Epidemiology of Congenital Cholesteatoma: Surveys of the Last 17 Years in Japan.

(1) Background: The incidence of congenital cholesteatoma (CC) has rarely been discussed, particularly from a demographic viewpoint. Therefore, we conducted an epidemiological study of CC using local medical characteristics. (2) Methods: The participants were 100 patients (101 ears) who underwent initial surgical treatment at university hospitals in two rural prefectures between 2006 and 2022. A total of 68% of the patients were males and 32% were females, with a median age of 5 years. We reviewed the medical records for the date of birth, date of surgery, stage of disease, and first symptoms of the disease. (3) Results: The total incidence of CC was calculated to be 26.44 per 100,000 births and tended to increase. No significant difference was found between the incidences in the two prefectures. The number of surgeries performed was higher in the second half of the study period. No difference in the stage of progress was observed based on age. (4) Conclusions: The incidence of CC was estimated to be 26.44 per 100,000 newborn births. The number of patients with CC tended to increase; however, this can be attributed to an increase in the detection rate rather than the incidence.

Systematic Analysis of 2'-O-Alkyl Modified Analogs for Enzymatic Synthesis and Their Oligonucleotide Properties.

Enzymatic oligonucleotide synthesis is used for the development of functional oligonucleotides selected by in vitro selection. Expanding available sugar modifications for in vitro selection helps the functional oligonucleotides to be used as therapeutics reagents. We previously developed a KOD DNA polymerase mutant, KOD DGLNK, that enzymatically synthesized fully-LNA- or 2'-O-methyl-modified oligonucleotides. Here, we report a further expansion of the available 2'-O-alkyl-modified nucleotide for enzymatic synthesis by KOD DGLNK. We chemically synthesized five 2'-O-alkyl-5-methyluridine triphosphates and incorporated them into the oligonucleotides. We also enzymatically synthesized a 2'-O-alkyl-modified oligonucleotide with a random region (oligonucleotide libraries). The 2'-O-alkyl-modified oligonucleotide libraries showed high nuclease resistance and a wide range of hydrophobicity. Our synthesized 2'-O-alkyl-modified oligonucleotide libraries provide novel possibilities that can promote the development of functional molecules for therapeutic use.

A SAM analogue-utilizing ribozyme for site-specific RNA alkylation in living cells.

Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes.

Ribozyme-Catalyzed Late-Stage Functionalization and Fluorogenic Labeling of RNA.

Site-specific introduction of bioorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post-synthetic bioconjugation reactions. Here, we report a ribozyme-based method for the synthesis of aldehyde-functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site-specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5-amino-4-formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde-reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3-trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme's area of application from a methyltransferase to a tool for site-specific late-stage functionalization of RNA.

Speech Perception of Second Cochlear Implant after 6 Years of Age in Prelingually Deaf Children.

Sequential bilateral cochlear implantation in children becomes less effective as the inter-implant interval increases. However, the cause of this and the age at which speech perception becomes impossible are unclear. We examined the cases of 11 prelingually deaf children who underwent unilateral cochlear implantation at our hospitals before the age of 5 years old, followed by a second implantation procedure on the contralateral side when they were aged ≥ 6 years old (6-12 years old). The subjects' hearing thresholds and speech discrimination scores for the second cochlear implant were evaluated at 3 postoperative months and 1-7 years. All of the subjects demonstrated improvements in their hearing thresholds to a mean of 30 dB HL at 1 year. Regarding speech perception, one patient (a 12-year-old), who had developed bilateral hearing loss at 30 months of age after contracting mumps, demonstrated a 90% improvement in his speech discrimination score at 1 year. However, among the other congenitally deaf children, there were two patients whose speech discrimination scores had improved by ≥ 80% at > 4 postoperative years. The congenitally deaf children exhibited poor speech perception despite showing improved hearing thresholds in the ears that received second cochlear implants. Assuming that the auditory pathway beyond the superior olivary complex remained functional, the reduced speech perception abilities associated with the second cochlear implants may have been attributable to the loss of the spiral ganglion and cochlear nucleus cells due to a lack of auditory input since birth.

Real-world treatment patterns and outcomes in Japanese patients with cervical esophageal cancer.

