Differential regulation of viral entry-associated genes modulated by inflammatory cytokines in the nasal epithelium.

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections.

Interpretation of the nine-step test for Eustachian tube function should consider mastoid cavity volume.

INTRODUCTION: The modified nine-step test is a classical method for evaluating Eustachian tube function. However, clinical interpretation of the increased maximal difference in middle ear pressure (mdMEP) in the modified nine-step test is unknown. We hypothesised that the different reservoir effects of the mastoid cavity can bias the results of the modified nine-step test. METHODS: A total of 108 consecutive participants (216 ears) were retrospectively screened. Of these, 55 participants (82 ears) who met the inclusion/exclusion criteria were enrolled. The volumetric results of the mastoid cavity, parameters of the modified nine-step test (mdMEP, middle ear pressure, tympanic membrane compliance), and demographic data were analysed. RESULTS: A significant negative correlation was found between mdMEP and mastoid cavity volume (R = .467, p < .001). Ears with mdMEP >70 daPa showed poor pneumatization in the mastoid cavity, with volumes less than 3000 mm3 (10th percentile of all ears analysed). Ears with mastoid cavity volumes lower than the 25th percentile showed a significantly higher mdMEP (p < .001). Patients with mastoid cavity volumes higher than the 75th percentile were significantly younger (p < .001). Multivariate regression analysis for mdMEP showed a good fit (R = .854) using factors including middle ear pressure, admittance and, most importantly, the reciprocal of mastoid volume (Beta = 0.752, p < .001). CONCLUSIONS: The mdMEP, the main parameter of the modified nine-step test, was negatively correlated with the mastoid cavity volume. Therefore, the results of the modified nine-step test should be interpreted with consideration of mastoid cavity volume.

Diagnostic Value of the Air-Bone Threshold Gap in Stapes Fixation.

INTRODUCTION: The Carhart notch is a well-known sign of stapes fixation. However, previous studies have reported that the Carhart notch is not specific to stapes fixation and is also present in other middle ear diseases. Therefore, this study investigated the diagnostic value of threshold gap between air conduction and bone conduction (ABG) for stapes fixation, instead of the bone conduction dip representing the Carhart notch. METHODS: A total of 199 ears that underwent exploratory tympanotomy were enrolled in this retrospective study. They were categorized into three groups according to surgical findings: stapes fixation (SF), other ossicle fixation (OF), and chain disconnection (CD). Preoperative pure-tone audiograms and impedance audiograms were compared between the groups. RESULTS: The incidence of the Carhart notch did not differ between the groups. The ABG at 2,000 Hz showed a good diagnostic performance for distinguishing between the SF and CD groups (area under the curve, AUC = 0.816, p < 0.001), but poor performance for distinguishing between the SF and OF groups (AUC = 0.662, p = 0.003). Bone conduction at 2,000 Hz showed a moderate performance for distinguishing between the SF and CD groups (AUC = 0.707, p < 0.001) and did not show statistically significant results for distinguishing between the SF and OF groups (AUC = 0.594, p = 0.080). The tympanic membrane compliance was significantly higher in the CD group than in the SF group (p = 0.001). CONCLUSIONS: The Carhart notch was not a specific finding of SF. The sensitivity and specificity of ABG ≤15 dB at 2,000 Hz for distinguishing between SF and CD were 60.4% and 89.2%, respectively. To prepare for surgical interventions in patients with conductive hearing loss but a normal tympanic membrane, clinicians should comprehensively consider these results.

Genomic profiling of the residual disease of advanced high-grade serous ovarian cancer after neoadjuvant chemotherapy.

The goal of our study was to demonstrate the spectrum of genomic alterations present in the residual disease of patients with advanced high-grade serous ovarian cancer (HGSOC) after neoadjuvant chemotherapy (NAC), including matched pretreatment biopsies. During the study period between 2006 and 2017, we collected pre-NAC and post-NAC tumor tissue samples from patients with advanced HGSOC. We performed combined next-generation sequencing and immunohistochemistry to identify actionable targets and pathway activation in post-NAC residual tumors. We also examined whether post-NAC profiling of residual HGSOC identified targetable molecular lesions in the chemotherapy-resistant component of tumors. Among 102 post-NAC samples, 41 (40%) of patients had mutations in homologous recombination repair (HRR) genes (HRR deficiency). Patients with HRR mutations had higher tumor mutation burdens (p < 0.001) and higher alterations in the PI3K-AKT-mTOR pathway (p = 0.004) than patients without these HRR mutations. Nevertheless, we found no significant differences in progression-free survival (p = 0.662) and overall survival (OS; p = 0.828) between the two groups. Most patients (91%) had alterations in at least one of the targetable pathways, and those patients with cell cycle (p = 0.004) and PI3K-AKT-mTOR signaling (p = 0.005) pathway alterations had poorer OS (Bonferroni-corrected threshold = 0.0083, 0.05/6). We showed the genomic landscape of tumor cells remaining in advanced HGSOC after NAC. Once validated, these data can help inform biomarker-driven adjuvant studies in targeting residual tumors to improve the outcomes of patients with advanced HGSOC after NAC.

