[Next generation sequencing technology for susceptible gene screening in familial non-medullary thyroid carcinoma].

Objective: To screen genes related to familial non-medullary thyroid carcinoma (FNMTC) using next-generation sequencing (NGS). Methods: A panel of NGS was designed and sequencing was performed for DNA samples extracted from peripheral blood leukocytes of FNMTC patients and sporadic non-medullary thyroid carcinoma (SNMTC) cases, respectively, and gene mutations were screened. In addition, the clinicopathological characteristics, including tumor size, extension of surgery, lymph node metastasis and extra-thyroidal extension, were compared between patients with or without mutations. Results: In 63 NMTC samples, 45 mutations were detected on 13 genes. 37 germline mutations were detected in 47 FNMTC patients, while 8 germline mutations were detected in 16 SNMTC patients. In 8 FNMTC family lineages, the same mutations were carried by FNMTC patients from the same pedigree. The number of carriers of mutations was 29 in the 47 FNMTC patients and 6 in the 16 SNMTC patients, with a non-significant difference (P= 0.092). Among the FNMTC patients, there were 22 patients with central lymph node metastasis in the 29 mutation-positive patients, significantly more than 7 in the 16 mutation-negative cases (P= 0.031). As for the parentage, there were 3 patients with central lymph node involvement among the 7 patients of parent generation, while all the 9 patients of offspring generation had central lymph node metastasis (P=0.019). Conclusions: This panel of NGS can be used to screen mutant susceptibility gene of FNMTC patients, and the findings may be helpful for early detection of FNMTC patients and predicting the disease risk to familial members of FNMTC patients.

[Use of alveolar bone mucosa-periosteal bone flap in dental implantation].

To introduce a method of increasing the width of alveolar bone. The patient, who needed dental implantation and had narrow alveolar bone, was selected. The preparation of mucosa-periosteal bone flap included two surgeries. The first surgery was corticotomy, which made a square cortex cut on narrow alveolar bone region. The second surgery was performed four weeks after the first one, which split the alveolar bone and inserted implants. Artificial bone and/or autologous bone was filled between inner and outer bone plate, and collagen membrane and platelet-rich fibrin membrane were used to cover the wound. This technique maintained the blood supply of labial(buccal) alveolar bone completely. Artificial bone and/or autologous bone graft contacted with cancellous bone directly and led to better bone growth and bone formation. Alveolar bone mucosa-periosteal bone flap can maintain the labial(buccal) alveolar bone effectively and avoid bone resorption.

Protective effect of naringenin-7-O-glucoside against oxidative stress induced by doxorubicin in H9c2 cardiomyocytes.

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits its clinical use. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) and cardiomyocyte apoptosis appear to be involved in DOX cardiotoxicity. Protection or alleviation of DOX cardiotoxicity can be achieved by administration of natural phenolic compounds via activating endogenous defense systems and antiapoptosis. Naringenin-7-O-glucoside (NARG), isolated from Dracocephalum rupestre Hance, could protect from cardiomyocyte apoptosis and induce endogenous antioxidant enzymes against DOX toxicity, but the effects on intracellular ROS generation and cell membrane stability were not demonstrated. In the present study, we investigated the effects of NARG on H9c2 cell morphology, viability, lactate dehydrogenase (LDH) and creatine kinase (CK) leakage, glutathine peroxidase (GSH-Px) activity, intracellular Ca2+ concentration, and ROS generation. Compared with DOX alone treatment group, the morphological injury of the cells in groups treated by DOX plus NARG was alleviated, cell viability was increased, the amount of released LDH and CK was significantly decreased, the activity of GSH-Px was increased, the content of intracellular Ca2+ and ROS generation was lowered remarkably. These results suggest that NARG could prevent cardiomyocytes from DOX-induced toxicity by their property of stabilizing the cell membrane and reducing ROS generation.

Valsartan attenuated oxidative stress, decreased MCP-1 and TGF-ß1 expression in glomerular mesangial and epithelial cells induced by high-glucose levels.

