[Study on the shift of ocular vestibular evoked myogenic potentials frequency tuning of Meniere's disease].

Objective: To study the shift of oVEMP frequency tuning of unilateral Meniere's disease, by analyzing oVEMP of air-conducted tone bursts in various frequencies. Methods: From May 2016 to October 2017, 33 patients with unilateral Meniere's from Aerospace Center Hospital were tested for oVEMP in 500 Hz and 1 000 Hz air-conducted tone bursts respectively, and 20 healthy subjects(40 ears), matched for age and sex, were used as healthy control. The amplitudes of the N1-P1 wave and the frequency amplitude ratios of 500/1 000 Hz in affected ears, unaffected ears and normal ears were compared; and receiver operating characteristic curve was analyzed for frequency amplitude ratios of 500/1 000 Hz. Results: By the 500 Hz tone-burst stimulus, the provocation rates of the oVEMP were 84.9%(28/33), 93.9%(31/33) and 97.5%(39/40) in affected ears, contralateral ears and the ears of normal controls, respectively. By the stimulus of 1 000 Hz tone-burst, the provocation rates of the oVEMP were 81.8%(27/33), 87.9%(29/33) and 82.5% (33/40) in affected ears, contralateral ears and normal control ears, respectively. Amplitudes of N1-P1 waveforms in 500 Hz air-conducted tone bursts in affected ears were under normal control ears and contralateral ears. There was significant difference between affected ears and healthy control ears (P<0.05). Amplitudes of N1-P1 waveforms in contralateral ears were also significantly smaller than those in normal control ears (P<0.05). There were no significant differences in amplitudes of N1-P1 waveforms between affected ears, contralateral ears and normal control ears in 1 000 Hz tone bursts stimulus(P>0.05). Frequency amplitude ratios of 500/1 000 Hz in affected ears were significantly under contralateral and normal control ears (P<0.05). According to ROC, the frequency amplitude ratio critical value of frequency tuning was set as 1.17, the positive rate of frequency tuning shift in affected ears was 54.5%(18/33) and significantly higher than in contralateral ears(18.2%, 6/33, χ(2)=9.429, P=0.002) and normal control ears (7.5%, 3/40, χ(2)=19.530, P=0.000). In contrast, there was no significant difference in the incidence of frequency tuning shift between contralateral ears and normal ears(χ(2)=1.909, P=0.167). Conclusions: Frequency tuning oVEMP with Meniere's disease will be changed.Frequency tuning of oVEMP with Meniere's disease shifts from low frequency region to high frequency region in comparison with healthy people. Frequency amplitude ratio is helpful for diagnosis of Meniere's disease.

[Features of vestibular evoked myogenic potential in patients with residual dizziness after canalith repositioning procedures for benign paroxysmal positional vertigo].

Objective: To ananlyze the characteristics of the vestibular evoked myogenic action potential of patients with residual dizziness after successful canalith repositioning procedures(CRP)for idiopathic benign paroxysmal positional vertigo(IBPPV).Method: Ninety-seven consecutive patients with IBPPV of the unilateral posterior semicircular canal and health controls were initially enrolled. The patients received 500 Hz tone burst evoked ocular vestibular evoked myogenic potentials(oVEMP) and cervical vestibular evoked myogenic potentials (cVEMP). After successfully CRP, the patients were divided into two groups: group with residual symptoms and group without residual symptoms. The abnormality of results of both oVEMP and cVEMP were compared between three groups.Result:In the group with residual symptoms, the abnormal rate were 64.5% for oVEMP and 28.1% for cVEMP. In the group without residual symptoms, the abnormal rate were 17.9% for oVEMP and 12.5% for cVEMP. The abnormal rate of oVEMP in the group with residual symptoms was significantly higher than that in the group without residual symptoms(χ²=19.228, P=0.000). However, there was no significant difference in the abnormal rate of cVEMP between these two groups(χ²=3.342, P=0.068).Conclusion: In patients with IBPPV underwent successful CRP,the occurrence of residual symptoms is correlated to the abnormalities of oVEMP. It is indicated that the dysfunction of the utricle is related to the presence of residual symptoms after the CRP for IBPPV.

