Detection of copy number variations in epilepsy using exome data.

Epilepsies are common neurological disorders and genetic factors contribute to their pathogenesis. Copy number variations (CNVs) are increasingly recognized as an important etiology of many human diseases including epilepsy. Whole-exome sequencing (WES) is becoming a standard tool for detecting pathogenic mutations and has recently been applied to detecting CNVs. Here, we analyzed 294 families with epilepsy using WES, and focused on 168 families with no causative single nucleotide variants in known epilepsy-associated genes to further validate CNVs using 2 different CNV detection tools using WES data. We confirmed 18 pathogenic CNVs, and 2 deletions and 2 duplications at chr15q11.2 of clinically unknown significance. Of note, we were able to identify small CNVs less than 10 kb in size, which might be difficult to detect by conventional microarray. We revealed 2 cases with pathogenic CNVs that one of the 2 CNV detection tools failed to find, suggesting that using different CNV tools is recommended to increase diagnostic yield. Considering a relatively high discovery rate of CNVs (18 out of 168 families, 10.7%) and successful detection of CNV with <10 kb in size, CNV detection by WES may be able to surrogate, or at least complement, conventional microarray analysis.

Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses.

In this chapter, we introduce the usage of pHluorin-mKate2-human LC3 for monitoring autophagy. Using EGFP and RFP, tandem fluorescent protein-tagged LC3 has been generated for monitoring autophagic structures. A critical point for this purpose is the sensitivity of the green fluorescent protein to acidic pH. A super-ecliptic pHluorin is most sensitive to acidic pH among EGFP, mWasabi, and pHluorin, indicating pHluorin is most suitable for monitoring autophagic structures. During autophagy, green-positive and red-positive fluorescent puncta of pHluorin-mKate2-human LC3 indicate signals of preautophagosomes and autophagosomes. After fusion of autophagosomes with lysosomes to form autolysosomes, green fluorescence of this intraautophagosomal protein is abolished according to acidification of autolysosomes. Therefore, these green-negative and red-positive fluorescent puncta reflect autolysosomes, in which intraluminal proteins are finally degraded by lysosomal proteases. To monitor autophagic flux, the accumulation of its green-negative and red-positive fluorescent puncta is monitored by inhibiting major lysosomal proteases, cathepsins. In addition, a mutant pHluorin-mKate2-human LC3△G is also introduced as a negative control probe.

Influence of balancing-side occlusal interference on smoothness of working-side condylar movement and intra-articular space in chewing efforts.

Response of temporomandibular joint (TMJ) articulation adapting to occlusal alteration has been sparsely known. For 10 healthy adults with acceptably good occlusion, an artificial occlusal interference (OI) was introduced to the lower molar on the balancing side of unilateral chewing. Subjects were asked to chew a gum on their preferred side. The chewing jaw movements with/without the OI were recorded using a video-based optoelectronic system. The mandibular movements were generated in each individual's TMJ model reconstructed by magnetic resonance images. The smoothness of local condylar point movements towards the normal direction of the condylar surface and interarticular space on the working side was measured. Overall, the smoothness of condylar point movements in the closing phase was impaired immediately after introduction of the OI. In the intercuspal phase, the OI increased the joint space. After about 60 chewing cycles, the movement smoothness and joint space began to recover. These findings suggest that OI on the balancing side induced irregular stress field translation on the working-side condylar surface followed by acute recovery process.

Temporomandibular joint articulations on working side during chewing in adult females with cross-bite and mandibular asymmetry.

Influence of mandibular asymmetry and cross-bite on temporomandibular joint (TMJ) articulation remained unknown. This study aimed to investigate whether/how the working-side condylar movement irregularity and articular spaces during chewing differ between patients with mandibular asymmetry/cross-bite and control subjects. The cross-bite group and the control group consisted of 10 adult female patients and 10 adult female subjects, respectively. They performed unilateral gum-chewing. The mandibular movements were recorded using a video-based opto-electronic system. The 3D articular surface of the TMJ for each individual was reconstructed using CT/MRI data. For local condylar points, the normalised jerk cost (NJC) towards normal direction to the condylar surface, the angle between tangential velocity vector and condylar long axis and intra-articular space were measured. Three rotatory angles at centre of the condyle were also measured. During closing and intercuspation, (i) movements of posterior portion of the deviated side condyle showed significantly less smoothness as compared with those for the non-deviated side and control subjects, (ii) the rotations of the condyle on the deviated side induced greater intra-articular space at posterior and lateral portions. These findings suggest that chewing on the side of mandibular deviation/cross-bite may cause irregular movement and enlarged intra-articular space at posterior portion of the deviated side condyle.

