Central and mixed apneas in children with obstructive sleep apnea: effect of adenotonsillectomy.

PURPOSE: Investigate the effect of adenotonsillectomy on mixed apnea index (MAI) and central apnea index (CAI) in children with moderate-to-severe obstructive sleep apnea syndrome (OSAS). METHODS: Observational retrospective analysis of polysomnographic data in children diagnosed with moderate-to-severe OSAS and without comorbidity, submitted to adenotonsillectomy. RESULTS: Data were available for 80 children, 55 boys and 25 girls, with a median age of 3.6 years (2.1-5.9). Before surgery AHI was 14.1 (11.0-18.4) per hour, with a median preoperative OAI of 7.1 (4.1-10.6), MAI of 1.2 (0.6-1.6) and CAI of 1.0 (0.4-2.0). Adenotonsillectomy caused significant improvements in MAI, from 1.2 (0.6-1.6) to 0.5 (0.1-0.8) (p < 0.001) and CAI from 1.0 (0.4-2.0) to 0.5 (0.1-0.9) (p < 0.001). This represents a normalization of MAI in 91.7% and CAI in 75.6% of children that had an abnormal value prior surgery. CONCLUSION: Non obstructive apneas are common in children with OSAS. Adenotonsillectomy caused significant decrease not only in OAI, but also in MAI and CAI in children with moderate-to-severe OSAS.

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

Surgical treatment for obstructive sleep apnea: effect on sleep architecture.

PURPOSE: Investigate the effect of surgical treatment of obstructive sleep apnea syndrome (OSA) on sleep architecture. METHODS: Observational retrospective analysis of polysomnographic data of adults diagnosed with OSA, submitted to surgical treatment. Median (25-75th percentile) was used to present the data. RESULTS: Data were available for 76 adults, 55 men and 21 women, with median age of 49.0 years (41.0-62.0), body mass index of 27.3 kg/m2 (25.3-29.3) and AHI of 17.4 per hour (11.3-22.9) before surgeries. Preoperatively, 93.4% of patients had an abnormal distribution of at least one of the sleep phases. After surgical treatment, we found a significant increase in median N3 sleep percent from 16.9% (8.3-22-7) to 18.9% (15.5-25.4) (p = 0.003). Postoperatively, 18.6% patients that had an abnormal preoperative N1 sleep phase distribution had a normalization of this sleep phase, as also occurred to N2, N3 and REM sleep phases in 44.0%, 23.3% and 63.6% of patients, respectively. CONCLUSION: This study aims to show the impact of OSA treatment, not only on respiratory events but also on other polysomnographic data often underestimated. Upper airway surgeries have shown to be effective in sleep architecture improvements. There is a trend for sleep distribution normalization, with increase of time spend in profound sleep.

Severe Pediatric Sleep Apnea: Drug-Induced Sleep Endoscopy Based Surgery.

Although adenotonsillectomy is the recommended treatment of obstructive sleep apnea (OSA) in children, some patients with preoperative severe OSA (Apnea-hypopnea index/AHI > 10) remain symptomatic after surgery and may need further workup. This study aims to: (1) analyse preoperative factors and its relation with surgical failure/persistent OSA (AHI > 5 after adenotonsillectomy) in severe pediatric OSA; (2) determine the levels of airway collapse during DISE (drug induced sleep endoscopy) in cases of surgical failure; (3) evaluate the efficacy of targeted surgery based on DISE findings. This retrospective study was conducted between August and September 2020. Across 9 years (from 2011 to 2020), all children diagnosed with severe OSA in our Hospital underwent adenotonsillectomy and repeated type 1 polysomnography (PSG) 3 months after surgery. Cases of surgical failure underwent DISE for planning eventual directed surgery. Chi-square test was used to assess the relationship between persistent OSA and preoperative patients' characteristics. 80 cases of severe pediatric OSA were diagnosed (68.8% males; mean age: 4.3 years-standard deviation: 2.49; mean AHI: 16.3-standard deviation 7.14) in the aforementioned period. We found a significant association between surgical failure (11.3% of cases; mean AHI: 6.9-SD 0.91) and obesity (p = 0.002; confidence level of 95%). Neither preoperative AHI nor other PSG parameters were associated with surgical failure. In cases of surgical failure, epiglottis collapse was present in every DISEs and adenoid tissue was present in 66% of children. All cases of surgical failure had directed surgery and surgical cure (AHI ≤ 5) was obtained in 100% of cases. This study suggests that obesity is the strongest predictor of surgical failure in children with severe OSA who undergo adenotonsillectomy. Epiglottis collapse and presence of adenoid tissue are the most common findings in postoperative DISEs of children with persistent OSA after primary surgery. DISE based surgery seems a safe and effective tool to manage persistent OSA after adenotonsillectomy.

