Changes to the practice of pediatric otolaryngology as a consequence of the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic has shifted medical practice globally. The objective of this study was to examine the changes to the practice of pediatric otolaryngology internationally due to the COVID-19 pandemic and examine potential contributors. METHOD: An online survey was designed to assess practice demographics, patterns of COVID-19 related restrictions in communities, and changes to practice and referrals. This was disseminated via an international Covid-19 WhatsApp™ group of pediatric otolaryngologists. RESULTS: There were 45 respondents of 177 group members (25.4%) from 15 countries. The mean estimated time spent under strictest lockdown measures was 16.2 (±10.7) weeks (range: 1-45 weeks). Operating room time was reduced for 82.9%, with an average reported reduction of 41.5%. Almost all (>75%) of respondents reported reduced referrals for five common conditions: otitis media with effusion (average reported decrease - 56.1%); acute otitis media (average decrease 62.8%); acute mastoiditis (average decrease 66.6%); recurrent pharyngotonsillitis (average decrease 51.0%); and peritonsillar abscess (average decrease 52.1%). COVID-19 cases per million population significantly influenced the acuity of referrals received (p < .05). No conditions were reported as increased in frequency and the acuity of most conditions was reported as unchanged by the majority of respondents. CONCLUSION: The measures taken to reduce the spread of COVID-19 have resulted in many changes to pediatric otolaryngology practice and the referral patterns of common conditions. Some of these changes may have enduring sequelae.

Infant Midnasal Stenosis: Reliability of Nasal Metrics.

BACKGROUND AND PURPOSE: Midnasal stenosis is a poorly defined entity that may be a component of other conditions of nasal obstruction contributing to respiratory distress in infants. We sought to establish whether midnasal vault narrowing is a component of well-defined syndromes of nasal narrowing, such as bilateral choanal atresia and pyriform aperture stenosis, and to characterize the nasal anatomy of patients with syndromic craniosynostosis. MATERIALS AND METHODS: A convenience sample of patients with pyriform aperture stenosis, bilateral choanal atresia, and Apert and Crouzon syndromes with maxillofacial CT scans was identified. Patients with Pierre Robin Sequence were used as controls. Nasal measurements were performed at the pyriform aperture, choana, and defined midnasal points on axial and coronal CT scans. Intra- and interrater reliability was quantified with the intraclass correlation coefficient. T tests with Bonferroni adjustment were used to assess differences from controls. RESULTS: The study included 50 patients: Eleven had pyriform aperture stenosis, 10 had Apert and Crouzon syndromes, 9 had choanal atresia, and 20 were controls. Measurements in patients with pyriform aperture stenosis and Apert and Crouzon syndromes were narrower than those of controls at all measured points (P < .001). Measurements in patients with choanal atresia were only narrow in the posterior half of the nose (P < .001). The intra- and interrater reliability of midnasal and pyriform measurements was very good to excellent (intraclass correlation coefficient > 0.87). The choanal measurement was good (intraclass correlation coefficient = 0.76-0.77). CONCLUSIONS: Pyriform aperture stenosis, Apert and Crouzon patients were narrower at all measured points compared to controls. Bilateral choanal atresia patients were only narrower in the posterior half of the nose. More research is needed to evaluate the clinical implications of these radiographic findings.

Experimental study and analytical model of deformation of magnetostrictive films as applied to mirrors for x-ray space telescopes.

The desire for continuously gaining new knowledge in astronomy has pushed the frontier of engineering methods to deliver lighter, thinner, higher quality mirrors at an affordable cost for use in an x-ray observatory. To address these needs, we have been investigating the application of magnetic smart materials (MSMs) deposited as a thin film on mirror substrates. MSMs have some interesting properties that make the application of MSMs to mirror substrates a promising solution for making the next generation of x-ray telescopes. Due to the ability to hold a shape with an impressed permanent magnetic field, MSMs have the potential to be the method used to make light weight, affordable x-ray telescope mirrors. This paper presents the experimental setup for measuring the deformation of the magnetostrictive bimorph specimens under an applied magnetic field, and the analytical and numerical analysis of the deformation. As a first step in the development of tools to predict deflections, we deposited Terfenol-D on the glass substrates. We then made measurements that were compared with the results from the analytical and numerical analysis. The surface profiles of thin-film specimens were measured under an external magnetic field with white light interferometry (WLI). The analytical model provides good predictions of film deformation behavior under various magnetic field strengths. This work establishes a solid foundation for further research to analyze the full three-dimensional deformation behavior of magnetostrictive thin films.

Chronic plantar ulcer secondary to congenital indifference to pain.

Congenital indifference to pain (CIP) is a rare condition characterised by painless injuries beginning in early life, with normal sensory exam findings. Young people with inexplicable, painless chronic wounds may present to the plastic surgeon for surgical management. Given the young age of onset and high likelihood of postoperative failure, alternative options for closure of non-healing wounds should be considered. We present the case of a 17-year-old boy with congenital indifference to pain and successful management of his longstanding plantar ulcer.