Effect of vitrification solutions on survival rate of cryopreserved Epinephelus moara embryos.

Embryo cryopreservation is important for long-term preservation of germplasm and assisted reproduction. However, it is still very difficult to obtain viable embryos from cryopreserved fish embryos. In this study, embryos of Epinephelus moara were used to investigate the effects of various cryopreservation methods. Embryos in stages 10 pairs somite (10S), 18 pairs somite (18S), 22 pairs somite (22S), tail-bud (TB), embryo twitching (ET) and pre-hatch (PH) were treated with five-step equilibrium penetration in 40% PMG3T vitrification solution, which contained 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Trehalose. We found that 18S, 22S, TB and ET stage embryos had higher survival rates and were more tolerant to the vitrification solution. Five-step equilibrium treatments on the embryos at the tail-bud stage were performed using two vitrification solutions: 40% PMG3T and 40% PMG3S, which consisted of 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Sucrose. The embryonic survival rate under PMG3S treatment (63.36%) was significantly higher than PMG3T treatment (43.93%) (P < 0.05). PMG3S and PMG3T with concentrations of 35%, 40% and 45% were tested on tail-bud stage embryos. Higher concentration of the vitrification solution led to significantly lower embryonic survival rate (P < 0.05). The survival rate was 36.79-72.05% in PMG3S, and 37.11-55.18% in PMG3T, and there were non-significant differences in embryonic development and malformation rates among the groups treated with different concentrations. The embryonic normal development rates in PMG3S and PMG3T were 21.27% and 11.04%, and the malformation rates were 36.13% and 31.04%, respectively. The optimum treatment condition was 40 min using 40% PMG3S on embryos at the tail-bud stage. Both PMG3S and PMG3T were used for cryopreserving embryos at 16 pairs somite, tail-bud and ET stage in liquid nitrogen, where we obtained 190 surviving embryos, and 44 fishes underwent normal development and hatched. The survival rate of cryopreserved embryos was 5.15%, the normal development rate was 1.31%, and the malformation rate was 3.66%. We found that PMG3S and PMG3T were effective for cryopreservation of Epinephelus moara embryos. The results provide a foundation for further explorations of fish embryo cryopreservation techniques.

[Experimental study of autologous fibroblast cell transplantation in the treatment of vocal fold scar].

Objective:To study the change characteristics of vocal cords scar after the implantation of autologous fibroblast cells into the vocal cords. Method:Forty New Zealand rabbits were used for the production of vocal fold scar model by creating a unilateral vocal fold wounds, we take the skin tissues of each rabbits and cultured them via enzyme digestion method to obtain autologous fibroblast cells. The models were separated into the experimental group and the control group, the third generation fibroblast cells with CM DIL labeled were then injected into the surrounding area of the vocal fold scar in the experimental group, the survival situation of the injected cells were observed by fluorescence microscopy after 1 week, histological examination was performed after 1 months to observe the morphological changes of vocal fold scar tissues, RT-PCR method was used to measure the expression variation of typeⅠcollagen and type Ⅲ collagen in the vocal cords and the normal vocal cords, respectively. Result:After 1 week of autologous fibroblast transplantation into the vocal folds, the fluorescence labeled cells were observed.1 month after transplantation, the collagen protein in the vocal fold scar tissues was similar to that in the normal vocal folds，the expression of typeⅠcollagen and type Ⅲ collagen in vocal fold scars is similar to those in the normal vocal folds. Conclusion:Autologous fibroblast migration into the vocal cords can survive well inside the vocal cords, and the vocal scar can be recovered to normal vocal cord structure after transplantation of autologous fibroblast cells.

Two triterpeniods from Cyclocarya paliurus (Batal) Iljinsk (Juglandaceae) promote glucose uptake in 3T3-L1 adipocytes: The relationship to AMPK activation.

