Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Objective To investigate the effects of output power,action time and radiofrequency(RF) needle on the cool-tip radiofrequency ablation(RFA) by experimental tools and to determine the value of ultrasonography in size evaluation of RFA zone.Methods The cool-tip RFA to fresh calf liver were monitored by ultrasound.The experiments by single electrode needle were performed with different combination of output power (80 W,120 W) and time (5 min,8 min,10 min).The cluster needle was used for assessment at 5 min with different output power(80 W,120 W).After the end of trial,the longitudinal specimens were cut open.The view and size of the ablation zone were recorded with naked eyes.The pathological changes displayed by optical microscope were recorded as well.Results The measurement of ablation zone with naked eyes showed with the ablation zone expanded with time in 80 W-power cases,but the pace of expansion slowed down,but in 120 W-power cases,expansion of the ablation zone was not obvious; the ablation zone in 120 W-power was bigger than that in 80 W-power at 5 min,their difference decreased with time,and the ablation zones were similar at 10 min.The cluster needle can produce ablation zone with lesser aspect ratio than that of single electrode needle,consequently similar to circle.Ultrasonic measurement of the ablation zone had real discrepancy.Most of longitudinal diameters were greater than the real ones,while in large ablation lesions,vertical diameters were often less than the real ones.Under optical microscope,no change could be found in shape and structure of the cells in ablation zone.Conclusion The output power and performing time have impact on ablation.The high-power output increased heat production as well as reduction of heat conduction.Compared with single electrode needle,the cluster needle produced ablation zone closer to real hepatic tumor,thus has more reliable effect to small hepatocellular carcinoma with diameter around 2 cm.The ultrasond has a great significance in RFA guidance,but it could not accurately define the border of ablation zone.

Objective To identify the disease-causing gene mutation and investigate the genotypephenotype correlation in a Chinese pedigree with familial hypertrophic cardiomyopathy.Methods In this study we collected a large multigenerational Chinese family with FHCM.Total genome DNA was extracted from 67 subjects' peripheral leucocytes.The exons and boundary introns of MYH7 gene was amplified by PCR and directly sequenced by ABI PRISM 3700 DNA sequencer.Then,the mutation was examined.Results Fourteen family members had hypertrophic cardiomyopathy,including 4 deceased 2 of whom died from sudden death at young age.Analysis by echocardiography showed all the 10 living affected individuals have a maximal leftventricular-wall thickness of at least 13 mm.Three single nucleotide polymorphisms (SNP) which had been reported in NCBI SNP database,were found mutated.No mutation co-seperated with the disease was identified.Conclusion FHCM of this family was not caused by MYH7 mutation.Other genes should be screened to further identify the disease-causing gene mutation in hypertrophic cardiomyopathy.

Objective:To investigate the influencing factors of hospitalization for pregnant women with influenza A.Methods:From December 2018 to February 2019, 261 pregnant women with influenza A were admitted to Beijing Ditan Hospital, Capital Medical University. The clinical data of age, gestational period, underlying diseases, time from onset to treatment, white blood cell count and lymphocyte count of these patients were collected. Data of out-patients were compared with those of inpatients. Chi-square test and multivariate logistic regression were used to analyze the influencing factors of hospitalization in pregnant women with influenza A.Results:Among the 261 cases of pregnancy with influenza A, 36 cases (13.79%) were hospitalized, of which 10 (27.78%) were hospitalized due to severe influenza complications, the other 26 cases (72.22%) were hospitalized due to pregnancy related adverse events. The proportions of hospitalized patients with age ≥30 years old, gestational period ≥28 weeks, combined with underlying diseases and lymphocyte count <1×10 9/L were 75.00%(27/36), 83.33%(30/36), 16.67%(6/36) and 50.00%(18/36), respectively, which were significantly higher than those of out-patients (47.11%(106/225), 35.56%(80/225), 0.89%(2/225) and 13.22%(16/121), respectively; χ2=9.66, 29.05, 26.00 and 22.12, respectively, all P<0.05). The proportions of inpatients and out-patients with white blood cell count ≥4×10 9/L were 97.22%(35/36) and 97.52%(118/121), respectively, and there was no significant difference ( χ2=0.01, P=0.921). Multivariate logistic regression analysis showed that age ≥30 years (odds ratio ( OR)=5.181, 95% confidence interval ( CI) 1.628-16.489, P=0.005), gestational period ≥28 weeks ( OR=11.054, 95% CI 3.233-37.796, P<0.01), lymphocyte count <1×10 9/L ( OR=6.864, 95% CI 2.237-20.729, P=0.001), and time from onset to treatment <24 h ( OR=0.076, 95% CI 0.012-0.468, P=0.005) were the influencing factors for hospitalization of pregnant women with influenza A. Conclusion:Age ≥30 years old, gestational period ≥28 weeks, lymphocyte count <1×10 9/L and time from onset to treatment <24 h are the influencing factors for hospitalization of pregnant women with influenza A.

