Objective To investigate the oxidative stress and inflammation in trophoblast cells stimulated by different chain length fatty acids.MethodsSerum-free trophoblast cells cultured in vitro were divided into five groups,which were incubated with DMEM medium without free fatty acid (F-FFA),short chain fatty acids (SC-FFA),medium chain fatty acids (MC-FFA),long chain fatty acids (LC-FFA),very long chain fatty acids (VLC-FFA).Then cells in each group were stimulated by DMEM medium,reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (apocynin) and p38 mitogen-activated protein kinases (p38MAPK) inhibitor (SB203580) and were subdivided as each FFA plus-DMEM group, plus-NADPH-Ⅰ and plus-p38MAPK-Ⅰ groups.Expressions of mRNA and protein of p38MAPK and cyclooxygenase 2 (COX-2) in trophoblast cells were detected by real-time PCR and western blot.Results (1) The mRNA expression of p38MAPK in LC-FFA + DMEM,VLC-FFA + DMEM,LC-FFA + NADPH-Ⅰ,LC-FFA + p38MAPK-Ⅰ,VLC-FFA + NADPH-Ⅰ,VLC-FFA + p38MAPK-Ⅰ group were 4.56 ±0.28,22.65 ±2.40,0.87 ±0.06,1.02 ±0.15,19.87 ± 1.93,10.22 ±0.75 separately,and the protein expressions were 0.79 ± 0.02,0.93 ± 0.10,0.43 ± 0.06,0.44 ± 0.19,0.79 ± 0.10,0.81 ±0.14.Compared with other groups,the mRNA and protein expressions of p38MAPK in LC-FFA + DMEM,VLC-FFA + DMEM group were increased ( P ＜ 0.05 ).Compared with LC-FFA + DMEM group,mRNA and protein expressions of p38MAPK in LC-FFA + NADPH-Ⅰ and LC-FFA + p38MAPK-Ⅰ group were significantly decreased (P ＜ 0.05 ).Compared with VLC-FFA + DMEM group,mRNA and protein expressions of p38MAPK had no difference in VLC-FFA + NADPH-Ⅰ group (P ＞ 0.05 ),mRNA expression of p38MAPK in VLC-FFA + p38MAPK-Ⅰ group was significantly decreased (P ＜ 0.05 ),but there was no difference in protein expression ( P ＞ 0.05).(2) The mRNA expression of COX-2 in LC-FFA + DMEM,VLC-FFA +DMEM,LC-FFA + NADPH-Ⅰ,LC-FFA + p38MAPK-Ⅰ,VLC-FFA + NADPH-Ⅰ,VLC-FFA + p38MAPK-Ⅰ group were 3.97 ±0.03,39.08 ±0.63,0.99 ±0.13,0.98 ±0.18,20.93 ±3.70,13.46 ± 2.31 separately,and the protein expressions were 1.32 ± 0.20,1.33 ± 0.25,0.59 ± 0.13,0.58 ± 0.30,0.88 ± 0.18,0.91 ± 0.24.Compared with other groups,mRNA and protein expressions of COX-2 in LC-FFA + DMEM and VLC-FFA + DMEM group were significantly increased ( P ＜ 0.05 ).Compared with LC-FFA + DMEM group,mRNA and protein expressions of COX-2 in LC-FFA + NADPH-Ⅰ and LC-FFA +p38MAPK-Ⅰ group were decreased ( P ＜ 0.05 ).Compared with VLC-FFA + DMEM group,mRNA and protein expressions of COX-2 in VLC-FFA + NADPH-Ⅰ and VLC-FFA + p38MAPK-Ⅰ group were all decreased ( P ＜ 0.05 ).( 3 ) The correlation analysis showed that there were significantly positive correlations between the mRNA and protein expressions of p38MAPK and COX-2 in LC-FFA group ( P ＜ 0.05 ).There were significantly positive correlations in protein expression ( P ＜ 0.05 ),but no conrelation in the mRNA expression between p38MAPK and COX-2 in the F-FFA,SC-FFA,MC-FFA,VLC-FFA groups (P ＞ 0.05).ConclusionsThe oxidative stress and inflammation may exist in trophoblast cells which were stimulated by LC-FFA and VLC-FFA.p38MAPK signal transduction pathway may contributed in this process.

