The impact of maxillary dimensions on determining surgical approach of fungal ball in the maxillary sinus.

Endoscopic middle meatal antrostomy (MMA) is commonly used for maxillary sinus (MS) fungal ball removal. For challenging cases involving anterior or inferior recess, an additional inferior meatal approach (IMA) might be needed. We analyzed the differences in MS dimensions on CT scans according to the surgical approach to suggest preoperative variables that could facilitate an additional IMA. CT scans of 281 adult patients who underwent ESS for the MS fungal ball (139 MMA, 62 MMA & IMA) were evaluated for comparative analysis of 8 MS measurements based on the surgical approach. Complete removal was achieved in all cases. Age and sex didn't differ significantly (p > 0.05). The maximum distances between the anterior-posterior walls, the inferior ostium border to the lateral recess, and the ostium to the inferior wall of the MS were statistically greater in the MMA & IMA group compared to the MMA group (p = 0.003, p = 0.005, and p = 0.010, respectively), especially among females. This study underscores the clinical importance of specific measurements-anterior to posterior wall, medial wall to lateral recess, and ostium to inferior wall of the maxillary sinus-for guiding optimal surgical approaches in MS lesions.

Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Emerging insights into transcriptional condensates.

Eukaryotic transcription, a fundamental process that governs cell-specific gene expression, has long been the subject of extensive investigations in the fields of molecular biology, biochemistry, and structural biology. Recent advances in microscopy techniques have led to a fascinating concept known as "transcriptional condensates." These dynamic assemblies are the result of a phenomenon called liquid‒liquid phase separation, which is driven by multivalent interactions between the constituent proteins in cells. The essential proteins associated with transcription are concentrated in transcriptional condensates. Recent studies have shed light on the temporal dynamics of transcriptional condensates and their potential role in enhancing the efficiency of transcription. In this article, we explore the properties of transcriptional condensates, investigate how they evolve over time, and evaluate the significant impact they have on the process of transcription. Furthermore, we highlight innovative techniques that allow us to manipulate these condensates, thus demonstrating their responsiveness to cellular signals and their connection to transcriptional bursting. As our understanding of transcriptional condensates continues to grow, they are poised to revolutionize our understanding of eukaryotic gene regulation.

Direct observation of a condensate effect on super-enhancer controlled gene bursting.

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.

YTHDF2 facilitates aggresome formation via UPF1 in an m6A-independent manner.

YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N6-methyladenosine (m6A), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the m6A-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.

The role of cellular traction forces in deciphering nuclear mechanics.

Cellular forces exerted on the extracellular matrix (ECM) during adhesion and migration under physiological and pathological conditions regulate not only the overall cell morphology but also nuclear deformation. Nuclear deformation can alter gene expression, integrity of the nuclear envelope, nucleus-cytoskeletal connection, chromatin architecture, and, in some cases, DNA damage responses. Although nuclear deformation is caused by the transfer of forces from the ECM to the nucleus, the role of intracellular organelles in force transfer remains unclear and a challenging area of study. To elucidate nuclear mechanics, various factors such as appropriate biomaterial properties, processing route, cellular force measurement technique, and micromanipulation of nuclear forces must be understood. In the initial phase of this review, we focused on various engineered biomaterials (natural and synthetic extracellular matrices) and their manufacturing routes along with the properties required to mimic the tumor microenvironment. Furthermore, we discussed the principle of tools used to measure the cellular traction force generated during cell adhesion and migration, followed by recently developed techniques to gauge nuclear mechanics. In the last phase of this review, we outlined the principle of traction force microscopy (TFM), challenges in the remodeling of traction forces, microbead displacement tracking algorithm, data transformation from bead movement, and extension of 2-dimensional TFM to multiscale TFM.

Effects of Transcription-Dependent Physical Perturbations on the Chromosome Dynamics in Living Cells.