BLH1 and KNAT3 modulate ABA responses during germination and early seedling development in Arabidopsis.

The signal transduction pathway governed by the phytohormone abscisic acid (ABA) regulates not only abiotic stress responses but also early developmental programs such as seed dormancy, germination and seedling growth in response to environmental signals. Optimal plant growth and development depend on the integration of environmental stimuli and intrinsic developmental programs. Here, we show that the homeodomain transcription factors BLH1 and KNAT3, previously implicated in embryo sac development, have additional functions in ABA-mediated seed dormancy and early seedling development. The ABA-dependent induction of BLH1 and KNAT3 expression required the presence of functional PYR/PYL/RCAR receptors. The blh1 and knat3 mutants were less sensitive than the wild-type to ABA or salinity exposure during seed germination and early seedling development. In contrast, BLH1 over-expressing lines were hypersensitive to ABA and salinity, and exhibited increased expression of ABA-responsive genes, such as ABI3 and ABI5. BLH1 interacted with KNAT3 and enhanced the retention of KNAT3 in the nucleus. BLH1 and KNAT3 synergistically increased the ABA responses by binding to and subsequently activating the ABI3 promoter. Taken together, we propose that BLH1 and KNAT3 together modulate seed germination and early seedling development by directly regulating ABI3 expression.

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-ß and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher (~two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

Clinical Factors Associated With Prognosis of Tinnitus and Aural Fullness After Sudden Sensorineural Hearing Loss.

OBJECTIVES: We evaluated the long-term prognosis and risk factors associated with tinnitus and aural fullness, which occurred with sudden sensorineural hearing loss. STUDY DESIGN: Retrospective cross-sectional review. SETTING: Tertiary referral center. PATIENTS: Those who visited our clinic for sudden hearing loss from January 2016 to May 2020, diagnosed with sudden sensorineural hearing loss based on pure-tone audiometry, and underwent magnetic resonance imaging to rule out other cause of hearing loss. In total, 106 patients were enrolled in this study. INTERVENTION: All patients were treated with oral glucocorticoids. Salvage intratympanic dexamethasone injection therapy was performed for the patients whose hearing was not fully recovered. MAIN OUTCOME AND MEASURES: We scored the loudness of tinnitus and the intensity of aural fullness using the numerical rating scale. We used a mixed-effects model for repeatedly measured tinnitus and aural fullness scores. RESULTS: The time after the onset of sudden sensorineural hearing loss (SSNHL; ß = -0.07; 95% confidence interval, -0.09 to -0.05; p < 0.001) and hearing outcome after treatment (overall p = 0.003) were significant factors associated with the prognosis of tinnitus. Concerning aural fullness, the time after the onset of SSNHL was a significant prognosis factor ( ß = -0.08; 95% confidence interval, -0.09 to -0.06; p < 0.001), unlike hearing outcome (overall p = 0.261). Pretreatment pure-tone audiometry average threshold and mainly affected frequencies were not significant factors for tinnitus and aural fullness recovery, respectively. CONCLUSION: The persistence of tinnitus with SSNHL was significantly affected by hearing recovery after treatment, whereas aural fullness was not associated with hearing recovery. However, both symptoms were improved over time after SSNHL.

Diagnosis of Eosinophilic Otitis Media Using Blood Eosinophil Levels.