Objective To investigate the changes of fatty acid oxidase in the placenta of preeclampsia cases with different clinical features, and the relationship with oxidative stress and inflammatory response. To study the correlation of serum free fatty acid (FFA) and triglycerides (TG) level in early second trimester with the molecular changes of the long-chain fatty acid oxidase in the third trimester. Methods This was prospective cohort study, in which cases with singleton pregnancies who archived in Haidian Maternal and Children′s Hospital, Beijing, from January 1st 2012 to May 31st, with regular prenatal care were included. Doppler ultrasound was used for screening for the presence of early diastolic notch of uterine artery at 22-24 weeks of gestation. All the 101 cases with the early diastolic notch of uterine artery were included as the notch group, and 377 cases without the early diastolic notch of uterine artery were included as the non-notch group. The perinatal outcomes and the incidence of hypertensive disorders in pregnancy of the two groups were observed. The serum level of FFA and TG was tested, and the mRNA and protein expression of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), P47-phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p38 mitogen-activated protein kinase α (p38MAPK-α) and cyclooxygenase-2 (COX-2) were detected using real-time quantitative PCR and western blot. The relationship between serum level of FFA and TG and the mRNA and protein expression of LCHAD, NADPH P47-phox,p38MAPK-α and COX-2 of the placental tissue specimens were analyzed. Results (1) In the notch group, there were 9 cases of early-onset preeclampsia,15 cases of late-onset preeclampsia and 10 cases of gestational hypertension;and there were 8 cases of late-onset preeclampsia and 18 cases of gestational hypertension in the non-notch group. 15 cases with normal blood pressure in each group were randomly selected as the control group.(2)The serum level of TG of cases of early-onset preeclampsia, late-onset preeclampsia and gestational hypertension in the notch group were(2.0±0.8),(1.8±0.6)and (1.9±0.7)mmol/L, and that of FFA were(0.68±0.26),(0.52±0.10)and(0.52±0.17)mmol/L, respectively. The serum level of TG of cases of late-onset preeclampsia and gestational hypertension in the non-notch group were(1.6±0.6)and(1.4±0.4)mmol/L, and that of FFA were(0.49±0.11)and(0.48±0.05)mmol/L, respectively. The serum level of TG and FFA in the control group were(1.4±0.5)and(0.52±0.06)mmol/L, respectively. The TG level of the notch group was higher than that of the control group, and the difference was statistically significant (P<0.05). The FFA level of the early-onset preeclampsia in the notch group was higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group, and the difference was statistically significant (P<0.05).(3) The mRNA expression of LCHAD in the placenta of early-onset preeclampsia in the notch group was significantly lower than that of the late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P<0.01). The mRNA expression of NADPH P47-phox of the early-onset preeclampsia in the notch group were significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group(P<0.01). The mRNA expression of p38MAPK-α of the early-onset preeclampsia in the notch group were significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P<0.01). The mRNA expression of COX-2 of the early-onset preeclampsia in the notch group were significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P<0.01).