Insulin Resistance-Varying Associations of Adiposity Indices with Cerebral Perfusion in Older Adults: A Population-Based Study.

OBJECTIVES: Excessive accumulation of adipose tissue may accelerate brain aging, but the underlying mechanisms are poorly understood. Several adiposity indices were proposed to assess obesity, while their linkage with brain health in older adults remained unclear. Here we aimed to examine the associations of adiposity indices with global and regional cerebral blood flow (CBF) in older adults, while considering insulin resistance. DESIGN: This was a cross-sectional population-based study that included older adults derived from the baseline participants in the ongoing Multimodal Interventions to Delay Dementia and Disability in rural China (MIND-China) study. SETTING AND PARTICIPANTS: The study included 103 Chinese rural-dwelling older adults (age≥60 years; 69.9% women) who underwent brain magnetic resonance imaging scans. METHODS: We estimated eight adiposity indices based on anthropometric measures. We automatically quantified global and regional CBF using the arterial spin labeling scans. Insulin resistance was assessed using the triglyceride-glucose index and then dichotomized into high and low levels according to the median. Data were analyzed using general linear model and voxel-wise analysis. RESULTS: Of the eight examined adiposity indices, only higher waist-to-height ratio (WHtR) and body roundness index (BRI) were associated with reduced global CBF (multivariable-adjusted ß-coefficients and 95%CI: -1.76; -3.25, -0.27 and -1.77; -3.25, -0.30, respectively) and hypoperfusion in bilateral middle temporal gyri, angular gyri and superior temporal gyri, left middle cingulum and precuneus (P<0.05). There were statistical interactions of WHtR and BRI with levels of insulin resistance on CBF, such that the significant associations of higher WHtR and BRI with lower global and regional CBF existed only in people with high insulin resistance (P<0.05). CONCLUSION: Higher WHtR and BRI are associated with cerebral hypoperfusion in older adults, especially in people with high insulin resistance. This may highlight the pathological role of visceral fat in vascular brain aging.

[Study on the correlation between subgroup of blood lipid with degree of coronary artery stenosis in patients with coronary atherosclerotic heart disease].

Objective: To study the correlation between low-density lipoprotein particles (LDL-P) with other lipoprotein indexes. To explore the correlation between LDL-P and its subgroup particles(LDL1-P-LDL6-P) with the degree of coronary artery stenosis in patients with coronary atherosclerotic heart disease(CHD) combining with the result of coronary arteriography. To explore the value of lipoprotein subgroup granules in preventing the severity of coronary artery stenosis in CHD patients. Methods: Cross-sectional study. A total of 259 patients without lipid-lowering drugs for coronary angiography in the department of cardiology of TEDA International Cardiovascular Hospital during 3 months from August 2019 to December 2019 were collected, and 52 healthy subjects were recruited during the same period. The level of high sensitivity C-reactive protein (hs-CRP) and other biochemical indexes were detected by automatic biochemical analyzer. The level of LDL-P and other biochemical indexes were detected by nuclear magnetic resonance spectroscopy(NMRS). The relation between various biomarkers levels with coronary artery stenosis degree was analyzed. Analysis of variance and nonparametric tests were used to compare the differences of indexes among each group. Pearson correlation analysis was used to determine the correlation among the measured indexes. Logistic regression was used for multi-factor analysis, ROC curve was used to evaluate the diagnostic value of related indexes. Results: LDL-P was highly correlated with low-density lipoprotein cholesterol (LDL-C),apolipoprotein B (ApoB) and total cholesterol (TC) (r= 0.927, P<0.001; r=0.921, P<0.001; r=0.844, P<0.001). LDL-P, LDL4-P, LDL5-P and LDL6-P in patients with severe coronary stenosis were higher than those in patients with mild coronary stenosis(U=4 172.000, Z=4.256, P<0.001; t=2.573, P=0.011; U=3 995.000, Z=4.621, P<0.001;t=5.223, P<0.001), LDL-P and LDL6-P were higher than those of patients with moderate coronary stenosis (U=1 159.000, Z=2.294, P=0.022; t=2.075, P=0.041). High levels of hs-CRP, LDL5-P and LDL6-P were risk factors for the degree of coronary stenosis(OR=1.095, P=0.036;OR=1.015, P=0.046;OR=1.012, P=0.039). ROC analysis showed that the AUC of LDL-P, LDL5-P and LDL6-P on coronary stenosis was 0.67, 0.68 and 0.69, respectively. Hs-CRP combined with LDL5-P and LDL6-P had the greatest effect on the degree of coronary stenosis (AUC= 0.70). Conclusions: LDL-P is highly correlated with LDL-C. The levels of LDL-P and LDL6-P were significantly higher in patients with severe stenosis than in patients with mild and moderate stenosis. hs-CRP, LDL5-P and LDL6-P can be used as new risk factors for the degree of coronary stenosis and may be further used as risk predictors. The combined detection of hs-CRP, LDL5-P and LDL6-P is helpful for the diagnosis of the severity of coronary stenosis, and may further become risk predictors.

