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Effects of gain of function of Notch ligand杕ediated signals on EPC differentiation. results

Circulation. 2008;118:157-165

Figure 4. Effects of gain of function of Notch ligand杕ediated signals on EPC differentiation. A, Scheme of the insert culture system with 0.4-祄 pores, in which receptors on the surface of the target stem cells in the upper chamber are capable of directly interacting with ligands on the cells in the bottom chamber. In the present study, BM-Lin?/SUP> cells were seeded in the upper chamber, whereas 3T3 stromal cells overexpressing a specific Notch ligand, Jag-1 or Dll-1, were placed in the bottom chamber. B, Reverse-transcription polymerase chain reaction revealed enhanced expression of the specific Notch ligand in the stromal cells stimulated by Jag-1 or Dll-1 signal compared with those transduced with empty vector. C, Reverse-transcription polymerase chain reaction revealed expression of Hes-1 and Hes-5, target effector genes of active Notch, in BM-Lin?/SUP> cells, which were cocultured with 3T3 stromal cells specifically expressing the target Notch ligand gene. D, Fluorescence-activated cell sorting analysis revealed more frequent expression of Flk-1 and CD31 in BM-Lin?/SUP> cells stimulated by Jag-1杕ediated signals, but not Dll-1杕ediated signals, than in those stimulated by empty vector. E, Reverse-transcription polymerase chain reaction to detect expression of typical EPC surface markers and vascular endothelial growth factor in BM-Lin?/SUP> cells stimulated by specific Notch ligand or empty vector. The cellular mRNA level of CD31, Flk-1, and vascular endothelial cadherin (VE-cadherin) was elevated in the Jag-1 group compared with the Dll-1 and empty-vector groups. In contrast, the cellular mRNA level of vascular endothelial growth factor and Flt-1 was similar in all groups. PECAM indicates platelet and endothelial cell adhesion molecule. F, EPC colony-forming assay using BM-KSLs stimulated by specific Notch ligand杕ediated signals revealed significant augmentation of vasculogenic capacity in the Jag-1 group, but not the Dll-1 group, compared with the empty-vector group. CFU indicates colony-forming units. **P<0.01 (n=3 in each group). G, Frequency of apoptotic cells (TUNEL-positive cells) in BM Sca-1+/Lin?/SUP> cells in vitro was significantly lower in EPC-enriched cells stimulated by Jag-1 signal than in those stimulated by Dll-1 signal or empty vector. *P<0.05 (n=3 in each group).

TLR3 and TLR4 protein is localised to endometrial cells during the menstrual cycle results

Reproductive Biology and Endocrinology 2008, 6:40

TLR3 and TLR4 protein is localised to endometrial cells during the menstrual cycle. TLR3 protein staining in healthy late proliferative (LP) tissue was high in luminal and glandular tissue (A, brown precipitate) and lower in LP endometriotic tissue (B). Late secretory (LS) endometrium showed highly expressed TLR3 in the epithelium (C), but weakly in endometriosis (D). Intense staining of TLR4 proteins was shown in mid proliferative (MP) tissue (E). In late proliferative phase of endometriosis, TLR4 proteins were comparably lower (F). TLR4 protein was high in mid secretory (MS) normal endometrium (G), whereas it was decreased in endometriotic MS tissue (H). During the menstrual phase, both TLR3 (I) and TLR4 (J) were highly expressed. Co-immunostaining for TLR4 (green), CD14 (K, red) and CD163 (L, red) demonstrated that TLR4 proteins were expressed by CD14 positive dendritic cells and monocytes (K, yellow) and by CD163 positive macrophages (L, yellow). Localisation of TLR4 to immune cells is marked by a black arrow (J) and by white arrows (K, L). Allhorn et al. Reproductive Biology and Endocrinology 2008 6:40

ROP2 Gene Expression in Arabidopsis Guard Cells. results

Plant Cell. 2008 January; 20(1): 75–87.

Figure 1. ROP2 Gene Expression in Arabidopsis Guard Cells. (A) ROP2 PCR products were amplified from 1 μg of total RNA from guard cell (G) and mesophyll cell (M) protoplasts. PCR was repeated twice with similar results. Actin was used as a loading control. (B) Histochemical assay of GUS activity in 4-week-old Arabidopsis seedlings expressing ROP2 promoter:GUS fusions. Bar = 50 μm. Plant Cell. 2008 January; 20(1): 75–87.

