RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens.

OBJECTIVE: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. METHODS: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID 50 per µL and copies per µL, and the newly established methods were tested in clinical specimens collected in recent years. RESULTS: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID 50 per µL and 10 virus copies per µL, and for the complete VP1 gene was 1 CCID 50 per µL and 100 virus copies per µL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. CONCLUSION: This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Cadmium induces ovarian granulosa cell damage by activating PERK-eIF2α-ATF4 through endoplasmic reticulum stress.

This study aimed to investigate whether cadmium induces ovarian granulosa cell damage by activating protein kinase R-like endoplasmic reticulum kinase (PERK)-eIF2α-ATF4 through endoplasmic reticulum (ER) stress and to elucidate the underlying regulation mechanism. Two models of cadmium exposure were established. In one model, ovarian granulosa cells isolated from 21-day-old female Sprague Dawley rats were cultured in vitro for 36 h and exposed to CdCl2 (0, 5, 10, and 20 µM), and in another model, a human ovarian granulosa tumor cell line (COV434) was used to construct the binding immunoglobulin protein (BIP)-knockdown cell line sh-BIP and exposed to 0 and 20 µM CdCl2. After exposure to cadmium for 12 h, the expression mRNA and protein levels of BIP, p-PERK, and p-eIF2α were determined in the two models. miRNAs related to BIP were also detected in granulosa cells after cadmium exposure. We found that mRNA and protein levels of all factors were upregulated in each cadmium-dose group, except for BIP mRNA expression in the 5 µM Cd group. The BIP gene was knocked down in COV434 cells before exposure to cadmium. All factors were upregulated in COV434 cells exposed to Cd, and the expression of the p-eIF2α protein was downregulated in sh-BIP cells exposed to Cd. In addition, no differences in BIP-related miRNAs were detected in cadmium-exposed rat ovarian granulosa cells versus the control group. Cadmium induces ovarian granulosa cell damage by inducing ER stress.

Construction and evaluation of two computational models for predicting the incidence of influenza in Nagasaki Prefecture, Japan.

It remains challenging to forecast local, seasonal outbreaks of influenza. The goal of this study was to construct a computational model for predicting influenza incidence. We built two computational models including an Autoregressive Distributed Lag (ARDL) model and a hybrid model integrating ARDL with a Generalized Regression Neural Network (GRNN), to assess meteorological factors associated with temporal trends in influenza incidence. The modelling and forecasting performance of these two models were compared using observations collected between 2006 and 2015 in Nagasaki Prefecture, Japan. In both the training and forecasting stages, the hybrid model showed lower error rates, including a lower residual mean square error (RMSE) and mean absolute error (MAE) than the ARDL model. The lag of log-incidence, weekly average barometric pressure, and weekly average of air temperature were 4, 1, and 3, respectively in the ARDL model. The ARDL-GRNN hybrid model can serve as a tool to better understand the characteristics of influenza epidemic, and facilitate their prevention and control.

N-hexane alters the maturation of oocytes and induces apoptosis in mice.

OBJECTIVE: This study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes. METHODS: Cell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis. RESULTS: Germinal vesicle breakdown (GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials (ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes. CONCLUSION: N-hexane might have affected the maturation of oocytes, causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms.

[Effects of bisphenol A on OCT4 and SOX2 genes expression in mouse embryonic stem cells].

OBJECTIVE: To explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC). METHODS: mESC were cultured, and treated with the doses of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L respectively of BPA and DMSO (the solvent control group)for 24 hours, and three groups of cells were treated with the same method. The morphological changes of mESC in the control and exposure groups were observed through an inverted microscope. Cell counting kit 8 (CCK8) was used to detect the effects of BPA on proliferation of mESC, and based on the results, the half inhibitory concentration (IC50) was calculated. Real-time fluorescent quantitative polymerase chain reaction (RT-QPCR) and western blotting were used to detect the expression of OCT4 and SOX2. RESULTS: BPA had certain toxicity on mESC, the treatment of BPA significantly increased cell toxicity in a concentration-dependent manner, and the IC50 was 4.3×10(-4) mol/L, combined with the BPA exposure concentration of the environment and the related literature, eventually taking the five concentrations of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L as the experimental groups. The mESC morphology were effected after the treatment of BPA for 24 h, compared with the control group, the number of cells decreased, appearing some floating cells, and the cell cloning became irregular and differentiation in the higher concentration groups. The OCT4 mRNA expression level in the 10(-7) mol/L (1.146 ± 0.087), 10(-6) mol/L (1.156 ± 0.030), 10(-5) mol/L (1.158 ± 0.103) and the 10(-4) mol/L (1.374 ± 0.053) dose group were all significantly higher than the control group (1.000 ± 0.000) (t values were -2.384, -2.953, -3.203, -4.021 respectively, P value all < 0.05). Meanwhile, the SOX2 mRNA expression level in the 10(-4) mol/L (1.113 ± 0.052) were higher than the control group (1.000 ± 0.000) (t value was -2.765, P value < 0.05). Moreover, the OCT4 protein expression level in the 10(-5) mol/L (1.360 ± 0.168) and 10(-4) mol/L (1.602 ± 0.151) were all significantly higher than the control group (1.000 ± 0.000) (t values were -3.538, -4.002 respectively, P value all < 0.05), while no obvious change of the SOX2 protein expression level was detected in all treated groups. CONCLUSION: BPA in a certain dose range could upregulate the expression of OCT4 gene in mouse embryonic stem cells while had no significant effect on the expression of SOX2 gene.