Using breast radiographers' reports as a second opinion for radiologists' readings of microcalcifications in digital mammography.

OBJECTIVE: The aim of this study was to investigate a practical method for incorporating radiographers' reports with radiologists' readings of digital mammograms. METHODS: This simulation study was conducted using data from a free-response receiver operating characteristic observer study obtained with 75 cases (25 malignant, 25 benign and 25 normal cases) of digital mammograms. Each of the rating scores obtained by six breast radiographers was utilized as a second opinion for four radiologists' readings with the radiographers' reports. A logical "OR" operation with various criteria settings was simulated for deciding an appropriate method to select a radiographer's report in all combinations of radiologists and radiographers. The average figure of merit (FOM) of the radiologists' performances was statistically analysed using a jackknife procedure (JAFROC) to verify the clinical utility of using radiographers' reports. RESULTS: Potential improvement of the average FOM of the radiologists' performances for identifying malignant microcalcifications could be expected when using radiographers' reports as a second opinion. When the threshold value of 2.6 in Breast Imaging-Reporting and Data System (BI-RADS®) assessment was applied to adopt/reject a radiographer's report, FOMs of radiologists' performances were further improved. CONCLUSION: When using breast radiographers' reports as a second opinion, radiologists' performances potentially improved when reading digital mammograms. It could be anticipated that radiologists' performances were improved further by setting a threshold value on the BI-RADS assessment provided by the radiographers. ADVANCES IN KNOWLEDGE: For the effective use of a radiographer's report as a second opinion, radiographers' rating scores and its criteria setting for adoption/rejection would be necessary.

Physical impairment and walking function required for community ambulation in patients with cervical incomplete spinal cord injury.

STUDY DESIGN: Cross-sectional study. OBJECTIVE: To identify the physical impairments and walking function required for community ambulation in patients with cervical incomplete spinal cord injury (ISCI). SETTING: Chubu Rosai Hospital, Nagoya, Japan. METHODS: Forty patients with cervical ISCI (mean age: 49.9 years, American Spinal Injury Association Impairment Scale D) were included. The primary outcome measure was community ambulation based on Spinal Cord Independence Measure outdoor scores for a distance of >480 m. We measured the upper- and lower-extremity motor scores (UEMS and LEMS), sensory and spasticity. The walking tests included 10 m of walking at a comfortable- and maximum-walking speed (CWS and MWS; m s(-1)), 6 min walking test (6 MWT; m) and the walking index for spinal cord injury II (WISCI II). Multivariate logistic regression models were used to assess the physical impairments associated with community ambulation. Receiver operating characteristic curves were analyzed to determine the cutoff points for physical impairment and walking function. RESULT: The LEMS (beta coefficient (ß)=0.71) and UEMS (ß=0.41) were independently associated with community ambulation in patients with cervical ISCI. The cutoff points of the LEMS, UEMS, CWS, MWS, 6MWT and WISCI II were 41.5, 36.5, 1.00 m s(-1), 1.32 m s(-1), 472.5 m and 17.5, respectively, which suggests moderate to high accuracy. CONCLUSION: The LEMS and UEMS were the most important factors affecting community ambulation in patients with cervical ISCI. The cutoff points of the walking function tests were highly accurate; therefore, these points can serve as targets for walking training in the future.

MRI in a case of osteosarcoma in the temporomandibular joint.

Osteosarcoma of the temporomandibular joint (TMJ) is rare. We report a case of osteosarcoma in the TMJ of a 62-year-old female, pre-operatively diagnosed to have a benign tumour, and discuss the usefulness and limits of MRI using a TMJ coil as a diagnosis.

CT and MR imaging features of a case of calcifying epithelial odontogenic tumor.

Calcifying epithelial odontogenic tumor is a rare lesion. We report the imaging features of a calcifying epithelial odontogenic tumor. The imaging including conventional radiograph, CT and MR imaging revealed a well-defined lesion in the alveolar bone of the left maxilla, which contained an impacted tooth and some small radiopacities. CT and MR imaging demonstrated a contrast enhancement mainly at the central portion of the lesion.

Calcifying cystic odontogenic tumour: CT imaging.

