Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s -1 ) was increased approximately 10-fold compared to that in the absence of CaM (22 s -1 ). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.

Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulincontrolled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH ⅐ radical signals in inducible nitricoxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/ semiquione couple ‫؍‬ ؉70 mV, FMN semiquinone/hydroquinone couple ‫؍‬ ؊180 mV, and heme ‫؍‬ ؊180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents. Nitric-oxide synthases (NOSs)3 are a family of enzymes that generate nitric oxide (NO) from arginine, requiring 2 mol of O 2 and 1.5 mol of NADPH/mol of NO produced (1-3). The constitutive isoforms, eNOS and nNOS, are regulated by calcium/calmodulin (Ca ϩ2 /CaM) (4, 5) and additional inputs including phosphorylation of specific residues (6); the NO produced by constitutive isoforms functions as a molecular signal. A cytokine inducible isoform (iNOS) is calcium insensitive, and produces much larger fluxes of NO as a cytotoxin in immune response (4). Eukaryotic NOS isoforms are large modular enzymes. The monomer molecular mass is 120 -161 kDa; the dimer is the active form (7), and the dimerization interface includes the tetrahydrobiopterin (H 4 B) binding site in the oxygenase domain (8). The common elements are the heme and H 4 B containing the oxygenase domain and a complex reductase unit homologous to NADPH P450 reductase that consists of a NADPH binding domain, a FAD binding domain, and an FMN binding domain (9 -11). The reductase and oxygenase regions are linked by a polypeptide segment containing a CaM binding site (12). Evidence suggests that the oxygenase domain of one monomer is reduced by the reductase unit of the other (13) through an oxygenase domain surface that exposes the corner of the hem...

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In our cohort, ABR, caloric response, cVEMP, and elevated intralabyrinthine protein correlated with tumor volume, but 4f-PTA did not. Abnormal ABR and 4f-PTA correlated with elevated intralabyrinthine protein. These findings may provide insight on the effect of small CVS on the inner ear and cochleovestibular nerves, which may aid in their optimal management.

ith each passing day, the novel coronavirus disease 2019 (COVID-19) pandemic escalates around the world. First identified in Wuhan, China, the disease is caused by a novel coronavirus: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1 Globally, health care professionals have been infected at high rates (63% of all cases in Wuhan as of February 11, 2020, were health care professionals; in Italy, 20% of health care professionals responding to the pandemic were reported to be infected) and are also dying of COVID-19. [1][2][3][4][5] These high rates are likely due to transmission of the virus through droplet, contact, and even airborne modalities, similar to the 2002-2003 SARS pandemic, 6 as well as by asymptomatic individuals. A study by Zou et al 7 has shown similar viral loads present in both asymptomatic and symptomatic patients, with the highest viral load being identified in the nasal cavity. Subsequently, it is not surprising that otolaryngologists have been identified as a high-risk group among health care professionals, given their level of exposure to the upper aerodigestive tract. [8][9][10] The risk of infection for health care professionals is compounded by shortages of personal protective equipment (PPE), including filtering facepiece respirators (FFRs), in particular, N95 respirators. 2,11 Early studies report that adequate PPE and hand hygiene provide good protection from infection, especially in cases of high-risk procedures in which SARS-CoV-2 can be aerosolized. 12,13 The N95 respirators were widely used in previous SARS and influenza outbreaks to prevent both droplet and airborne transmission. 6,14 At UCLA Health, the use of N95 respirators along with a gown and eye protection or face shield is required for any invasive otolaryngologic procedure involving mucosal surfaces, in line with recent guidelines based on international experience. 8 In the US, the supply of necessary PPE has been unable to keep up with the rapid increase in the number of patients with COVID-19 owing to the disruption of the global supply chain. 2,15 The Centers for Disease Control and Prevention (CDC) has estimated that more than 90 million respirators would be required for health care professionals alone in a pandemic lasting 42 days. 16 As of March 2020, the US Department of Health and Human Services estimated that 3.5 billion N95 respirators would be needed in a severe event, but only 35 million were available. 17 Previously, the CDC set guidance for extending the use of single-use PPE in times of high demand and more recently released new guidelines on decontamination methods for N95 IMPORTANCE The novel coronavirus disease 2019 (COVID-19) has proven to be highly infectious, putting health care professionals around the world at increased risk. Furthermore, there are widespread shortages of necessary personal protective equipment (PPE) for these individuals. Filtering facepiece respirators, such as the N95 respirator, intended for single use, can be reused in times of need. We explore the evidence for dec...

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Importance: This is the first description of 3-dimensional (3D) pediatric airway endoscopy in the otolaryngology literature detailing the superior visualization with this technology. Ultimately, enhanced optics may further improve the treatment of airway pathology.Objective: To report the first case series examining the use of 3D direct laryngoscopy and bronchoscopy (DLB) in the diagnosis and management of laryngotracheal pathology.Design: Case series.Setting: Tertiary care pediatric hospital.Participants: Three patients underwent both telescopic 2-dimensional (2D) and 3D DLB for comparison purposes: a 12-year-old boy for visualization of complete tracheal rings, a 23-year-old man for dilation of tracheal stenosis, and a 4-month-old boy for resection of subglottic cysts.Main Outcome Measures: Enhanced visualization of laryngotracheal pathology and facilitated endoscopic surgery.Results: To our knowledge, this is the first case series in the otolaryngology literature examining the use of 3D DLB for the resection of subglottic cysts, dilation of tracheal stenosis, and visualization of complete tracheal rings. We believe that the 3D view offers qualitatively improved depth perception, accuracy of balloon placement, and appraisal of subglottic cyst resection margins. Conclusions and Relevance:This emerging technology has vast potential for improving endoscopy, surgical precision in airway interventions, tissue preservation, and methods of teaching. More research is needed in this area regarding the benefits and advantages of 3D compared with 2D endoscopy.