Recent studies with single-particle tracking in live cells have revealed that chromatin dynamics are directly affected by transcription. However, how transcription alters the chromatin movements followed by changes in the physical properties of chromatin has not been elucidated. Here, we measured diffusion characteristics of chromatin by targeting telomeric DNA repeats with CRISPR-labeling. We found that transcription inhibitors that directly block transcription factors globally increased the movements of chromatin, while the other inhibitor that blocks transcription by DNA intercalating showed an opposite effect. We hypothesized that the increased mobility of chromatin by transcription inhibition and the decreased chromatin movement by a DNA intercalating inhibitor is due to alterations in chromatin rigidity. We also tested how volume confinement of nuclear space affects chromatin movements. We observed decreased chromatin movements under osmotic pressure and with overexpressed chromatin architectural proteins that compact chromatin.

Preoperative Magnetic Resonance Image and Computerized Tomography Findings Predictive of Facial Nerve Invasion in Patients with Parotid Cancer without Preoperative Facial Weakness-A Retrospective Observational Study.

(1) Background: Facial nerve resection with reconstruction helps achieve optimal outcomes in the treatment of facial nerve invasion (FNI) of parotid cancer. Preoperative imaging is crucial to predict facial nerve reconstruction. The radiological findings of CT or MRI may predict FNI in the parotid cancer even without facial paralysis. Methods: We retrospectively reviewed the records of 151 patients without facial nerve paralysis before surgery who had undergone tumor resection. Previously untreated parotid cancers were included. (2) Results: The median follow-up duration was 62 months (range: 24-120 months). The FNI (+) group (n = 30) showed a significantly worse 5-year overall survival compared with the FNI (-) group (75.5 vs. 93.9%; hazard ratio = 4.19; 95% confidence interval: 1.74-10.08; p = 0.001). The tumor margin, tumor size, presence in the anterolateral parotid region (area 3), retromandibular vein involvement, distance from the stylomastoid foramen to the upper tumor margin, and a high tumor grade were significant factors related to FNI in the univariate analysis. A spiculated tumor margin, the tumor size (2.2 cm), and presence in area 3 were factors predicting FNI in the logistic regression model (p = 0.020, 0.005, and 0.050, respectively; odds ratio: 4.02, 6.40, and 8.16, respectively). (3) Conclusions: The tumor size (≥2.2 cm), spiculated margin, and presence in area 3 as presented in CT and MRI may help clinicians preoperatively predict FNI in patients with parotid cancer and establish an appropriate surgical plan.

Suture Ligation of Pyriform Sinus Fistulas Following Cauterization to Treat Branchial Cleft Anomalies.

OBJECTIVES: Obliteration with cauterization of the internal opening of pyriform sinus fistulas, with or without marsupialization, has been a mainstay for preventing recurrence. However, this procedure predisposes patients to recurrence caused by the reopening of the cauterized orifice. We applied suture ligation to secure the closure of the internal orifice following cauterization and evaluated treatment. METHODS: A total of 42 patients were diagnosed with third or fourth branchial cleft anomaly with internal pyriform sinus fistula and treated either with cauterization or with cauterization and suture ligation, between January 2008 and December 2020. The medical records were reviewed to assess demographic characteristics, clinical presentations, diagnoses, surgical treatment, and outcomes. Treatment flow characteristics for intractable patients were analyzed. RESULTS: The median age of onset was 9 years (range, 0-57 years). Neck swelling (n = 32, 76.2%) was commonly encountered symptom, and a history of neck infection was found in 27 patients (64.3%). After initial treatment, 11 cases (56.2%) recurred. Younger age (≤9 years) and thyroid involvement were associated with recurrence (P = .034 and P = .046, respectively). Cauterization with suture ligation presented lower recurrence rate (19.2%) than cauterization only (37.5%, P = .037). Cauterization with suture ligation for recurrent cases (n = 11) showed 81.8% (9/11) of cure rate. Intractable fistulas (n = 2) from both groups were also salvaged by suture ligation. CONCLUSIONS: Suture ligation with cauterization for an internal orifice of branchial anomaly showed lower recurrence rate than cauterization only. This method was beneficial for refractory cases.

Comparison of Convolutional Neural Network Models for Determination of Vocal Fold Normality in Laryngoscopic Images.

