Sex differences in the relationship of sleep-disordered breathing and asthma control among children with severe asthma.

OBJECTIVE: Children with severe asthma are underrepresented in studies of the relationship of sleep-disordered breathing (SDB) and asthma and little is known about sex differences of these relationships. We sought to determine the relationship of SDB with asthma control and lung function among boys and girls within a pediatric severe asthma cohort. METHODS: Patients attending clinic visits at the Boston Children's Hospital Pediatric Severe Asthma Program completed the Pediatric Sleep Questionnaire (PSQ), Asthma Control Test (ACT) and Spirometry. The prevalence of SDB was defined as a PSQ score >0.33. We analyzed the association between PSQ score and both ACT score and spirometry values in mixed effect models, testing interactions for age and sex. RESULTS: Among 37 subjects, mean age was 11.8 years (4.4) and 23 (62.2%) were male, the prevalence of SDB was 43.2% (16/37). Including all 80 observations, there was a moderate negative correlation between PSQ and ACT scores (r=-0.46, p < 0.001). Multivariable linear regression models revealed a significant sex interaction with PSQ on asthma control (p = 0.003), such that for each 0.10 point increase in PSQ there was a 1.88 point decrease in ACT score for females but only 0.21 point decrease in ACT score for males. A positive PSQ screen was associated with a 9.44 point (CI 5.54, 13.34, p < 0.001) lower ACT score for females and a 3.22 point (CI 0.56, 5.88, p = 0.02) lower score for males. CONCLUSIONS: SDB is common among children with severe asthma. Among children with severe asthma, SDB in girls portends to significantly worse asthma control than boys.Supplemental data for this article is available online at https://doi.org/10.1080/02770903.2021.1897838.

Cerebral aneurysm flow diverter modeled as a thin inhomogeneous porous medium in hemodynamic simulations.

Rapid and accurate simulation of cerebral aneurysm flow modifications by flow diverters (FDs) can help improving patient-specific intervention and predicting treatment outcome. However, when FD devices are explicitly represented in computational fluid dynamics (CFD) simulations, flow around the stent wires must be resolved, leading to high computational cost. Classic porous medium (PM) methods can reduce computational expense but cannot capture the inhomogeneous FD wire distribution once implanted on a cerebral artery and thus cannot accurately model the post-stenting aneurysmal flow. We report a novel approach that models the FD flow modification as a thin inhomogeneous porous medium (iPM). It improves over the classic PM approaches in two ways. First, the FD is more appropriately treated as a thin screen, which is more accurate than the classic 3D-PM-based Darcy-Forchheimer relation. Second, pressure drop is calculated cell-by-cell using the local FD geometric parameters across an inhomogeneous PM. We applied the iPM technique to simulating the post-stenting hemodynamics of three patient-specific aneurysms. To test its accuracy and speed, we compared the results from the iPM technique against CFD simulations with explicit FD devices. The iPM CFD ran 500% faster than the explicit CFD while achieving 94%-99% accuracy; thus, iPM is a promising clinical bedside modeling tool to assist endovascular interventions with FD and stents.

Fast virtual coiling algorithm for intracranial aneurysms using pre-shape path planning.

To aid in predicting and improving treatment outcome of endovascular coiling of intracranial aneurysms, simulation of patient-specific coil deployment should be both accurate and fast. We developed a fast virtual coiling algorithm called Pre-shape Path Planning (P3). It captures the mechanical propensity of a released coil to restore its pre-shape for bending energy minimization, producing coils without unrealistic kinks and bends. A coil is discretized into finite-length segments and extruded from the delivery catheter segment-by-segment following a generic coil pre-shape. With the release of each segment, coil-wall and coil-coil collisions are detected and resolved. Modeling of each case took seconds to minutes. To test the algorithm, we evaluated its output against the literature, experiments, and patient angiograms. The periphery-to-core ratio of coils deployed by P3 decreased with increasing coil packing density, consistent with observations in the literature. Coils deployed by P3 compared well with in vitro experiments, free from unphysical kinks and loops that arose from previous virtual coiling algorithms. Simulations of coiling in four patient-specific aneurysms agreed well with the patient angiograms. To test the influence of coil pre-shape on P3, we performed hemodynamic simulations in aneurysms with coils deployed by P3 using the generic pre-shape, P3 using a coil-specific pre-shape, and full finite-element-method simulation. We found that the generic pre-shape was sufficient to produce results comparable to virtual coiling by finite element modeling. Based on these findings, P3 can rapidly simulate coiling in patient-specific aneurysms with good accuracy and is thus a potential candidate for clinical treatment planning.