OBJECTIVES: Calcifying cystic odontogenic tumour (CCOT) is a rare disorder of the jaw. A comparison between conventional radiographs and CT images in CCOTs has not been reported. The purposes of this study were to analyse conventional radiographs and CT images of CCOTs, establish CT images of CCOTs and assess the utility of CT in the diagnosis of CCOTs. METHODS: Nine patients with a histopathologically confirmed CCOT who had both conventional radiographs and CT images were enrolled. RESULTS: CT was superior to conventional radiographs in detecting buccolingual expansion, odontomas and radio-opaque bodies. CONCLUSION: The characteristic CT appearances of CCOT were that radio-opaque bodies were typically located in the periphery of the lesion and the shape of radio-opaque bodies was linear and/or spotted. CT was useful in diagnosing a CCOT.

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy.

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.

Radiation dose measurement with newly developed head phantom for interventional neuroradiology.

Although it is important to measure and record radiation dose to a patient's skin, no standardized measuring method is available to compare the performance of digital subtraction angiography (DSA) systems with those of other institutions. We developed a new bidirectional information exchange system using a web page and e-mail. Measurements are obtained using an original head phantom with skin dose monitors. This system was sent to institutions ordering it from the web page. The data obtained with this system by each institution were evaluated and displayed anonymously on a graph on the web page for comparison. This method for measuring radiation dose allows the comparison of DSA systems under identical conditions. This system can alert the user that the radiation dose delivered using ordinary parameters differs from that of other institutions or DSA systems. To promote the use of this system, we developed a web page with information about radiation dose measuring and initiated an online lending operation. This head phantom and standardized measuring method allows the accurate comparison of skin radiation dose and DSA performance under the same conditions.

A pilot study of EUS-guided hot saline injection for induction of pancreatic tissue necrosis.

BACKGROUND AND STUDY AIMS: Hot saline may be potentially useful for inducing necrosis of pancreatic tissue. However, the local and systemic effects are largely unknown. This pilot study aimed to evaluate the feasibility and safety of EUS-guided injection of hot saline into the pancreas in the porcine model. METHODS: Boiling hot saline was injected into the tail of normal porcine pancreas under EUS guidance in six pigs via a transgastric approach. Three pigs were killed 4 hours later to study the acute effect of the hot saline injection (acute study). The remaining three pigs were killed after 7 days of clinical observation (survival study). RESULT: Injection of 5 mL, 2 mL and 1 mL of hot saline produced localized necrosis (7 - 10 mm) of pancreatic tissue in the acute study. However, there was pooling of hot saline on the surface of the pancreas when 5mL was injected. On the basis of the results of the acute study, the volume of hot saline injected in the survival study was 1 mL. One milliliter of hot saline produced localized or sporadic necrosis of pancreatic tissue without any signs of pancreatitis in all three pigs in the survival study; hot saline was observed to pool on the pancreatic surface of one pig. There was no histological evidence of necrosis in the pancreatic tissue adjacent to the pooled hot saline in either the acute or the survival study. CONCLUSION: EUS-guided hot saline injection of pancreatic tissue in the porcine model was technically successful and led to localized necrosis of pancreatic tissue without any sign of pancreatitis.

The utility of FDG-PET for detecting multiple primary cancers in hypopharyngeal cancer patients.

AIM: To examine the utility of 2'-[18F]-fluoro-2'-deoxy-D-glucose positron emission tomography (FDG-PET) for detecting multiple primary cancers (MPC) in patients with hypopharyngeal cancer (HPC). PATIENTS, METHODS: Seventy patients with HPC underwent FDG-PET to determine the staging. Routine clinical examinations were carried out, including computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and oesophagealgastroduodenoscopy (EGDS). The detection rate of synchronous and metachronous cancer was calculated based on FDG-PET alone or FDG-PET combined with clinical routine examination. Sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and accuracy were used to diagnose oesophageal cancer using FDG-PET. RESULTS: Of the 70 patients, 12 (17.1%) had 15 synchronous tumours, and 2 of the 58 remaining patients (3.4%) had metachronous tumours. Oesophageal cancer was discovered most frequently: superficial type (n=6), advanced type (n=4). On a per-patient basis, 11 of 12 patients (91.6%) were diagnosed with synchronous tumours, and on a per-lesion basis, 12 of 15 lesions (80.0%) were detected by FDG-PET. The sensitivity, specificity, accuracy, PPV, and NPV of FDG-PET regarding oesophageal cancer were 70%, 100%, 95.7%, 100%, and 95.2% respectively. Three of the six superficial types were positive on FDG-PET. Both of the metachronous tumour lesions were detected by FDG-PET. CONCLUSION: FDG-PET is useful for estimating the MPC in HPC patients. Since 3 of 10 synchronous oesophageal cancer were missed with PET alone, a combination with EGDS should be considered to exclude synchronous oesophageal cancer.

