Dysferlin-deficient myotubes show tethering of different membrane compartments characterized by TMEM16E and DHPRα.

TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.

Major non-cardiac surgery is a risk factor for rapid hemodynamic progression of non-rheumatic aortic stenosis.

BACKGROUND: Inflammatory processes are suggested to play a pathogenic role in the development and progression of non-rheumatic aortic stenosis (AS). Major surgery causes an inflammatory reaction. With the increasing prevalence of non-rheumatic AS, the number of affected patients undergoing major surgery increases. We hypothesized that major non-cardiac surgery (MNCS) could accelerate the progression of non-rheumatic AS. METHODS AND RESULTS: We enrolled 218 consecutive patients with non-rheumatic AS who underwent transthoracic echocardiography (TTE) at least twice more than 6 months apart. Study patients were divided into the MNCS group and the non-MNCS group. The MNCS group consisted of patients who underwent MNCS during the TTE follow-up interval. At baseline, peak pressure gradient across the aortic valve (AVG) was similar between the groups. Also baseline clinical characteristics and TTE follow-up interval were similar. The annual rate of peak AVG increase was much higher in the MNCS group than in the non-MNCS group. The proportion of patients with rapid hemodynamic progression was much higher in the MNCS group than in the non-MNCS group. Multiple logistic regression analysis showed that MNCS was an independent predictor of rapid hemodynamic progression of non-rheumatic AS. CONCLUSIONS: The present study indicates for the first time that MNCS is associated with the rapid progression of non-rheumatic AS.

Echocardiographic characteristics of patients with acute heart failure requiring tolvaptan: a retrospective study.

BACKGROUND: No study has investigated the admission echocardiographic characteristics of acute heart failure (AHF) patients who are resistant to conventional diuretics and require tolvaptan. METHODS: We retrospectively analyzed the echocardiographic characteristics of AHF patients who were resistant to conventional diuretics and took tolvaptan (tolvaptan group: 26 patients), and compared them to those who were sensitive to conventional diuretics (conventional group: 180 patients). RESULTS: The tolvaptan group had a higher left atrial volume index (96.0 ± 85.0 mL/m2 vs. 45.8 ± 25.9 mL/m2, p < 0.0001), maximum inferior vena cava diameter (20.7 ± 6.9 mm vs. 18.1 ± 4.2 mm, p < 0.01), and higher tricuspid regurgitation grade (1.1 ± 0.8 vs. 0.8 ± 0.6, p < 0.05) than the conventional group. However, the left ventricular ejection fraction and end diastolic diameter were similar between the groups. Responders of tolvaptan had no significant echocardiographic differences compared to the non-responders. CONCLUSIONS: The admission echocardiographic characteristics of AHF patients requiring tolvaptan included a larger left atrium, inferior vena cava, and more severe tricuspid regurgitation. Echocardiography may provide useful information for the early and appropriate initiation of tolvaptan.

TMEM16E (GDD1) exhibits protein instability and distinct characteristics in chloride channel/pore forming ability.

TMEM16E/GDD1 has been shown to be responsible for the bone-related late-onset disease gnathodiaphyseal dysplasia (GDD), with the dominant allele (TMEM16E(gdd) ) encoding a missense mutation at Cys356. Additionally, several recessive loss-of-function alleles of TMEM16E also cause late-onset limb girdle muscular dystrophy. In this study, we found that TMEM16E was rapidly degraded via the proteasome pathway, which was rescued by inhibition of the PI3K pathway and by the chemical chaperone, sodium butyrate. Moreover, TMEM16E(gdd) exhibited lower stability than TMEM16E, but showed similar propensity to be rescued. TMEM16E did not exhibit cell surface calcium-dependent chloride channel (CaCC) activity, which was originally identified in TMEM16A and TMEM16B, due to their intracellular vesicle distribution. A putative pore-forming domain of TMEM16E, which shared 39.8% similarity in 98 amino acids with TMEM16A, disrupted CaCC activity of TMEM16A via domain swapping. However, the Thr611Cys mutation in the swapped domain, which mimicked conserved cysteine residues between TMEM16A and TMEM16B, reconstituted CaCC activity. In addition, the GDD-causing cysteine mutation made in TMEM16A drastically altered CaCC activity. Based on these findings, TMEM16E possesses distinct function other than CaCC and another protein-stabilizing machinery toward the TMEM16E and TMEM16E(gdd) proteins should be considered for the on-set regulation of their phenotypes in tissues.