Promotion of in vivo neovascularization by transplantation of putative EPCs stimulated by Jag-1–mediated signals. results

Circulation. 2008;118:157-165

Figure 5. Promotion of in vivo neovascularization by transplantation of putative EPCs stimulated by Jag-1–mediated signals. A, Representative laser Doppler perfusion imaging findings in nude mice receiving PBS (no cells) or BM-Lin– cells cocultured with empty-vector– or specific Notch ligand (Jag-1, Dll-1)–transfected 3T3 stromal cells at days 0 and 14 (upper panel). Hindlimb perfusion recovery was significantly enhanced in the Jag-1 group compared with the Dll-1, empty-vector, and PBS groups (n=6 per group, lower panel). *P<0.05 vs PBS; **P<0.01 vs PBS; ***P<0.001 vs PBS; P<0.05 vs vector; P<0.01 vs vector; P<0.05 vs Dll-1; P<0.01 vs Dll-1. B, Histological capillary density by isolectin B4 staining revealed augmented neovascularization in the Jag-1 group but not the Dll-1 group compared with the PBS group. *P<0.05; **P<0.01 (n=4 per group). HPF indicates high-power field. C, Histological density of putative EPCs (BM-Lin– cells obtained from GFP transgenic mice) incorporating into vasculature of ischemic tissue. The density of the incorporating EPCs identified as CD31+/GFP+ cells was significantly greater in the Jag-1 group than in the Dll-1, empty-vector, and PBS groups. Green fluorescence indicates GFP; red signal, CD31. *P<0.05; **P<0.01 (n=4 per group).

Localization of ROP2 in V. faba Guard Cells. results

Plant Cell. 2008 January; 20(1): 75–87.

Figure 2. Localization of ROP2 in V. faba Guard Cells. (A) An intact V. faba guard cell transformed with RFP-CA-ROP2. (B) An intact V. faba guard cell transformed with RFP-DN-ROP2. (C) An intact V. faba guard cell transformed with RFP alone. (D) An intact V. faba guard cell transformed with GFP-ROP2. (E) An intact V. faba guard cell transformed with GFP alone. In all panels, the guard cells at left were transformed by biolistic bombardment and the cells at right were not. A fluorescence image (right) and the corresponding bright-field image (left) of the same cell are shown. The guard cells shown in (D) and (E) were kept under darkness after bombardment until observation, and those in (A) to (C) were irradiated with white light for 3 h before observation. Black dots in bright-field images are gold particles. Focus was on the mid-plane of the cells. Bars = 10 μm. Plant Cell. 2008 January; 20(1): 75–87.

cohort studies produced lower estimated sensitivity and specificity than studies of other designs results

Circulation. 2008;118:166-175

Figure 3 shows that cohort studies produced lower estimated sensitivity and specificity than studies of other designs. The pooled diagnostic odds ratio was 9 (95% confidence interval 5 to 16) for cohort studies and 213 (85 to 535) for studies of other designs (P < 0.001, permutation test). Further analysis was restricted to the 15 cohort studies that used a diagnosis of clinically definite multiple sclerosis, arrived at by clinical information alone, as the reference standard. Fig 3 Receiver operating characteristic plots for cohort studies and for studies of other designs BMJ. 2006 April 15; 332(7546): 875–884.