Our previous studies revealed that valsartan, an angiotensin II type I receptor blocker, exhibited renoprotective effects through decreasing urine protein excretion levels due to improving glomerular permeability in rats with diabetic nephropathy (DN). In this study, we sought to investigate the underlying mechanisms in perspectives of oxidative stress, transforming growth factor beta-1 (TGF-ß1) and monocyte chemoattractant protein-1 (MCP-1) expressions in glomerular mesangial cells (GMCs) and glomerular epithelial cells (GECs) since their roles are well-established in the development and progression of DN. High-glucose levels significantly increased oxidative stress in GMCs and GECs, as evidenced by enhanced generation of reactive reactive oxygen species (ROS), reduced levels of glutathione (GSH) and antioxidant enzyme superoxide dismutase (SOD), and increased production of malondialdehyde (MDA). Treatment with valsartan significantly restored the levels of those oxidative stress relevant molecules. Furthermore, valsartan obviously diminished the expression of proinflammatory cytokine MCP-1 in GMCs and GECs induced by high-glucose levels both at mRNA and protein levels, as determined by real-time PCR, immunocytochemistry, western blotting, and ELISA. In addition, the increased expressions of TGF-ß1 mRNA and protein induced by high-glucose level were also abrogated by valsartan treatment in GMCs, as evaluated by real-time PCR and ELISA. These results suggest that the renoprotective effects of valsartan may be related to its potential in decreasing oxidative stress and the expressions of MCP-1 and TGF-ß1 in GMCs and GECs.

A low-dose combination of valsartan and low molecular weight heparin better improved glomerular permeability than did high-dose monotherapy in rats with diabetic nephropathy.

Diabetic nephropathy is the most common and severe renal complication of diabetes mellitus. The present study sought to investigate the renoprotective effects of a combination therapy of valsartan and low molecular weight heparin (LMWH) in rats with diabetic nephropathy induced by uninephrectomy and streptozotocin. The animals were divided into five groups as follows: sham-operated rats, diabetic control rats, diabetic rats treated with 20 mg/kg/day valsartan, diabetic rats treated with 600 IU/kg/day LMWH, diabetic rats treated with a combination of valsartan and LMWH (valsartan 10 mg/kg/day and LMWH 300 IU/kg/day). The treatment regimen was maintained for 8 weeks. Treatment with valsartan, LMWH, or a combination of the two had no significant effect on blood glucose levels. However, the urine protein excretion levels significantly decreased for the three drug treatment groups; the most dramatic decreases were observed in the combination treatment group. Kidney histology was examined using periodic acid-Schiff staining and immunohistochemical staining of extracellular matrix proteins. Results indicated that histopathology improved markedly in the three drug treatment groups; combination therapy had an equal or better effect than monotherapy in terms of decreasing the abnormal thickness of the glomerular basal membrane, the ratio of the area of the mesangial region with respect to the total area of renal glomeruli, and the accumulation of collagen IV and laminin in kidney tissue. In addition, serum levels of transforming growth factor-ß1 (TGF-ß1) also markedly decreased in the drug treatment groups according to ELISA. However, there were no significant differences between the combination therapy group and monotherapy group. These results suggest that a combination of valsartan and LMWH at half the dose used in monotherapy is better at improving glomerular permeability in rats with diabetic nephropathy.

Effects of KT-362, a new antiarrhythmic agent, on membrane ionic currents of guinea pig ventricular myocytes.

