Mesencephalic astrocyte-derived neurotrophic factor (MANF), a newly discovered secreted neurotrophic factor, has been proven to not only protect dopaminergic neurons and other cell types but also regulate neuroinflammation and the immune response to promote tissue repair and regeneration. However, to date, there is no information regarding the relationship between MANF and retinal ganglion cells (RGCs) in the eye. In the current study, we first determined the expression of MANF in the retina and vitreous. Then, we examined the effect of MANF on RGCs using both in vivo and in vitro models and simultaneously explored the underlying neuroprotective mechanisms of MANF. Finally, we measured the concentrations of MANF in the vitreous of patients with different retinopathies. We demonstrated that MANF was highly expressed in RGCs and that exogenous MANF could protect RGCs from hypoxia-induced cell injury and apoptosis both in vitro and in vivo by preventing endoplasmic reticulum stress-mediated apoptosis. Furthermore, MANF can be detected in the vitreous humor, and the concentration changed under pathological conditions. Our results provide important evidence that MANF may be a potential therapeutic protein for a range of retinal pathologies in either the preclinical stage or after diagnosis to promote the survival of RGCs. Vitreous MANF may be a promising protein biomarker for the indirect assessment of retinal disorders, which could provide indirect evidence of retinal pathology.
Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known as Armet, was first isolated from a rat type-1 astrocyte ventral mesencephalic cell line. MANF is widely expressed in the nervous system and non-neuronal tissue, such as the cerebral cortex, liver, and heart (Lindholm et al., 2008). Because MANF is a secreted protein, its expression is especially abundant in secretory cells and tissues, including human serum, salivary glands, and embryonic pancreas. Recently, studies conducted at the University of Helsinki demonstrated that the concentration of MANF is increased in the serum in children with type 1 diabetes, which indicated that MANF may be used as a potential therapeutic protein for diabetes (Galli et al., 2016). Furthermore, our recent studies showed that MANF could be robustly expressed in RGCs and that hypoxia can stimulate the up-regulation of MANF expression over time, which suggested that MANF may play a vital role in the functional regulation of RGCs in both health and disease (Gao et al., 2017).
Recent advances have indicated that MANF could protect dopaminergic neurons and cell types in animal models of Parkinson's disease, Alzheimer's disease, and brain ataxia (Petrova et al., 2003; Lindholm et al., 2007). MANF is a new secretory neurotrophic factor, and its expression and secretion are regulated by endoplasmic reticulum stress (ERS) (Apostolou et al., 2008; Tadimalla et al., 2008). MANF not only modifies ER homeostasis to protect cells against ERS-induced cell death but also regulates neuroinflammation and the immune response to promote tissue repair and regeneration (Neves et al., 2016; Zhu et al., 2016). The mechanisms of the neuroprotective effects of MANF in rodent models of cerebral ischemia and spinocerebellar ataxia are mediated by inhibition of ischemia-induced apoptosis (Yang et al., 2014). Numerous eye diseases are associated with neuronal apoptosis, ERS, inflammation, and ischemia, such as diabetic retinopathy (Ikesugi et al., 2006; Li et al., 2009; Tang and Kern, 2011; Zhang et al., 2015), glaucomatous optic neuropathy (Yang et al., 2011; Ha et al., 2015), retinal detachment (Liu et al., 2010; Zhu et al., 2013), and age-related macular degeneration (Li et al., 2008; Libby and Gould, 2010). These findings suggest that MANF may be a promising therapeutic strategy for promoting the survival of retinal neurons. Therefore, we hypothesized that MANF may have therapeutic potential for the treatment of retinopathy. However, there is little research available on this subject. The only study on MANF in the retina was reported in Science and demonstrated for the first time that MANF is expressed in the mouse retina and exerts immune modulatory function to promote tissue repair and successful regeneration in the retina (Neves et al., 2016). These results provided a strong foundation for our current study and revealed that further attention and investigation of the expression, function, and effect of MANF in retinopathy are imperative.