Cardiac Allograft Vasculopathy results

Circulation Research. 2004; 94: 46-52

Results Cardiac Allograft Vasculopathy Mice treated with fasudil were well tolerated and showed no side effects, such as weight loss, hair loss, or diarrhea. A total of 2369 coronary arteries were evaluated by computer-assisted analysis in terms of the severity of CAV. Four weeks after the cardiac transplantation from AKR to C3H/He mice, neointima formation (evaluated by intima/vascular area ratio) and perivascular fibrosis of coronary arteries were markedly enhanced in the control allograft group compared with the isograft group (Figures 1 and 2⇓). By contrast, coronary veins were resistant to those changes (Figure 1E). In the isograft group, perivascular fibrosis was also developed probably due to a reperfusion injury alone (Figure 2B). Both neointima formation and perivascular fibrosis of the allografts were dose-dependently attenuated in the fasudil groups (Figures 1 and 2⇓). The high dose of fasudil inhibited the perivascular fibrosis to the level seen in the native hearts (Figure 2B). The medial area of the coronary artery was reduced only in the high-dose fasudil group compared with the control group (Figure 2C). The medial area in the high-dose fasudil group (0.31±0.01, n=9) was equal to that seen in the native hearts (0.36±0.01, n=10). At 4 weeks after the treatment with fasudil, plasma concentrations of hydroxyfasudil (ng/mL) increased from 0 (control animals) to 5.53±2.08 and 37.24±19.12 in the low-dose (n=6) and the high-dose (n=5) fasudil groups, respectively, specific therapeutic ranges of the Rho-kinase inhibitor.12 By contrast, fasudil was not detected in any groups. View larger version:In this pageIn a new window Download as PowerPoint Slide Figure 1. Representative photomicrographs of a mouse coronary artery from an isograft (A) and from an allograft in the control (B), the low-dose (C) and the high-dose fasudil group (D) at 4 weeks after the transplantation (Masson’s trichrome staining, ×200). In the control allograft group, intimal thickening and perivascular fibrosis were noted, both of which were dose-dependently suppressed by fasudil treatment. Coronary vein, indicated by an asterisk, in the allograft is shown for comparison with coronary arteries (E). Bars=100 μm. View larger version: Figure 2. Long-term treatment with hydroxyfasudil inhibits the development of CAV in mice. In the control allograft group, intimal thickening (A, as expressed by intima/vascular area ratio) and perivascular fibrosis (B) developed at 4 weeks after the transplantation, both of which were dose-dependently suppressed by the fasudil treatment. By contrast, medial thickness was reduced only in the high-dose fasudil group (C).

Obesity, diabetes mellitus, and the risk of female breast cancer in Eastern China results

World J Surg Oncol. 2013; 11: 71.

The demographic characteristics of breast-cancer case and control groupsThe mean age (± standard deviation; SD) was 49.94 (±9.44) for the entire population (123 breast-cancer cases and 369 controls), 49.95 ± 9.48 for the cases alone, and 49.92 ± 9.37 for the controls alone. No significant differences were found between the breast-cancer case and control groups for age distribution (χ2 = 0.659, P = 0.417), residence (χ2 = 2.517, P = 0.113), or education level (χ2 = 0.042, P = 0.838). However, there were significant differences between these two groups for family history of breast cancer (χ2 = 3.828, P<0.001), total life satisfaction (χ2 = 15.990, P<0.001), current life satisfaction (χ2 = 12.461, P<0.001), and frequency of exercise (χ2 = 5.207, P = 0.022), and the P-value for annual family income was close to significance (χ2 = 3.828, P = 0.05) (Table1).Table 1Demographic characteristics of breast cancer and control groups

Light-Dependent Translocation of GFP-ROP2 to the Plasma Membrane in Guard Cells. results

Plant Cell. 2008 January; 20(1): 75–87.

Figure 3. Light-Dependent Translocation of GFP-ROP2 to the Plasma Membrane in Guard Cells. (A) Cartoon describing how GFP-ROP2 localization to the plasma membrane (PM) and cytosol was measured. From the fluorescent images of guard cells expressing GFP-ROP2, GFP intensity was line-scanned along two lines drawn at right angles to the long axis of the cells, at ∼25% distance from both ends (white dotted lines in the left panel). The average pixel intensities of the plasma membrane (gray bar) and the cytosol (white bar) were obtained from the GFP intensity profiles as indicated. (B) Light illumination induces GFP-ROP2 translocation to the plasma membrane. The intensity of GFP-ROP2 localized to the plasma membrane was measured as described for (A) before and after 2 h of illumination from individual guard cells. The resulting pixel intensity of plasma membrane/cytosol is displayed (right panel; means ± se; n = 11, P < 0.02). Stomatal movements are inhibited by mounting the epidermal pieces between the cover slip and the slide glass during observations. Focus was on the mid-plane of the cells. Bar = 10 μm. (C) and (D) Intact V. faba guard cells transformed with GFP-ROP2 and RFP-RhoGDI1 (C) or GFP-ROP2 and RFP alone (D) and incubated under light for 3 h. Bars = 10 μm. (E) Relative pixel intensity at the plasma membrane of GFP-At ROP2 from guard cells transiently transformed with GFP-ROP2 together with RFP-RhoGDI1 (C) or with RFP (D) in darkness or after 3 h of illumination. Results from three independent experiments are shown (means ± se; n = 32 to 60). Plant Cell. 2008 January; 20(1): 75–87.