BACKGROUND: Cervical esophageal cancer (CEC) carries a poor prognosis; however, due to its low incidence, optimal treatment for CEC remains to be established. The purpose of this study was to clarify the current status of treatment of CEC in Japan and obtain evidence for establishing the appropriate treatment method. PATIENTS AND METHODS: We asked specialist training facilities accredited by the Japanese Broncho-Esophageal Society to register data on CEC cases that received curative treatment from January 2009 to December 2014, and conducted a retrospective review of the clinical data of 302 cases registered from 27 facilities. RESULTS: In regard to the initial therapy, of the 302 patients, 33 had undergone endoscopic resection, 41 had undergone surgery, 67 had received induction chemotherapy (IC), and 143 had received chemoradiotherapy (CRT). There were no significant differences in the 5-year overall survival rates among the patient groups that had received surgery, IC or CRT as the initial treatment; advanced stage and recurrent nerve invasion were identified as independent poor prognostic factors. Among the patients who had received IC or CRT as laryngeal-preserving surgery was not indicated at the time of the initial diagnosis, the functional laryngeal preservation rate at the end of the observation period was 34.8%. CONCLUSION: Even in patients with advanced CEC, there is the possibility of preserving the larynx by adopting IC or CRT. However, if the laryngeal function cannot be preserved, there is a risk of complications from aspiration pneumonia, so that the choice of treatment should be made carefully.

Combined Electric acoustic stimulation in a patient with otitis media with antineutrophil cytoplasmic antibody-associated vasculitis.

There are few reports of the treatment for severe hearing loss due to otitis media with antineutrophil cytoplasmic antibody-associated vasculitis (OMAAV) achieved by cochlear implantation (CI). Here, we have reported the case of a patient with severe bilateral sensorineural hearing loss with low-frequency residual hearing by OMAAV. CI was performed in her right ear based on the results of contrast-enhanced magnetic resonance imaging (CE-MRI) and promontory stimulation test (PST). The residual hearing in her right ear was preserved after CI and utilized for combined electric acoustic stimulation (EAS). The combined EAS was used for 3 years until the residual hearing became stabilized. However, the usable hearing in low frequency worsened gradually, and the fitting strategy of cochlear implant was changed from combined EAS to CI alone 4 years after CI. Even when the speech discrimination score with CI no longer exceeds 50 %, the patient continued using CI because of its advantages in maintaining the quality of life of the patient. The combined EAS was found to be a feasible option even in an OMAAV patient with residual hearing. CE-MRI and PST may thus be helpful in deciding the side of CI surgery in a patient with OMAAV.

Comparison of speech perception lists used to evaluate Japanese single syllable hearing.

OBJECTIVE: In Japan, the 57S and 67S single syllable lists have been used to test speech perception in cochlear implant (CI) users. However, due to advances in implants and processors, these lists have become too simple for CI users. In 2019, the Japan Otological Society created a new list, referred to as the iCI2004. The objective of this study was to compare the performance of these lists. METHODS: The Japanese single syllable speech perception tests, iCI2004, 57S, and 67S, were administered to 70 patients with CI users. The effects of single syllable characteristics on the test scores were examined. The type and number of single syllables used in each list were different. Therefore, we compared the scores for normal lists, shared single syllables, and non-shared single syllables. RESULTS: The average test results were 52% for iCI2004, 64% for 57S, and 77% for 67S; 67S performed the best, followed by 57S and iCI2004. The test results were significantly different. In a comparison of shared single syllables, the average scores were 58% for iCI2004 and 63% for 57S (45 pieces). A comparison of iCI2004 and 67S (17 pieces) showed that the average scores were 63% for iCI2004 and 75% for 67S. A comparison of 57S and 67S (20 pieces) showed that the average score for 57S was 70% and the average scored for 67S was 77%. No significant differences were detected under all conditions. Based on these results, the type of single syllable adopted affected the result. CONCLUSION: The data gives no clear indication that the selection of the word table or presentation of sound pressure affects listening to sound for CI users in Japan. Based on the type and number of single syllables used, iCI2004 seems appropriate for evaluation of hearing in patients using CI.

A Case of Amelanotic Malignant Melanoma of the Lingual Base That Was Diagnosed Based on a Biopsy of Late Metastasis to a Lumbar Vertebra after Being Misdiagnosed as HPV-Positive Oropharyngeal Anterior Wall Squamous Cell Carcinoma.

