Synthetic Circular RNA for microRNA-1269a Suppresses Tumor Progression in Oral Squamous Cell Carcinoma.

microRNAs (miRs) function in cancer progression as post-transcriptional regulators. We previously reported that endogenous circular RNAs (circRNAs) function as efficient miR sponges and could act as novel gene regulators in oral squamous cell carcinoma (OSCC). In this study, we carried out cellular and luciferase reporter assays to examine competitive inhibition of miR-1269a, which is upregulated expression in several cancers, by circRNA-1269a, a synthetic circRNA that contains miR-1269a binding sequences. We also used data-independent acquisition (DIA) proteomics and in silico analyses to determine how circRNA-1269a treatment affects molecules downstream of miR-1269a. First, we confirmed the circularization of the linear miR-1269a binding site sequence using RT-PCR with divergent/convergent primers and direct sequencing of the head-to-tail circRNA junction point. In luciferase reporter and cellular functional assays, circRNA-1269a significantly reduced miR-1269a function, leading to a significant decrease in cell proliferation and migration. DIA proteomics and gene set enrichment analysis of OSCC cells treated with circRNA-1269a indicated high differential expression for 284 proteins that were mainly enriched in apoptosis pathways. In particular, phospholipase C gamma 2 (PLCG2), which is related to OSCC clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Taken together, synthetic circRNA-1269a inhibits tumor progression via miR-1269a and its downstream targets, indicating that artificial circRNAs could represent an effective OSCC therapeutic.

[Novel Application of Post-contrast T1map for Detection of Subendocardial Infarction].

In cardiac magnetic resonance (CMR) for myocardial infarction, there have been quite a few cases of obscure image contrast between subendocardial lesion and left ventricular (LV) blood pool on late gadolinium enhancement (LGE) images. This study was motivated by confirmation of usefulness of post-contrast T1map for detection of subendocardial infarction. From June 2017 to May 2018, forty-eight consecutive patients who underwent contrast-enhanced CMR to assess myocardial infarction were reviewed. We measured the contrast ratio (CR) between the infarcted myocardium and LV blood pool on LGE and on post-contrast T1map images, and compared them. The CR (mean±standard deviation) was -0.04±0.11 for LGE images and 0.02±0.04 for post-contrast T1map images (P<0.05). These results suggest that the post-contrast T1map, which uses the difference in T1 value as image contrast rather than magnitude image, can clearly depict the boundary between the infarcted myocardium and LV blood pool. The addition of post-contrast T1map to image interpretation might provide valuable information in the evaluation of subendocardial infarction.

A novel cell-based transplantation method using a Rho kinase inhibitor and a specific catheter device for the treatment of salivary gland damage after head and neck radiotherapy.

Radiotherapy (RT) for head and neck cancer results in irreversible damage to salivary glands (SGs) and decreases saliva production, leading to a dry mouth. To date, there are no satisfactory therapies to solve this problem. We recently established a novel culturing method using a Rho kinase inhibitor (RI), Y-27632, that maintained cellular morphology and function for a prolonged period of time. In the present study, we investigated whether cell-based transplantation using our culturing method ameliorated the dysfunction of irradiated SGs. First, rat SG cells were cultured in a medium with RI. Cells were characterized by morphological findings and mRNA expression analysis. We also assessed features of SG cells in three-dimensional (3-D) culture by scanning electron microscopy and immunohistochemistry (IHC). The RI-containing medium led to higher cell proliferation of rat SG cells with preservation of cell morphology and higher alpha-amylase (AMY) expression in both 2-D and 3-D culture systems. To establish the atrophic-SG models, external RT at a dose of 15 Gy was delivered to the head and neck fields of nude rats. The SG cells derived from GFP-rats were cultured in medium with RI, after which they were transplanted into the submandibular glands of atrophic-SG rats using a catheter placed into Wharton's duct. IHC and salivary flow rate (SFR) analyses were measured 12 weeks after the transplantation. Following transplantation, donor cells (GFP-SG cells) were primarily located in the ductal region of the SG, and AMY expression in SGs and the SFR were increased in the SG cell transplantation group compared with the control. Those data indicated that cell-based therapy using RI-treated SG cells could restore salivary hypofunction of irradiated SGs by direct integration of the donor cells in the duct of SGs. We propose that these data support future clinical plans in which SG cells would be excised from the labial minor SGs of the patients with head and neck cancers prior to RT, cultured during RT, and auto-transplanted into SGs using a catheter into the Wharton's duct. We believe that our culturing and transplantation methods can be applied to SG cells, constituting a therapeutic approach for the treatment of patients with dry mouth after not only RT but also aging and Sjögren's syndrome.