Regulation of exocytosis by protein kinase C.

PKC (protein kinase C) has been known for many years to modulate regulated exocytosis in a wide variety of cell types. In neurons and neuroendocrine cells, PKC regulates several different stages of the exocytotic process, suggesting that these multiple actions of PKC are mediated by phosphorylation of distinct protein targets. In recent years, a variety of exocytotic proteins have been identified as PKC substrates, the best characterized of which are SNAP-25 (25 kDa synaptosome-associated protein) and Munc18. In the present study, we review recent evidence suggesting that site-specific phosphorylation of SNAP-25 and Munc18 by PKC regulates distinct stages of exocytosis.

Reaching impaired populations with HIV prevention programs: a clinical trial for homeless mentally ill African-American men.

This study tested an intervention to reduce sexual risk behaviors in a high risk impaired population: homeless African-American, Caucasian and Hispanic men with mental illness. In a comparison group clinical trial, men were assigned to an experimental cognitive-behavioral or a control intervention and followed up over 16 months. Men were recruited from a psychiatric program in two shelters for homeless men in Nashville, Tennessee. An ethnically mixed cohort of subjects (54% African-American, 42% Caucasian and 4% Hispanic) were included in the study. Most had a chronic psychiatric disorder and a co-morbid substance abuse disorder. The 257 participants who were sexually active (130 experimental, 127 control) prior to the trial were the main target of the intervention. An experimental intervention (SexG), adapted from Susser and Associates (51), comprised 6 group sessions. The control intervention was a 6-session HIV educational program. Sexual risk behavior was the primary outcome. The experimental and control groups were compared with respect to the mean score on a sexual risk index. Complete follow-up data were obtained on 257 men (100%) for the initial six-month follow-up. These individuals have been followed for the remainder of the 16-month follow-up. This intervention, (SexG), successfully reduced sexual risk behaviors of homeless mentally ill African-American, Caucasian and Hispanic men. Similar approaches may be effective in other impaired high-risk populations.

Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis.

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-alpha (alpha-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular alpha-SNAP failed to detect (32)P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser(10). PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser(10) phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.

Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40).

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

Control of membrane fusion dynamics during regulated exocytosis.

The study of regulated exocytosis uniquely allows the direct measurement of intracellular membrane fusion events in real time. We have exploited this to examine factors that regulate not only the extent but also the dynamics of single fusion/release events. The general strategy used has been to assess exocytosis in transiently transfected PC12 or adrenal chromaffin cells. We aimed to design mutant constructs based on in vitro biochemistry, in some cases informed by knowledge of protein structure. Using this approach we have demonstrated an inhibitory role for the putative Rab3 effector Noc2 that requires interaction with Rab3. Using carbon-fibre amperometry on adrenal chromaffin cells, we have demonstrated regulation of the kinetics of single granule release events consistent with changes in fusion pore dynamics and switches between full fusion and 'kiss-and-run' fusion. These studies have demonstrated a late role for cysteine string protein in exocytosis. In addition, they have focused attention on a key role for Munc18 in the regulation of post-fusion events that affect fusion pore dynamics.

The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4.

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.

SNAP-25 with mutations in the zero layer supports normal membrane fusion kinetics.

Considerable data support the idea that intracellular membrane fusion involves a conserved machinery containing the SNARE proteins. SNAREs assembled in vitro form a stable 4-helix bundle and it has been suggested that formation of this complex provides the driving force for bilayer fusion. We have tested this possibility in assays of exocytosis in cells expressing a botulinum neurotoxin E (BoNT/E)-resistant mutant of SNAP-25 in which additional disruptive mutations have been introduced. Single or double mutations of glutamine to glutamate or to arginine in the central zero layer residues of SNAP-25 did not impair the extent, time course or Ca2+-dependency of exocytosis in PC12 cells. Using adrenal chromaffin cells, we found that exocytosis could be reconstituted in cells transfected to express BoNT/E. A double Q-->E mutation did not prevent reconstitution and the kinetics of single granule release events were indistinguishable from control cells. This shows a high level of tolerance of changes in the zero layer indicating that the conservation of these residues is not due to an essential requirement in vesicle docking or fusion and suggests that formation of a fully stable SNARE complex may not be required to drive membrane fusion.

Determination of phosphorylation levels of tyrosine hydroxylase by electrospray mass spectrometry.

A novel approach has been developed to quantify the extent of phosphorylation of tyrosine hydroxylase (TH). The strategy consists of a chemical cleavage and characterization of the products using electrospray mass spectrometry (ESMS). The chemical cleavage involves selective hydrolysis of the aspartyl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixtures of TH-P/TH. The calibration yields a straight line with an R2 of 0.996, which is valid within the 10-90% range. The ESMS protocol has been used to determine the extent of phosphorylation of TH in the presence of CaM-PKII. The experimental conditions were designed to produce low levels of phosphorylation. Nevertheless, the ESMS analysis yielded single, double, and nonphosphorylation forms of TH. With respect to in vivo measurements, this ESMS protocol may be a generic procedure for determining the extent of phosphorylation of proteins.