Rapid Implementation of Telehealth Services in a Pediatric Pulmonary Clinic During COVID-19.

OBJECTIVES: The coronavirus pandemic created significant, abrupt challenges to the delivery of ambulatory health care. Because tertiary medical centers limited elective in-person services, telehealth was rapidly enacted in settings with minimal previous experience to allow continued access to care. With this quality improvement (QI) initiative, we aimed to achieve a virtual visit volume of at least 75% of our prepandemic volume. We also describe patient and provider experience with telehealth services. METHODS: Our QI team identified the primary drivers contributing to low telehealth volume and developed a telehealth scheduling protocol and data tracking system using QI-based strategies. Patients and providers were surveyed on their telehealth experience. RESULTS: At the onset of the pandemic, weekly visit volume dropped by 65% (99 weekly visits; historical average of 281). Over the subsequent 3 weeks, using rapid Plan-Do-Study-Act cycles, we achieved our goal volume. In surveys, it was indicated that most participants had never before used telehealth (71% of patients; 82% of providers) yet reported high satisfaction (90% of patients; 81% of providers). Both groups expressed concern over the lack of in-person assessments. Most respondents were interested in future use of telehealth. CONCLUSIONS: With a QI-based approach, we successfully maintained access to care via telehealth services for pediatric pulmonary patients during the coronavirus pandemic and found high rates of satisfaction among patients and providers. Telehealth will likely continue to be a part of our health care delivery platform, expanding the reach of our services. Further work is needed to understand the effects on clinical outcomes.

Asthma control and psychological health in pediatric severe asthma.

OBJECTIVES: Psychological comorbidities have been associated with asthma in adults and children, but have not been studied in a population of children with severe asthma. The aim of this study was to test the hypothesis that symptoms of anxiety or depression are highly prevalent in pediatric severe asthma and negatively effects asthma control. METHODS: Longitudinal assessments of anxiety or depression symptoms (Patient Health Questionnaire-4 [PHQ-4]), asthma control (Asthma Control Test [ACT]), and lung function were performed in a single-center pediatric severe asthma clinic. Participant data were collected during routine clinical care. Primary outcomes were ACT and forced expiratory volume in 1 s per forced vital capacity (FEV1/FVC). RESULTS: Among 43 subjects (with total 93 observations), 58.1% reported at least one anxious or depressive symptom and 18.6% had a PHQ-4 more than 2, the threshold for an abnormal test result. After adjusting for age, sex, race, and asthma medication step, there was a significant reduction in ACT for girls with PHQ-4 more than 2 (adjusted mean [SE] ACT for PHQ-4 > 2: 13.64 [0.59], ACT for PHQ-4 ≤ 2: 20.64 [1.25], p = .02) but not boys. Moreover, there was a significant differential effect of mental health impairment for girls than boys. ACT for girls with PHQ more than 2: 13.64 (0.59) compared with boys with PHQ-4 more than 2: 17.82 (0.95), adjusted mean difference ACT by sex = 4.18 points; 95% confidence interval, 0.63-7.73; p = .033. In adjusted models, there was no association between PHQ-4 more than 2 and FEV1/FVC. CONCLUSIONS: Symptoms of anxiety and depression are common. In children with severe asthma, a PHQ-4 score more than 2 is associated with worse asthma symptom control in girls, but not boys.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.

Pollen performance traits reveal prezygotic nonrandom mating and interference competition in Arabidopsis thaliana.