The antioxidant activity and active component of Gnaphalium affine extract.

The antioxidant activity of Gnaphalium affine extract (GAE) against H2O2-induced oxidative injury in Caco-2 cells was evaluated, and the main antioxidant component was isolated and identified by column chromatography, high performance liquid chromatography, time-of-flight mass spectrometer and nuclear magnetic resonance. In vitro assays, GAE showed remarkable antioxidant activity to scavenge free radicals (ABTS, DPPH, superoxide and hydroxyl radicals), inhibit lipid peroxidation and show reducing power. In food system, GAE exhibited the obvious capacity to inhibit the oxidation of peanut oil and lard, which may be attributed to its high content of phenolic compounds. Moreover, GAE could effectively protect Caco-2 cell against H2O2-induced oxidative injury. With the isolation and purification by chromatography, quercetin was identified as the main antioxidant component of GAE, which was capable of scavenging ABTS, DPPH, superoxide and hydroxyl radicals. These results suggest that G. affine is a potential source for preparing functional foods and nutraceuticals in food industry.

The effect of n-hexane on the gonad toxicity of female mice.

OBJECTIVE: To investigate the toxic effects of n-hexane on the Ganod of female mice. METHODS: n-Hexane was administered to four groups of mice by inhalation at doses of 0, 3.0, 15.1, and 75.8 mL/m3 respectivelyfor five weeks. Each group consisted of 10 mice, of which half were injected in first with 10 IU of pregnant mare serum gonadotrophin (PMSG) on the 33rd days, and then with 10 IU of human chorionic gonadotrophin (HCG) 48 hrs later. After the treatment, mouse sera were sampled and ovulating hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and progesterone (P4) levels were measured by electrochemiluminescence immunoassays (ECLIA). In each group, the right ovaries of the non-super-ovulated mice were stained with hematoxylin and eosin while ovaries on the left side were prepared with the TUNEL method in order to detect apoptotic cells. RESULTS: The duration of the diestrus stage decreased significantly (P < 0.05) in the 75.8 mL/m3 group. All super-ovulated mice in each treatment group produced fewer eggs than those in the control group (P < 0.05). The number of follicles in ovaries in the 75.8 mL/m3 group was smaller compared with the control group (P < 0.05).The serum P4 levels in each treatment group were lower than those in the control group (F = 6.196, P < 0.01). The cell apoptotic rate in the 75.8 mL/m3 group was higher (P < 0.05). CONCLUSION: n-Hexane may have directly mediated via alterations hormone secretion and promoted granulosal cell apoptotic, which may be one of the important mechanisms for n-hexane induced mouse ovary impairment.

Blastocysts cloned from the Putian Black pig ear tissues frozen without cryoprotectant at -80 and -196 degrees Celsius for 3 yrs.

The Putian Black pig, as one of elite cultivars of endemic species in China, has been on the verge of extinction and urgently needs protection. Somatic cell nuclear transfer (SCNT) and noncryoprotected frozen tissue technology have successfully resurrected several mammalian species. Therefore, this study explored the primary feasibility of conserving this breed using a combination of both technologies. Skin tissues obtained from the ears of adult Putian Black boars were frozen without cryoprotectant at -20, -80, or -196 °C and stored for 3 yrs. Primary cell culture, passage and subculture were performed on frozen samples after being rapidly thawed at 39 °C and on fresh pig ear tissues (control). Cloned embryos were reconstructed using fibroblasts (from frozen and fresh tissues) with enucleated oocytes. Live cell lines were obtained from tissues frozen at -80 and at -196 °C and appeared to have normal proliferative activity after passage; furthermore, they directed cloned embryos to develop to the blastocyst stage after nuclear transfer. We concluded that the population of Putian Black pig might be increased in the future by transferring cloned blastocysts into synchronized recipient pigs.

