Objective To establish the murine congenital infection model by MCMV and observe the pathological changes and infection status of brain tissue.Methods After anesthesia,mice who were pregnant 11-13.5 days (E11-13.5 d) were intra-amniotic injected one uterus by one with virus (MCMV K181 suspension,1 μl,1×103 PFU).The control group of the same period was intra-anmiotic injected with culture medium DMEM (1 μl).Carefully reset the uteruses and close the abdomen.After 5 days of separated feeding,kill the pregnant mice,take the fetus out of the uterus,anesthetize and kill them.Make frozen sections of these fetal brains.Some sections were stained using conventional HE method,to observe the pathological changes under the light microscope.Detect MCMV early antigen in the brain tissue by immunohistochemistry staining and immunofluorescence assay.Results The survival rates of the infected group were 71.9％.Compared with the control group,intra-amniotic inoculation of MCMV does not affect the rate of fetal survival,fetal absorption,fetal death and the average weight of the heads,but decrease their average weight of the bodies.The pathological changes are found in the brain tissue of the mouse in the infection group.Through enzyme immunohistochemistry assay,there are many MCMV infected cells in brain-ventricular zone,brain subependymal zone,cerebral cortex and hippocampus area in the infection group.Similar findings were observed by immunofluorescence method.Conclusion By intra-amniotic injection of MCMV suspension,murine model of MCMV congenital infection can be successfully established.This model could be used to study the mechanisms of encephalodysplasia caused by congenital CMV infection in vivo.

Objective To investigate the influence of murine cytomegalovirus(MCMV) infection on differentiation and differentiation gene expression of neural stem cells (NSCs) in vitro for studying the mechanisms of brain abnormalities calmed by congenital cytomegalovirns infection. Methods NSCs were separated from fetal BALB/c mouse and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunnfluorescence. The NSCs infected by MCMV at dosage of multiplicity of infection (MOI) equaled to 5, I and 0. 1, respectively, were cultured in differentiation medium. The morphological changes of the cells were observed by inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence ( MOI = 1 ). The expression of early antigen (EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key differentiation genes Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs at early phage of differentiation culture. Results NSCs isolated from embryonic mouse brains could proliferate to form neurnspheres and strongly express Nestin and differentiate into NF-200 positive neurons or GFAP positive astrocytes. The NSCs of the infected groups couldn't adhere to the wall and appear differentia-tion growth, but showed swollen gradually after differentiation culture. The nostin expression of the infected groups downregulated slowly and was higher than that of the control groups ( P < 0.05 ). The GFAP and NSE expression of the infected groups were lower than that of the control groups (P <0.05). The EA of MCMV could be always detected in the cells of the infected groups. The ratios of nestin positive cells of the infected groups were higher than that of the control groups, but the ratios of GFAP and NSE positive cells of the for-mer were lower than that of the latter from 3rd to 9th day after differentiation culture ( P < 0.05 ). The levels of Wnt-3 mRNA and Wnt-7a mRNA of the infected groups were markedly lower than that of the control groups from 1st to 2nd clay and from 12th hour to 2nd day after differentiation culture respectively ( P < 0.05 ) . These changes of the infected groups became more obvious as MCMV MOI increased . Conclusion MCMV could inhibit significantly NSCs differentiate to neurons and astrocytes and lead to the decrease of dif-ferentiated cells. MCMV could inhibit or interfere with the gene expression of Wnt-3 and Wnt-7a in Wnt sig-nal pathway of NSCs. The effect that MCMV inhibited the differentiation and the differentiation gene expres-sion of NSCs showed dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering the differentiation gene expression of NSCs, which is possibly the one of primary causes of brain development disorders caused by congenital CMV infection.