In the current study, we first determined the expression of MANF in the retina and vitreous. Then, we assessed the effect of MANF on retinal ganglion cells (RGCs) using both in vivo and in vitro models and simultaneously explored the underlying neuroprotective mechanisms. Finally, we measured the concentrations of MANF in the vitreous of patients with different retinopathies. We demonstrated that MANF was highly expressed in the RGCs and that exogenous MANF could protect RGCs from hypoxia-induced cell injury and apoptosis both in vitro and in vivo by preventing ERS-mediated apoptosis. MANF can be detected in the vitreous humor, and the concentration of MANF changed under pathological conditions.
A total of 92 male Sprague-Dawley rats (150–200 g) and 150 newborn Sprague-Dawley rats (1–4 days) (SLAC Laboratory Animal Co., Ltd. Shanghai, China) were used. This study was carried out in accordance with the guidelines of Shanghai Jiaotong University Affiliated Sixth People's on the ethical use of animals. The protocol was reviewed and approved by the animal experimental ethics committee of Shanghai Jiaotong University Affiliated Sixth People's. The animal handling and experimental protocols were carried out in accordance with the approved guidelines of Animal Care and Use Committee of Research Ethical Committee, Shanghai Jiaotong University Affiliated Sixth People's and the Association Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were kept in standard cages at room temperature (15–25°C) and 12-h light/12-h dark schedule with food and water available ad libitum.
Sprague-Dawley rats were allocated randomly into normal control (NC) group (n = 26), chronic ocular hypertension (COHT) group (n = 32), COHT + PBS group (n = 8, intravitreous injection of 2 μl PBS) and COHT + MANF group (n = 26, intravitreous injection of 2 μl, 1 μg/ml). In this study, all animals were anesthetized by intraperitoneal injection of 10% chloral hydrate.
In this study, vitreous samples were obtained from 55 patients undergoing vitreoretinal surgery, including 21 proliferative diabetic retinopathy, 16 retinal detachment and 18 macular hole patients. The mean age was 48.71 ± 11.83 years (range 29–65 years). This study was carried out in accordance with the recommendations of Shanghai Jiaotong University Affiliated Sixth People's Hospital with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Office of Research Ethical Committee, Shanghai Jiaotong University Affiliated Sixth People's Hospital. Undiluted vitreous samples were obtained at the onset of vitrectomy and frozen at -80°C immediately until assayed. Healthy vitreous was obtained from the Shanghai Jiaotong University Affiliated Sixth People's Hospital following the guidelines of the Shanghai Jiaotong University Clinical Human Research Ethics Committee (n = 2). Both were from donors who had been screened to make sure there was no underlying ocular disease.
Primary cultured RGCs were purified by two-step immunopanning method as we described previously (Gao et al., 2016). RGCs were collected and seeded into 96, 24 and 6-well plates pretreated with mouse-laminin (Trevigen Inc., Gaithersburg, MD, USA) and poly-D-lysine (Sigma–Aldrich, St. Louis, MO, USA). Plates were incubated in a humidified incubator with 5% CO2 at 37° C. RGCs purity was checked by Thy1.1 and Brn 3b immunocytochemical staining (about 85%) (Gao et al., 2016). Forty-eight hours after seeding, RGCs were incubated with 200 μM cobalt chloride (CoCl2, Sigma–Aldrich, St. Louis, MO, USA) to induce hypoxia and apoptosis (Kim et al., 2013), combined with 0, 10, 20, 50, or 100 ng/ml MANF for 24 h, then incubated 12 h, 24 and 48 h under optimal concentration. Half of the RGC medium was replaced with fresh RGC medium every 3 days.