Rho-Kinase Activity results

Circulation Research. 2004; 94: 46-52

Rho-Kinase Activity The extent of ERM phosphorylation, as normalized by that of total actin, was significantly enhanced in the control allograft group compared with the isograft group (Figure 3). The long-term treatment with fasudil dose-dependently suppressed the increase in Rho-kinase activity in the allograft group (Figure 3). The total amount of ERM did not change among the 4 groups (Figure 3). The actin density was significantly less in the allograft group than any other groups because of the abundant ECM in the equal amount of the sample. View larger version:In this pageIn a new window Download as PowerPoint Slide Figure 3. Representative images and quantified analysis of Western blotting for phosphorylated ERM, a marker of Rho-kinase activity, total ERM, and actin, in cardiac grafts at 4 weeks after cardiac transplantation. The Rho-kinase activity was increased in the control allograft group, which was dose-dependently suppressed by the fasudil treatment.

Obesity, diabetes mellitus, and the risk of female breast cancer in Eastern China results

World J Surg Oncol. 2013; 11: 71.

The association between indexes related to obesity and female breast cancerOf the four common indexes of obesity, no significant differences were found between the breast-cancer case and control groups for waist to hip ratio (WHR) (χ2 = 0.121, P = 0.727) or hip circumference (HC) (χ2 = 1.169, P = 0.280), but there were significant differences in the distribution of BMI (χ2 = 6.603, P = 0.010) and waist circumference (WC) (χ2 = 7.255, P = 0.007) (Table2).Table 2Distribution of obesity-related indexes between the female breast-cancer case and control groupsWhen the results were stratified by different menopausal status of the women in the breast-cancer case and control groups, there were no significant differences in BMI in the pre-menopausal group, WC in the post-menopausal group, and WHR and HC in both the pre-menopausal and post-menopausal groups. However, there were significant differences in BMI for the post-menopausal group (χ2 = 9.645, P = 0.008), and WC for the pre-menopausal group (χ2 = 4.701, P = 0.030) (Table3).Table 3Association between obesity-related indexes and female breast cancer in patients with different menopausal status

The ROC and linear regression curves. results

Reproductive Biology and Endocrinology 2008, 6:37

The ROC and linear regression curves. Al-Ghamdi et al. Reproductive Biology and Endocrinology 2008 6:37

Light-Induced Stomatal Opening in Wild-Type and Mutant rop2 Arabidopsis. results

Plant Cell. 2008 January; 20(1): 75–87.

Figure 5. Light-Induced Stomatal Opening in Wild-Type and Mutant rop2 Arabidopsis. (A) Light-induced stomatal opening in CA-rop2, wild-type (ecotype Columbia [Col-0]), and DN-rop2 Arabidopsis. (B) Gene structure of ROP2 and site of the T-DNA insertion in the rop2 knockout line in rop2-1. Boxes represent exons. Arrows indicate the regions of ROP2 from which the ROP2-specific primers were designed for RT-PCR. pROK2, T-DNA present in the SALK Arabidopsis mutants. (C) RT-PCR amplification of ROP2 mRNA. ROP2 transcript was amplified from wild-type and three independent complemented lines (ROP2-1, ROP2-2, and ROP2-3) but not from rop2-1. Tubulin was amplified as a positive control. (D) Light-induced stomatal opening in wild-type (Col-0), rop2-1, and three independent lines of ROP2-complemented rop2-1 Arabidopsis. White light with an intensity of 250 μmol·m−2·s−1 was used to irradiate the plants. Results (means ± se; n > 250) from four independent experiments are shown. In each data set, the extent of increase in stomatal aperture and the time to reach half-maximum stomatal aperture (t1/2) were obtained by fitting the data from illuminated samples to a first-order exponential function. Plant Cell. 2008 January; 20(1): 75–87.