Swallowing evaluation in patients who underwent Partial CO2 LASER Epiglottectomy for Sleep Apnea treatment.

OBJECTIVE: To evaluate swallowing in patients who underwent CO2 LASER Partial Epiglottectomy (CO2-LPE) for Obstructive Sleep Apnea Syndrome and to assess the risk of aspiration with this technique. MATERIAL & METHODS: Chart review of adult patients who underwent CO2-LPE between 2016 and 2020, in a secondary care hospital. Patients underwent surgery for OSAS, in accordance to Drug Induced Sleep Endoscopy findings and an objective swallowing evaluation was done at least 6 months after surgery. Eating Assessment Tool (EAT-10) questionnaire was applied and Volume-Viscosity Swallow Test (V-VST) and Fiberoptic Endoscopic Evaluation of Swallowing (FEES) were performed. Dysphagia was classified according to Dysphagia Outcome Severity Scale (DOSS). RESULTS: Eight patients were included in the study. The mean time between surgery and the swallowing evaluation was 50 (±13,2) months. Only 3 patients presented ≥ 3 points on EAT-10 questionnaire. Two patients presented signs of decreased efficacy of swallowing (piecemeal deglutition) but none had a decrease in safety, according to V-VST. Although 50% of the patients presented some pharyngeal residue on FEES, it was classified as trace to mild in most of the cases. No evidence of penetration or aspiration was identified (DOSS ≥ 6 in all patients). CONCLUSION: The CO2-LPE is a potential treatment for OSAS patients with epiglottic collapse and no evidence of swallowing safety compromise was found.

Treatment of otomycosis with clotrimazole: results accordingly with the fungus isolated.

BACKGROUND: Otomycosis is usually caused by Candida spp or Aspergillus spp. While Candida is usually multissensitive to available antifungals, Aspergillus is not. Topical antifungals for otomycosis that are available in Portugal are scarce, and systemic treatments have too many interactions and contraindications. OBJECTIVES: Determine otomycosis epidemiology, microbiology and treatment results. METHODS: Observational study that included patients followed in Professor Doutor Fernando Fonseca Hospital, between 2011 and 2020. Otomycosis diagnosis was obtained through ear drainage culture, and every case was treated with 1% clotrimazole ear drops plus ear cleaning once per week. RESULTS: Aspergillus was found in ear drainage culture in 43.9% of patients and Candida in the remaining. There was a significant statistical difference between patients with otomycosis caused by Aspergillus versus Candida in treatment duration from 25.0 days (16.5-43.0) versus 14.0 days (7.0-18.5) (p < .001), respectively. CONCLUSIONS: Otomycosis was more frequently caused by Candida, and this type of otomycosis is treated faster with clotrimazole 10 mg/dL plus ear cleaning, when compared with otomycosis by Aspergillus. SIGNIFICANCE: If otomycosis causative agent is identified or suspected, a prediction of the time needed till the resolution of otomycosis can be made, when clotrimazole ear drops are used.

Unilateral vocal fold paralysis as a port complication.