MiR-222-5p promotes the growth and migration of trophoblasts by targeting AHNAK.

OBJECTIVE: The purpose of this study was to detect microRNA-222-5p (miR-222-5p) levels in placental tissues of preeclampsia (PE) pregnancies, and to explore the role of miR-222-5p in the proliferative and migratory potentials of trophoblast cell line HTR-8/SVneo. PATIENTS AND METHODS: Expression levels of miR-222-5p and AHNAK in placental tissues of PE pregnancies (n=24) and healthy pregnancies (n=24) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Potential influences of miR-222-5p and AHNAK on proliferative, migratory and apoptotic potentials in HTR-8/SVneo cells were examined. At last, Luciferase assay was conducted to illustrate the interaction between miR-222-5p and AHNAK in trophoblasts. RESULTS: It was found that miR-222-5p was downregulated in placental tissues of PE pregnancies. Overexpression of miR-222-5p stimulated proliferative and migratory potentials, and inhibited apoptosis in HTR-8/SVneo cells. Moreover, AHNAK was the target gene binding to miR-222-5p, and overexpression of AHNAK inhibited proliferative and migratory potentials and promoted apoptosis in HTR-8/SVneo cells. CONCLUSIONS: MiR-222-5p stimulates proliferative and migratory potentials and inhibits apoptosis in HTR-8/SVneo cells by negatively regulating AHNAK.

[Association between soluble receptor for advanced glycation end products (sRAGE) and coronary and cerebral atherosclerosis].

Objective: To investigate the Association between soluble receptor for advanced glycation end products (sRAGE) and coronary and cerebral atherosclerosis. Methods: A total of 232 consecutive patients who synchronously undertook coronary angiography and craniocerebral CT angiography (or total cerebral angiography) were included between May 2018 and December 2018 in Beijing Tiantan Hospital, Capital Medical University in this study. Patients were divided into the control group (without coronary artery disease (CAD) and cerebrovascular stenosis (CVS), n=55), CAD group (n=118), CVS group (n=11), concomitant CAD and CVS group (CAD+CVS, n=48). Plasma sRAGE level was measured by enzyme-linked immunosorbent assay (ELISA) and compared among the four groups. The relationship between sRAGE and Gensini Score (GS) and cerebrovascular stenosis severity was assayed. sRAGE levels were compared among low, middle and high GS group as well as between extracranial and intracranial arteries stenosis. Results: The levels of sRAGE in CAD group (1.96 µg/L) were higher than those in the control group (1.66 µg/L, P=0.025) or the CVS group (1.53 µg/L, P=0.013). However, no significant difference in sRAGE level was found between the groups of CAD and CAD+CVS (1.89 µg/L, P>0.05). Meanwhile, sRAGE was positively associated with GS in the entire study population (r=0.153, P=0.023) or in the diabetic patients (r=0.242, P=0.017). The sRAGE leves in both middle GS and high GS groups were higher than those in low GS group (P<0.05). No association between sRAGE and CVS severity and vascular count. Additionally, no significant difference in levels of sRAGE was found between extracranial (1.84 µg/L) and intracranial arteries stenosis (1.66 µg/L, P=0.523). Conclusion: Plasma sRAGE level is positively associated withseverity of CAD, but its association with cerebral atherosclerosis needs further studies.

