Seven genes (fabD, fabG, fabH, fabA, fabZ, fabB and fabI) of E.coli fatty acid biosynthetic enzymes were cloned by PCR amplifying and appropriate expression vectors were constructed. Under induction assay expression of plasmid encoded proteins was carried out in strain BL21(DE3) and seven enzymes were purified using Ni-NTA agarose resin. In the absence of [2-14C] malonyl-CoA fatty acid synthetic reaction was reconstituted in vitro by adding seven enzymes and co-factors. And several model reactions were established for identification of special fatty acid biosynthetic enzymes. Meanwhile Clostridium acetobutylicium FabZ function was characterized by this method.

Objective To explore the role of brain derived neurotrophic factor(BDNF) promoter methylation for the morbidity of depression patients,and to investigate the relevance between BDNF promoter methylation and the suicidal ideation in patients with first-episode major depressive disorder.Methods The peripheral blood samples from 23 cases of depression patients with suicide ideation ,24 cases of depression patients without suicide ideation,and 27 healthy controls were grouped using simple randomization method, and the methylation status of CpG island of BDNF promoter were subsequently evaluated using bisulphite sequencing.Results The methylation rates(median,quartile interval) of depression patients with suicidal ideation, depression patients without suicidal attempt, and the healthy control were detected as follows, CPG-1 : 2.60 (0.28), 2.70 (0.60), 6.40 (1.00);CPG-2:3.35 (1.15),3.20 (1.00),14.50 (2.60);CPG-3:1.50 (0.40),1.50 (0.60),4.60 (0.90);CPG-4:1.75 (0.90) ,1.50 (0.50),3.90 (0.90);CPG-5:0.80 (0.58),0.90 (1.00),5.70 (2.10);CPG-6:6.05 (1.03),6.20 (1.70), 14.50 (1.70);CPG-7:10.55 (1.68), 10.10 (1.50), 14.70 (1.70);CPG-8:16.35 (0.90), 16.40 (1.00),4.80 (1.20);CPG-9:4.20 (4.80),4.70 (1.30),28.50 (19.30);CPG-10:10.25 (1.08),10.30 (1.20) ,4.30 (0.90);CPG-11:62.80 (4.05) ,62.30 (4.00) ,37.20 (2.60);CPG-12:5.60 (1.05) ,5.60 (1.30) ,12.30 (1.80);CPG-13:4.15 (1.10) ,4.10 (1.10) ,59.70 (4.90);CPG-14:5.45 (0.78) ,5.70 (0.90),6.30 (0.60);CPG-15:3.70 (0.70) ,3.90 (1.00) ,62.40 (6.50);CPG-16:8.05 (1.75) ,8.10 (1.60) ,74.30 (50.90);CPG-17:3.20 (0.78),3.20 (0.80),32.40 (13.20);CPG-18:1.90 (0.48),1.80 (0.40),2.30 (0.30);CPG-19 : 35.00 (6.27), 35.20 (3.00), 64.20 (44.70).There was an obvious difference in methylation rate among three groups,which was statistically significant (P＜0.01).However, no statistical significance for the methylation rates was observed between the depression patients with suicidal ideation and without suicidal ideation.Interestingly, statistical significance for the methylation rates were observed when compared the depression patients with suicidal ideation with healthy control (P＜ 0.05), and the depression patients without suicidal ideation with healthy control (P＜0.05).Conclusion The results show that the association is observed between BDNF promoter methylation and the morbidity of depressive disorder,but not between BDNF promoter methylation and the suicidal ideation of depression patients.