We report a case of amelanotic malignant melanoma (AMM) in a 66-year-old female. AMM of the lingual base was diagnosed based on a biopsy of late metastasis to the bone marrow of the L4 lumbar vertebra. The patient was initially treated with chemoradiotherapy after being misdiagnosed with poorly differentiated human papillomavirus- (HPV-) related squamous cell carcinoma of the oropharyngeal anterior wall. p16 immunostaining is used to diagnose HPV-related oropharyngeal cancer. However, while p16 expression is used as a surrogate marker of HPV infection, it is important to be aware that p16 protein overexpression can also be caused by other factors. Malignant melanoma is known to express the p16 protein. Morphologically differentiating between AMM and poorly differentiated squamous cell carcinoma based on hematoxylin-eosin staining is difficult. Therefore, in cases that are pathologically diagnosed as p16-positive poorly differentiated oropharyngeal squamous cell carcinoma, it is important to rule out AMM.

Hybrid-Type SELEX for the Selection of Artificial Nucleic Acid Aptamers Exhibiting Cell Internalization Activity.

Nucleic acid aptamers have attracted considerable attention as next-generation pharmaceutical agents and delivery vehicles for small molecule drugs and therapeutic oligonucleotides. Chemical modification is an effective approach for improving the functionality of aptamers. However, the process of selecting appropriately modified aptamers is laborious because of many possible modification patterns. Here, we describe a hybrid-type systematic evolution of ligands by exponential enrichment (SELEX) approach for the generation of the artificial nucleic acid aptamers effective against human TROP2, a cell surface protein identified by drug discovery as a promising target for cancer therapy. Capillary electrophoresis SELEX was used for the pre-screening of multiple modified nucleic acid libraries and enrichment of TROP2 binding aptamers in the first step, followed by functional screening using cell-SELEX in the second step for the generation of cell-internalizing aptamers. One representative aptamer, Tac-B1, had a nanomolar-level affinity to human TROP2 and exhibited elevated capacity for internalization by cells. Because of the growing interest in the application of aptamers for drug delivery, our hybrid selection approach has great potential for the generation of functional artificial nucleic acid aptamers with ideal modification patterns in vitro.

Base-modified aptamers obtained by cell-internalization SELEX facilitate cellular uptake of an antisense oligonucleotide.

Intracellular delivery of oligonucleotides is important for their use as therapeutic drugs. The conjugation of molecules interacting with cell membrane proteins to enhance their internalization into cells is an effective strategy for delivering oligonucleotides. In the present study, we focused on creating aptamers, which are single-stranded oligonucleotides that bind target molecules with high affinity and specificity, as membrane protein-binding molecules. With an evolutionary selection approach using a random DNA library containing a uracil derivative with a hydrophobic functional group at the 5 position, we successfully obtained aptamers that are efficiently internalized into A549 cells. The efficacies of the aptamers were tested by further conjugation with MALAT1-targeting antisense oligonucleotides (ASOs), and the expression levels of MALAT1 RNA were examined. The aptamer-ASO conjugates were taken up by A549 cells, although there was no observable reduction in MALAT1 RNA levels. In contrast, the activity of the aptamer-ASO conjugate was potentiated when endosomal/lysosomal escape was enhanced by the addition of chloroquine. Thus, we showed that the hydrophobic modification of the nucleobase moiety is useful for developing highly internalizing aptamers and that endosomal/lysosomal escape is important for the intracellular delivery of ASOs by aptamers.

Discovery of cell-internalizing artificial nucleic acid aptamers for lung fibroblasts and targeted drug delivery.

Lung fibroblasts play major roles in the lung repair/fibrosis process through synthesis and remodeling of extracellular matrix. Those aberrant activations and elevated proliferations are associated with several fibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Targeting fibroblasts is a promising approach for preventing aberrant remodeling of lung architecture and protect irreversible pulmonary fibrosis. In this study, we developed an aptamer that can target lung fibroblasts and explored its potential as a delivery vehicle of cytotoxic agents intracellularly. The aptamer was discovered from artificial nucleic acid libraries through cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). This indole-modified aptamer can bind to LL97A cells, a fibroblast cell line derived from IPF patients, with high affinity (Kd = 70 nM). It also showed affinity to other lung fibroblasts, while cross-reactivity to epithelial cells was minimal. An aptamer-monomethyl auristatin F (MMAF) conjugate was generated by hybridizing with complementary DNA linked to MMAF. The resulting aptamer-MMAF conjugate inhibited proliferation of fibroblasts but appeared non-toxic to non-targeted epithelial cells. Our results show that artificial nucleic acid aptamer may potentially be used for fibroblast-specific therapy and diagnostic applications.

Synthesis and Evaluation of Artificial Nucleic Acid Bearing an Oxanorbornane Scaffold.