OBJECTIVETo analyze the effects of long-term microwave exposure on hippocampal structure and function in the rat.

METHODSExperiments were performed on 184 male Wistar rats (three exposure groups and a sham group). Microwaves were applied daily for 6 min over 1 month at average power densities of 2.5, 5, and 10 mW/cm2. Learning and memory abilities were assessed by Morris water maze. High performance liquid chromatography was used to detect neurotransmitter concentrations in the hippocampus. Hippocampal structures were observed by histopathological analysis.

RESULTSFollowing long-term microwave exposure there was a significant decrease in learning and memory activity in the 7 d, 14 d, and 1 m in all three microwave exposure groups. Neurotransmitter concentrations of four amino acids (glutamate, aspartic acid, glycine, and gamma-aminobutyric acid) in hippocampus were increased in the 2.5 and 5 mW/cm2 groups and decreased in the 10 mW/cm2 group. There was evidence of neuronal degeneration and enlarged perivascular spaces in the hippocampus in the microwave exposure groups. Further, mitochondria became swollen and cristae were disordered. The rough endoplasmic reticulum exhibited sacculated distension and there was a decrease in the quantity of synaptic vesicles.

CONCLUSIONThese data suggest that the hippocampus can be injured by long-term microwave exposure, which might result in impairment of cognitive function due to neurotransmitter disruption.

Animals ; Chromatography, High Pressure Liquid ; Cognition ; Hippocampus ; pathology ; physiopathology ; radiation effects ; Learning ; Male ; Memory ; Microscopy, Electron, Transmission ; Microwaves ; Rats ; Rats, Wistar

Objective To investigate the effect of high power microwave(HPM) irradiation on neuropeptide Y (NPY) and neural nitric oxide synthase (nNOS) expression in the cerebral cortex and hippoeampus of Wistar rats. Methods A total of 110 Wistar rats were used for this study.Three groups of 30 Wistar rats were exposed to HPM irradiation at intensities of 3,10,30 and 100 mW/cm~2,respectively.Twenty rats served as controls and were ex- posed to sham HPM irradiation.At 6 h,and at 1,3,7,14 and 28 d after irradiation,five rats from each group were sacrificed,and their cerebral cortices and hippocampi were harvested.HE staining was used to highlight any change in the structure of the cerebral cortex or hippocampus.Immunohistochemistry techniques and image analysis were used to study the changes in NPY and nNOS expression.Results 10 to 100 mW/cm~2 HPM irradiation caused pyc- nosis and deep staining of some neurons in the cerebral cortex and hippocampus.The increase in nNOS expression and decrease in NPY expression observed were significant at 3 days after irradiation.Conclusion HPM irradiation can induce injury in neurons of the cerebral cortex and hippoeampus,and abnormal NPY and nNOS expression.

OBJECTIVETo investigate the expression of aquaporin 4 (AQP4) after microwave exposure and the correlation with the brain injury by radiation.

METHODS70 male rats were exposed to microwave whose average power density was 0, 10, 30 and 100 mW/cm(2) respectively. Rats were sacrificed at 6 h, 1 d, 3 d and 7 d after exposure. Immunohistochemistry and Western blot were used to detect the expression of AQP4 in protein level in rat hippocampus, and the expression of AQP4 in gene level was measured by in situ hybridization and RT-PCR.

RESULTSThe expression of AQP4 in rat hippocampus was abnormal after 10, 30, 100 mW/cm(2) microwave exposure. The protein level showed increased at first and then recovered at 10 and 30 mW/cm(2) groups, while increased progressively in 100 mW/cm(2) group within 14 d (P < 0.01). The gene expression of AQP4 was increased (0.51 +/- 0.02) at the beginning (6 h) and then regained after 10 mW/cm(2) microwave exposure, while in 30 and 100 mW/cm(2) groups, it rose to the peak at 7 d (0.46 +/- 0.02 and 0.43 +/- 0.08) and didn't get back (P = 0.004; P = 0.012).

CONCLUSIONMicrowave radiation can increase the expression of AQP4 in rat hippocampus. The change might participate in the process of increasing permeability of blood-brain barrier and lead to the brain edema after microwave radiation.

Animals ; Aquaporin 4 ; genetics ; metabolism ; Hippocampus ; metabolism ; radiation effects ; Male ; Microwaves ; adverse effects ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of PI-3K and p38MAPK signal pathways on the cyclooxygenase-2 (COX-2) expression induced by the epidermal growth factor (EGF) in PC-3 cells.