Detection of latent anthracycline-induced cardiotoxicity using left ventricular end-systolic wall stress-velocity of circumferential fiber-shortening relationship.

Although cardiotoxicity is a well-known side effect of anthracycline, detection of subclinical impairment of myocardial contractility at the latent stage is difficult. The left ventricular end-systolic wall stress (WS)-velocity of circumferential fiber-shortening (VCF) relationship reflects the load-independent myocardial contractility and can detect sensitively intrinsic abnormalities in myocardial contractility. Usefulness of this relationship in detecting subclinical anthracycline-induced cardiotoxicity has not yet been established. We investigated whether latent anthracycline-induced cardiotoxicity at the subclinical state can be detected by using the WS-VCF relationship in patients receiving anthracycline therapy. We studied 45 patients who had received anthracycline therapy and 40 healthy controls. All patients had preserved left ventricular ejection fraction (LVEF). WS and VCF were measured using echocardiography. VCF was corrected by heart rate. The WS-VCF relationship was derived by linear regression. Patients with data points lying below -2 SD derived from controls were regarded as having impaired intrinsic myocardial contractility. Although VCF was within normal limits in all patients, it was significantly reduced in the patient group overall compared with the control group. On the other hand, WS was significantly increased in the patient group overall compared with the control group. The WS-VCF relationship demonstrated impaired intrinsic myocardial contractility in 24 patients (53.3 %). In more than half of patients with preserved LVEF, impairment of intrinsic myocardial contractility was detected using the WS-VCF relationship, suggesting the presence of latent anthracycline-induced cardiotoxicity. The WS-VCF relationship may be able to detect sensitively latent anthracycline-induced cardiotoxicity at the subclinical stage.

Aesthetic recovery of alveolar atrophy following autogenous onlay bone grafting using interconnected porous hydroxyapatite ceramics (IP-CHA) and resorbable poly-L-lactic/polyglycolic acid screws: case report.

BACKGROUND: Onlay bone grafting techniques have some problems related to the limited volume of autogenous grafted bone and need for surgery to remove bone fixing screws. Here, we report a case of horizontal alveolar ridge atrophy following resection of a maxillary bone cyst, in which autogenous onlay bone grafting with interconnected porous hydroxyapatite ceramics (IP-CHA) and bioresorbable poly-L-lactic/polyglycolic acid (PLLA-PGA) screws was utilized. CASE PRESENTATION: A 51-year-old man had aesthetic complications related to alveolar atrophy following maxillary bone cyst extraction. We performed onlay grafting for aesthetic alveolar bone recovery using IP-CHA to provide adequate horizontal bone volume and PLLA-PGA screws for bone fixing to avoid later damage to host bone during surgical removal. During the operation, an autogenous cortical bone block was collected from the ramus mandibular and fixed to the alveolar ridge with PLLA-PGA screws, then the gap between the bone block and recipient bone was filled with a granular type of IP-CHA. Post-surgery orthopantomograph and CT scan findings showed no abnormal resorption of the grafted bone, and increased radiopacity, which indicated new bone formation in the area implanted with IP-CHA. CONCLUSION: Our results show that IP-CHA and resorbable PLLA-PGA screws are useful materials for autogenous onlay bone grafting.

[Development of an magnetic resonance imaging safety management system for metallic biomedical products using an magnetic resonance compatibility database and inquiry-based patient records].