OBJECTIVES: Deep learning using convolutional neural networks (CNNs) is widely used in medical imaging research. This study was performed to investigate if vocal fold normality in laryngoscopic images can be determined by CNN-based deep learning and to compare accuracy of CNN models and explore the feasibility of application of deep learning on laryngoscopy. METHODS: Laryngoscopy videos were screen-captured and each image was cropped to include abducted vocal fold regions. A total of 2216 image (899 normal, 1317 abnormal) were allocated to training, validation, and test sets. Augmentation of training sets was used to train a constructed CNN model with six layers (CNN6), VGG16, Inception V3, and Xception models. Trained models were applied to the test set; for each model, receiver operating characteristic curves and cutoff values were obtained. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated. The best model was employed in video-streams and localization of features was attempted using Grad-CAM. RESULTS: All of the trained models showed high area under the receiver operating characteristic curve and the most discriminative cutoff levels of probability of normality were determined to be 35.6%, 61.8%, 13.5%, 39.7% for CNN6, VGG16, Inception V3, and Xception models, respectively. Accuracy of the CNN models selecting normal and abnormal vocal folds in the test set was 82.3%, 99.7%, 99.1%, and 83.8%, respectively. CONCLUSION: All four models showed acceptable diagnostic accuracy. Performance of VGG16 and Inception V3 was better than the simple CNN6 model and the recently published Xception model. Real-time classification with a combination of the VGG16 model, OpenCV, and Grad-CAM on a video stream showed the potential clinical applications of the deep learning model in laryngoscopy.

CTCF-mediated chromatin looping provides a topological framework for the formation of phase-separated transcriptional condensates.

CTCF is crucial to the organization of mammalian genomes into loop structures. According to recent studies, the transcription apparatus is compartmentalized and concentrated at super-enhancers to form phase-separated condensates and drive the expression of cell-identity genes. However, it remains unclear whether and how transcriptional condensates are coupled to higher-order chromatin organization. Here, we show that CTCF is essential for RNA polymerase II (Pol II)-mediated chromatin interactions, which occur as hyperconnected spatial clusters at super-enhancers. We also demonstrate that CTCF clustering, unlike Pol II clustering, is independent of liquid-liquid phase-separation and resistant to perturbation of transcription. Interestingly, clusters of Pol II, BRD4, and MED1 were found to dissolve upon CTCF depletion, but were reinstated upon restoration of CTCF, suggesting a potent instructive function for CTCF in the formation of transcriptional condensates. Overall, we provide evidence suggesting that CTCF-mediated chromatin looping acts as an architectural prerequisite for the assembly of phase-separated transcriptional condensates.

Visualization of chromatin higher-order structures and dynamics in live cells.

Chromatin has highly organized structures in the nucleus, and these higher-order structures are proposed to regulate gene activities and cellular processes. Sequencing-based techniques, such as Hi-C, and fluorescent in situ hybridization (FISH) have revealed a spatial segregation of active and inactive compartments of chromatin, as well as the non-random positioning of chromosomes in the nucleus, respectively. However, regardless of their efficiency in capturing target genomic sites, these techniques are limited to fixed cells. Since chromatin has dynamic structures, live cell imaging techniques are highlighted for their ability to detect conformational changes in chromatin at a specific time point, or to track various arrangements of chromatin through long-term imaging. Given that the imaging approaches to study live cells are dramatically advanced, we recapitulate methods that are widely used to visualize the dynamics of higher-order chromatin structures. [BMB Reports 2021; 54(10): 489-496].

Diagnostic Accuracies of Laryngeal Diseases Using a Convolutional Neural Network-Based Image Classification System.