(4)The protein expression of LCHAD in the placenta of early-onset preeclampsia in the notch group, late-onset preeclampsia in the notch group and gestational hypertension in the notch group were significantly lower than that of the control group (P<0.01); and the protein expression of LCHAD in the placenta of early-onset preeclampsia in the notch group was significantly lower than that of late-onset preeclampsia in the non-notch group (P<0.01). The protein expression of NADPH P47-phox in the placenta of early-onset preeclampsia in the notch group was significantly higher than that of late-onset preeclampsia in the non-notch group and control group (P<0.05). The protein expression of p38MAPK-α in the placenta of early-onset preeclampsia in the notch group was significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and control group (P<0.01). The protein expression of COX-2 in the placenta of early-onset preeclampsia in the notch group, late-onset preeclampsia in the notch group, gestational hypertension in the notch group, late-onset preeclampsia in the non-notch group, and gestational hypertension in the non-notch group, were significantly higher than that of control group (P<0.01).(5)The blood concentration of maternal FFA in the early-onset preeclampsia in the notch group was significantly negatively correlated with the mRNA and protein expression of placental LCHAD (r=-0.810,-0.932,P<0.01). There was no correlation between maternal TG level and the mRNA and protein expression of placental LCHAD in each group(P> 0.05).(6)The mRNA expression of placental LCHAD in the early-onset preeclampsia in the notch group was significantly negatively correlated with the mRNA expression of placental NADPH P47-phox and COX-2 (r=- 0.877,-0.762, P<0.05). The mRNA expression of placental LCHAD in the control group was significantly negatively correlated with the mRNA expression of placental COX-2 (r=- 0.565, P<0.01). The protein expression of placental LCHAD in the early-onset preeclampsia in the notch group was significantly negatively correlated with the protein expression of NADPH P47-phox (r=- 0.818, P<0.01). The protein expression of placental LCHAD in the control group was significantly negatively correlated with the protein expression of COX-2 (r=- 0.502,P<0.01). Conclusions The placental mRNA and protein expression of long-chain fatty acid oxidation enzymes were different in different clinical features of preeclampsia, which were reduced more obviously in the early-onset preeclampsia in the notch group than that of the late-onset preeclampsia in the notch group, and were negatively correlated with the elevated serum FFA level, significantly enhanced oxidative stress and inflammatory response, but with no correlation with serum TG level.

In order to understand metabolic functions essential for methane assimilation, we investigate dribulose monophosphate pathway and adjacent pathways in gammaproteobacterial Methylomicrobium alcaliphilum 20Z by using combined approaches of RNA-seq, LC-MS, and 13C-labeled techniques. The absolute quantification of metabolome showed that the concentrations of intermediates, such as glucose-6-phosphate and 2-dehydro-3-deoxy-phosphogluconate, involved in Entner-Doudoroff (EDD) pathway were (150.95 +/- 28.75) micromol/L and below the limit of detection of mass spectrometry. In contrast, fructose-1, 6-bisphosphate, glyceraldehyde-3-phosphate/dihydroxyacetone and phosphoenolpyruvate in Embden-Meyerhof-Parnas (EMP) pathway had significantly higher concentrations with (1 142.02 +/- 302.88) micromol/L, (1 866.76 +/- 388.55) micromol/L and (3 067.57 +/- 898.13) micromol/L, respectively. 13C-labeling experiment further indicated that the enrichment of [3-13C1]-pyruvate involved in EMP pathway was 4-6 fold higher than [1,13C1]-pyruvate in EDD pathway in a dynamic course. Moreover, gene expression profile showed that the expression levels of genes in EMP pathway (e.g. fbaA, tpiA, gap and pykA) were 2 479.2, 2 493.9, 2 274.6 and 1 846.0, respectively, but gene expressionlevels in EDD pathway (e.g. pgi, eda and edd) were only 263.8, 341.2 and 225.4, respectively. Overall our current results demonstrated that EMP pathway was the main route for methane assimilation in M. alcaliphilum 20Z. This discovery challenged our understanding of methane assimilation pathway in gammaproteobacterial methanotrophic bacteria, and further provided an important insight for efficient methane biocatalysis in the future.