Several incidents involving magnetic resonance imaging (MRI) examinations of patients with unchecked MR-unsafe metallic products have been reported. To improve patient safety, we developed a new MRI safety management system for metallic biomedical products and evaluated its efficiency in clinical practice. Our system was integrated into the picture archiving and communication system (PACS) and comprised an MR compatibility database and inquiry-based patient records of internal metallic biomedical products, enabling hospital staff to check MR compatibility by product name. A total of 6,637 biomedical implants and devices were listed in this system, including product names and their MR compatibilities. Furthermore, MRI histories for each patient at our hospital were also recorded. Using this system, it was possible to confirm the MR compatibility of the patients' metallic biomedical products effectively and to reduce the number of unchecked internal products through systematic patient inquiry. In conclusion, our new system enhanced metallic biomedical product checking procedures, and improved patient safety during clinical MRI examinations.

Evaluation of left ventricular diastolic function by fractional area change using cine cardiovascular magnetic resonance: a feasibility study.

BACKGROUND: Evaluation of left ventricular (LV) diastolic function is essential for the management of heart failure. We verified whether LV diastolic function could be evaluated by measuring the fractional area change (FAC) using cine cardiovascular magnetic resonance (CMR). METHODS: We collected clinical data from 59 patients who underwent echocardiography and cine CMR. Normal, impaired relaxation, pseudonormal, and restrictive LV filling were observed in 15, 28, 11, and 5 patients, respectively. We calculated FAC during the first 30% of diastole (diastolic-index%) in the short-axis view, by tracing the contours on only three MR cine images. RESULTS: The diastolic index was significantly lower (p < 0.0001) in patients with impaired relaxation (32.4 ± 7.5), pseudonormal filling (25.4 ± 5.6), and restrictive filling (9.5 ± 1.5) compared to those with normal diastolic function (67.7 ± 10.8), and the index decreased significantly with worsening of diastolic dysfunction. The diastolic index correlated positively with early diastolic mitral annular velocity measured by tissue Doppler imaging (r = 0.75, p < 0.0001), respectively. CONCLUSIONS: Measurement of FAC can be useful for the evaluation of LV diastolic function using cine CMR.

Primary cardiac lymphoma: echocardiography and F-18-fluorodeoxyglucose positron emission tomography in selection of a biopsy site.

A 63-year-old man was referred to our hospital because of a cardiac tumor. Transthoracic echocardiography revealed a rough, mobile tumor in the dilated right atrium, and transesophageal echocardiography showed that the tumor consisted of small, botryoidal masses. Catheter-based biopsy carried a high risk of embolism. Therefore, we used F-18-fluorodeoxyglucose positron emission tomography (FDG-PET), which revealed an abnormal accumulation in the right cervical lymph nodes, as well as in the heart. We safely performed biopsy of the lymph nodes and diagnosed the patient with primary cardiac lymphoma. We concluded that echocardiography and FDG-PET are useful for selecting an appropriate biopsy site in primary cardiac lymphoma.

Development of Kampo（traditional Japanese medicine）e-learning program: evaluation of the flipped classroom for medical students.

In May 2019, the World Health Assembly, in an unprecedented move, endorsed the inclusion of traditional medicine in the International Classification of Diseases, 11th Revision. In Japan, traditional medicine (known as Kampo) is regulated by the government and prescribed by over 90% of physicians along with modern medicine under the national health insurance system. Although Kampo education must be included in Japan's core medical curricula, there are significant challenges to implementation. In the educational context, the flipped classroom teaching method has received considerable attention in recent years. This study developed a Kampo e-learning program and verified the effectiveness of a flipped classroom using Kampo e-learning. The Kampo e-learning Committee determined three courses and assigned an administrator for each. The administrators appointed lecturers who developed Kampo e-learning lessons. Physicians, pharmacists, medical students, and pharmacy students were asked to participate in the e-learning program, and their comments and suggestions were collected after program completion. The flipped classroom was evaluated by implementing Kampo e-learning in the Kampo session with fourth-year students at Keio University School of Medicine in Japan. Seven courses were created, including four based on volunteer suggestions. The 'Systematic Kampo Curricula' featured 88 lessons developed by 54 Kampo specialists. Out of 118 fourth-year medical students who participated in the flipped classroom, 113 registered for the Kampo e-learning program, 100 attended the session, and 88 answered the post-session questionnaire. Among the students who answered the questionnaire, 86.4% were satisfied with the flipped classroom, 79.5% replied that the program made them understand Kampo and 80.7% stated that it should be adopted. The flipped classroom using Kampo e-learning program was shown to be attractive in one medical school. Further expanded study is necessary in the near future to reveal the usefulness of the flipped classroom of Kampo learning.