Elevation of IOP was induced by injection of 5 μl of paramagnetic polystyrene microbeads (FluoSpheres; Invitrogen, Carlsbad, CA, USA; 15-μm diameter) into the anterior chamber of the right eye of each animal under a surgical microscope, as previously reported (Sappington et al., 2010). IOP was measured every 3 days after the injection using a rebound tonometer (Icare® Tonolab, TioLat, Helsinki, Finland). All the measurements were performed between 9 and 10 am by the same operator, and the IOP is reported as the mean ± standard deviation (SD) of the average of three independent readings.
A dose of 2 μl of MANF (1 μg/ml, PeproTech, Rocky Hill, NJ, USA) was intravitreally injected 2 days before increased IOP was induced and then once a week for 2 weeks.
Immunofluorescence staining was conducted as previously described Wu et al. (2013). The eyes and RGCs from different groups were fixed with 4% paraformaldehyde (PFA) for 2 h and 20 min at room temperature, respectively. Then, the eyes were cut into 8-μm-thick sections after graded dehydration with 20 and 30% sucrose solution and then stored at -80°C until use. Sections and RGCs were incubated in 0.1% Triton X-100 and 3% (w/v) bovine serum albumin (BSA) (Sigma–Aldrich, St. Louis, MO, USA) for 30 min sequentially at room temperature to prevent non-specific background. Then, sections and RGCs were incubated with rabbit anti-MANF (1:200, Abcam, Cambridge, MA, USA) and anti-survivin (1:200, Abcam, Cambridge, MA, USA) antibody at 4°C overnight, followed by successive incubations with fluorescein-conjugated goat anti-rabbit secondary antibody (1:400, Molecular Probes, USA) and Hoechst staining. Negative controls were routinely prepared by incubating the cells and retinal sections in normal buffered serum instead of the primary antibody. The stained sections and cells were visualized and imaged using confocal microscopy (Leica SP8, Hamburg, Germany).
CCK8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to 96-well plates (10 μl/well). After a 4-h incubation at 37°C, the plates were analyzed at 450 nm with the Tecan Genios microplate reader (Synergy H1, BioTAK). All values are expressed as the mean ± SD of at least three wells and at least three separate experiments. As the initial cell density was the same, higher OD values represent increased cell viability over time. We compared the changes in the OD values at different times to evaluate the RGC viability.
Approximately 10,000 purified RGCs were seeded into 96-well plates. The caspase 3/7 activation in the different groups was evaluated by the Caspase-Glo 3/7 assay (Promega, Milano, Italy), as previously reported Park et al. (2016). The luminescence signals from the cell lysate solution were determined with the Tecan Genios microplate reader (Synergy H1, BioTAK). All experiments were performed in triplicate.
The proportion of apoptotic cells was measured using a FACSCalibur platform according to the manufacturer's instructions for the Annexin V-FITC/PI flow cytometric assay kit (Becton Dickinson, San Jose, CA, USA). Briefly, after each treatment, the cells were collected, washed, and resuspended in phosphate-buffered saline (PBS) containing Annexin V-FITC and PI for 20 min in the dark at 37°C in 5% CO2. Then, the stained cells were analyzed using a FACSCalibur platform. The percentage of apoptotic cells was determined by calculating the number of annexin V+/PI-cells. All experiments were performed in triplicate.
To obtain a high protein concentration from the vitreous samples for Western blot analysis, protein precipitation must be the first step. In the current study, the vitreous was concentrated using the acetone method as described by Jiang et al. (2004). Primary cultured RGCs and retinal protein extractions were performed as previously described Wu et al. (2015). Then, the protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA); the proteins were separated by SDS-polyacrylamide gel electrophoresis; and the separated proteins were electrotransferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk for 30 min, the membranes were incubated overnight at 4°C with primary antibodies against MANF, survivin, ERS-associated protein CCAAT-enhancer-binding protein (C/EBP), homologous protein (CHOP), cleaved caspase-3 antibody, and β-actin (Abcam, Cambridge, MA, USA). The secondary antibodies included HRP-conjugated goat anti-rabbit antibody (Millipore, Billerica, MA, USA) and HRP-conjugated goat anti-mouse antibody (Millipore, Billerica, MA, USA). The Western blots were imaged with the Kodak Imaging System (Kodak 440CF) using the ECL Western blotting substrate (Hyperfilm ECL, Thermo Fisher Scientific, Rockford, IL, USA). The intensity of the band was quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).