[Effect of Baicalin on the expression of connexin 36 in the cerebral cortex and striatum area of Parkinson's disease model rat].

Objective: To investigate the effect of Baicalin on the expression of connexin 36 (Cx36) in cerebral cortex and striatum area of 6-OHDA-induced Parkinson's (PD) model rats and its significance. Methods: Male Sprague-Dawley (SD) rats were randomly divided into 6 groups, 12 in each group: normal control group, PD model group (untreated group), PD model (Medopa group), PD model (Baicalin low dose group) PD model (Baicalin medium dose group) and PD model (Baicalin high dose group). Except for the normal control group, 6-OHDA was injected using microinjection under the mouse brain stereotaxic apparatus to establish the hemiparkinsonian PD model. On the basis of the success of making model, the rats were treated by Medopa and Baicalin (low, medium and high dose). Immunohistochemistry was used to detect tyrosine hydroxylase (TH) and Cx36 expression in cerebral cortex and striatum of the 6 groups. Western-Blot technique was used to detect the cerebral cortex and striatum Cx36 expression changes, and to preliminarily study the effect of Baicalin on rat cerebral cortex and striatum Cx36 expression levels. Results: Immunohistochemical staining and Western blotting showed that the expression of TH-positive neurons and Cx36 in the cerebral cortex and striatum of the PD model group was lower than that of the normal control group (828±188). While expressions of Cx36 in the low, medium and high dose PD model groups of Baicalin (733±118, 759±134, 779±125) were up-regulated, compared with the untreated PD model group (487±125), and the differences were statistically significant (all P<0.05), but the difference between the doses was not statistically significant (P>0.05). Conclusion: The expression of Cx36 decreases in cerebral cortex and striatum area of 6-OHDA-induced Parkinson's disease model rats, and the expressions of TH and Cx36 in cerebral cortex and striatum increase after treatment with Baicalin, which may provide new drug research direction for Parkinson's disease.

[A clinical retrospective study on 160 cases of multiple umbilical cord around the neck].

Objective: To compare the neonatal and maternal outcomes between the patients with umbilical cord around the neck (≥3 loops) and with (1 or 2 loops). Methods: A retrospective analysis was conducted on the clinical data of 160 cases with multiple umbilical cord around the neck (≥3 loops) in the Department of Obstetrics, Women's Hospital School of Medicine Zhejiang University between January 2014 and April 2017.For each case, two control women who gave birth at the same day with vertex position and singletons were selected.The neonatal and maternal outcomes were compared. Result: (1) The incidence of cord multiple cord around the neck (≥3 loops) in our hospital was 0.45%. (2) Comparison between groups: The rate of abnormal fetal movement or abnormal cardiotocography in case group was higher than those of the control group, (33.13%, 53/160) vs (8.13%, 26/320), with significant difference, P=0.000.The Umbilical Artery Systolic/diastolic (S/D) ratio of the case group was lower than that of the control group, 2.00(0.40) vs 2.14(0.40), with significant difference, P=0.000.The cesarean section rate of the case group was higher than that of the control group, (81.25%, 130/160) vs (7.50%, 24/320), and the difference was statistically significant, P=0.000.Birth Weight of the case group was lower than that of the control group, (3 143±367) g vs (3 323±349) g, with significant difference, P=0.000.(3) Comparison between subgroups: The rate of lateral incision or obstetrical forceps in the subgroup of virginal delivery among the case group (n=30) was higher than that in the control group (n=296), (30.00%, 9/30) vs (12.50%, 37/296), with significant difference, P=0.009.While, the Apgar score at 1 and 5 min of the virginal delivery case in the case group were lower than that in the control group, 10(1.25) vs 10(0) and 10(0) vs 10(0), there were both significant difference, P=0.000, 0.012, respectively.The rate of meconium-stained amniotic fluid, 1 min Apgar score of ≤7 and NICU admission were showed no significance, all P>0.05.(4) After Logistic regression, the four factors most closely associated with meconium-stained amniotic fluid in patients with multiple cord around the neck (≥3 loops), which were gestational age ≥39 weeks, Birth Weight >3 500 g, umbilical cord around the neck ≥4 loops, and trial of labor. Conclusion: (1) Multiple umbilical cord around the neck (≥3 loops) had a more positive treatment. Vaginal delivery led to lower APGAR score, but didn't increase the incidence of neonatal asphyxia.(2) Independent risk factors for meconium-stained amniotic fluid were gestational age ≥39 weeks, Birth Weight>3 500 g, umbilical cord around the neck ≥4 loops and trial of labor.

