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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.

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.

The intricate physical interaction of transcription factors with specific target genes in the genome has long been regarded as a fundamental mechanism of cell-type specific gene expression. However, due to insufficient spatiotemporal resolution of microscopy techniques, direct visualization of protein dynamics within the nuclei of living cells had not been achieved for decades. Resolving existing limitations, recent advances in imaging techniques enabled the direct observation of protein dynamics, even at a single-molecule level. In addition, the new imaging techniques accomplished capturing higher-order chromatin structures that influence gene expression regulation along with transcription factor dynamics. This review discusses the recent applications of microscopy techniques to investigate the dynamics of nuclear proteins in living cells and their achievements.

Summary ParagraphInside the nucleus, there are special compartments with characterized functions, some of which are involved in gene expression1. These compartments include transcriptional condensates and nuclear speckles, which contain factors required for transcription and splicing, respectively. While the characteristics of these intranuclear compartments were extensively investigated, spatial relationship between them is yet unclear. Meanwhile, RNA-protein structural network named the nuclear matrix has been observed inside the nucleus and suggested as a framework for the spatial segregation of the intranuclear space2–6. However, concept of the nuclear matrix was not widely accepted due to its lack of in vivo evidence7. Here, we report visualization of the nuclear matrix and intranuclear compartments using super-resolution fluorescence microscopy. We found the nuclear matrix is dynamic in live cells and easily disrupted upon transcription inhibition. Remarkably, we observed an orderly layered distribution of transcriptional condensates and nuclear speckles relative to the nuclear matrix. We also observed a separation of chromosome territories from transcriptional condensates and nuclear speckles by the nuclear matrix. Based on our findings, we propose a topological model for the regulation of transcription across the nuclear matrix.

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.

(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.

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).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).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.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.

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. 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. 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

Summary 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.

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. Keywords: CRISPR; Stem cell; MS2; Single RNA imaging; Quantitative microscopy; Live cell imaging

Phase separation and gene control Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science , this issue p. eaar2555 , p. eaar3958 , p. 412 ; see also p. 329

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.

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 P P 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.

We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell., National Institutes of Health (U.S.), National Cancer Institute (U.S.) (NIH Director’s New Innovator Award DP2-CA195769), Massachusetts Institute of Technology. Department of Physics

We present a software for quantitative analysis of single molecule based super-resolution data. The software serves as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living, or fixed cells.

RNA Polymerase I (Pol I) is responsible for over 60% of transcriptional output in human cells, yet basic questions concerning the spatial and temporal organization of the polymerase remain unanswered. Here we investigate how mammalian cells rely on Pol I organization throughout the cell cycle to balance different needs, from complete transcription shut down to massive increase in protein synthesis (and thus ribosomal RNA synthesis) before cell division. In contrast to our previous reports on RNA Polymerase II, Pol I clusters are stable with active transcription, and the presence of transient Pol I clusters correlates with inactive ribosomal transcription. Our results suggest that both stable and transient populations Pol I clusters co-exist in individual living cells, and their relative fraction may directly reflect the global gene expression need of the cell.

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that Taq-MutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.

Summary The αɛθ core of Escherichia coli DNA polymerase III (Pol III) associates with the β 2 sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The α subunit is the polymerase while ɛ is the 3′ to 5′ proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood. We used single-molecule assays to determine the excision rate and processivity of the β 2 -associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ɛ and β 2 . Thus, the ɛ-β 2 contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between ɛ and β 2 .

Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging., National Cancer Institute (U.S.) (Award DP2CA195769), Massachusetts Institute of Technology. Department of Physics

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output., National Cancer Institute (U.S.) (DP2CA195769)

SummaryThe mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (∼1 s) and an unusually stable (∼600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR.

Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ssDNA excision tracts.

The toner type paper‐like display (PLD) has been developed with two polymer particles having opposite polarity composed of polymer, colorant and external additives (nano‐sized silica). Nano‐sized silica with triboelectric charge was used for the charge control agent (CCA) and influenced on the electrostatic properties of the silica‐coated polymer particles. The surface morphology and the cohesiveness of silica‐coated polymer particles were changed with the silica coating time. From these results, it was verified that the PLD cell using silica‐coated particles (200 seconds) shows a good white appearance and low driving voltage.