Unilateral vocal fold paralysis (UVFP) is a frequent finding in otorhinolaryngology practice, but its occurrence as a port complication was very rarely described in English Literature. The authors report a 55-year-old woman with a pancreatic adenocarcinoma who presented a left vocal fold paralysis that occurred concurrently with a venous thrombosis of the left subclavian vein, where a totally implantable venous-access had been previously placed. Although the patient's oncologic disease, that could mislead to a neoplastic cause of the UVFP, the authors came across with an unusual etiology and to their best knowledge, it is the first case of irreversible UVFP associated with onsite thrombosis of the vessel where a port was implanted. The objective of this article is to present and discuss this rare case of UVFP secondary to a port complication and to review the main mechanisms of iatrogenic vocal fold paralysis related to these devices.

Optimization of TEAD P-Site Binding Fragment Hit into In Vivo Active Lead MSC-4106.

The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable. The discovery and optimization of a P-site binding fragment (1) are described. Utilizing structure-based design, enhancement in target potency was engineered into the hit, capitalizing on the established X-ray structure of TEAD1. The efforts culminated in the optimized in vivo tool MSC-4106, which exhibited desirable potency, mouse pharmacokinetic properties, and in vivo efficacy. In close correlation to compound exposure, the time- and dose-dependent downregulation of a proximal biomarker could be shown.

Predictors of OSA following adenotonsillectomy in children with trisomy 21.

OBJECTIVES: Given that 30%-50% of children with trisomy 21 have persistent obstructive sleep apnoea (OSA) after adenotonsillectomy, we evaluated whether demographic, clinical and polysomnographic factors predicted persistent OSA and OSA severity after adenotonsillectomy. DESIGN: Retrospective study. SETTING: Secondary care hospital. PARTICIPANTS: Retrospective review of 32 children with the diagnosis of trisomy 21 and OSA by polysomnography who underwent adenotonsillectomy, from January 2010 to December 2018. MAIN OUTCOME AND MEASURE: Non-parametric analysis was used to compare pre- and postoperative factors, and regression was used to model persistent OSA and OSA severity. RESULTS: Thirty-two children were included (17 males, median age 10.00 ± 8.00 years, median body mass index z-score 0.89 ± 1.25). Overall, adenotonsillectomy resulted in a significant improvement in median obstructive apnoea-hypopnoea index (oAHI) from 7.5 ± 8.95 to 4.40 ± 4.38 events per hour (P < .001) and in median OSA-18 score from 85.00 ± 12.00 to 61.00 ± 37.75 (P < .001). Persistent OSA was found in 56.25% of the children. Univariate regression suggests that postoperative OSA-18 score was associated with persistent OSA after adenotonsillectomy. Preoperative oAHI, preoperative oxygen desaturation index, pre- and postoperative OSA-18 scores correlated with OSA severity after adenotonsillectomy. However, in a multivariate model only the postoperative OSA-18 score correlated with OSA severity after adenotonsillectomy. CONCLUSIONS: Although adenotonsillectomy results in a significant improvement of OSA in children with trisomy 21, more than half of the children had persistent OSA. The postoperative OSA-18 score was associated both with persistent OSA and OSA severity after adenotonsillectomy.

Auditory Brainstem Response in Preterm Infants in the Neonatal Intensive Care Unit.