TUNEL staining was performed according to the manufacturer's instructions (In Situ Cell Detection Kit; Roche, Mannheim, Germany) as we previously reported Fu et al. (2016). Cultured RGCs were fixed in 4% (w/v) PFA at 4°C for 30 min and then blocked with 3% BSA and 0.1% Triton X-100 for 1 h at room temperature. The TUNEL reaction mixture was added to the samples and incubated for 60 min at 37°C in the dark. After 3 rinses with PBS, the samples were stained with DAPI (1:2000; Life Technologies, Molecular Probes, NucBlue Fixed Cell Stain, Eugene OR, USA). The TUNEL-positive cell nuclei were visualized using a confocal microscope (Leica SP8, Hamburg, Germany) and quantified using ImageJ software. Six microscope fields of view from six different wells were analyzed per treatment condition.
The PhNR was recorded to evaluate RGC function at baseline and after 3, 7, and 14 days of IOP elevation as previously reported Rangaswamy et al. (2007) and Porciatti (2015). After the rats were anesthetized, the pupils were dilated with phenylephrine hydrochloride and tropicamide, and two 3-mm platinum wire loop electrodes were then placed on the corneal surface of the eyes. One subdermal needle electrode inserted at the base of the right leg served as the ground, and the other subdermal needle electrode placed over the nasal bone acted as the common reference. Light stimuli were delivered using a ColorDome unit. Four different stimuli of 11.38 cd.s/m2-0.33 Hz, 11.38 cd.s/m2-1 Hz, 22.76 cd.s/m2-0.33 Hz, and 22.76 cd.s/m2-0.33 Hz were used in a four-step examination. In each step, the stimulus frequency was 2 Hz, with a 4-ms exposure to a green light on a green background with an intensity of 10 cd/m2 displayed by the Espion Visual Electrophysiology System (Espion E3, Diagnosys, Diagnosys UK Ltd, UK). The PhNR amplitude was recorded from the baseline to the trough of the negative response.
The procedures were performed as previously reported (Wu et al., 2013). Briefly, Fluoro-Gold (FG, Sigma–Aldrich, St. Louis, MO, USA) was injected into the superior colliculus on each side after the rats in each of the four groups were deeply anesthetized. One week post-injection, the rat eyes were harvested and placed in 4% PFA for 2 h at room temperature, and the whole retina was then carefully dissected and flat-mounted on a slide. The RGCs were counted and averaged per eight microscopic fields of identical size at a distance of 1–2 mm from the optic nerve head where the RGC densities were comparable at 200 × magnification. Each group contained at least three rats for measurement of the mean density. The RGCs were manually counted by two operators in a blinded manner using ImageJ software (NIH, Bethesda, MD, USA). The density of RGCs is expressed as the number of cells per mm2.
The levels of vitreous MANF expression were measured by human ELISA kits (CUSBIO Life Science, College Park, MD, USA) according to the manufacturer's instructions. The vitreous samples were diluted 10 times before analysis. The acceptance criteria of the assay followed the previously published recommendations (DeSilva et al., 2003). The MANF concentration was calculated from the standard curve of six individual assays.
All data are expressed as the means ± SD and were analyzed using SigmaStat software. All data were assessed by the Shapiro–Wilk test, and only variables that were normally distributed were tested; we also analyzed the variance of the data, followed by performance of the two-tailed unpaired Student's test. Values of p < 0.05 were considered the threshold for statistical significance.