Assay for transposase-accessible chromatin with high-throughput sequencing reveals radioresistance-related genes in oral squamous cell carcinoma cells.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), and radioresistance is a critical factor resulting in poor outcomes. Several genes have been reported to be therapeutic targets for radioresistance; however, the involvement of chromatin accessibility in radioresistance has not been clarified in OSCC cells. Accordingly, in this study, we evaluated chromatin accessibility in radioresistant (HSC-3) and radiosensitive (KOSC-2) cells, identified from nine OSCC cell lines using clonogenic survival assays after irradiation. Chromatin accessibility in radioresistant OSCC cells was assessed using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Gene expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot analysis. Viability was assessed by MTS assay. We found 1273 peaks (open chromatin regions by ATAC-seq) related to 8 Gy irradiation in HSC-3 but not KOSC-2 cells, among which 235 genes located around the chromatin open peaks were identified by ChIPpeakAnno analysis. Subsequently, 12 genes were selected as signal transduction-related genes by Gene Ontology analysis, and gene expression was confirmed by RT-qPCR. Among these genes, adenylate cyclase 2 (ADCY2) was significantly upregulated after treatment with irradiation in HSC-3 but not KOSC-2 cells. To further evaluate ADCY2 function in radioresistant cells, we performed ADCY2 knockdown by transfection of HSC-3 cells with small interfering RNA (siADCY2). Cell viability after irradiation was significantly decreased in siADCY2-transfected cells compared with that in control cells. These results suggested that ADCY2 expression was related to the open chromatin region in radioresistant OSCC cells and that ADCY2 may have therapeutic efficacy when used in combination with radiotherapy in patients with OSCC.

Ginkgolide B Regulates CDDP Chemoresistance in Oral Cancer via the Platelet-Activating Factor Receptor Pathway.

The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways, including regulating the activation of kinases. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). We first analyzed the correlation between PAFR expression and CDDP sensitivity in seven OSCC-derived cell lines based upon cell viability assays. Among them, we isolated 2 CDDP-resistant cell lines (Ca9-22 and Ho-1-N-1). In addition to conducting PAFR-knockdown (siPAFR) experiments, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. We next evaluated the downstream signaling pathway of PAFR in siPAFR-treated cells and GB-treated cells after CDDP treatment. In both cases, we observed decreased phosphorylation of ERK and Akt and increased expression of cleaved caspase-3. These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients.

Tumor Suppressive Circular RNA-102450: Development of a Novel Diagnostic Procedure for Lymph Node Metastasis from Oral Cancer.

Circular RNAs (circRNAs), which form as covalently closed loop structures, have several biological functions such as regulation of cellular behavior by adsorbing microRNAs. However, there is limited information of circRNAs in oral squamous cell carcinoma (OSCC). Here, we aimed to elucidate the roles of aberrantly expressed circRNAs in OSCC. CircRNA microarray showed that circRNA-102450 was down-regulated in OSCC cells. Clinical validation of circRNA-102450 was performed using highly sensitive droplet digital PCR in preoperative liquid biopsy samples from 30 OSCC patients. Interestingly, none of 16 studied patients with high circRNA-102450 had regional lymph node metastasis (RLNM), whereas 4 of 14 studied patients (28.5%) with low expression had pathologically proven RLNM. Overexpressed circRNA-102450 significantly inhibited the tumor metastatic properties of cell proliferation, migration, and invasion. Furthermore, circRNA-102450 directly bound to, and consequently down-regulated, miR-1178 in OSCC cells. Taken together, circRNA-102450 has a tumor suppressive effect via the circRNA-102450/miR-1178 axis and may be a novel potential marker of RLNM in OSCC patients.

Engineered exosomes delivering specific tumor-suppressive RNAi attenuate oral cancer progression.