Exercise-induced delayed onset of left ventricular early relaxation in association with coronary microcirculatory dysfunction in patients with diabetes mellitus.

BACKGROUND: The initiation of ventricular diastole is an energy-dependent phase of cardiac cycle. Delayed onset of left ventricular (LV) relaxation has been proposed to identify myocardial ischemia. Diabetes mellitus (DM) is known to be associated with coronary microangiopathy, but its influence on LV early relaxation is not established. METHODS AND RESULTS: Ninety-two subjects consisting of 70 DM patients without overt cardiac disease and 22 normal controls were evaluated. Using strain rate imaging, time from R-wave on the electrocardiogram to onset of LV relaxation (Tr) was measured at rest and peak exercise. Using myocardial contrast echocardiography, myocardial blood flow (MBF) was measured at rest and peak exercise, enabling MBF reserve. Tr at rest was similar between DM patients and controls, but Tr at peak exercise was significantly longer in DM patients than controls. MBF reserve was significantly reduced in DM patients compared with controls. There was a significant negative correlation between Tr at peak exercise and MBF reserve. In a multivariate analysis, MBF reserve was an independent determinant of Tr at peak exercise. CONCLUSIONS: This study demonstrates that DM patients have exercise-induced delayed onset of LV relaxation in association with impaired coronary microcirculatory function in the absence of coexistent heart disease.

Overexpression of the receptor for hyaluronan-mediated motility, correlates with expression of microtubule-associated protein in human oral squamous cell carcinomas.

Receptor for hyaluronan-mediated motility (RHAMM) has previously been characterized as a cell surface receptor for hyaluronan and a microtubule-associated intracellular hyaluronan binding protein. We examined the expression of RHAMM mRNA in 43 oral squamous cell carcinomas (SCCs) and 7 normal gingivae by real-time RT-PCR. The expression level of RHAMM mRNA was significantly higher in oral SCCs than normal gingivae (P=0.0047). Forty out of 43 oral SCCs showed expression of RHAMM splice variant (48 bp deletion). We immunohistochemically confirmed the protein expression of RHAMM in oral SCCs with higher levels of RHAMM mRNA. Patients with oral SCC who had high RHAMM expression had shorter survival rates than patients with low expression. However, it was not statistically significant. It has been reported that RHAMM interacts with spindle assembly factors such as microtubule-associated protein (TPX2). To investigate the expression of microtubule-associated protein in oral SCCs, mRNA expression of TPX2 was also examined by real-time RT-PCR. The expression level of TPX2 mRNA was significantly higher in oral SCCs than normal gingivae (P=0.046). Furthermore, a significant correlation between the mRNA expression levels of TPX2 and RHAMM was recognized (P=0.011). The results indicate that there is a strong correlation between the mRNA expression levels of TPX2 and RHAMM in oral SCCs. Our observations suggest that the up-regulations of human RHAMM and TPX2 gene correlate with the malignant condition and might be linked to the increased or abnormal cell proliferation in human oral SCCs.

Expression of TPX2 in salivary gland carcinomas.

TPX2 is a microtubule-associated protein and is required for microtubule formation at kinetochores in mammalian cells. The purpose of this study was to clarify the expression of TPX2 mRNA and correlation between TPX2 and clinicopathological factors in salivary gland carcinomas. The expression of TPX2 mRNA was investigated in 20 human salivary gland carcinomas (8 mucoepidermoid carcinomas, 7 adenoid cystic carcinomas, 5 acinic cell carcinomas) and 6 normal submandibular glands using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of TPX2 mRNA was higher in mucoepidermoid carcinomas (0.53+/-0.51) than in normal submandibular glands (0.047+/-0.029); a significant association was found (Mann-Whitney U test, P=0.0067). The mean expression levels of TPX2 were also higher in acinic cell carcinomas (0.45+/-0.49) and adenoid cystic carcinomas (0.28+/-0.22) than in normal submandibular glands. Statistical correlations were found (Mann-Whitney U test, P=0.028 and P=0.003, respectively). Correlation between expression of TPX2 and receptor for hyaluronan-mediated motility (RHAMM) was also investigated in this study. A significant association was found between the mRNA expression levels of TPX and RHAMM (Pearson's correlation coefficient by rank test, P=0.020). These results indicate that human TPX2 mRNA is closely linked to increased or abnormal cell proliferation in malignant salivary gland tumors.