Objective To explore the clinical characteristics of neonatal-onset carbamoyl phosphate synthetase I deficiency (CPS1D). Methods Clinical data and result of genetic detection of one neonate with CPS1D were retrospectively analyzed. The pertinent literature was reviewed. Results A 3-day old girl, with onset symptoms of nonspecific performance, such as poor feeding, less activity, tachypnea, and seizures. After fasting, anti-infection, and respiratory support etc. the condition was improved. However, the condition deteriorated and developed rapidly after feeding restarted. MRI showed extensive cerebral white matter lesions. Blood ammonia?>?500 μmol/L. Gene detection found two heterozygous mutations in pathogenic gene CPS1 in twentieth exon of c.2407C?>?G (p.803, R, G) and fourth exon C.323G?>?A (p.108, G, E), according to which CPS1D was diagnosed finally. Conclusions For neonate with normal birth, had feeding difficulty, seizures, and consciousness disorder after establishment of normal feeding, if blood ammonia level significantly increased, the blood and urine amino acids analysis and gene detection should be performed to confirm the diagnosis.

Objective To investigate and analyze cognitive similarities and differences of nurses factors that lead to the tense nurse-patient relationship from both attitudes of nurses and patients. Methods By using the method of questionnaire, 284 nurses and 305 patients were investigated respectively in a top'three hospital of a city. Results There was a significant difference in cognitive scores of nurses factors that lead to the tense nurse- patient relationship (P < 0.01), patients' scores were (3.90±0.47) points which were higher than (3.52±0.35) points of the nurses′. In dimensions of prioritization, both nurses and patients believed that technical ability, professional knowledge and service attitude were the main factors leading to the tense relationship between nurses and patients. While nurses held that communication ability was more important, patients held that the nurse′s own personality was more important. Conclusions There are similarities and differences of attitudes to nurs's factors between nurses and patients regarding tension in nurse- patient relationship. The tense relationship between nurses and patients is affected to a great extent by nurses' professional knowledge, technical ability and service attitude. Managers should objectively evaluate and fully pay attention to cognitive similarities and differences of both sides, strengthen nurses′theoretical knowledge and clinical skills training, improve service attitude, and develop good character,in order to provide high quality nursing service for patients.

Objective To investigate the effects of stress on the methylation of brain derived neurotrophic factor gene in the patients with major depressive disorder (MDD),and to investigate the relationship between BDNF gene methylation and MDD.Methods 47 cases of MDD were divided into MDD stress group (n=24) and MDD non stress group(n=23) while 27 health subjects were collected as normal control group.The methylation status of CpG island in the promoter region of BDNF gene in peripheral blood was detected by the method of heavy hydrogen and hydrogen sulfate.SPSS17.0 statistical software package was used to analyze the data.The differences of 19 CpG methylation rates and the overall level of methylation rates of three groups were analyzed.Results The CpG overall methylation rates (median,interquartile range) of MDD stress group,MDD non stress group and normal control group was 189.150 (7.575),188.500 (400)and 480.200(770) respectively,and the difference was statistically significant (P＜0.01).There was no significant difference in the overall methylation rate of CpG in the MDD stress group compared with MDD non stress group (P＞0.05).The CpG methylation rates (mean± SD or median,interquartile range) of three groups were detected as follows:CpG-1:2.600(0.275),2.700 (0.400),6.500(0.800);CpG-2:3.350(0.650),3.300(0.800),14.600(1.500);CpG-3:1.596±0.363,1.543±0.400,4.581 ±0.437;CpG-4:1.779±0.516,1.522±0.329,4.033 ±0.529;CpG-5:0.900 (0.575),0.800 (0.600),5.700 (1.500);CpG-6:6.258 ± 0.805,6.213 ±0.944,14.589±0.819;CpG-7:10.667±0.894,10.283± 1.006,15.000±0.763;CpG-8:16.421 ±0.697,16.330±0.775,24.796±0.547;CpG-9:4.713±0.565,4.891 ±0.554,28.826±0.679;CpG-10:10.254±0.902,10.378±0.777,11.381±0.538;CpG-11:24.125±2.301,24.170±2.613,37.474± 1.579;CpG-12:5.442±0.641,5.596±1.117,12.141 ±0.940;CpG-13:4.150(1.150),4.200(1.000),61.700(4.800);CpG-14:5.500±0.544,5.717±0.568,6.378±0.397;CpG-15:3.700 (0.700),4.100(1.000),63.300(2.500);CpG-16:8.200 (1.775),8.100(1.500),75.200(3.300);CpG-17:3.250(0.550),3.300(0.800),34.600(5.000);CpG-18:1.988±0.279,1.939±0.259,2.330±0.207;CpG-19:35.338±2.421,35.187±2.259,65.941 ±2.692.16 CpG methylation rates of 19 CpG were higher in MDD stress group and MDD non stress group.Compared with the normal control group,the difference was statistically significant (P＜0.01).There was no significant difference in CpG methylation rate between MDD stress group and MDD non stress group (P＞0.05).Conclusion The overall methylation rate of CpG in BDNF gene promoter region is closely related with MDD,which may affect the incidence of MDD.There was no correlation between CpG methylation in BDNF gene promoter region and MDD,and stressful life events may not be the direct cause of CpG methylation in BDNF gene promoter region in patients with MDD.