Eosinophilic otitis media (EOM) is a rare middle ear disease with unfavorable outcomes. Under the current diagnostic criteria of EOM, it is challenging to suspect EOM before tympanostomy. Therefore, this study attempted to use blood eosinophil levels for the differential diagnosis of EOM from other conditions. Three disease groups with features of recurrent otorrhea were categorized, which included the following: EOM (n = 9), granulomatosis with polyangiitis (GPA, n = 12), and primary ciliary dyskinesia (PCD, n = 6). Clinical and radiological characteristics were analyzed in the three groups. Patients who underwent ventilation tube insertion due to serous otitis media were enrolled as the control group (n = 225) to evaluate the diagnostic validity of blood eosinophilia. The EOM group showed a significantly higher blood eosinophil concentration (p < 0.001) and blood eosinophil count (p < 0.001) compared to the GPA and PCD groups. The estimated sensitivity and specificity for diagnosing EOM from OME patients who underwent ventilation tube insertion were 100% and 95.6%, respectively. In addition, EOM tended to have protympanic space soft tissue density and a relatively clear retrotympanic space in temporal bone computerized tomography. Blood eosinophil evaluation is a significant clinical indicator of EOM. Furthermore, the assessment of exclusive protympanic soft tissue density can provide an additional diagnostic clue.

Robot-Assisted Total Thyroidectomy with or without Robot-Assisted Neck Dissection in Pediatric Patients with Differentiated Thyroid Cancer.

Pediatric thyroid cancer more frequently develops cervical node metastasis than adult thyroid cancer, even in differentiated thyroid carcinoma (DTC). Thus, cervical neck dissection often needs to be performed simultaneously with thyroidectomy in pediatric patients. Herein, we describe our experience with robot-assisted total thyroidectomy with/without robot-assisted neck dissection in pediatric patients compared with the conventional operated group. A total of 30 pediatric patients who underwent thyroidectomy for DTC between July 2011 and December 2019 were retrospectively reviewed. Among them, 22 underwent robot-assisted operation, whereas 8 underwent conventional operation. There was no statistical difference in the mean operation times, blood loss, drainage amounts, and hospital stay length between the robot-assisted and conventional operation groups; however, the operation time was less in the retroauricular approach subgroup (robot-assisted operation group) with better satisfaction on cosmesis. No postoperative complications, such as seromas, hemorrhages, or hematomas were observed. Our experience suggested that robot-assisted thyroidectomy with or without neck dissection through the retroauricular approach is a feasible and safe alternative treatment, producing outstanding esthetic results compared to the conventional approach, especially in pediatric patients with DTC.

The use of technical replication for detection of low-level somatic mutations in next-generation sequencing.

Accurate genome-wide detection of somatic mutations with low variant allele frequency (VAF, <1%) has proven difficult, for which generalized, scalable methods are lacking. Herein, we describe a new computational method, called RePlow, that we developed to detect low-VAF somatic mutations based on simple, library-level replicates for next-generation sequencing on any platform. Through joint analysis of replicates, RePlow is able to remove prevailing background errors in next-generation sequencing analysis, facilitating remarkable improvement in the detection accuracy for low-VAF somatic mutations (up to ~99% reduction in false positives). The method is validated in independent cancer panel and brain tissue sequencing data. Our study suggests a new paradigm with which to exploit an overwhelming abundance of sequencing data for accurate variant detection.

Integrating a Next Generation Sequencing Panel into Clinical Practice in Ovarian Cancer.

PURPOSE: Few efforts have been made to integrate a next generation sequencing (NGS) panel into standard clinical treatment of ovarian cancer. The aim of this study was to investigate the clinical utility of NGS and to identify clinically impactful information beyond targetable alterations. MATERIALS AND METHODS: We conducted a retrospective review of 84 patients with ovarian cancer who underwent NGS between March 1, 2017, and July 31, 2018, at the Yonsei Cancer Hospital. We extracted DNA from formalin-fixed, paraffin-embedded tissue samples of ovarian cancer. The TruSight Tumor 170 gene panel was used to prepare libraries, and the MiSeq instrument was used for NGS. RESULTS: Of the 84 patients, 55 (65.1%) had high-grade serous carcinomas. Seventy-three (86.7%) patients underwent NGS at the time of diagnosis, and 11 (13.3%) underwent NGS upon relapse. The most common genetic alterations were in TP53 (64%), PIK3CA (15%), and BRCA1/2 (13%), arising as single nucleotide variants and indels. MYC amplification (27%) was the most common copy number variation and fusion. Fifty-seven (67.9%) patients had more than one actionable alteration other than TP53. Seven (8.3%) cases received matched-target therapy based on the following sequencing results: BRCA1 or 2 mutation, poly ADP ribose polymerase inhibitor (n=5); PIK3CA mutation, AKT inhibitor (n=1); and MLH1 mutation, PD-1 inhibitor (n=1). Fifty-three (63.0%) patients had a possibility of treatment change, and 8 (9.5%) patients received genetic counseling. CONCLUSION: Implementation of NGS may help in identifying patients who might benefit from targeted treatment therapies and genetic counseling.