Effects of microbubbles on ultrasound-mediated gene transfer in human prostate cancer PC3 cells: comparison among Levovist, YM454, and MRX-815H.

We compared the enhancement effects of three different echo contrast agents (ECAs); Levovist, YM454, and MRX-815H as artificial microbubbles on ultrasound mediated gene transfection (USMGT) with 1MHz ultrasound at 0.2MPa using a luciferase expression vector in PC3 cells and elucidated the mechanisms of differences of USMGT facilitation by these ECAs. At a concentration of each ECA that induced iso-survival, ECAs with lipid shell (YM454 and MRX-815H) facilitated USMGT higher than those without shell (Levovist), and the order of the ECAs facilitating free radical formation by sonication was; YM454>MRX-815H>Levovist. These results suggested that the lipid shell type ECAs facilitated gene transfer higher than that by the non-shell type ECA.

Expression of epiregulin, a novel epidermal growth factor ligand associated with prognosis in human oral squamous cell carcinomas.

We examined the expression of epiregulin and amphiregulin mRNA in 39 oral SCCs, 2 epithelial dysplasias and 7 normal gingivae by real-time RT-PCR. The mean expression level of epiregulin mRNA was higher in oral SCCs (0.29+/-0.50) than normal gingivae (0.01+/-0.007) and epithelial dysplasias (0.01+/-0.001). The expression level of epiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.023). Epiregulin mRNA was higher in stage III/IV than in stage I/II oral SCCs. However, a significant association was not found. The mean expression level of amphiregulin mRNA was higher in oral SCCs (0.18+/-0.24) than normal gingivae (0.002+/-0.003) and epithelial dysplasias (0.01+/-0.001). Amphiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.001). We then examined the expression of four EGF receptor mRNA in oral SCCs. The expression levels of HER1, HER2, HER3 and HER4 mRNA in oral oral SCCs were increased compared to those in normal gingivae. A significant correlation was found between the mRNA expression levels of epiregulin and HER2, HER3 and HER4 (Spearman's correlation coefficient by rank test, P=0.031, P=0.004 and P=0.027, respectively). Patients with oral SCC that have a high expression of epiregulin had a significantly shorter survival than those with low a expression (log-rank test, P<0.05). These results indicate that human epiregulin is closely linked to the increased or abnormal cell proliferation in human oral SCC.

Effects of ultrasound on apoptosis induced by anti-CD20 antibody in CD20-positive B lymphoma cells.

AIM: The present study was conducted to examine the thermal and non-thermal effects of ultrasound on apoptosis induced by anti-CD20 monoclonal antibody (rituximab). MATERIALS AND METHODS: SU-DHL-4 cells, a CD20-positive cell line derived from B cell lymphomas with a BCL2 gene rearrangement, were exposed to continuous 1 MHz ultrasound for therapeutic use under an air- or CO(2)-saturated condition to control cavitation. Early apoptosis (EA) and secondary necrosis (SN) were examined by flow cytometry. Cavitation was determined by detecting the hydroxyl radicals derived from pyrolysis of water molecules using electron paramagnetic resonance-spin trapping. To assess thermal effects, cells were treated in a temperature-controlled water bath. RESULTS: There was a significant additive increase in EA and EA+SN observed in cells treated with rituximab combined with heat at 42 degrees C or non-thermal ultrasound at 0.5 W/cm(2) under an air-saturated condition, where heat or ultrasound induced some cell death. A significant synergistic increase in EA and EA+SN was observed in cells treated with rituximab and ultrasound at 2.5 W/cm(2) under CO(2)-saturated conditions, where inertial cavitations were completely suppressed. No enhancement was observed at a temperature less than 40 degrees C or ultrasound at 0.5 W/cm(2) under CO(2)-saturated conditions. CONCLUSION: These results suggest that the immuno-therapeutic application of ultrasound at relatively high-intensities combined with rituximab thus produces synergistic effects under conditions where the non-thermal and non-cavitational effects are predominant.