Exosomes are involved in a wide range of biological processes in human cells. Considerable evidence suggests that engineered exosomes (eExosomes) containing therapeutic agents can attenuate the oncogenic activity of human cancer cells. Despite its biomedical relevance, no information has been available for oral squamous cell carcinoma (OSCC), and therefore the development of specific OSCC-targeting eExosomes (octExosomes) is urgently needed. We demonstrated that exosomes from normal fibroblasts transfected with Epstein-Barr Virus Induced-3 (EBI3) cDNA were electroporated with siRNA of lymphocyte cytoplasmic protein 1 (LCP1), as octExosomes, and a series of experiments were performed to evaluate the loading specificity/effectiveness and their anti-oral cancer cell activities after administration of octExosomes. These experiments revealed that octExosomes were stable, effective for transferring siLCP1 into OSCC cells and LCP1 was downregulated in OSCC cells with octExosomes as compared with their counterparts, leading to a significant tumor-suppressive effect in vitro and in vivo. Here we report the development of a new valuable tool for inhibiting tumor cells. By engineering exosomes, siLCP1 was transferred to specifically suppress oncogenic activity of OSCC cells. Inhibition of other types of human malignant cells merits further study.

Clinical significance of gadobutrol in magnetic resonance imaging for the detection of myocardial infarction: matched-pair cohort study to compare with gadopentetate dimeglumine at standard dose.

We aimed to confirm whether gadobutrol is more useful for late gadolinium enhancement (LGE) imaging than gadopentetate dimeglumine (Gd-DTPA) at the standard dose. Patients who underwent LGE imaging to assess myocardial infarction were retrospectively enrolled: gadobutrol, 51 cases; Gd-DTPA, 49 cases. Contrast ratios of infarcted lesion to remote myocardium (CRremote) and to left ventricular blood (CRblood) were compared. Patient characteristics that might affect image contrast did not differ between groups. CRremote (median, (interquartile range)) at 10 and 15 min after administration was 0.79 (0.08) and 0.70 (0.09) for gadobutrol, and 0.74 (0.13) and 0.65 (0.16) for Gd-DTPA (P < 0.05 and < 0.05), respectively. CRblood was - 0.05 (0.17) and - 0.002 (0.15) for gadobutrol, and - 0.05 (0.18) and 0.01 (0.16) for Gd-DTPA (P = 0.29 and = 0.22), respectively. Gadobutrol provided significantly better delineation of infarcted from normal myocardium than Gd-DTPA. Meanwhile, there was no difference in image contrast between infarcted myocardium and left ventricular blood.

[Clinical Usefulness of Ultra-short TE MRA for Follow-up Imaging after Cerebral Aneurysm Clipping].

The aims of this study were to elucidate signal pattern of cerebral aneurysm clip in brain magnetic resonance angiography (MRA) using non-contrast enhanced ultra-short echo time (UTE) sequence and to explore effective utilization of this novel technique for patients, who underwent cerebral aneurysm clipping. The clip was embedded in homemade phantom and scanned using UTE sequence. We investigated characteristic features of the artifacts derived from the clip. Besides, we compared the volume of signal loss between conventional time-of-flight (TOF) and UTE-MRA in 50 patients with the cerebral aneurysm clip. In phantom study, the clip was delineated as signal void area fully surrounded by high signal on original images. On reconstructed short-axial views for the clip, four-leaf clover pattern of artifact was observed when clip was arranged orthogonal to the static magnetic field. On the other hand, this artifact disappeared when the clip was arranged in parallel with the static magnetic field. The volume of signal loss in clinical cases was significantly reduced in UTE-MRA (P < 0.05): 1.30, 0.52-2.77 cm3 for TOF; 0.84, 0.28-1.74 cm3 for UTE (median, range). The scan time for UTE-MRA was 2 minutes and 52 seconds. To understand the characteristic feature of the artifacts from the clip could contribute to define vascular structure in image interpretation. Adding UTE-MRA to routine protocol is useful approach for follow-up imaging after cerebral aneurysm clipping with clinically acceptable prolongation of the scan time.

[A Case of Small Cell Lung Cancer Diagnosed with Metastasis-Induced Acute Pancreatitis].

A 56-year-old man was admitted to our hospital for management of acute epigastric abdominal pain and elevation of pancreatic enzymes. The CT scan revealed enlargement ofthe pancreatic body as well as the lung tumor ofthe right hilar and superior mediastinum. Therefore, bronchoscopy was performed and a diagnosis of small cell lung cancer with metastasisinduced acute pancreatitis(MIAP)was made. Prompt improvement in pancreatic findings was observed following chemotherapy. MIAP, which is a rare complication of lung cancer may affect the prognosis and quality of life of the patients; therefore, rapid diagnosis and appropriate treatment are important.

Quantification of the radiation dose to the pyramidal tract using tractography in treatment planning for stereotactic radiosurgery.