Glycolysis ; Industrial Microbiology ; Methane ; metabolism ; Methylococcaceae ; metabolism ; Pyruvic Acid ; metabolism

Objective To analyze the heterogeneous variation of serum free fatty acid (FFA) and lipids during early second trimester in women with or without uterine artery notch in pre-eclampsia (PE).Methods This is a prospective cohort study of 4 000 women with singleton pregnancies registered in early pregnancy and in whom regular check-ups were performed in Haidian Maternal & Child Health Hospital.Blood specimens were collected at gestational age 14-18 weeks at the same time of screening for Down's syndrome.One hundred and one cases with early diastolic notch of the uterine artery were included in the N+ group,and 172 cases without notch but at high risk of PE were included in the N-group at 22-24 weeks.In addition,205 women who were selected randomly at a ratio of 1 ∶ 5,without notch or PE high-risk factors,were also included in the N group.Both groups were subgrouped according to the outcomes of pregnancy complications:early-onset PE group EPE,late-onset PE (LPE),gestational hypertension (GH) group,gestational diabetes mellitus (GDM) group with normal blood pressure,and no complications (NC) group.The variation in FFA and other lipid metabolism indicators in the PE subgroups were compared and analyzed by two independent-sample t-test,one-factor analysis of variance,Chi-square test (or Fisher's exact) and Logistic regression.Results History of PE and pre-hypertension at first visit differed significantly between the N+ and N-groups [3.9％ (4/101) vs.0.8％ (3/377),x2=5.52,P＜0.05; pre-hypertension at first visit,42.2％ (43/101) vs.25.7％ (97/377),x2=10.91,P＜0.05].In the N+ group,23.8％ (n=24) of women had PE,of which 37.5％ (n=8) were early onset.In the N group,2.1％ (n=8) had PE,and all were late onset.The incidence of PE differed significantly between the N+ and N-groups (x2=59.72,P＜0.05).In the N+ group,FFA gradually decreased among the ePE,IPE,GH and NC groups [(0.68±0.27),(0.58±0.21),(0.57±0.21) and (0.49±0.19) mmol/L,F=2.78,P＜0.05]; Multivariate regression analysis showed that FFA (OR=135.68,95％CI:3.78-4 873.00) and PE history (OR=123.25,95％CI:9.27-i 638.00) were risk factors of ePE.Pre-hypertension at registration (OR=4.69,95％CI:2.08-10.58) and pre-pregnancy body mass index (BMI) 24-28 (OR=3.69,95％CI:1.26-10.83) were risk factors ofGH.FFA (OR=9.08,95％CI:2.49-33.01) and pre-pregnancy BMI ≥ 28 (OR=5.08,95％CI:2.16-11.92) were risk factors for GDM.Conclusions Serum FFA and TG levels in early second trimester are correlated with PE,especially the early-onset PE.The onset of PE is heterogeneous and affected by many factors,and occurs in patients with or without early diastolic notch of the uterine artery in the second trimester.Patients with notch are more likely to have early-onset PE,which is correlated with blood FFA and TG levels.

Objective To investigate the effects of expression of mitochondria long-chain fatty acid oxidative enzyme (long-chain 3 hyroxyacyl CoA dehydrogenase,LCHAD) and p38 mitogen activated proteinkinase (p38MAPK) signal transduction pathway in severe preeclampsia.Methods Serum-free trophoblast cells cultured in vitro were stimulated by early onset severe preeclampsia serum (E-PE group),late onset severe preeclampsia serum (L-PE group),HELLP syndrome serum (HELLP group),and normal pregnancy serum (NP group) respectively; each group was added DMEM/F12 medium,reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (NADPH-Ⅰ) and p38 MAPK inhibitor (p38-Ⅰ)to stimulate cells.Expression of mRNA and protein of LCHAD in trophoblast cells were detected by real-time PCR and western blot.Results (1) The expression of mRNA of LCHAD:the level of mRNA of LCHAD