[Female genital toxicities of high-frequency electromagnetic field on rats].

OBJECTIVE: To study on the effects of sub-chronic exposures to high-frequency electromagnetic field (HF-EMF) on the estrous cycle, ovarian pathological changes and related hormones and preliminarily investigate the female genital toxicities of HF-EMF in rats. METHODS: 60 Wistar female adult rats were randomly divided into five groups based on body weight and radiated with different levels of 30 MHz electromagnetic field (0, 25, 100, 400 and 1600 V/m) for eight hours daily. Weekly the rats were continuously exposed five days. From the 48th day the four stage of estrus cycle were observed with smear method of the vaginal cell. Fifty-six days later the serum levels of sexual hormones were detected with the radioimmunoassay on estrus stage. The constituent ratio of the distinct follicle number on ovaries were observed with the HE staining and the ultrastructure was observed with the transmission electron microscope. Meanwhile, the ovarian humid weight and organ coefficient were observed. RESULTS: There was no significant difference in ovarian humid weight and organ coefficient between the exposure and the control groups. The time of proestrus in the 100 V/m group, the 400 V/m group and the 1600 V/m group was decreased significantly [(15.00 +/- 5.06), (11.40 +/- 2.05) and (10.56 +/- 0.96) h in the exposure group compared with (18.70 +/- 2.96) h in the control group, P < 0.01], and the time of Anestrum in the 400 V/m group and the 1600 V/m group were increased significantly [(101.20 +/- 17.81) and (115.33 +/- 19.28) h in the exposure group compared with (69.80 +/- 11.42) h in the control group, P < 0.01)]. Serum LH in the 400 V/m and 1600 V/m group was increased significantly [(11.02 +/- 1.11) and (14.70 +/- 1.94) mU/ml in the exposure groups compared with (8.70 +/- 0.53) mU/ml in the control group, P < 0.01], and serum E2 was decreased significantly [(57.16 +/- 31.56) and (50.57 +/- 25.16) pg/ml in the exposure groups compared with (95.04 +/- 32.62) pg/ml in the control group, P < 0.01]. The composition ratio of the corpus luteum/albicans number in the 400 V/m group and the 1600 V/m group was increased significantly (19.75% and 19.04% in the exposure groups compared with 14.01% in the control group, P < 0.05). The composition ratio of the atretic follicle number was increased significantly in the 100 V/m, the 400 V/m and the 1600 V/m group (8.45%, 9.95% and 11.70% in the exposure groups compared with 7.72% in the control group, P < 0.01). The composition ratio of the mature follicle and the pri/sec follicle was decreased significantly in the 400 V/m and the 1600 V/m group (1.50% and 1.55% in the exposure groups compared with 3.36% in the control group. 22.24% and 21.09% in the exposure groups compared with 26.60% in the control group, P < 0.01). Along with the increase of radiation dose, the ultrastructure of cell on the ovaries appeared more abnormal. CONCLUSIONS: The toxicities of female gonads are closely associated with exposures to HF-EMF. The nonage damage of female gonadal toxicities might emerge on the ovaries.

[Protective effect of tert-butylhydroquinone on PC12 cells from neurotoxicity induced by manganese in vitro].

OBJECTIVE: To investigate the protective effect of the tert-butylhydroquinone (tBHQ) on PC12 cells from neurotoxicity induced by manganese. METHODS: Cytotoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of MnCl2 (300, 600, 900 µmol/L) for 24, 48 or 72 h. PC12 cells were pretreated with 40 µmol/L tBHQ for 12 h, followed by the treatment of 600 micromol/L or 300 µmol/L MnCl2 for 72 h. Cytotoxicity of PC12 cells was measured by MTT assay, and cell apoptosis was examined by the method of Annexin V-FITC/PI in flow cytometry (FCM). RESULTS: The proliferation of PC12 cells treated with 300, 600, 900 µmol/L MnCl2 was suppressed in the dose dependent pattern (P < 0.01). Proliferation of PC12 cells treated with 600 µmol/L MnCl2 was suppressed to 40% of that in control group (P < 0.01), but the proliferation rate of PC12 cell pretreated with 40 µmol/L tBHQ was 180% of that in control group (P < 0.01). Apoptotic rate of PC12 cells treated with 300 micromol/L MnCl2 was higher than the vehicle control group (P < 0.01). Apoptotic rate of 40 µmol/L tBHQ pretreatment followed by 300 µmol/L MnCl2 treatment was lower than that of MnCl2 treatment group (P < 0.01). The inhibition rate of apoptosis was 61%. CONCLUSIONS: Manganese may suppress PC12 cells proliferation and induce apoptosis. tBHQ can reduce PC12 cells proliferation suppressed by manganese and attenuate the apoptosis induced by manganese.