We studied the effects of KT-362, (5-[3[2-(3,4-dimethoxyphenyl)ethyl]amino]-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate), a newly synthesized vasodilating and antiarrhythmic agent, on membrane currents of single guinea pig ventricular cells, using whole-cell voltage-clamp techniques. In the steady state with a stimulation frequency of 0.5 Hz, KT-362 at concentrations of 10 and 30 microM decreased the peak sodium current (INa) in a concentration-dependent manner, i.e., by 27% and 49%, respectively. The inhibition of INa by this agent was use-dependent: KT-362 (30 microM) inhibited INa by 21% at 0.2 Hz and by 51% at 1 Hz. In addition, KT-362 (10-30 microM) decreased the L-type Ca current (ICa) in a concentration-dependent fashion. The delayed rectifier potassium current and the inward rectifier potassium current were also inhibited by KT-362. The effects of KT-362 on INa and ICa were confirmed in experiments using ventricular papillary muscle preparations and microelectrode techniques. KT-362 (10-300 microM) decreased the maximum rate of rise of action potentials provoked at normal (2.7 mM) K+ concentration and that provoked at high (20 mM) K+ concentration. KT-362 at concentrations over 100 microM significantly depolarized the resting membrane, and the action potential duration remained unaltered. From these findings, we conclude that apart from the alleged inhibitory effects of this agent on the release of calcium from sarcoplasmic reticulum (it is therefore termed "an intracellular Ca++ blocker"), KT-362 suppresses a variety of membrane ionic currents of cardiac cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine reverses isoproterenol-induced depression of Vmax in residual Na channel-dependent action potentials of guinea-pig cardiac muscles.

We examined effects of acetylcholine (ACh) on isoproterenol (ISP)-induced changes of the upstroke velocity of the action potential (Vmax) in isolated 13.5 mM K(+)-depolarized atrial muscles from guinea-pigs, using conventional glass microelectrode techniques. In some experiments, ventricular muscles were also used, for purposes of comparison. ISP (0.1 microM) decreased the fast component of Vmax (Vmax,fast) and increased the slow component of Vmax (Vmax, slow) of the atrial muscle, as has been noted in ventricular muscle. ACh (0.1 microM) reversed or antagonized these effects of ISP. However, in the presence of atropine (0.1 microM), the antagonism disappeared. In the presence of the Ca2+ channel blocker, D600 (1 microM), the depressant effect of ISP on the Vmax,fast was augmented while ACh exerted a much less restorative effect on the ISP-induced, depressed Vmax,fast. Similar findings were obtained in low (0.36 and 0.072 mM) Ca2+ media. To investigate the possible involvement of GTP-binding protein (Gi) on these ACh effects, we performed similar experiments using atrial muscles obtained from guinea pigs pre-treated with pertussis toxin (150 micrograms/kg) for 48 h. In these preparations, the depressant effect of ISP on the Vmax,fast remained unaffected, while the reversing effect of ACh on the ISP-induced depression of Vmax,fast either specifically diminished or disappeared. These results show that ACh antagonizes the ISP-induced Vmax changes via stimulation of muscarinic ACh receptors and that this effect is presumably mediated by Gi and modified by intracellular Ca2+. Clinical implications are discussed.

Transmembrane voltage changes produced by real and virtual electrodes during monophasic defibrillation shock delivered by an implantable electrode.

INTRODUCTION: Epicardial point stimulation produces nonuniform changes in the transmembrane voltage of surrounding cells with simultaneous occurrence of areas of transient positive and negative polarization. This is the phenomenon of virtual electrode. We sought to characterize the responses of epicardial ventricular tissue to the application of monophasic electric shocks from an internal transvenous implantable cardioverter defibrillator (ICD) lead. METHODS AND RESULTS: Langendorff-perfused rabbit hearts (n = 12) were stained with di-4-ANEPPS. A 9-mm-long distal electrode was placed in the right ventricle. A 6-cm proximal electrode was positioned horizontally 3 cm posteriorly and 1 cm superiorly with respect to the heart. Monophasic anodal and cathodal pulses were produced by discharging a 150-microF capacitor. Shocks were applied either during the plateau phase of an action potential (AP) or during ventricular fibrillation. Leading-edge voltage of the pulse was 50 to 150 V, and the pulse duration was 10 msec. Transmembrane voltage was optically recorded during application of the shock, simultaneously from 256 sites on a 11 x 11 mm area of the anterior right ventricular epicardium directly transmural to the distal electrode. The shock effect was evaluated by determining the difference between the AP affected by the shock and the normal AP. During cathodal stimulation an area of depolarization near the electrode was observed, surrounded by areas of hyperpolarization. The amplitude of polarization gradually decreased in areas far from the electrode. Inverting shock polarity reversed this effect. CONCLUSION: ICD monophasic defibrillation shocks create large dynamically interacting areas of both negative and positive polarization.

