)O(2)-halide and the xanthine oxidase systems.

Preincubation of human polymorphonuclear leukocytes (PMNLs) with Legionella pneumophila toxin impaired activation of the superoxide-generating complex induced by latex particles and by the Ca++ ionophore A23187. The toxin had no effect, however, on activation of the complex induced by phorbol myristate acetate (PMA), concanavalin A, valinomycin, or bromolasalocid. The toxin prevented PMNL plasma membrane depolarization induced by A23187 but failed to influence the membrane depolarization induced by PMA. These observations indicate that Legionella pneumophila toxin selectively impairs activation of the phagocyte superoxide-generating complex without affecting the functional integrity of components of the complex.

The effect of Legionella pneumophila toxin on selected functions of human polymorphonuclear leukocytes was investigated. Amounts of L. pneumophila toxin that had no effect on leukocyte viability or phagocytosis significantly decreased hexose monophosphate shunt activity and O2 consumption during phagocytosis and bacterial iodination and killing in a dose-dependent fashion. The mechanism of action of this toxin appears to be unique among bacterial products thus far studied.

all-trans-Retinal was previously shown to stimulate high levels of superoxide release by guinea pig neutrophils. When the cells, previously labeled with [3H]inositol, are treated with all-trans-retinal, they exhibit a decrease in the levels of [3H]inositol phospholipids and an increase in the accumulation of [3H]inositol phosphates. The maximal accumulation of inositol phosphates and the optimal rate of superoxide release occurred together at approximately 7 min after stimulation. The levels of [3H]inositol phosphates accumulated were comparable to those observed when the cells were stimulated with a chemotactic peptide. In direct measurements, using concentrations that stimulate intact cells maximally, all-trans-retinal was found not to inhibit protein kinase C from the cytosol of neutrophils significantly. This contrasts with the situation with this kinase obtained from other sources. These observations represent additional effects of vitamin A on cells.

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.