In stereotactic radiosurgery for intracranial lesions, optimization of the dose to the at-risk organs is important to avoid neurological complications. We aimed to quantify the dose to the pyramidal tract (PT) and improve treatment planning for gamma knife radiosurgery by combining tractography. Pyramidal tractography images were depicted in 23 patients with lesions adjacent to the PT and fused with stereotactic magnetic resonance images. We regarded the PT as an at-risk organ and performed dose planning. To assess the efficacy of this process, we compared clinical parameters between plans with and without tractography. In the plans with tractography, the maximum PT dose was significantly reduced, although the irradiation time was prolonged by 3.5 min. There was no significant difference in the dose covering 95% of the lesion volume (D95). This result suggests that the PT dose can be reduced while maintaining the D95 with clinically acceptable prolongation of the irradiation time.

Frequency Extension to the THz Range in the High Pressure ESR System and Its Application to the Shastry-Sutherland Model Compound SrCu2(BO3)2.

We have made a survey of ceramics for the inner parts of the transmission-type pressure cell to achieve the high pressure and the high transmission in the THz range. By using the optimal combination of ZrO2-based ceramic and Al2O3 ceramic, we have succeeded in obtaining a pressure up to 1.5 GPa and a frequency region up to 700 GHz simultaneously. We show the high-pressure ESR results of the Shastry-Sutherland compound SrCu2(BO3)2 as an application. We observed the direct ESR transition modes between the singlet ground state and the triplet excited states up to a pressure of 1.51 GPa successfully, and obtained the precise pressure dependence of the gap energy. The gap energy is directly proved to be suppressed by the pressure. Moreover, we found that the system approaches the quantum critical point with pressure by comparing the obtained data with the theory. This result also shows the usefulness of high-pressure ESR measurement in the THz region to study quantum spin systems.

A new reference point for patient dose estimation in neurovascular interventional radiology.

In interventional radiology, dose estimation using the interventional reference point (IRP) is a practical method for obtaining the real-time skin dose of a patient. However, the IRP is defined in terms of adult cardiovascular radiology and is not suitable for dosimetry of the head. In the present study, we defined a new reference point (neuro-IRP) for neuro-interventional procedures. The neuro-IRP was located on the central ray of the X-ray beam, 9 cm from the isocenter, toward the focal spot. To verify whether the neuro-IRP was accurate in dose estimation, we compared calculated doses at the neuro-IRP and actual measured doses at the surface of the head phantom for various directions of the X-ray projection. The resulting calculated doses were fairly consistent with actual measured doses, with the error in this estimation within approximately 15%. These data suggest that dose estimation using the neuro-IRP for the head is valid.

Ultrahigh-Q one-dimensional photonic crystal nanocavities with modulated mode-gap barriers on SiO2 claddings and on air claddings.

We report designs for a silicon-on-insulator (SOI) one-dimensional (1D) photonic crystal (PhC) nanocavity with modulated mode-gap barriers based on the lowest dielectric band. These cavities have an ultrahigh theoretical quality factor (Q) of 10(7)-10(8) while maintaining a very small modal volume of 0.6-2.0 (lambda/n)(3), which are the highest Q for any nanocavities with SiO(2) under-cladding. We have fabricated these SOI 1D-PhC cavities and confirmed that they exhibited a Q of 3.6 x 10(5), which is also the highest measured Q for SOI-type PhC nanocavities. We have also applied the same design to 1D PhC cavities with air claddings, and found that they exhibit a theoretical quality factor higher than 10(9). The fabricated air-cladding 1D Si PhC cavities have showed a quality factor of 7.2 x 10(5), which is close to the highest Q value for 1D PhC cavities.

Oxidation of unsaturated carboxylic acids under hydrothermal conditions.

Hydrothermal oxidation pathways of high molecular weight unsaturated carboxylic acids were investigated for the potential use of chemoselectivity to improve the efficiency of the desired products from biomasses directly containing or easily producing unsaturated carboxylic acids. Hock cleavage, which frequently occur at general chemical, was observed in the absence of any acid catalyst and may be a potential major oxidation cleavage mechanism, which leads to the cleavage at both the carbon-carbon double bond and the single bond near a double bond. The addition of a peroxyl radical to the double bond may be also a potential major oxidation mechanism, which leads to the oxidation cleavage mainly at the carbon-carbon double bond. Cleavage at the carbon-carbon bond near the double bond by the addition of a peroxyl radical to the double bond may also occur. However, oxidation at either alpha-, beta-, or gamma-carbon to the -COOH group hardly occurred. These results may help to selectively produce desired products from biomasses, such as lignin and oils.