Objective To examine the effects of maternal rats exposed to chronic unpredictable stress before pregnancy on the behaviors and brain monamine of their adult male offspring.Methods Sixteen SD rats were divided into chronic unpredictable stress (CUS) group and controls.CUS rats were exposed to 21 days chronic unpredictable stressors ,and the controls were stress-free.Ten days after the last stressor, all the female rats were caged with sexually experienced males of the same strain.Then we performed the following experiments on the two months male progeny, sucrose consumption measuring anhedonia, Morris water maze measuring cognitive function and high performance liquid chromatography detecting the contents of monoamine.Results The sucrose consumption showed that both sucrose intake and sucrose consumption percentage of the control progeny were higher than those of the CUS progeny ( sucrose consumption: ( 10.23 ± 4.12 ) g vs ( 6.48 ± 3.19 ) g; sucrose consumption percentage: ( 85.43 ± 20.15 ) % vs (60.98 ± 24.65 ) % ) (P ＜ 0.05 ).The number of times crossing the removed hidden platform in the CUS progeny ( 1.64 ± 1.69) was significantly fewer than that in the control progeny (4.17±2.29 ) in Morris water maze (P ＜ 0.05 ).The contents of serotonin in the hypothalamus of CUS progeny ( ( 500.17 ± 80.94 ) ng/g tissue) was lower than that of the control progeny ( ( 569.63 ± 50.91 ) ng/g tissue) (P ＜0.05) ,while the norepinephrine in the hippocampus of CUS progeny( (2315.01 ± 1397.12) ng/g tissue) was higher than that of the control progeny( (907.56 ± 207.27) ng/g tissue) (P＜0.05) by high performance liquid chromatography.Conclusions Depression or stressful events before pregnancy of dams result in anhedonia, decreased spatial memory and abnormalities in brain monoamine of their adult male progeny.

Objective To investigate the relationship between the homocysteine,sperm DNA fragmentation index and sperm counts of male with severe impaired spermatoqenesis.Methods From December 2015 to February 2017,56 male patients with severe impaired spermatoqenesis were enrolled in the study.The patients were divided into two groups according to the WHO criteria:severe oligozoospermia and azoospermia group (n =25) and oligoasthenoteratozoospermia group (n =31),and the control group was a male with no reproductive impairment (n=27).The sperm parameters were analyzed by using the computer automatic semen analyzer,sperm DNA fragmentation index and serum Hcy level were detected by sperm chromatin diffusion method and enzyme colorimetric method.Results The median of Sperm DNA fragmentation index and homocysteine level in control groups were 33％ [95％CI(29.0％ ～34.4％)] and 13.2 μmol/L [95％CI(12.4 μmol/L～14.2 μmol/L)],and in severe spermatogenesis groups in these two indicators were 21％ [95％CI(19.0％ ～24.0％)] and 8.9 mol/L [95％CI(8.4 μmol/L～ 9.4 μmol/L)],respectively.The results of these two items were higher than the control group,the difference was statistically significant (t=6.793～7.543,P=0.000).Sperm survival rate in normal control group and severe spermatogenesis group was 71％ [95％ CI(67.8％ ～75.1％)] and 57％[95％CI(52.3％ ～58.0％)],respectively,and the difference was statistically significant (t=-8.475,P=0.000).Sperm DNA fragmentation index was positively correlated with serum Hcy level and sperm concentration,Passing-Bablok regression analysis was:Y=10.705 +0.053X,Y=21.071+0.286X,and Hcy level was negatively correlated with sperm concentration.Conclusion The increase of Hcy level and sperm DNA fragmentation index may be an importantcause of male with severe impaired spermatoqenesis,but the specific mechanism remains to be further studied.