Effect of sodium selenite and selenium yeast on performance, egg quality, antioxidant capacity, and selenium deposition of laying hens.

This study compared the effects of sodium selenite and selenium yeast and their combination on laying performance, egg quality, antioxidant capacity, and selenium (Se) contents in tissues and eggs. Two-hundred-eighty-eight Jing Hong layers that were similar in laying rate (87.5 ± 0.38%) and body weight (1.70 ± 0.02 kg) were randomly distributed into 4 treatments for 11 wk (from 203 d old to 279 d old) with 9 replicates of 8 hens per replicate. The diets (corn-soybean meal diet) were supplemented with 0 [blank control (BC)], 0.3 mg/kg Se from sodium selenite (SS), 0.15 mg/kg Se from sodium selenite and 0.15 mg/kg Se from Se yeast (SS+SY), or 0.3 mg/kg Se from Se yeast (SY). Results showed that the laying rate of the SS+SY group increased significantly (P < 0.05) compared to the BC and SY groups. There were no differences (P > 0.05) in egg quality between the Se-supplemented diets and the BC diet. The serum glutathione peroxidase (GSH-Px) activity was increased (P < 0.01) in hens fed Se-supplemented diets compared to the BC diet. The liver superoxide dismutase (SOD) activity of the SY group was increased significantly (P < 0.05) compared to the BC group. Significant increase (P < 0.01) due to SY supplementation was noted in the serum vitamin E content compared to BC and SS. Layers fed Se-supplemented diets had higher (P < 0.01) contents of Se in the serum, liver, and kidney compared to the BC diet. Compared to BC, Se content in eggs was significantly increased (P < 0.05) by feeding supplementary Se. In conclusion, the effects of SS and Se yeast were approximately equal in promoting antioxidant capacity of laying hens, while Se yeast is easier to deposit into eggs and tissues. The diet with added equal amounts of the 2 sources of Se was more cost effective and affordable than a comparable amount of Se yeast to obtain the promising production performance and nearly similar Se deposition.

Relationship between lesion patterns of single small infarct and early neurological deterioration in the perforating territory.

OBJECTIVE: To investigate the relationship between lesion patterns of single small infarct (SSI) in perforating territory of the vertebral-basilar artery and early neurological deterioration (end)/short-term functional outcome. PATIENTS AND METHODS: 126 patients with acute SSI in the perforating territory of the vertebral-basilar artery, admitted within 24 h after symptom onset, were recruited between August 2010 and May 2013. The patients were divided into proximal SSI and distal SSI according to the relationship between their lesion location and their parent artery. Early neurological deterioration (END) was defined as an increase in the National Institutes of Health Stroke Scale (NIHSS) ≥ 2 within 3 days after admission. Functional outcome at 30 days after onset was assessed using the modified Rankin Score (mRS) and dichotomized as good (0-2), and poor (≥ 3). RESULTS: Out of 126 patients, proximal SSI was found in 70 (55.56%) patients and distal SSI in 56 (44.44%) patients. After standard treatment, 36 (28.57%) patients experienced END within 3 days after admission, and 19 (15.70%) patients had a poor outcome at 30 days. Univariate analysis revealed that lesion size, baseline NIHSS score, parent artery disease, diabetes mellitus, and asymptomatic cerebral arterial atherosclerosis were significantly associated with END (with either p<005 or p<0.01), while the short-term outcome was just as significantly associated with proximal SSI, the baseline NIHSS score and END (with either p<005 or p<0.01). Results from multiple logistic regression analysis revealed that proximal SSI was an independent predictor of END (odd ratio [OR] 3.222, 95% CI 1.170-8.874, p=0.024) and that the END independently predicted a poor outcome (OR 4.126, 95% CI: 1.241-13.713, p=0.021) at 30 days after onset. CONCLUSIONS: Proximal SSI in the perforating territory of the vertebral-basilar artery was closely related to the presence of END, and the END independently predicted the subsequent poor outcome at 30 days after onset.