Amphotericin B ; administration & dosage ; therapeutic use ; Antifungal Agents ; administration & dosage ; therapeutic use ; Biomarkers ; blood ; Child ; Cough ; diagnosis ; drug therapy ; etiology ; Cryptococcosis ; Fever ; diagnosis ; drug therapy ; etiology ; Fluconazole ; administration & dosage ; therapeutic use ; Humans ; Lung ; diagnostic imaging ; pathology ; Lung Diseases, Fungal ; complications ; diagnosis ; drug therapy ; Male ; Radiography, Thoracic ; Tomography, X-Ray Computed

Objective To explore the effects of Buyang Huanwu decoction(BYHWD)on expressions of nuclear factor-κB p65(NF-κBp65)and its inhibitor( I-κB)in signal transduction of NF-κB in brain tissue of rats with focal cerebral ischemia injury. Methods 180 Sprague-Dawley(SD)rats were randomly divided into normal group,sham-operated group,model group,pynolidine dithiocarbamate(PDTC)group,minocycline(MC)group and BYHWD treatment group,each group 30 rats. The rats of PDTC group were given PDTC 100 mg?kg-1?d-1 by intra-peritoneal injection. In MC group,MC was given by filling the stomach,the dose was 2.35 g?kg-1?d-1,the drug solution was prepared by adding the distilled water,and the total volume of drug solution to fill the stomach was kept at the same volume in various groups,thus the concentration of the drug was different. In BYHWD group,BYHWD was given,the dose was reduced to 5 g?kg-1?d-1 according to the body surface area dose conversion formula about people and animals. In sham-operated group and model group,the distilled water was given in the same volume as other drug solution. The protein expression levels of NF-κBp65 and I-κB in ischemic tissues were examined by using immunohistochemical method on the time points 7,14 and 21 days after treatment in each group. Results Compared with model group, the cell numbers with expression of NF-κBp65 in PDTC group,MC group and BYHWD group were significantly decreased along with the prolongation of therapy time,the decrease in number was more and more,until 21 days,it reached the valley level(cell/400 times HP:44.00±6.91,45.33±6.55,18.67±2.14 vs. 126.00±5.78,all P<0.05);the number of cells with expression of I-κB was obviously increased,the differences being statistically significant(all P<0.05),but the differences in expression of NF-κBp65 among the treatment groups at the different time points were not statistically significant(all P>0.05). After treatment for 7 days,the number of cells with positive expression of I-κB protein in BYHWD group was less than that in MC group(cell/400 times HP:55.00±3.40 vs. 72.50±4.29,P<0.05);after treatment for 14 days,the number in BYHWD group was approximately the same as that in the MC group, the difference being not statistically significant(93.50±6.15 vs. 93.00±6.20,P>0.05),and after treatment for 21 days,the number in BYHWD group was significantly higher than that in MC group(88.83±4.95 vs. 71.17±7.16, P<0.05). Conclusion BYHWD can regulate the expressions of inflammatory cytokine I-κB and NF-κB in signal transduction of NF-κB in ischemic brain tissue to inhibit the inflammatory reaction,thus it has the protective effect on cerebral ischemia.

Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.

Objective To explore the role of CD4+CD25+regulatory T cells(Treg)in the co-culture svstem consisting of T cells and mouse embryo fibroblasts(MEF)infected with murine cytomegalovirus (MCMV)in vitro.Methods A co-culture system of T cells and MCMV infected MEF(MEFMCMV)was established.The viral load in the supernatants was determined by plaque assay.TH1/TH2 differentiation-specific transcription factors T-bet/GATA-3 were assayed by Western blot.The levels of cytokines IL-4,IL-10and IFN-γ in the co-culture supenatants were measured by double-antibody sandwich ELISA.Results After 3 d incubation,the viral load in the supernatants of TdepTreg-MEFMCMC group,in which the T cells depleted Treg(TdepTreg)were co-cultured together with MEFMVMV,was significantly lower than that in MEFMCMV group.And in the co-cultivation of MEFMCMV and T cells without Treg the expressions of T-bet/GATA-3,IL-4,IL-10 and IFN-γ incteased significantly.Compared with the virus content in the TdepTreg-MEFMCMV group,the MCMV load in the"add-on Treg group"increased and the levels of T-bet/GATA-3 and IFN-γ were lower,and the expression of IL-4 didn't show any significant difference. Compared with the level of IL-10 in the TdepTreg-MEFMCMV group,the IL-10 level in the"add-on TREG group"didn't show any significant differece when the Treg ratio among total T cells was 1%～2%,and increased significantly when the Treg ratio was more than 5%.These eflfects were all correlated with Treg ratios in a dose-dependent manner.Conclusion MCMV infected MEF can induce the proliferation and activation of effector T cells,but Treg can inhibit the T cell-mediated protective effect on MCMV infection.