Objective To investigate the transcriptional autoregulation of PhoP/PhoQ under different growth conditions in Yersinia pestis.Methods The entire promoter region of YPO1635 was amplified and cloned into the pRW50 vector containing a promoterless lacZ reporter gene.The recombinant LacZ reporter plasmid was transformed into the wild-type strain (WT) and the phoP mutant strain (ΔphoP),respectively,to measure the promoter activity (the β-galactosidase activity) of the target gene in WT and ΔphoP by using the β-galactosidase enzyme assay system.Total RNAs were extracted from WT and ΔphoP strains,and primer extension assay was employed to detect the promoter activity by examining the amount of primer extension products of YPO1635 in WT and ΔphoP.Results The LacZ fusion results showed that the transcription of YPO1635 was positively regulated by PhoP under L-TMH and brain-heart infusion(BHI) conditions,but it was not regulated in H-TMH medium.The primer extension assay detected two transcriptional start sites located at 90 and 118 bp upstream of the translation initiation site of phoP,named P1 and P2,respectively.Under low Mg2+ TMH conditions,the promoter activity of P1 rather than P2 was positively regulated by PhoP.Under high Mg2+ TMH conditions,the promoter activities of both P1 and P2 showed no obvious difference in the WT and ΔphoP strains.Under rich BHI conditions,both promoters were under negative control of PhoP.Conclusion Different autoregualtion patterns of PhoP/PhoQ under different growth conditions would help Y.pestis to quickly adapt to the changing living environment.

Objective This study aimed to establish a reliable primary culture protocol for preparing murine spleen-derived mesenchymal stem cells ( MSCs) by tissue explant culture.Methods Healthy mouse spleens were crushed by syringe handle to harvest spleen mesenchymal tissues.Then the tiny pieces of spleen tissue were digested by collagenase II before seeded into culture flasks.The morphological characteristics of spleen tissue-derived cells were observed under the inverted microscope.Further, the surface antigen profile of the cells was analyzed by flow cytometry (FACS).The cells were induced to differentiate into osteoblasts and adipocytes.Results The murine spleen-derived MSCs exhibited a spindle-shaped appearance.The FACS results showed that the spleen-derived MSCs highly expressed CD29, CD44, CD105 and Sca-1, but weakly expressed CD11b, CD34, CD45 and Ia. In addition, the spleen-derived MSCs steadily differentiated into osteoblasts and adipocytes in the induction medium.Conclusions A method of primary culture of murine spleen-derived MSCs by explant culture is successfully established.The harvested MSCs exhibit high purity and cell proliferation ability, and provide a reliable cell model for related researches.

OBJECTIVE: To analyze the clinical characteristics and genetic variant of a child featuring X-linked mental retardation. METHODS: Whole exome sequencing and Sanger sequencing were used for the detection of variant and pedigree validation, respectively. Clinical manifestation of patients with DDX3X gene variants were also reviewed. RESULTS: The child was found to harbor a heterozygous NM_001193416.3: c.1332_1333delCT (p.Leu445Serfs*19) variant of the DDX3X gene. The same variant was not found in either of her parents. CONCLUSION The child was diagnosed with X-linked mental retardation due to variant of the DDX3X gene. Above finding has enriched the spectrum of DDX3X gene variants and provided a basis for clinical diagnosis and prenatal diagnosis for this pedigrees.
Child ; DEAD-box RNA Helicases/genetics* ; Female ; Heterozygote ; Humans ; Intellectual Disability/genetics* ; Mental Retardation, X-Linked/genetics* ; Mutation ; Pedigree ; Pregnancy ; Exome Sequencing