PREMISE: The lack of ability to measure pollen performance traits in mixed pollinations has been a major hurdle in understanding the mechanisms of differential success of compatible pollen donors. In previous work, we demonstrated that nonrandom mating between two accessions of Arabidopsis thaliana, Columbia (Col) and Landsberg (Ler), is mediated by the male genotype. Despite these genetic insights, it was unclear at what stage of reproduction these genes were acting. Here, we used an experimental strategy that allowed us to differentiate different pollen populations in mixed pollinations to ask: (1) What pollen performance traits differed between Col and Ler accessions that direct nonrandom mating? (2) Is there evidence of interference competition? METHODS: We used genetically marked pollen that can be visualized colorimetrically to quantify pollen performance of single populations of pollen in mixed pollinations. We used this and other assays to measure pollen viability, germination, tube growth, patterns of fertilization, and seed abortion. Finally, we assessed interference competition. RESULTS: In mixed pollinations on Col pistils, Col pollen sired significantly more seeds than Ler pollen. Col pollen displayed higher pollen viability, faster and greater pollen germination, and faster pollen tube growth. We saw no evidence of nonrandom seed abortion. Finally, we found interference competition occurs in mixed pollinations. CONCLUSION: The lack of differences in postzygotic processes coupled with direct observation of pollen performance traits indicates that nonrandom mating in Arabidopsis thaliana is prezygotic, due mostly to differential pollen germination and pollen tube growth rates. Finally, this study unambiguously demonstrates the existence of interference competition.

High-quality immunofluorescence of cultured cells.

Immunofluorescence microscopy of cultured cells often gives poor preservation of delicate structures. We have obtained dramatically improved results with a simple modification of a standard protocol. Cells growing on a coverslip are rapidly dehydrated in a cold organic solvent and then are rehydrated in a solution containing a homobifunctional crosslinker. The crosslinking reaction stabilizes cellular structures during subsequent incubation and wash steps, usually without compromising antigenicity. This method reproducibly yields high-quality images of endomembrane compartments and cytoskeletal elements.

LAP3, a novel plant protein required for pollen development, is essential for proper exine formation.

We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in beta-propeller enzymes. Insertion mutations in LAP3 lead to male sterility. To investigate possible roles for LAP3 in pollen development, we assayed the metabolite profile of anther tissues containing developing pollen grains and found that the lap3-2 defect leads to a broad range of metabolic changes. The largest changes were seen in levels of a straight-chain hydrocarbon nonacosane and in naringenin chalcone, an obligate compound in the flavonoid biosynthesis pathway.

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles.

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.

Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below approximately 25 degrees C. At 37 degrees C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcepsilonRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.

Lipid segregation and IgE receptor signaling: a decade of progress.

Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment. Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation. These and other results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and other cell surface receptors.

Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells.

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

De novo formation of transitional ER sites and Golgi structures in Pichia pastoris.

Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.

Paz2 and 13 other PAZ gene products regulate vacuolar engulfment of peroxisomes during micropexophagy.

BACKGROUND: In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded through direct engulfment by the vacuole in a process known as micropexophagy, but the mechanism of micropexophagy is not known. RESULTS: To gain molecular insights into micropexophagy, we used fluorescence time-lapse microscopy, coupled with gene-tagging mutagenesis to isolate P. pastoris mutants defective in micropexophagy. The relevant genes have been designated PAZ genes. Morphological and genetic analyses enabled us to postulate a schematic model for micropexophagy. This new model invokes the generation of new vacuolar compartments as an intermediate structure during micropexophagy. Different classes of paz mutants arrest micropexophagy at distinct stages of the process. Most of APG-related paz mutants ceased micropexophagy at Stage 1c and that GCN-family paz mutants ceased micropexophagy at Stage 2. The paz2Delta strain shows a unique phenotype. Paz2 is the homologue of Saccharomyces cerevisiae Apg8, which is necessary for macroautophagy in that yeast. Our analysis revealed that in P. pastoris, Paz2 plays a key role in repressing the engulfment of peroxisomes by the vacuole before the onset of micropexophagy. Paz2 is proteolytically processed by another autophagy-related Paz protein Paz8, but this processing is not required for the ability of Paz2 to suppress aberrant micropexophagy. CONCLUSION: Micropexophagy has been dissected into a multistep reaction that involves 14 identified Paz gene products. Our studies indicate that Paz2 controls the engulfment of peroxisomes by the vacuole, pointing to a novel early function of this protein.