[Effect of endoplasmic reticulum stress in trophocytes on the pathogenesis of intrahepatic cholestasis of pregnancy].

Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, P<0.01). (2) The volume of endoplasmie reticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 µmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 µmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 µmol/L cholylglycine experimental group were 1.01±0.04, 1.17±0.15, 1.33±0.13, 1.73±0.13, respectively. The expression of GRP-78 mRNA and protein in 100 and 50 µmol/L cholylglycine experimental group were significantly higher than 0 µmol/L (all P<0.01). Conclusion: The obvious expansion of endoplasmic reticulum and the increased expression of GRP-78 in trophocytes indicated that endoplasmic reticulum stress of trophocytes may be involved in the pathogenesis of ICP.

High temperature breakdown of the Stokes-Einstein relation in a computer simulated Cu-Zr melt.

Transport properties and the Stokes-Einstein (SE) relation in liquid Cu8Zr3 are studied by molecular dynamics simulation with a modified embedded atom potential. The critical temperature Tc of mode coupling theory (MCT) is derived as 930 K from the self-diffusion coefficient D and viscosity η. The SE relation breaks down around TSE = 1900 K, which is far above Tc. At temperatures below TSE, the product of D and η fluctuates around a constant value, similar to the prediction of MCT near Tc. The influence of the microscopic atomic motion on macroscopic properties is investigated by analyzing the time dependent liquid structure and the self-hole filling process. The self-holes for the two components are preferentially filled by atoms of the same component. The self-hole filling dynamics explains the different breakdown behaviors of the SE relation in Zr-rich liquid CuZr2 compared to Cu-rich Cu8Zr3. At TSE, a kink is found in the temperature dependence of both partial and total coordination numbers for the three atomic pair combinations and of the typical time of self-hole filling. This indicates a strong correlation between liquid structure, atomic dynamics, and the breakdown of SE relation. The previously suggested usefulness of the parameter d(D1/D2)/dT to predict TSE is confirmed. Additionally we propose a viscosity criterion to predict TSE in the absence of diffusion data.

Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.

The neomycin-resistance (neo(r)) gene is widely used as a selectable marker in eukaryotic expression vectors; however, its expression often affects that of target genes. Cre recombinase recognizes LoxP sites, leading to site-specific recombination and deletion of DNA and RNA between two LoxP sites. In the present study, a humanized Fat-1 gene (hFat-1) was generated by DNA Works and used to construct a pC-PGK-neo(r)-hfat-1 expression vector, in which PGK-neo(r) was flanked by two LoxP sites. The pC-PGK-neo(r)-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 µg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neo(r), which was integrated into the genome. hFat-1-neo(r) negative cells were obtained following puromycin selection. Real-time quantitative polymerase chain reaction data indicated that neomycin-resistant cells had higher hFat-1 expression than neomycin-sensitive cells. High performance gas chromatography data suggested that the n-6/n-3 ratio was significantly lower in transfected cells than in wild-type cells. The n-6/n-3 ratio in Cre-treated hFat-1-transfected cells was higher than that in untreated cells, suggesting that deletion of PGK-neo(r) decreased hFat-1 expression.

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency.