PURPOSE: The current study investigated the efficacy of Cyclocarya paliurus chloroform extract (CPEC) and its two specific triterpenoids (cyclocaric acid B and cyclocarioside H) on the regulation of glucose disposal and the underlying mechanisms in 3T3-L1 adipocytes. METHODS: Mice and adipocytes were stimulated by macrophages-derived conditioned medium (Mac-CM) to induce insulin resistance. CPEC was evaluated in mice for its ability by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). To investigate the hypoglycemic mechanisms of CPEC and its two triterpenoids, glucose uptake, AMP-activated protein kinase (AMPK) activation, inhibitor of NF-κB kinase ß (IKKß) phosphorylation and insulin signaling transduction were detected in 3T3-L1 adipocytes using 2-NBDG uptake assay and Western blot analysis. RESULTS: Mac-CM, an inflammatory stimulus which induced the glucose and insulin intolerance, increased phosphorylation of IKKß, reduced glucose uptake and impaired insulin sensitivity. CPEC and two triterpenoids improved glucose consumption and increased AMPK phosphorylation under basal and inflammatory conditions. Moreover, CPEC and its two triterpenoids not only enhanced glucose uptake in an insulin-independent manner, but also restored insulin-mediated protein kinase B (Akt) phosphorylation by reducing the activation of IKKß and regulating insulin receptor substrate-1 (IRS-1) serine/tyrosine phosphorylation. These beneficial effects were attenuated by AMPK inhibitor compound C, implying that the effects may be associated with AMPK activation. CONCLUSIONS: CPEC and its two triterpenoids promoted glucose uptake in the absence of insulin, as well as ameliorated IRS-1/PI3K/Akt pathway by inhibiting inflammation. These effects were related to the regulation of AMPK activity.

Pharmacological postconditioning with diazoxide attenuates ischemia/reperfusion-induced injury in rat liver.

It has been demonstrated that ischemic postconditioning (IPO) is capable of attenuating ischemia/reperfusion (I/R) injury in the heart. However, the novel role of pharmacological postconditioning in the liver remains unclear. In this study, the hypothesis that diazoxide postconditioning reduces I/R-induced injury in rat liver was tested. Rats were assigned randomly to the sham-operated control, I/R (occlusion of the porta hepatis for 60 min, followed by a persistent reperfusion for 120 min), diazoxide ischemic postconditioning (DIPO; occlusion of the porta hepatis for 60 min, then treatment with diazoxide for 10 min reperfusion, followed by a persistent reperfusion for 110 min) or 5-hydroxydecanoate (5-HD)+DIPO (occlusion of the porta hepatis for 60 min, then treatment with diazoxide and 5-HD for 10 min reperfusion, followed by a persistent reperfusion for 110 min) groups. The alanine aminotransferase (ALT) and aspartate transaminase (AST) levels were assayed. The expression levels of protein kinase c-ε (pkc-ε), cytochrome c (cyt-c), caspase-3 and bcl-2 protein were determined by western blotting. The serum levels of ALT and AST and expression levels of cyt-c and caspase-3 were significantly lower in the DIPO group (P<0.05). However, the protein expression levels of pkc-ε and bcl-2 were markedly increased in the DIPO group (P<0.05). 5-HD abrogated the protective effects of DIPO. The data of the present study provide the first evidence that DIPO protects the liver from I/R injury by opening the mitochondrial KATP channels, activating and upregulating pkc-ε and inhibiting the activation of the apoptotic pathway by decreasing the release of cyt-c and the expression of caspase-3 and increasing bcl-2 expression.

[Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris].