Relationship between frequency spectrum of heart rate variability and autonomic nervous activities during sleep in newborns.

INTRODUCTION: We analyzed the frequency spectrum of two neonatal sleep stages, namely active sleep and quiet sleep, and the relationship between these sleep stages and autonomic nervous activity in 74 newborns and 16 adults as a comparison. METHOD: Active and quiet sleep were differentiated by electroencephalogram (EEG) patterns, eye movements, and respiratory wave patterns; autonomic activity was analyzed using the RR interval of simultaneously recorded electrocardiogram (ECG) signals. Power values (LFa, absolute low frequency; HFa, absolute high frequency), LFa/HFa ratio, and the values of LFn (normalized low frequency) and HFn (normalized high frequency) were obtained. Synchronicity between the power value of HFa and the LFa/HFa ratio during active and quiet sleep was also examined by a new method of chronological demonstration of the power values of HFa and LFa/HFa. RESULTS: We found that LFa, HFa and the LFa/HFa ratio during active sleep were significantly higher than those during quiet sleep in newborns; in adults, on the other hand, the LFa/HFa ratio during rapid eye movement (REM) sleep, considered as active sleep, was significantly higher than that during non-REM sleep, considered as quiet sleep, and HFa values during REM sleep were significantly lower than those during non-REM sleep. LFn during quiet sleep in newborns was significantly lower than that during active sleep. Conversely, HFn during quiet sleep was significantly higher than that during active sleep. Analysis of the four classes of gestational age groups at birth indicated that autonomic nervous activity in a few preterm newborns did not reach the level seen in full-term newborns. Furthermore, the power value of HFa and the LFa/HFa ratio exhibited reverse synchronicity. CONCLUSION: These results indicate that the autonomic patterns in active and quiet sleep of newborns are different from those in REM and non-REM sleep of adults and may be develop to the autonomic patterns in adults, and that parasympathetic activity is dominant during quiet sleep as compared to active sleep from the results of LFn and HFn in newborns. In addition, in some preterm infants, delayed development of the autonomic nervous system can be determined by classifying the autonomic nervous activity pattern of sleep stages.

Non-invasive quantitation of myocardial fibrosis using combined tissue harmonic imaging and integrated backscatter analysis in dilated cardiomyopathy.

BACKGROUND: Echocardiographic modalities have challenged the myocardial tissue characterization, but this reliability has not reached to the clinical use. This study investigated whether combined tissue harmonic imaging (THI) and integrated backscatter analysis (IB) provide the reliable and quantitative information about myocardial fibrosis in idiopathic dilated cardiomyopathy (DCM) in comparison with myocardial biopsy findings. METHODS: We studied 56 patients with DCM. All patients underwent left ventricular endomyocardial biopsy and IB with fundamental imaging (FI) and THI. RESULTS: In patients with good echocardiographic image quality, excellent correlations between the percentage of area occupied by myocardial fibrosis (% fibrosis) and the mean of integrated backscatter during a cardiac cycle (m-IB) measured with FI or THI were seen, and the correlation was closer with THI compared with FI. While in patients with poor image quality the correlation between m-IB and % fibrosis was only modest with FI, but the correlation was excellent with THI. Four cut-off values of m-IB with THI obtained from receiver operating characteristic curve discriminated between % fibrosis of more and less than 25, 30, 35, and 40% with high sensitivity and specificity. Multivariate analysis revealed that m-IB with THI was an independent predictor for discrimination of the severity of myocardial fibrosis. CONCLUSION: Combined IB and THI are a clinically applicable method and may be an alternative to myocardial biopsy in evaluating quantitatively myocardial fibrosis in DCM.