In Vivo Gene Transfer of DN-Rho-Kinase results

Circulation Research. 2004; 94: 46-52

In Vivo Gene Transfer of DN-Rho-Kinase To confirm the specificity of the inhibitory effect of hydroxyfasudil on CAV, adenovirus-mediated gene transfer of DN-Rho-kinase was performed while LacZ transfection was used as a control. X-gal staining demonstrated that LacZ was expressed widely in the cardiac grafts (Figure 4A). Histological analysis showed that the gene transfer of DN-Rho-kinase suppressed both intimal thickening (evaluated by intima/vascular area ratio) and perivascular fibrosis compared with that of LacZ (Figures 4B and 4C). In this experiment, since the extent of myocardial fibrosis was too high to identify some small blood vessels, we examined only relatively larger arteries where intimal thickening was prominent while perivascular fibrosis was less prominent. Thus, compared with the results obtained in the fasudil protocol (Figure 2), the extent of intimal thickening was relatively greater while that of perivascular fibrosis was relatively smaller (Figure 4). View larger version:In this pageIn a new window Download as PowerPoint Slide Figure 4. Inhibitory effects of in vivo gene transfer of DN-Rho-kinase on CAV in mice. DN-Rho-kinase and LacZ were transfected into allografts immediately after transplantation. A, X-gal staining performed 1 week after the transfection confirmed the expression of LacZ. B, Representative photomicrographs of a coronary artery in the allograft groups transfected with LacZ or DN-Rho-kinase. The intimal thickening (as expressed by the intima/vascular area ratio) (C) and perivascular fibrosis/vascular area ratio (D) were suppressed by in vivo gene transfer of DN-Rho-kinase. Bars=100 μm.

Obesity, diabetes mellitus, and the risk of female breast cancer in Eastern China results

World J Surg Oncol. 2013; 11: 71.

The association between breast cancer and physiological, reproductive, clinical, behavioral, and dietary factorsOther possible factors that might be related to breast cancer, including physiological, reproductive, clinical, behavioral, and dietary factors, were also analyzed. We did not find any significant differences between the breast-cancer case and control groups for menstrual history, including age at menarche (χ2 = 1.496, P = 0.221), menstrual pattern (χ2 = 2.446, P = 0.118), dysmenorrhea (χ2 = 0.382, P = 0.536), or menopausal status (χ2 = 0.643, P = 0.423); reproductive history, including age at first birth (χ2 = 1.613, P = 0.204), number of births (χ2 = 1.433, P = 0.231), or breastfeeding status (χ2 = 0.057, P = 0.811); or history of hypertension (χ2 = 1.246, P = 0.264), cardiovascular disease (χ2 = 1.712, P = 0.191), nephritis (χ2 = 0.333, P = 0.564), or gynecologic tumors (χ2 = 0.914, P = 0.339). However, significant differences between the case and control groups were found for number of miscarriages (χ2 = 8.660, P = 0.003), history of benign breast tumor (χ2 = 12.075, P = 0.001) and presence of DM (χ2 = 4.459, P = 0.035).No significant differences were found for other factors including oral contraception (χ2 = 0.000, P = 0.997), inverted nipple (χ2 = 1.731, P = 0.188), nipple discharge (χ2 = 0.150, P = 0.699), orgalactophore hyperplasia (χ2 = 0.441, P = 0.507), or for dietary habits, including intake of bean products (χ2 = 2.578, P = 0.108), fresh beans (χ2 = 0.022 P = 0.883), red meat (χ2 = 0.010, P = 0.919), milk products (χ2 = 0.422, P = 0.516), corn (χ2 = 543, P = 0.461), carrots (χ2 = 0.319, P = 0.572), fried food (χ2 = 0.721, P = 0.396), vegetables or fruit (χ2 = 0.630, P = 0.427), garlic (χ2 = 3.316, P = 0.069), hams (χ2 = 0.763, P = 0.383), or pickled foods (χ2 = 0.583, P = 0.445).

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