Catalase is well known to eliminate H2O2 in cells and reduces the toxicity of peroxide compounds. A catalase gene HpCat1 of methylotrophic yeast Hansenula polymorpha without the part coding the native signal peptide was cloned into expression vector pYM3165 and then integrated into genome of Pichia pastoris GS115 by electroporation. The result of the enzyme activity assay and SDS-PAGE demonstrated that the recombinant protein (HpCAT1) of H. polymorpha was extracellularly expressed in P. pastoris. The expressed catalase was recovered from the culture supernatant of P. pastoris GS 115 and purified by (NH4) 2SO4 fractionation and Ni-NTA affinity chromatography. The main biochemical properties of the recombinant protein HpCAT1, such as thermodependence and thermostability, pH optimum and pH stability, as well as the effect of metal ions and chemicals, were characterized. With H2O2 as the substrate, HpCAT1 displayed pH and tem- perature optima of approximately 2.6 and 45°C,respectively. The recombinant HpCAT1 activity was inhibited by 1 mM Hg2+ and Cu2+, but was highly enhanced by 1.0 mM Fe2+.

Development and characterization of a new marine fish cell line from turbot (Scophthalmus maximus).

A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n=44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.

Microsatellite-centromere mapping in Cynoglossus semilaevis using gynogenetic diploid families produced by the use of homologous and non-homologous sperm.

Twenty-one microsatellite markers were studied in three meiogynogenetic families of Cynoglossus semilaevis gunther for centromere mapping using half-tetrad analysis. Among the 13 mapped loci, 10 were estimated to be located in the telomeric region, one in the centromeric region, and two in the intermediate region of the chromosome. This study provides a basis for constructing a linkage map of C. semilaevis.

Cryopreservation of flounder (Paralichthys olivaceus) embryos by vitrification.

Conventional cryopreservation of complex teleost embryos has been unsuccessful, possibly because their large size (1-7 mm diameter), multi-compartmental structure and low water permeability lead to intracellular ice formation and chilling injury. To overcome these obstacles, we have developed a vitrification procedure for cryopreservation of flounder (Paralichthys olivaceus) embryos. In initial toxicity tests, propylene glycol (PG) and methanol (MeOH) were less toxic to embryos than dimethylformamide (DMF) or dimethyl sulfoxide (Me2SO), whereas ethylene glycol (EG) and glycerol (Gly) were toxic to all tested embryos. Embryos between four-somite and tail bud stages were more tolerant to vitrifying solutions than embryos in other developmental stages. Four vitrifying solutions (FVS1-FVS4) were prepared by combining a basic saline solution (BS2) and cryoprotectants PG and MeOH in different proportions (FVS1: 67, 20 and 13%; FVS2: 60, 24 and 16%; FVS3: 55, 27 and 18%; FVS4: 50, 30 and 20% of BS2, PG and MeOH, respectively). Their impact on flounder embryos was then compared. FVS1 produced the highest survival rate; whereas deformation rate was highest for FVS4. Five-step equilibration of embryos in FVS2 resulted in higher survival rates than equilibration in 4, 3, 2 or 1 steps. Flounder embryos varying from the 14-somite to the pre-hatching stage were cryopreserved in the four vitrifying solutions in liquid nitrogen for 1-7 h. From eight experiments, 20 viable thawed embryos were recovered from 292 cryopreserved embryos. Fourteen larvae with normal morphology hatched successfully from the 20 surviving frozen-thawed embryos from five experiments. Embryos at the tail bud stage exhibited greater tolerance to vitrification than embryos at other stages. These results establish that cryopreservation of flounder embryos by vitrification is possible. The technology has many potential applications in teleost germplasm resource conservation.

[Differentiation between primary and metastatic ovarian cancers by monoclonal antibodies specific for gastrointestinal tract carcinomas].

Monoclonal antibodies MG7 and MC3, which are highly specific for gastrocolonic carcinomas, were used in immunohistochemical studies of detecting the relevant antigens present in the primary and metastatic carcinomas in ovaries. All 33 cases of primary ovarian carcinomas examined were negative, and positive reaction was obtained in 94.4% of 18 cases of metastatic carcinomas in ovaries. Results of this investigation confirmed the specificity of MG7 and MC3 in differential diagnosis of primary ovarian cancers from metastatic cancers in ovaries originating from the gastrointestinal tract.