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil (U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene. Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal denaturation experiments revealed that oligonucleotides modified with the conformationally restricted OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the Tm values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed to have extremely high stability against 3'-exonuclease activity, and its stability was even higher than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).

Inhibiting guanine oxidation and enhancing the excess-electron-transfer efficiency of a pyrene-modified oligonucleotide by introducing an electron-donating group on pyrene.

The introduction of an electron-donating group (e.g., methoxy or piperidinyl) onto the pyrene moiety of 5-(pyrenylethynyl)-2'-deoxyuridine (PyU) increases its DNA-mediated reductive electron-transfer efficiency. In addition, these modifications dramatically inhibit photoinduced guanine oxidation.

A Hydrogen Peroxide Activatable Gemcitabine Prodrug for the Selective Treatment of Pancreatic Ductal Adenocarcinoma.

The main concern in the use of anticancer chemotherapeutic drugs is host toxicity. Patients need to interrupt or change chemotherapy due to adverse effects. In this study, we aimed to decrease adverse events with gemcitabine (GEM) in the treatment of pancreatic ductal adenocarcinoma and focused on the difference of hydrogen peroxide levels in normal versus cancer cells. We designed and synthesized a novel boronate-ester-caged prodrug that is activated by the high H2 O2 concentrations found in cancer cells to release GEM. An H2 O2 -activatable GEM (A-GEM) has higher selectivity for H2 O2 over other reactive oxygen species (ROS) and cytotoxic effects corresponding to the H2 O2 concentration in vitro. A xenograft model of immunodeficient mice indicated that the effect of A-GEM was not inferior to that of GEM when administered in vivo. In particular, myelosuppression was significantly decreased following A-GEM treatment compared with that following GEM treatment.

1,3,9-Triaza-2-oxophenoxazine: An Artificial Nucleobase Forming Highly Stable Self-Base Pairs with Three AgI Ions in a Duplex.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies.

Correction: Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1.

Correction for 'Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1' by Keisuke Tanaka et al., Org. Biomol. Chem., 2018, 16, 4456-4463.

Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1.

The investigation of protein-protein interactions (PPIs) and the preparation of antagonists are important for determining whether certain proteins are suitable medical targets. In the present study, we used the capillary electrophoresis-systematic evolution of ligands by exponential enrichment to generate natural and artificial nucleic acid aptamers targeting Ebola virus protein 24 (eVP24), demonstrating that artificial aptamers, synthesised utilising a uridine analogue with an adenine residue at its C5 position, exhibited activities exceeding those of natural ones. To confirm the functionality of the as-prepared aptamers, their abilities to inhibit the PPIs of eVP24 were determined by capillary electrophoresis and bio-layer interferometry, and the obtained results unambiguously demonstrated that these aptamers interacted with the functional site of eVP24 and were thus good antagonists.

Hydrogen peroxide-triggered gene silencing in mammalian cells through boronated antisense oligonucleotides.

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) involved in various diseases, including neurodegeneration, diabetes, and cancer. Here, we introduce a new approach to use H2O2 to modulate specific gene expression in mammalian cells. H2O2-responsive nucleoside analogues, in which the Watson-Crick faces of the nucleobases are caged by arylboronate moieties, were synthesized. One of these analogues, boronated thymidine (dTB ), was incorporated into oligodeoxynucleotides (ODNs) using an automated DNA synthesizer. The hybridization ability of this boronated ODN to complementary RNA was clearly switched in the off-to-on direction upon H2O2 addition. Furthermore, we demonstrated H2O2-triggered gene silencing in mammalian cells using antisense oligonucleotides (ASOs) modified with dTB . Our approach can be used for the regulation of any gene of interest by the sequence design of boronated ASOs and will contribute to the development of targeted disease therapeutics.

Case Report of Atlantoaxial Rotatory Fixation after Cochlear Implantation.

Atlantoaxial rotatory fixation (AARF) is a relatively rare condition and is mainly seen in children. We report of a 7-year-old girl suffering from AARF after cochlear implantation (CI). Fortunately, early diagnosis based on three-dimensional computed tomography (3DCT) was made, and the patient was cured with conservative therapy. Nontraumatic AARF, which is also known as Grisel's syndrome and occurs subsequent to neck infections or ear, nose, and throat (ENT) surgery, represents only a small fraction of AARF cases. Two factors are mainly thought to contribute to the pathogenesis of the condition estimated, namely, (i) neck immaturity in children and (ii) infiltration by inflammatory mediators around the upper neck joint, easily permitted by the neck vasculature. AARF should be suspected in case of torticollis developing after ENT surgery.