METHODSPC-3 cell proliferation was detected by methylthiazolyl tetrazolium (MTT) assay after stimulated by EGF (0 microg/L), EGF (10 microg/L), EGF (10 microg/L) + LY294002 (20 micromol/L) and EGF (10 microg/L) + SC203580 (20 micromol/L), and so was the COX-2 expression in the PC-3 cells by RT-PCR and Western blot assay after stimulated the same way for 24 hours. ELISA was used to determine the changes of PGE2 in the culture medium.

RESULTSLY294002 and SC203580 signficantly inhibited PC-3 cell proliferation (P < 0.05), COX-2 expression and PGE2 production after EGF stimulation (P < 0.05).

CONCLUSIONEGF can stimulate PC-3 cells into proliferation and induce COX-2 mRNA and the upregulation of its protein expression, while LY294002 and SC203580 can inhibit EGF from the above effects on PC-3 cells.

Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 ; genetics ; metabolism ; Dinoprostone ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; pharmacology ; Gene Expression ; drug effects ; Humans ; Male ; Phosphatidylinositol 3-Kinases ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effect of long-term microwave radiation on male reproduction in rats.

METHODSA total of 100 male Wistar rats were exposed to microwave radiation with average power density of 0, 2.5, 5 and 10 mW/cm2 for 4 weeks, 5 times a week and 6 minutes per time. Changes in serum testosterone, testicular index, histology and ultrastructure, and the percentage of teratospermia in the epididymis were observed dynamically at 6 h, 7 d, 14 d, 28 d and 60 d after the exposure.

RESULTSThere was a significant decrease in serum testosterone concentration at 28 d after microwave radiation at 2.5, 5 and 10 mW/cm2 ([10.20 +/- 4.31] ng/ml, [5.56 +/- 3.47] ng/ml and [7.53 +/- 4.54] ng/ml) and at 60 d at 10 mW/cm2 ( [15.95 +/- 9.54] ng/ml), as compared with the control group ([23.35 +/- 8.06] ng/ml and [31.40 +/- 9.56] ng/ml) (P < 0.05 or P < 0.01). No significant changes were found in the testis index at 6 h -60 d after microwave radiation at the three doses, but different degrees of degeneration, necrosis and shedding of spermatogenic cells, thinning of spermatogenic epithelia, and decrease or deletion of spermatozoa were observed, and more obvious at 28 d and 60 d. Swelling and cavitation of mitochondria in all spermatogenic cells, agglutination and margin translocation of nuclear chromatin in the spermatogonial and Leydig cells were seen at 7 d and 60 d after 5 mW/cm2 microwave radiation. The rate of teratospermia of the epididymis was increased, more obviously at 7 d after 2.5, 5 mW/cm2, 60 d after 5 mW/cm2, and 7 d, 28 d and 60 d after 10 mW/cm2 microwave radiation (P < 0.05 or P < 0.01).

CONCLUSIONLong-term microwave radiation may cause injury to male reproduction, which is positively correlated with the radiation dose, and has an obvious late effect.

Animals ; Dose-Response Relationship, Radiation ; Male ; Microwaves ; adverse effects ; Rats ; Rats, Wistar ; Reproduction ; radiation effects ; Sperm Head ; radiation effects ; Testis ; radiation effects

OBJECTIVETo investigate the effects of pentoxifylline on rats and mice with learning and memory dysfunctions.

METHODSMorris water maze test was used to observe the effects of pentoxifylline on learning and memory of naturally aging rats, and jumping stand test was performed to examine its effects in promoting the learning and memory functions in mice with scopolamine- and ethanol-induced memory dysfunctions.

RESULTSIn aging rats, pentoxifylline at high, moderate and low doses all significantly reduced the latency of platform finding in the place navigation test (P<0.01 or P<0.05 ), and increased the quadrant searching frequency in the spatial probe test (P<0.05). Pentoxifylline at the 3 doses significantly increased the latency of electrification (P<0.01 or P<0.05) and decreased the times of error (P<0.05) of the mice as compared with scopolamine- treated group. Pentoxifylline also improved ethanol-induced memory dysfunction in the mice, but the changes in the performance of the mice were not statistically significant.

CONCLUSIONPentoxifylline can improve the learning and memory abilities of rats and mice.

Aging ; Animals ; Behavior, Animal ; drug effects ; Ethanol ; Maze Learning ; drug effects ; Memory ; drug effects ; Memory Disorders ; chemically induced ; Mice ; Pentoxifylline ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Scopolamine Hydrobromide