With the development of gene targeting approaches, genomic mutation technologies in livestock animals such as gene trapping, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats and their associated systems have been improved. Although ZFNs have been used for gene targeting in many species, the off-target sites are still present. Using gene trapping, the workload of screening of targeted clones was decreased by generating a smaller number of drug-resistant clones. Determining whether the efficiency of gene trapping is lower than that of ZFNs for a specific gene has been challenging. In this study, to knock out the bovine myostatin gene, we constructed a promoter trap vector and compared its efficiency with that of ZFNs. The promoter trap vector contained a green fluorescent protein sequence without the promoter and a neomycin phosphotransferase (neo(R)) cassette driven by the phosphoglycerate kinase promoter. When the trapping vector was inserted downstream of the endogenous promoter, the fluorescent protein gene was expressed. The targeted-positive cell clones were identified based on green fluorescence and G418 double selection, followed by polymerase chain reaction analysis and sequencing. The targeting efficiency reached 5%. Compared with the efficiency of ZFN pairs (5.17 and 2.86%), the promoter trap vector PIII-myostatin could knock out the bovine myostatin gene. Therefore, gene trapping may be an effective tool for genomic modification.

Expression of rat oligodendrocyte transcription factor 2 in COS-7 cells following its eukaryotic expression vector construction.

OBJECTIVES: The basic HLH transcription factor Olig is a key regulator for differentiating the oligodendrocyte lineage cells during development. Oligodendrocyte transcription factor 2 (Olig2) plays a crucial role in differentiating the oligodendrocytes in the spinal cord. We aimed to construct and investigate the eukaryotic expression recombinant plasmid in the rat Olig2. DESIGN, TIME AND SETTING: The experiment was performed at the Laboratory of Neurobiology, Xuzhou Medical College from October 2011 to March 2012. MATERIALS AND METHODS: The pEGFP-N1 vector was purchased from Invitrogen. JM101 competent cells and COS-7 cells were preserved at the Laboratory of Neurobiology, Xuzhou Medical College, China. The Olig2 cDNA fragment was cloned by RT-PCR with the total RNA from the neonatal rat spinal cord, and subsequently cloned into pGEM-T vector. The confirmed Olig2 fragment was then cloned into the pEGFP-N1 vector. The right recombinant was transfected into COS-7 cells by lipofectamine 2000. The expression of the Olig2 in COS-7 cells was detected by RT-PCR and immunoblot analysis. Enzyme digestion and sequencing of the recombinant plasmid; and expression of the Olig2 were analyzed by fluorescence microscope and western blot. RESULTS: The correct pEGFP-N1-Olig2 cloning was verified by restriction endonuclease digestion and sequencing. The western blot analysis indicated that the Olig2-GFP fusion protein was expressed in the COS-7/pEGFP-N1-Olig2 cells at 72 h. CONCLUSIONS: The pEGFP-N1-Olig2 vector was constructed successfully. The Olig2-GFP fusion protein was expressed in the COS-7/pEGFP-N1-Olig2 cells. This study lays the foundation for further research in gene therapy for central nervous system demyelinating diseases.

Expression of cyclinD1 and Ki-67 proteins in gliomas and its clinical significance.

OBJECTIVES: To investigate the expression of cyclinD1 and Ki-67 proteins in gliomas and its significance. PATIENTS AND METHODS: The immunohistochemistry was used to detect the expression of cyclinD1 and Ki-67 proteins in 18 cases of normal brain tissues, 32 cases of low-grade gliomas, and 24 cases of high-grade gliomas. RESULTS: The cyclinD1 positive ratio in normal brain tissues, low-grade gliomas, and high-grade gliomas were 4/18, 15/32, and 18/24, respectively, with statistically significant difference (p < 0.05). Differences were significant by pairwise comparison between normal brain tissue with high-grade gliomas and low-grade gliomas with high-grade glioma groups (p < 0.01). However, there was no significant differences between normal brain tissue with low-grade gliomas. The Ki-67 positive ratio in normal brain tissues, low-grade gliomas, and high-grade gliomas were 5/18, 21/32, and 20/24, respectively. The difference among three tissues was statistically significant (p < 0.05). Differences were significant by pairwise comparison between normal brain tissue with low-grade gliomas and normal brain tissue with high-grade glioma group (p < 0.01). There is no difference between low-grade gliomas and high-grade gliomas (p > 0.05). Spearman's rank correlation confirmed that cyclinD1 and Ki-67 was positively correlated in low-grade gliomas and high-level brain tumor (p < 0.05), but no correlation in the normal brain tissue (p > 0.05). CONCLUSIONS: The expression of CyclinD1 and Ki-67 increased in gliomas, suggesting that both may play an important role in the occurrence of gliomas.