Bisoprolol inhibits sodium current in ventricular myocytes of rats with diastolic heart failure.

1. Changes in sodium currents (I(Na)) in heart failure contribute to cardiac electrophysiological alterations and, thereby, to ventricular arrhythmias. Bisoprolol has anti-arrhythmic effects, but its direct effect on I(Na) in cardiac cells remains unclear. Accordingly, in the present study we investigated the effects of bisoprolol on ventricular I(Na) in diastolic heart failure (DHF) and normal rats. 2. The DHF model was produced by abdominal aortic coarctation for 4 weeks and single ventricular myocytes were isolated by enzymatic dissociation. The electrophysiological actions of bisoprolol on I(Na) currents were investigated using a whole-cell patch-clamp technique. 3. The membrane capacitance of rats in the DHF group was significantly greater than that of the control group and the current-voltage curve was simultaneously shifted downward. Bisoprolol concentration-dependently decreased I(Na) in ventricular myocytes of both groups (at -45 mV), with IC(50) values of 19.53 +/- 0.06 and 40.78 +/- 0.03 micromol/L in the control and DHF groups, respectively. 4. In both groups, the current-voltage curves were shifted upwards, whereas activation potentials, peak currents and reversal potentials showed no significant changes. At -45 mV, the descent ratio of current densities in the DHF group was lower than that of the control group. In both groups, inactivation curves were shifted to more negative potentials, but activation curves and recovery curves were not altered. Changes in the half-inactivation voltage, V(0.5), and the slope of the inactivation curve, S, were similar for both groups. 5. In conclusion, bisoprolol concentration-dependently decreases I(Na) in ventricular myocytes of DHF and normal rats, which could be responsible, at least in part, for its anti-arrhythmic effects.

[Exploitation of a signal collection system of a novel intelligent clinical patient monitor based on SoC].

A novel intelligent clinical monitoring system has been exploited based on SoC chip, with the integration of multiple parameters detecting techniques, the combination of the sensor technology and electric circuit technology.

[Effect of cadmium on estrogen receptor from rat uterus in vitro.].

OBJECTIVE: To investigate the effect of cadmium (Cd) on estrogen receptor and to assess its endocrine disrupting action. METHODS: The estrogen receptor rich supernatant was prepared from the ovariectomized Sprague-Dawley rats. The effects of cadmium on estrogen binding were performed using a sing-dose ligand-binding assay. Extract from uterus were treated with various concentrations of cadmium (0, 10(-3), 10(-5) or 10(-7) mol/L) for various pre-incubation time (0, 30, 60, 90 min) by means of orthogonal experimental design with orthogonal layout of L16(4(5)) (the experiment was repeated for 5 times). In addition to the radioinert competitor, each assay included a zero tube and a DES standard curve for quality control purpose. Data for cadmium and the DES standard curve were plotted as percent [3H]-E2 bound versus log (molar concentration), and the IC50 for cadmium was determined. The RBA for cadmium was calculated by dividing the IC50 of DES in terms of the IC50 of cadmium. RESULTS: Cadmium could not block the binding of estradiol to the receptor because hormone binding did not change with increasing cadmium concentration or increasing preincubation time. The results showed that the binding of [3H]-estradiol to uterine cytosols was not significant (P > 0.05). The Bmax (its unit is pmol/mg protein) of various concentrations of cadmium (0, 10(-3), 10(-5) or 10(-7) mol/L) for pre-incubating 0 min is 203.15 +/- 75.16, 203.41 +/- 22.78, 220.82 +/- 45.35, 209.10 +/- 49.66 respectively; The Bmax of them for pre-incubating 30 min is 215.67 +/- 92.97, 139.79 +/- 53.78, 205.27 +/- 23.60, 172.63 +/- 55.09 respectively. The Bmax of them for pre-incubating 60 min is 197.11 +/- 50.68, 203.24 +/- 66.33, 183.92 +/- 31.89, 183.33 +/- 32.70, respectively. The Bmax of them for pre-incubating 90 min is 229.69 +/- 76.88, 175.70 +/- 70.28, 164.26 +/- 24.46, 150.78 +/- 65.97 respectively. Mean IC50 for cadmium is 10(-4) - 10(-3) M. If the affinities of DES binding to estrogen receptors was taken to be 100%, the relative binding affinities of cadmium was 10(-6) - 10(-7). The results indicated that cadmium had only a very poor affinity with estrogen receptor. CONCLUSION: In vitro assay cadmium did not have distinct disrupting effect on binding of estradiol to estrogen receptors from rat uterine.