Measurement of exocytosis by amperometry in adrenal chromaffin cells: effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics.

We have used carbon-fibre amperometry to examine the kinetics of individual secretory granule fusion/release events in bovine adrenal chromaffin cells. Transfection with plasmids encoding the light chains of botulinum neurotoxins (BoNTs) was used to investigate the effects of cleavage of syntaxin or SNAP-25 on exocytosis. Expression of BoNT/C1 or BoNT/E inhibited the extent of exocytosis that was evoked by application of digitonin/Ca(2+) to permeabilise and stimulate single chromaffin cells. Following neurotoxin expression, the residual release events were no different from those of control cells in their magnitude and kinetics from analysis of the amperometric spikes. In contrast, activation of protein kinase C (PKC) resulted in a modification of the kinetics of single granule release events. Following phorbol ester treatment, the amperometric spikes showed a significant decrease in their total charge due to a decrease in their mean half-width with increases in the rate of the initial rise and also the fall to baseline of the spikes. These changes were prevented by pre-treatment with the PKC inhibitor bisindolylmaleimide. These results suggest that PKC regulates the rate of fusion pore expansion and also subsequent pore closure or granule retrieval. A PKC-mediated regulation of kiss-and-run fusion may, therefore, control the extent of catecholamine release from single secretory granules. The experimental approach used here may provide further information on the protein constituents and regulation of the fusion pore machinery.

Comparison of cysteine string protein (Csp) and mutant alpha-SNAP overexpression reveals a role for csp in late steps of membrane fusion in dense-core granule exocytosis in adrenal chromaffin cells.

Assembly of the SNARE complex and its disassembly caused by the action of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) and NSF is crucial for the maintenance of vesicular traffic, including fusion of regulated exocytotic vesicles. Various other proteins may also have important roles in the processes leading to membrane fusion via interaction with the SNARE proteins, including the secretory vesicle cysteine string protein (Csp). Here we have examined the effect of overexpression of a dominant negative alpha-SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to detect released catecholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granules was substantially inhibited by expression of alpha-SNAP(L294A). The amplitude and characteristics of the individual release events were unaffected by expression of alpha-SNAP(L294A), consistent with an essential role for alpha-SNAP in early steps of priming but not in the fusion process. In contrast, Csp overexpression, which also inhibited the extent of exocytosis, also modified the kinetics of the individual release events seen as an increase in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike alpha-SNAP, Csp plays a key role in the protein interactions close to the fusion process or fusion pore opening during Ca(2+)-regulated exocytosis.

Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis.

Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.

Immune-mediated haemolytic anaemia in two sibling cats associated with multicentric lymphoblastic infiltration.

Immune-mediated haemolytic anaemia associated with multicentric lymphoblastic infiltration is reported in two sibling cats. Both cats presented at under 16 months of age with clinical signs of acute anaemia. In each case there was autoagglutination, a positive Coombs' test and the anaemia was regenerative. At presentation, both cats were negative for FeLV antigen. In each case, the disease proved fatal within 2 months of the initial diagnosis. In both cases, T-lymphoblastic infiltration of bone marrow, liver and spleen was found at post-mortem examination.

Neuronal Ca2+ sensor 1, the mammalian homologue of frequenin, is expressed in chromaffin and PC12 cells and regulates neurosecretion from dense-core granules.

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.

Evidence against an acute inhibitory role of nSec-1 (munc-18) in late steps of regulated exocytosis in chromaffin and PC12 cells.

nSec-1 (munc-18) is a mammalian homologue of proteins implicated in constitutive exocytosis in yeast and neurotransmission in Caenorhabditis elegans and Drosophila. Mutant phenotypes in these species suggest that nSec-1 is likely to be required for neurotransmission. Various other data have been interpreted as suggesting that nSec-1 could also be a negative regulator of Ca2+-dependent exocytosis. We have tested this possibility by introducing exogenous nSec-1 into permeabilised chromaffin or PC12 cells and examining its effects on Ca2+-induced and alpha-soluble N-ethylmaleimide-sensitive fusion protein attachment protein-stimulated exocytosis. No effects of exogenous nSec-1 were observed in these assays. In addition, the effect of nSec-1 overexpression in transiently transfected PC12 cells on reporter growth hormone (GH) secretion was examined. Overexpression of nSec-1 resulted in a marked increase in GH production, reflected in an increase in both cell-associated and medium GH levels. The relative amounts retained in the cells were unaffected by nSec-1 overexpression, indicating that GH storage was unaffected and that the major effect was on its synthesis. In contrast, nSec-1 overexpression did not affect the proportion of GH that was released following stimulation in intact or permeabilised cells. These results suggest either that nSec-1 is already expressed at sufficient levels and remains so following permeabilisation or that nSec-1 may not be an acute inhibitory regulator of Ca2+-dependent exocytosis in chromaffin or PC12 cells.