Expression analysis of VfDGAT2 in various tissues of the tung tree and in transgenic yeast.

The tung tree (Vernicia fordii Hemsl.; Vf) has great potential as an industrial crop owning to its seed oil that has multiple uses. Diacylglycerol acyltransferases (DGATs) catalyze the last and most committed step of triacylglycerol (TAG) biosynthesis. In order to examine the physiological role of the VfDGAT2 gene in the tung tree, we characterized its expression profiles in different tung tissues/organs and seeds at different developmental stages. Oil content and α-eleostearic acid production during seed development were also examined. Expression studies showed that VfDGAT2 was expressed in all tissues tested, with the highest expression in developing seeds where the expression was about 19-fold more than that in leaves. VfDGAT2 showed temporal-specific expression during seed development and maturation. Notably, the expression of VfDGAT2 in developing seeds was found to be consistent with tung oil accumulation and α-eleostearic acid production. The expression level of VfDGAT2 was lower in the early stages of oil accumulation and α-eleostearic acid biosynthesis, rapidly increased during the peak periods of fatty acid synthesis in August, and then decreased during completion of the accumulation period at the end of September. When the VfDGAT2 gene was transferred to the oleaginous yeast Rhodotorula glutinis, its expression was detected along with fatty acid products. The results showed that VfDGAT2 was highly expressed in transgenic yeast clones, and the total fatty acid content in one of these clones, VfDGAT2-3, was 7.8-fold more than that in the control, indicating that VfDGAT2 contributed to fatty acid accumulation into TAG and might be a target gene for improving tung oil composition through genetic engineering.

[Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA].

qRT-PCR is becoming a routine tool in molecular biology to study gene expression. It is nec- essary to find stable reference genes when performing qRT-PCR. The expression of genes cloned in oil-tea camellia currently can't be accurately analyzed because of a lack of suitable reference genes. We collected different tissues (including roots, stems, leaves, flowers and seeds) from six oil-tea camellia species to determine stable reference genes. Five novel and ten traditional reference gene sequences were selected from the RNA-seq database of Camellia oleifera C. Abel seeds and specific PCR primers were designed for each. Cycle threshold (Ct) data were obtained from each reaction for all samples. Three different software tools, geNorm, NormFinder and BestKeeper were applied to calculate the expression stability of the candidate reference genes according to the Ct values. The results were similar between analyzed by the three software packages, and indicated that the traditional gene TUBa-3, AC17a and the novel gene CESA were relatively stable in all species and tissues. However, no genes were sufficiently stable across all species and tissues, thus the optimal number of reference genes required for accurate normalization varied from two to six. Finally, the relative expression ofsqualene synthase (SQS) and squalene epoxidase (SQE) genes related to important ingredients squalene and tea saponin in oil-tea camellia seeds were compared by using stable to less stable reference genes. The comparison results validated the selection of reference genes in the current study. In summary, different optimal numbers of suitable reference genes were found for the different tissues of six oil-tea camellia species.

Remodeling of synapses in the CA1 area of the hippocampus after transient global ischemia.