OBJECTIVES/HYPOTHESIS: There may be an interobserver variation in the diagnosis of laryngeal disease based on laryngoscopic images according to clinical experience. Therefore, this study is aimed to perform computer-assisted diagnosis for common laryngeal diseases using deep learning-based disease classification models. STUDY DESIGN: Experimental study with retrospective data METHODS: A total of 4106 images (cysts, nodules, polyps, leukoplakia, papillomas, Reinke's edema, granulomas, palsies, and normal cases) were analyzed. After equal distribution of diseases into ninefolds, stratified eightfold cross-validation was performed for training, validation process and remaining onefold was used as a test dataset. A trained model was applied to test sets, and model performance was assessed for precision (positive predictive value), recall (sensitivity), accuracy, F1 score, precision-recall (PR) curve, and PR-area under the receiver operating characteristic curve (PR-AUC). Outcomes were compared to those of visual assessments by four trainees. RESULTS: The trained deep neural networks (DNNs) outperformed trainees' visual assessments in discriminating cysts, granulomas, nodules, normal cases, palsies, papillomas, and polyps according to the PR-AUC and F1 score. The lowest F1 score and PR-AUC of DNNs were estimated for Reinke's edema (0.720, 0.800) and nodules (0.730, 0.780) but were comparable to the mean of the two trainees' F1 score with the best performances (0.765 and 0.675, respectively). In discriminating papillomas, the F1 score was much higher for DNNs (0.870) than for trainees (0.685). Overall, DNNs outperformed all trainees (micro-average PR-AUC = 0.95; macro-average PR-AUC = 0.91). CONCLUSIONS: DNN technology could be applied to laryngoscopy to supplement clinical assessment of examiners by providing additional diagnostic clues and having a role as a reference of diagnosis. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:2558-2566, 2021.

Interpreting auditory brainstem evoked responses and distortion product otoacoustic emissions in diabetic patients with normal hearing.

OBJECTIVE: Hearing impairment is a reported late complication of diabetes mellitus (DM). Previous studies have suggested that microangiopathic complications may cause cochlear nerve function deterioration. We evaluated the auditory brainstem evoked responses (ABRs) and distortion product otoacoustic emission (DPOAE) results according to the presence of DM in subjects with normal hearing. METHODS: A cross-sectional comparative study was conducted from January 2016 to January 2018. Auditory function tests including ABR and DPOAE were performed for outpatients complaining of unilateral tinnitus. All of analyses were conducted in ears without tinnitus on contralateral side of tinnitus ears. We included subjects showing hearing thresholds within 25 dB at 0.5, 1k, 2k, and 4k on pure tone audiometry. 45 ears in patients with type 2 diabetes mellitus and 85 ears in non-diabetic patients were finally enrolled in our study. RESULTS: Diabetic subjects showed significantly more prolonged absolute peak latencies (I, III, V) and inter-peak latencies (I-V, III-V) than non-diabetic subjects. However, there was no significant difference in the inter-peak latency (I-III) between these two groups. Diabetic subjects also showed significantly lower amplitudes at f2 frequencies of 1001, 1200, 1587, 4004, 5042, and 6348 Hz than non-diabetic subjects. Additionally, the prevalence of a DPOAE response, defined as 3 dB above the noise floor, was significantly lower in diabetic subjects than that in non-diabetic subjects. CONCLUSION: Diabetic patients with normal hearing can still have abnormal ABR and DPOAE results due to diabetic neuroangiopathy. ABR and DPOAE assessments can help in detecting subclinical auditory dysfunction, which precedes the manifestation of hearing impairment in diabetic patients.

Comparison of allergens and symptoms in patients with allergic rhinitis between 1990s and 2010s.