in NP+DMEM,E-PE + DMEM,E-PE + NADPH-Ⅰ,E-PE + p38-Ⅰ,L-PE + DMEM,L-PE + NADPH-Ⅰ,L-PE + p38-Ⅰ and HELLP + DMEM,HELLP + NADPH-Ⅰ,HELLP + p38-Ⅰ groups were 1.00 ± 0.03,0.14 ±0.08,0.95 ±0.20,1.43±1.02,0.37 ±0.18,1.51 ±0.36,1.60 ±0.31,0.10 ±0.04,0.49 ±0.10,0.44 ± 0.21,respectively.The relative expressions of mRNA of LCHAD were significantly reduced in E-PE + DMEM,L-PE + DMEM and HELLP + DMEM groups compared with the NP + DMEM group (P ＜0.05).Compared with the NP groups,the relative expressions of mRNA of LCHAD were significantly increased in L-PE + NADPH-Ⅰ and L-PE + p38-Ⅰ group (P ＜ 0.05),while reduced in HELLP groups (P ＜0.05).(2) The expression of protein of LCHAD:the relative expressions of protein of LCHAD in NP +DMEM,E-PE + DMEM,E-PE + NADPH-Ⅰ,E-PE + p38-Ⅰ,L-PE + DMEM,L-PE + NADPH-Ⅰ,L-PE +p38-Ⅰ and HELLP + DMEM,HELLP + NADPH-Ⅰ,HELLP + p38-Ⅰ groups were 19.4 ± 2.2,10.7 ± 1.1,17.9±3.3,19.1 ±2.9,16.4 ±2.3,20.3 ±2.3,20.9 ±4.3,12.4 ±2.3,17.6 ±2.6,17.7 ±2.0 respectively.Compared with the NP groups,the protein expressions of LCHAD were significantly remarkably reduced in E-PE + DMEM,L-PE + DMEM and HELLP groups (P ＜ 0.05).Compared with the DMEM groups,the protein expressions of LCHAD were significantly increased in NADPH-Ⅰ and p38-Ⅰ groups of E-PE,L-PE and HELLP groups (P ＜ 0.05).Conclusions These studies demonstrate that long chain fatty acid oxidation was involved in the pathogenesis and development of preeclampsia.The expressions of gene and protein of LCHAD were remarkably affected by early onset severe preeclampsia and HELLP syndrome.NADPH-Ⅰ and p38-Ⅰ may allay the disorder of fatty acid oxidation.p38MAPK signal transduction pathway may contributed in this process.

Objective:To establish cell lines stably expressing Rab5a and its the inactive mutant Rab5aN133I,analyze the effect of Rab5a on the expression of cytokines in LPS-stimulated RAW264.7 cells .Methods:RAW264.7 cells were transfected with Rab5a and its inactive mutant vector Rab5a N133I separately,and then screened by G418.Rab5a stable expressing cell lines were identified by Real time-PCR.The growth of the stable cell lines was analyzed by MTT assay.After the stable cell lines were stimulated by LPS for different time periods,the expression of iNOS,TNF-αand IL-6 was detected.Results:Rab5a and Rab5aN133I transfection resulted in elevated Rab5a mRNA expression compared with the control cells ( P<0.05 ).Rab5a overexpression enhanced the proliferation of RAW264.7 cells.However,the proliferation of Rab5aN133I cells was significantly slower than the control cells ( P<0.05).Overexpression of Rab5a promoted LPS-induced production of iNOS,TNF-αand IL-6 in RAW264.7 cells (P<0.01). Conversely,overexpression of Rab5aN133I abolished the stimulating effects of Rab5a.Conclusion: Rab5a promoted LPS-induced expression of iNOS,TNF-αand IL-6 in RAW264.7 macrophages in a GTP-binding ability-dependent manner.

To screen the interactive proteins with IBDV Gt VP2 protein from cDNA library of B Lymphoid cells of the bursa of Fabricius. The expression cDNA library plasmids was transformed to the yeast competent cells, which have the bait plasmid-Gt VP2. After testing for growth in synthetic complete medium lacking histidine and uracil and for production of beta-galactosidase (X-gal), we obtained 16 positive clones. We searched the gene sequences of positive clones in the NCBI website. The blast results showed that five positive clones were the gallus sequences. They were Gallus gallus breed mitochondrial DNA, O_G1cNAc transferase, Tumor protein p53 binding protein, Stathmin and Chondroitin sulfate Ga1NAcT-2, respectively. This study is helpful for the further identifying the receptors of IBDV in B Lymphoid cells of the bursa of Fabricius.