As we have previously described Gao et al. (2017), immunofluorescence staining shows that MANF is predominantly localized in the ganglion cell layer and inner nuclear layer in both rat and human retinas (Figures 1A,B). To determine if the MANF expression in the ganglion cell layer was specific to the RGCs, primary cultured RGCs were used for in vitro validation. The results confirmed that MANF was robustly expressed in the RGC somas (Figure 1C). These observations suggest that MANF may play a potential role in the pathophysiology of the retina, especially of the RGCs.
FIGURE 1. Immunofluorescence staining of MANF protein in rat cultured RGCs (C), human retina (A), and Sprague-Dawley rat retina (B). MANF staining was mainly distributed throughout the GCL, with some labeling in the INL (indicated by arrowheads). The nuclei were labeled with DAPI (blue). Magnification 400X; Scale bar: 50 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
To determine whether MANF exerts neuroprotective effects on the RGCs, primary RGCs were cultured under hypoxia with or without exogenous MANF. We found that CoCl2 significantly inhibited cell viability (OD value:0.548 ± 0.086 vs. 0.438 ± 0.078, n = 9, p < 0.01, Figures 2A,B), while MANF rescued cell viability in a dose- and time-dependent manner in the RGCs treated with CoCl2. Because cell viability reached the optimum level at 50 ng/ml for 24 h (OD value:0.576 ± 0.069, p = 0.0081, <0.01, Figures 2A,B), we used 50 ng/ml as the optimal effective concentration in subsequent studies.
FIGURE 2. (A) The viability of RGCs treated with 0–100 ng/ml MANF with or without CoCl2 was measured by the CCK-8 assay. The bars represent the means ± SD, ∗p < 0.05, ∗∗p < 0.01. (B) The viability of RGCs treated with 50 ng/ml MANF and 200 μM CoCl2 for 0, 12, 24, and 48 h. The bars represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01. (C) Caspase 3/7 activities were measured by a luciferase assay. The bars represent the means ± SD. ∗∗p < 0.01. (D) Apoptosis was detected by Annexin V and propidium iodide (PI) staining in RGCs treated with 50 ng/ml MANF and 200 μM CoCl2 for 24 h. The R4 quadrant (Annexin V-/PI-), R5 quadrant (Annexin V+/PI-), and R3 quadrant (Annexin V+/PI+) indicate the percentage of viable cells, apoptotic cells, and necrotic cells, respectively. (E) The percentage of apoptotic cells following CoCl2 treatment with or without MANF. The values represent the mean ± SD of three independent experiments. ∗∗p < 0.01.
Furthermore, the protective effect of MANF was evaluated by measuring the caspase 3/7 activity using a luminescent caspase activity assay kit (Figure 2C). Caspase 3/7 activity was significantly increased under hypoxia (1.45 ± 0.48-fold, p = 0.0069, <0.01), whereas MANF treatment remarkably decreased caspase 3/7 activity by 0.65 ± 0.16-fold compared with the hypoxia group (p = 0.0076, <0.01). These results suggested that MANF could inhibit CoCl2-induced caspase 3/7 activation. RGC apoptosis and death were also evaluated by the flow cytometry-based Annexin V + PI assay and TUNEL staining. As shown in Figures 2D,E, 3, the numbers of annexin V (+) and TUNEL-positive RGCs were significantly increased under hypoxia (4.69 ± 0.78-fold, p < 0.01 vs. 5.98 ± 1.05-fold, p < 0.001), whereas MANF treatment remarkably decreased the number of apoptotic RGCs (decreased by 0.57 ± 0.12-fold, p < 0.01 vs. 0.48 ± 0.11-fold, p < 0.01). These results collectively demonstrated that MANF can protect RGCs from hypoxia-induced apoptosis in vitro.
FIGURE 3. (A) TUNEL analysis of RGCs from three different groups: NC group, CoCl2 treatment group (24 h), and CoCl2 with MANF treatment group (24 h). Red indicates TUNEL-positive cells, and blue indicates DAPI. All images were captured at the same magnification. Scale bar, 50 μm. (B) Quantitative analysis of TUNEL-positive cells in the different treatment groups. The data are presented as the means ± SD. ∗∗∗p < 0.001, the hypoxia group compared with the NC group; ∗∗p < 0.01, the hypoxia + MANF group compared with the hypoxia group.