BACKGROUND: The prevalence of allergic rhinitis (AR), an environment- and lifestyle-dependent condition, has been constantly increasing in Korea. Although the environment and lifestyle of the Korean people have recently undergone rapid changes, corresponding changes in the characteristics of AR patients have not been well documented. Therefore, we aimed to outline the changes in allergens and clinical manifestations of AR in Korean patients from the 1990s and 2010s. METHODS: We reviewed 1447 and 3388 AR patients who visited the same tertiary hospital in the 1990s and 2010s, respectively. All patients were diagnosed with AR based on the presence of characteristic symptoms, positive skin prick test results, and answered a symptom questionnaire at the time of visit. We compared differences in the allergens and results of the symptom questionnaire between the two sets of patients. RESULTS: When compared with the 1990s, the rate of sensitization to house dust mites, cockroaches, Aspergillus, Alternaria, and tree pollen significantly increased and that to cat fur significantly decreased in patients from the 2010s (all P < 0.05). Male predominance was observed with two peaks in the age distribution of patients from the 2010s. The proportion of patients with moderate-to-severe nasal obstruction and itching of the nose/eye increased (each P < 0.05) and that of patients with minor symptoms such as olfactory disturbances, cough, sore throat, and fatigue also increased (all P < 0.01) in the 2010s. CONCLUSIONS: Allergen reactivity and type and symptom severity in Korean AR patients significantly varied between the 1990s and 2010s. Our results may therefore be helpful for patient counseling and management.

Predictors of survival and recurrence after primary surgery for cervical metastasis of unknown primary.

PURPOSE: Cervical metastasis from unknown primary (CUP) is commonly classified as an advanced overall stage. P16 or human papillomavirus (HPV) positivity in metastatic lymph nodes (LN) might be associated with a favorable survival outcome of CUP. Therefore, we evaluated the prognostic values of p16 immuno-positivity in LN and other clinicopathological factors in patients with squamous cell carcinoma CUP (SCCUP). METHODS: This study involved 83 patients who underwent therapeutic neck dissection and panendoscopic examination and biopsy for suspected CUP. P16 immunostaining and HPV typing in LN were performed in 56 patients. Cox proportional hazard regression analyses were used to identify factors associated with overall survival (OS) and disease-free survival (DFS). RESULTS: Postoperatively, primary tumors (PT) were found in 32 (38.6%) patients, mainly (90.6%) in the oropharynx, and not found in 51 (61.4%) patients. The clinicopathological data (except for histological grade) and 5-year OS and DFS rates did not significantly differ between patients with and without PT identification (all P > 0.05). P16 positivity was associated with favorable OS and DFS outcomes in the patients with PT (P < 0.05) but not in those without PT (P > 0.1). Multivariate analyses showed that age (> 60 years) and LN ratio (≥ 0.1) were the independent predictors of OS and DFS outcomes (all P < 0.05). P16 positivity or other factors were not independent factors. CONCLUSION: Age and LN ratio are significant risk factors of survival and recurrence after primary surgery for SCCUP. Prognostic significance of LN p16 positivity should be further studied.

Lymph node ratio predictive of recurrence, distant metastasis, and survival in submandibular gland carcinoma patients.

PURPOSE: Submandibular gland (SMG) carcinoma has an unfavorable clinical course and a low survival rate. Specific tumor and nodal findings might help predict posttreatment recurrence and survival in SMG carcinoma patients. This study evaluated factors predictive of posttreatment recurrence and survival in SMG carcinoma patients. METHODS: This study enrolled 99 consecutive patients with previously untreated SMG carcinoma. All patients underwent tumor and metastatic lymph node resection along with or without radiation/chemo-radiation. The predictive clinical and pathological factors for disease-free survival (DFS), distant metastasis-free survival (DMFS), disease-specific survival (DSS), and overall survival (OS) were determined using univariate and multivariate Cox proportional hazards regression analyses. RESULTS: The median tumor size was 2.7 cm and 53 patients (53.5%) had high-grade tumors. The rates of initial nodal and distant metastasis at initial presentation or follow-up were 45.5% and 42.4%, respectively. The actuarial 5-year DFS, DMFS, DSS, and OS rates were 46.4%, 55.9%, 61.5%, and 59.7%, respectively. The independent factors associated with poor DFS, DMFS, DSS, and OS outcomes (all p < 0.05) were T3-T4 classification and lymph node ratio. A histological high-grade tumor was an independent prognostic factor predictive of poor DMFS, DSS, and OS outcomes (all p < 0.05). CONCLUSIONS: A high rate of distant site failure is associated with SMG carcinoma, resulting in a poor survival rate. Lymph node ratio might help predict recurrence, distant metastasis, and death due to SMG carcinoma.

A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells.

The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.

Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.