Synapses are essential to neuronal functions. Synaptic changes occur under physiological and pathological conditions. Here we report the remodeling of synapses in the CA1 area of the hippocampus after transient global ischemia using electron microscopy. Much electron-dense material appeared in the cytoplasm of dendrites at 24h after ischemia. Many dark axons or terminals were found in the CA1 neuropil; some of which were phagocytized by dendrites. Interestingly autophagosomes appeared in many axons or dendrites at 48 h after ischemia. In addition, postsynaptic density (PSD) - like structures or synaptic - like structures were found inside spines and dendrites. Statistical analysis demonstrated that the thickness of PSDs in the CA1 neuropil increased from 12 to 48 h after ischemia. The frequency of autophagosomes appeared to escalate from 12 to 48 h after ischemia. The frequency of asymmetric synapses was significantly increased at 12h and 24h after ischemia in stratum oriens, proximal and distal stratum radiatum. Among asymmetric synapses, the number of perforated synapses consistently increased and reached a peak (approximately 10-fold increase) at 48 h after ischemia. On the other hand, the number of multiple synaptic boutons decreased after ischemia reaching a two to fourfold decrease at 48 h after ischemia. These results have shown that ischemia induces an increase of asymmetric synapses as well as synaptic autophagy, which may contribute to the neuronal death in the CA1 area after transient global ischemia.

Altered dynamics of ubiquitin hybrid proteins during tumor cell apoptosis.

The ubiquitin hybrid genes Uba80 and Uba52 encode ubiquitin (Ub), which is fused to the ribosomal proteins S27a (RPS27a) and L40 (RPL40), respectively. Here, we show that these genes are preferentially over-expressed during hepatoma cell apoptosis. Experiments using the tet-inducible transgenic system revealed that over-expression of the ubiquitin hybrid genes sensitized the cells to apoptosis. Further analysis suggested that Ub, and not RPS27a or RPL40, was associated with apoptotic cell death. Cleavage-resistant mutation analysis revealed that the N-terminal portion and the last two amino acids (GG) of Ub are critical for cleavage at the junction between the two protein moieties. An apoptogenic stimulus enhances the nuclear targeting and aggregation of Ub in the nucleus, resulting in histone H2A deubiquitylation followed by abnormal ubiquitylation of the nuclear envelope and the lamina. These events accompany the apoptotic nuclear morphology in the late stage of apoptosis. Each fused RP is localized in the nucleoli. These results suggest a role for Ub hybrid proteins in the altered nuclear dynamics of Ub during tumor cell apoptosis induced by apoptogenic stimuli.

Antidepressant-like effect of sildenafil through oxytocin-dependent cyclic AMP response element-binding protein phosphorylation.

Oxytocin (OT) levels in plasma increase during sexual response and are significantly lower in patients with depression. A drug for the treatment of sexual dysfunction, sildenafil, enhances the electrically evoked release of OT from the posterior pituitary. In this study, we showed that sildenafil had an antidepressant-like effect through activation of an OT signaling pathway. Application of sildenafil reduced depression-related behavior in male mice. The antidepressant-like effect was blocked by an OT receptor (OTR) antagonist and was absent in OTR knockout (KO) mice. Sildenafil increased the phosphorylation of cAMP response element-binding protein (CREB) in the hippocampus. The OTR antagonist inhibited sildenafil-induced CREB phosphorylation and sildenafil had no effect on CREB phosphorylation in OTR KO mice. These results suggest sildenafil to have an antidepressant-like effect through the activation of OT signaling and to be a promising drug for the treatment of depression.

Clonal status and clinicopathological features of Langerhans cell histiocytosis.

Some studies have demonstrated that Langerhans cell histiocytosis (LCH) is a neoplastic hyperplasia of Langerhans cells, however some researchers consider that clonality should be assessed in more patients with LCH, both at disease presentation and during the disease course. Monoclonality is a major characteristic of most tumours, whereas normal tissue and reactive hyperplasia are polyclonal. To elucidate the nature of Langerhans cells further, the present study investigated the clinicopathological features and clonality of three cases of LCH in female patients using laser microdissection and a clonality assay, based on X-chromosomal inactivation mosaicism in somatic tissues and polymorphism of the androgen receptor gene. The results indicated that LCH was composed of Langerhans cells with a characteristic morphological appearance, eosinophils, giant cells, neutrophils and foamy cells. Immunohistochemically, the Langerhans cells were positive for CD1a, S-100 protein and vimentin. The clonality assay demonstrated that the Langerhans cells formed a monoclonal population, showing that LCH is neoplastic. We conclude that LCH is characterized by clonal proliferation, although additional studies with larger sample sizes are required to prove this conclusively.