Objective To observe the effect of a traditional Chinese medicine, BuyangHuanwu Decoction, on the ethology in rat models of post-stroke depression ( PSD) .Methods Sixty male SD rats, after open-field ethology scoring, were randomly divided into five groups ( 12 rats in each group):sham operation group, cerebral ischemic stroke group, post-stroke depression ( PSD) group, BuyangHuanwu Decoction group, and fluoxetine group.The rat PSD models were established with separately breeding and chronic unpredictable moderate stress ( CUMS ) after middle cerebral artery occlusion. Behavior changes of the rats were detected by body weight measurement, neurological scoring, sugar consumption test and open-field test.Results Compared with the sham operation group and cerebral ischemic stroke group, the body weight,sugar consumption and open-field spontaneous activity in the PSD model group were decreased, and the neurological function deficits became more severe ( P <0.01 or P <0.05 ) .Compared with the PSD group, Both BuyangHuanwu Decoction group and fluoxetine group got significantly higher scores of body weight,sugar consumption and spontaneous activity,and improved neurological function deficits(P<0.01 or P<0.05).Conclusions PSD rats exhibit abnormal behavior.BuyangHuanwu Decoction can significantly improve the behavioral impairment of PSD rats. The antidepressant effect of BuyangHuanwu Decoction is comparable to that of fluoxetine in the treatment of PSD rats.

AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells ( NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI( multiplicity of infection) equaled to 5, 1 and 0.1 .respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence( MOI = 1). The expression of early antigen ( EA ) of MCMV was detected to observe the infection process. Real - time RT - PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture. RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF - 200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups ( P < 0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups ( P <0.05). The early antigen ( EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P < 0.05 ). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively( P <0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d( P <0.05 ). These changes in infected groups became more obvious as MCMV MOI increased. CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Newog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose - dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.

Objective: To explore the genetic basis for a patient with autism. Methods: High-throughput sequencing was carried out to detect copy number variations in the patient. Results: DNA sequencing found that the patient has carried a 0.11 Mb deletion in distal 2p16.3 spanning from genomic position 50 820 001 to 50 922 000, which resulted removal of exon 6 and part of intron 7 of the NRXN1 gene. The same deletion was not found his parents and brother. Conclusion Partial deletion of the NRXN1 gene may underlie the disease in this patient.

Objective To set up and apply a standardized communication system of adolescents and young adults (AYAs) cancer patients,in order to improve AYAs cancer patients' psychological distress and other negative emotions,as well as promote social support and quality of life of patients.Methods A AYAs cancer patients standardized communication system,suitable for China's national conditions,was preliminarily built.Using randomized controlled trials,a total of 171 subjects,selected from 486 cases of AYAs cancer patients,in the Third Xiangya Hospital of Central South University and Hunan Cancer Hospital from August to September in 2016,were intervened with a set of standardized communication system.The Mental Distress Thermometer (DT),Hospital Anxiety and Depression Scale (HADS),Social Support Rating Scale (SSRS) and Concise Health Status Questionnaire (SF-36) were used as evaluation indexes to observe the psychological distress,emotion,social support and quality of life of the three groups of subjects before intervention,immediately after intervention,1 month after intervention and 3 months after intervention.Results At the follow-up of 3 months after intervention,38 cases were lost,14 cases in communication group (final n =43),11 cases in music therapy group (final n =46),and 13 cases in routine group (final n =44)..There was no significant difference in the evaluation indexes between the communication group,music treatment group and the routine group before intervention (P ＞ 0.05).There were statistically significant differences in scores of psychological pain,anxiety and depression,and social support in the 3 groups,before and immediately after intervention,1 month and 3 months after intervention (P ＜ 0.05).The scores of psychological pain,anxiety and depression,social support and quality of life in the communication group 1 month after intervention were statistically significant compared with those in the music group and the routine group (P ＜ 0.05).Conclusions Compared to music therapy and regular care,the standardized communication system has significant effect on improving the psychological distress of AYAs with cancer,and can also improve their social support level and quality of life.The clinical validation of the standardized communication system can provide reference for psychological rehabilitation of cancer survivors.