We have developed a paper‐like display using polymer particles charged triboelectrically. By using a toner‐type display with a simple structure, we confirmed that polymer particle movement is controlled by only a voltage difference between scan and data electrode. We fabricated a diagonal 2.4 inch panel on the glass substrate and successfully produced an image by the passive driving method with operating voltages of about ± 120 V. The contrast ratio was 4:1.

This paper for the first time reports the isolation of 5 compounds from Phellinus linteus. A sphingolipid (1) and two tyrosinase inhibitory compounds (2, 3) along with two carboxylic acids (4, 5), were isolated from the fruiting body of Phellinus linteus (BerkCurt) Aoshima. The structure of compound 1 was identified as 1-omicron-beta-D-glucopyranosyl-(2S, 3R, 4E, 8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol, known as cerebroside B, based on spectroscopic methods such as 1 D and 2D NMR as well as by acid hydrolysis. Compounds 2 -5 were identified as protocatechualdehyde (2), 5-hydroxymethyl-2-furaldehyde (HMF) (3), succinic acid (4), and fumaric acid (5) based on the spectroscopic evidence. Compounds 2 and 3 inhibited the oxidation of L-tyrosine catalyzed by mushroom tyrosinase with an IC50 of 0.40 and 90.8 microg/mL, respectively. The inhibitory kinetics, which were analyzed by the Lineweaver-Burk plots, were found to be competitive and noncompetitive inhibitors with a Ki of 1.1 microM and 1.4 mM, respectively.

MutS, a mismatch repair initiation protein, forms a transient clamp to search for mismatched nucleotides by rotational-coupled translational diffusion while in continuous but nonspecific contact with the helical duplex DNA. When MutS encounters a mismatch, ATP binding by MutS results in an extremely stable sliding clamp that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The DNA mismatch repair is a timing process to be operated after DNA is newly synthesized. The β-clamp protein, a subunit of the DNA polymerase III holoenzyme, encircles duplex DNA and stably diffuses on the DNA after a lagging strand is synthesized. The long dwell time of the sliding β-clamp on DNA results in its accumulation on the lagging strand, which may obstruct the way of the translocation of MutS to find a mismatch and to transmit its finding to a downstream site. We will present how the sliding β-clamp interacts with MutS on mismatched DNA. The single-molecule studies using FRET and a single particle tracking suggest that the β-clamp increases the searching time of MutS required to locate a mismatch site through the physical interaction with MutS and is separated from MutS bound to ATP at the mismatch.

This paper introduces a WDM system structure for protecting systems at optical channel layer and optical multiplexer layer respectively. The maintenance signal operation method for WDM systems is also presented for the case of system failure in comparison to SDH ones.

We have developed flexible and reflective paper-like display (PLD) using charged polymer particles. These particles are composed of polymer, pigment and charge control agent (CCA). In this paper, we demonstrated flexible display with PET (Polyethylene Terephthalate) film substrate, which had 320 × 240 pixels, 5.7 inch size panel with bending radius of 5cm. This panel achieved reflectivity of 32% and bi-stability over 6 months.

A new volcano-type triode field emitter array has been fabricated on a glass substrate using titanium nitride as the emitter material. It is based on the process development for the formation of uniform emitters over large areas using reactive ion etching and the sputtering method. In this study, the effects of the ratio of volcano emitter size to gate hole size on the emission characteristics were investigated with a fixed emitter–gate distance of 1.0 μm and compared with computer simulation results using a finite element method. As the ratio of volcano emitter size to gate hole size decreased, especially from a ratio of around 0.5 to 0.4, the anode current significantly increased. This behavior may result from the increase in the maximum electric field on a tip with a decrease in the ratio as is expected from computer simulation.

It has been studied that DNA repair proteins search a target via a 1-dimensional diffusion along naked DNA. Due to the absence of the target on DNA this single-molecule tracking approach lacked of understanding the catalytic function of the repair proteins and moreover the mechanism of the downstream transactions for the repair of an error on the DNA. We present the catalytic processes of MutS, mismatch repair initiation protein, on a mismatched DNA that bears an unpaired nucleotide. Our single-molecule analysis reveals that MutS undergoes two distinct conformational changes during DNA mismatch repair (MMR). MutS forms a transient clamp that scans duplex DNA for the unpaired nucleotide and a different sliding clamp that is induced by ATP binding to the mismatch-bound MutS with unusual stability on DNA. These observations suggest a mechanism of how MMR machinery can be recruited for the strand incision, which is highly controversial.