OBJECTIVE: To analyze auditory brainstem response (ABR) findings of preterm and term infants in the neonatal intensive care unit (NICU) with perinatal problems. STUDY DESIGN: Case series with chart review. SETTING: Secondary care hospital. METHODS: Analysis consisted of a consecutive series of 101 infants (69 preterm and 32 term) admitted in the NICU of Hospital Fernando Fonseca between 2016 and 2018 with perinatal problems who underwent an ABR evaluation. RESULTS: The major perinatal problems identified were hyperbilirubinemia, intravenous gentamicin >5 days, mechanical ventilation >5 days, congenital cytomegalovirus infection, meningitis, and periventricular hemorrhage. Gentamicin use significantly increased the absolute latency of wave I in preterm infants (95% CI, 0.01-0.37; P = .037). Mechanical ventilation significantly decreased the latency of wave V and intervals I-V and III-V in preterm infants (95% CI, -0.35 to -0.22; P = .026; 95% CI, -0.33 to -0.00; P = .001; 95% CI, -0.46 to 0.12; P = .049). Congenital cytomegalovirus significantly decreased interval III-V in preterm infants (95% CI, -0.36 to -0.01; P = .042).Multivariate analysis revealed that gentamicin use, lower gestational age, and lower birth weight predicted an increased ABR threshold in preterm infants (95% CI, 1.64-15.31; P = .016; 95% CI -1.72 to -0.09; P = .030; 95% CI, -14.55 to -0.63; P = .033). ABR measurements in term infants were not significantly altered, with the exception of an increased latency of wave III with a lower gestational age (95% CI, -0.49 to -0.01; P = .038). CONCLUSIONS: These findings suggest that perinatal problems in the NICU significantly impair the ABR threshold and the auditory pathway maturational process in preterm but not term infants.

Drug-Induced Sleep Endoscopy in Pediatric Obstructive Sleep Apnea.

OBJECTIVE: To describe drug-induced sleep endoscopy (DISE) findings in children with obstructive sleep apnea and to differentiate them between surgically naïve children and children who had adenotonsillectomy performed. STUDY DESIGN: Retrospective case series with chart review. SETTING: Secondary care hospital. SUBJECTS AND METHODS: A cohort of 56 children with the diagnosis of obstructive sleep apnea was submitted to DISE and subsequent upper airway surgery: 23 were surgically naïve, and 33 had persistent obstructive sleep apnea after adenotonsillectomy. Comparisons between groups were calculated with chi-square test and Student's t test. Simple linear regression was used to model polysomnographic indices. RESULTS: In surgically naïve children, the most common sites of obstruction were the adenoids (78.2%) and the lateral pharyngeal walls/tonsils (82.6%). In children with persistent obstructive sleep apnea after adenotonsillectomy, the most common sites of obstruction were the adenoids (54.5%), followed by the supraglottis (48.5%) and the tongue base (45.5%). No correlation was found between obstructive apnea-hypopnea index and DISE findings. Simple linear regression revealed that the degree of obstruction at the tongue base (ß = -0.73; 95% CI, -1.22 to -0.25; P = .004) and the presence of multilevel obstruction (ß = -1.75; 95% CI, -3.20 to -0.30; P = .02) predicted saturation nadir in children with persistent obstructive sleep apnea after adenotonsillectomy. CONCLUSION: DISE findings differed between surgically naïve children and children with persistent obstructive sleep apnea after adenotonsillectomy. Increased obstruction at the level of the tongue base and the presence of multilevel obstruction predicted a lower saturation nadir in children with persistent obstructive sleep apnea after adenotonsillectomy.

Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies.

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

Conservation in the mechanism of glucuronoxylan hydrolysis revealed by the structure of glucuronoxylan xylanohydrolase (CtXyn30A) from Clostridium thermocellum.