Morphological and biochemical signs of age-related neurodegenerative changes in klotho mutant mice.

Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported.

Influence of mouthguard on temporomandibular joint.

The aim of this study is to examine whether wearing a mouthguard (MG) has an influence on temporomandibular joint (TMJ) components (i.e., the condyle and the articular disk) and whether clenching with a resilient MG has an effect on the same components. Twenty-six healthy volunteers (15 females, 11 males) with an age range of 26-42 years old (median 28 years) participated in this study. Among all 52 joints in the 26 subjects, anterior disk displacement (AntDD) was recognized in 15. Thus, we classified the 52 joints into two groups, the AntDD group and the Normal group. Resilient thermoplastic materials were used to fabricate two types of MG, one that raised the vertical dimension height by 3 mm and another that raised it by 6 mm. Subjects were scanned with/without two types of MG with/without clenching by magnetic resonance imaging (MRI). The movement of the condylar head and articular disk was measured in the magnetic resonance images using the special subtraction technique. Wearing the MG without clenching did not have a negative influence on the TMJ and clenching in the Normal group. In the AntDD group, however, the relationship between the disk and condyle was affected by clenching and the degree of disk displacement was worsened by clenching with the thicker MG. From our results, we recommend that athletes with an internal derangement of the TMJ not wear the thicker MG and attention should be paid to its setting. And the subtraction technique using MRI was thought as a good tool for detecting a slight change in the TMJ.

[The efficacy of perioperative administration of steroid and erythromycin in the surgery for lung cancer complicated with interstitial pneumonia].

OBJECTIVES: We evaluated the efficacy of perioperative administration of steroid and erythromycin in surgery for lung cancer complicated with interstitial pneumonia (IP) to prevent postoperative acute exacerbation. PATIENTS AND METHODS: We operated on 21 lung cancer patients with IP for 10 years. The patients were given 400 mg of erythromycin over 1 week before surgery and re-administered on the 1st operative day. The patients were also given 125 mg of methylprednisolone intravenously just before operation and continued until the 2nd operative day. RESULTS: Lobectomy was performed in 16, segmentectomy or partial resection in 2 each, and completion pneumonectomy in 1. Three patients developed acute exacerbation of IP, but it occurred after the re-operation due to postoperative complications in 2. We experienced no operative death within 30 days, however, 2 died during the hospital stay due to multiple organ failure and sepsis. Seven of 21 patients had postoperative complications; air leakage over 1 week in 4, arrhythmia in 3, and atelectasis, postoperative bleeding, and pneumonia in 1 each, the morbidity rate was 33%. CONCLUSIONS: We conclude that the administration of steroid and erythromycin in surgery for lung cancer with IP was suspected the usefulness to prevent a postoperative acute exacerbation of IP.

Constitutive autophagy: vital role in clearance of unfavorable proteins in neurons.

Investigations pursued during the last decade on neurodegenerative diseases have revealed a common mechanism underlying the development of such diseases: conformational disorder of certain proteins leads to the formation of misfolded protein oligomers, which subsequently develop into large protein aggregates. These aggregates entangle other denatured proteins and lipids to form disease-specific inclusion bodies. The failure of the ubiquitin-proteasome system to shred the protein aggregates has led investigators to focus their attention to autophagy, a bulk degradative system coupled with lysosomes, which is involved in non-selective shredding of large amounts of cytoplasmic components. Research in this field has demonstrated the accumulation of autophagic vacuoles and intracytoplasmic protein aggregates in patients with various neurodegenerative diseases. Although autophagy fails to degrade large protein aggregates once they are formed in the cytoplasm, drug-induced activation of autophagy is effective in preventing aggregate deposition, indicating that autophagy significantly contributes to the clearance of aggregate-prone proteins. The pivotal role of autophagy in the clearance of aggregate-prone proteins has been confirmed by a deductive approach using a brain-specific autophagy-ablated mouse model. In this review, we discuss the consequences of autophagy deficiency in neurons.