Animals ; B-Lymphocytes ; metabolism ; virology ; Bursa of Fabricius ; metabolism ; Chickens ; DNA, Mitochondrial ; metabolism ; Gene Library ; Infectious bursal disease virus ; Protein Binding ; Protein Interaction Mapping ; Receptors, Virus ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Two-Hybrid System Techniques ; Viral Structural Proteins ; genetics ; metabolism

Objectives To explore the effect of fatigue intervention on biomechanics of lower extremity and performance during the stretch-shortening cycle(SSC) movement-drop jumps(DJ).Methods The Vicon motion capture system and 3D Kistler force plates were used to collect sagittal plane kinematics and ground reaction force data synchronously of a total of fifteen trained male athletes under pre-and post-fatigue conditions with shuttle running + vertical jumping fatigue protocol.The joint angle,joint moment,power,work,and leg/joint stiffness were compared before and after fatigue.Results After fatigue,the maximum height of DJ decreased(P＜0.05),while the touchdown angle and the range of motion(△θ) of knee and ankle joints,as well as the occurrence time for the eccentric,concentric and total phase increased(P＜0.05).However,the maximum push-off moment and power of knee reduced(P＜ 0.05).The stiffness of knee and ankle joints during the eccentric phase reduced,resulting in the reduction of the leg stiffness(P＜0.05).Moreover,the energy absorption and net energy of the ankle joint decreased;meanwhile,the energy contribution of the knee joint decreased during the eccentric phase of a DJ task.Conclusion Fatigue changes the movement pattern,decreasing the control ability of the lower extremity especially in the knee and ankle joint,and results in decreased performance.Moreover,the decrease of leg,ankle and knee stiffness and the corresponding energy can be used as sensitive indexes to evaluate the performance of drop jumps in a fatigued condition.

The research progress in fatigue protocols and biomechanics of lower extremity and its relationship with injury induced by exercise-induced fatigue was reviewed in this paper. At present, fatigue protocols can be divided into the traditional fatigue-induced protocol and the functional fatigue-induced protocol under laboratory condition. The former mainly includes power-cycling model, treadmill run model and step-ups model, while the latter is closer to what is experienced during competition, which is usually shown as multidirectional movements. In addition, the biomechanical measures of exercise-induced fatigue mainly include kinematics, ground reaction force, joint mechanics and electromyography. Different fatigue protocols do not uniformly produce alterations in lower limb biomechanical factors. The refinement of fatigue protocols and specific indicators should be considered in future studies, in order to compare the induced effects of fatigue protocols and provide references for the selection of fatigue protocols in laboratory tests. Meanwhile, the relationship between the response of brain-nerve system and the activation of musculoskeletal system for specific athletic task should be focused, so as to understand the difference of biomechanical mechanisms between fatigue protocols and further explore the effect of exercise-induced fatigue on sports injuries.

Objective To examine the effects of shoe cushioning on impact characteristics, kinematics and dynamics of lower extremities before and after neuromuscular fatigue during drop landing. Methods Fifteen trained male athletes were required to execute three successful trials of drop landing from a 60-cm platform before and after fatigue protocol. The impact force, loading rate, lower extremity kinematics, joint moment, joint stiffness were compared. Results Before fatigue, highly-cushioned shoes reduced the loading rate at heel before fatigue, and the time to peak loading rate of heel was significantly shorter than that of control shoes. After fatigue, highly-cushioned shoes significantly reduced the peak impact force and loading rate at heel, loading rate at forefoot. Meanwhile the time to peak impact force and peak loading rate at heel were significantly longer, and the contact and minimum angle of ankle were significantly greater. No significant changes were found in lower extremity stiffness, peak moment and joint stiffness of ankle. Conclusions In the situation where neuromuscular activity is reduced, highly-cushioned shoes can effectively attenuate the impact, thereby helping to avoid or reduce the potential impact damage after fatigue.