Glucuronoxylan endo-ß-1,4-xylanases cleave the xylan chain specifically at sites containing 4-O-methylglucuronic acid substitutions. These enzymes have recently received considerable attention owing to their importance in the cooperative hydrolysis of heteropolysaccharides. However, little is known about the hydrolysis of glucuronoxylans in extreme environments. Here, the structure of a thermostable family 30 glucuronoxylan endo-ß-1,4-xylanase (CtXyn30A) from Clostridium thermocellum is reported. CtXyn30A is part of the cellulosome, a highly elaborate multi-enzyme complex secreted by the bacterium to efficiently deconstruct plant cell-wall carbohydrates. CtXyn30A preferably hydrolyses glucuronoxylans and displays maximum activity at pH 6.0 and 70°C. The structure of CtXyn30A displays a (ß/α)8 TIM-barrel core with a side-associated ß-sheet domain. Structural analysis of the CtXyn30A mutant E225A, solved in the presence of xylotetraose, revealed xylotetraose-cleavage oligosaccharides partially occupying subsites -3 to +2. The sugar ring at the +1 subsite is held in place by hydrophobic stacking interactions between Tyr139 and Tyr200 and hydrogen bonds to the OH group of Tyr227. Although family 30 glycoside hydrolases are retaining enzymes, the xylopyranosyl ring at the -1 subsite of CtXyn30A-E225A appears in the α-anomeric configuration. A set of residues were found to be strictly conserved in glucuronoxylan endo-ß-1,4-xylanases and constitute the molecular determinants of the restricted specificity displayed by these enzymes. CtXyn30A is the first thermostable glucuronoxylan endo-ß-1,4-xylanase described to date. This work reveals that substrate recognition by both thermophilic and mesophilic glucuronoxylan endo-ß-1,4-xylanases is modulated by a conserved set of residues.

Extensive upper respiratory tract sarcoidosis.

Sarcoidosis is a chronic granulomatous disease of unknown aetiology. It can affect any part of the organism, although the lung is the most frequently affected organ. Upper airway involvement is rare, particularly if isolated. Sarcoidosis is a diagnosis of exclusion, established by histological evidence of non-caseating granulomas and the absence of other granulomatous diseases. The authors report a case of a man with sarcoidosis manifesting as a chronic inflammatory stenotic condition of the upper respiratory tract and trachea.

Phragmites sp. physiological changes in a constructed wetland treating an effluent contaminated with a diazo dye (DR81).

The role of Phragmites sp. in phytoremediation of wastewaters containing azo dyes is still, in many ways, at its initial stage of investigation. This plant response to the long-term exposure to a highly conjugated di-azo dye (Direct Red 81, DR81) was assessed using a vertical flow constructed wetland, at pilot scale. A reed bed fed with water was used as control. Changes in photosynthetic pigment content in response to the plant contact with synthetic DR81 effluent highlight Phragmites plasticity. Phragmites leaf enzymatic system responded rapidly to the stress imposed; in general, within 1 day, the up-regulation of foliar reactive oxygen species-scavenging enzymes (especially superoxide dismutase, ascorbate peroxidase (APX), glutathione peroxidase (GPX) and peroxidase) was noticed as plants entered in contact with synthetic DR81 effluent. This prompt activation decreased the endogenous levels of H2O2 and the malonyldialdehyde content beyond reference values. Glutathione S-transferase (GST) activity intensification was not enough to cope with stress imposed by DR81. GPX activity was pivotal for the detoxification pathways after a 24-h exposure. Carotenoid pool was depleted during this shock. After the imposed DR81 stress, plants were harvested. In the next vegetative cycle, Phragmites had already recovered from the chemical stress. Principal component analysis (PCA) highlights the role of GPX, GST, APX, and carotenoids along catalase (CAT) in the detoxification process.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum.

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.

Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11).

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11-cellohexaose and CtCBM11-cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand-protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

Preliminary structural characterization of human SOUL, a haem-binding protein.

Human SOUL (hSOUL) is a 23 kDa haem-binding protein that was first identified as the PP(23) protein isolated from human full-term placentas. Here, the overexpression, purification and crystallization of hSOUL are reported. The crystals belonged to space group P6(4)22, with unit-cell parameters a = b = 145, c = 60 A and one protein molecule in the asymmetric unit. X-ray diffraction data were collected to 3.5 A resolution at the ESRF. A preliminary model of the three-dimensional structure of hSOUL was obtained by molecular replacement using the structures of murine p22HBP (PDB codes 2gov and 2hva), obtained by solution NMR, as search models.