Relation of the atrial input sites to the dual atrioventricular nodal pathways: crossing of conduction curves generated with posterior and anterior pacing.

INTRODUCTION: The usually accepted definition of the dual pathway electrophysiology requires the presence of conduction curves with a discontinuity ("jump"). However, AV nodal reentrant tachycardia has been observed in patients with "smooth" conduction curves, whereas discontinuity of the conduction curve does not guarantee induction of stable reentry. We hypothesize that the duality of AV nodal conduction can be revealed by careful choice of stimulation sites during the generation of AV nodal conduction curves. METHODS AND RESULTS: In 21 rabbit heart atrial-AV nodal preparations, programmed electrical stimulation with S1-S2-S3 pacing protocol was applied either posteriorly at the crista terminalis input site (CrT) or anteriorly at the lower interatrial septum input site (IAS), or (in 8 preparations with surgically divided input sites) at both. We found that in intact preparations with "smooth" conduction curves, pacing at long coupling intervals produced shorter AV nodal conduction times from the IAS (56 +/- 9.8 msec vs 69 +/- 10.1 msec; P < 0.01). At short coupling intervals, in contrast, shorter conduction times were obtained from the CrT (173 +/- 21.8 msec vs 188 +/- 22.8 msec; P < 0.01). This resulted in a characteristic crossing of the superimposed IAS and CrT conduction curves. After division of the inputs, the IAS site had rapid conduction to the His bundle but a longer refractory period, whereas the CrT site had long conduction times and shorter refractory periods. Wavefronts entering the AV node from these two inputs can summate, resulting in improved conduction. CONCLUSION: Pacing protocols designed to accentuate the asymmetry between the AV nodal inputs can help to reveal the functional difference between the dual pathways and thus to better assess the properties of AV nodal conduction.

Effects of inhaled low molecular weight heparin on airway allergic inflammation in aerosol-ovalbumin-sensitized guinea pigs.

Low molecular weigh, heparin (LMWH) possesses multiple nonanticoagulant properties. In the present study, we observed its anti-airway allergic inflammatory effects by bronchoalveolar lavage in guinea pigs. Guinea pigs were sensitized by repeatedly inhaling aerosolized ovalbumin. LMWH (400 u/l, 800 u/l), dexamethasone (1.2 mg/1) or vehicle (normal saline) was inhaled for 7 days. Then the animals were sacrificed under anesthesia and then lavaged with ice-cold Hank's buffer immediately; bronchoalveolar lavage fluid (BALF) was prepared 24 h after the animals were challenged by antigen exposure. The effects of LMWH on total cell counts, absolute eosinophil counts and cell catalogues in BALF were studied; effects on the activity of eosinophil peroxidase (EPO) and the contents of histamine and eosinophil cationic protein (ECP) in BALF supernatant were detected. Our results showed that compared with the vehicle group, LMWH at 400 u/l and 800 u/1 could significantly reduce total cell counts, absolute eosinophil counts and percentage of eosinophils in BALF (P<0.05 and P<0.01, respectively); LMWH at 800 u/l markedly inhibited the activity of EPO in BALF supernatant (P<0.05); LMWH at 400 u/l and 800 u/l remarkably reduced the content of histamine in BALF supernatant (P<0.05 and P<0.01, respectively), LMWH at 800 u/l decreased the content of ECP (P<0.05) significantly. It suggested that LMWH exerted anti-airway allergic inflammatory action by inhibiting infiltration of inflammatory cells and reducing release of inflammatory mediators, as well as antagonizing their activities, and that LMWH could be developed as a potential anti-bronchial asthmatic drug.