For a further in-depth exploration of the mechanisms of the protective effect of MANF on RGCs under hypoxia, the levels of the anti-apoptotic survivin, apoptosis-related cleaved caspase-3, CHOP, and MANF proteins were analyzed by Western blotting (Figure 4). The results demonstrated that the expression levels of survivin were reduced (p = 0.0092, <0.01), while the levels of cleaved caspase-3, the activated form of caspase-3, were significantly upregulated (p = 0.0077, <0.01) after 24 h of CoCl2 treatment (Figures 4A,B). However, these changes were remarkably reversed by MANF treatment (p = 0.0056, <0.01). CHOP, a pro-apoptotic factor, was increased by >1.39 ± 0.12-fold and 1.49 ± 0.22-fold after 12 and 24 h of CoCl2 treatment, respectively, compared with the NC group, whereas MANF treatment markedly inhibited CHOP expression. Meanwhile, the expression levels of MANF were changed in a similar manner to CHOP (Figures 4C,D). These data indicated that MANF could protect against hypoxia-induced RGC apoptosis by upregulating the expression levels of survivin and suppressing the levels of cleaved caspase-3 and CHOP.
FIGURE 4. Western blot analysis was performed. The expression levels of cleaved caspase-3, survivin, MANF, and CHOP were determined in the RGCs from the three different treatment groups: NC group, CoCl2 treatment group, and CoCl2 with MANF treatment group. The bars represent the means ± SD. β-actin was used as an internal control. (A) and (C) Bands showing the protein expression. (B) and (D) Band density is represented as normalized ratios (β-actin used as control = 1). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, the hypoxia group compared with the NC group. #p < 0.05, ##p < 0.01, the hypoxia + MANF group compared with the hypoxia group.
To validate the protective effects of MANF on the RGCs, in vivo studies are needed. In the current study, a rat COHT model was used to establish the hypoxic injury to the RGCs. As shown in Figure 5A, significant IOP elevation (defined as 5 mmHg greater than the contralateral eye) was observed on the first day after the microbead injection (21.96 ± 3.64 mmHg vs. 9.82 ± 0.41 mmHg, p = 0.0016, <0.01), and the IOP remained elevated for an additional period of 2 weeks at significantly higher levels than those in the NC group at every time point over the entire 2-week study period (t-test, p = 0.028, <0.05 for each time point). MANF administration did not alter the IOP (n = 22, p = 0.32, >0.05). Therefore, the results demonstrated that the microbead injection resulted in COHT.
FIGURE 5. (A) The mean IOPs of the rats in the NC group (n = 22), COHT group (n = 28), COHT + PBS group (n = 8), and COHT + MANF group (n = 22). IOP was measured from 0 to 2 weeks after the microbead injections. The data are presented as the means ± standard deviations (SD). (B) The PhNR amplitudes at baseline (n = 22, 28, and 22), 3 days (n = 14, 25, and 22), and 2 weeks (n = 9, 16, and 15) in the NC group, COHT with MANF group, and COHT without MANF group, respectively. (C) Quantification of the mean amplitudes of the three groups. The data are expressed as the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
The function of the RGCs is impaired before death. To assess the functional changes in the RGCs, PhNR analysis was carried out (Figures 5B,C). The results showed that the PhNR amplitude was significantly reduced by approximately 48.52% after 3 days of COHT (n = 25, p = 0.0039, <0.01), which indicated that RGC function was significantly impaired in the early stage of COHT. With sustained COHT over time, the amplitudes of the PhNR declined further and were reduced by 69.43 ± 7.23% in the second week after COHT induction (n = 16, p = 0.0006, <0.001). As expected, MANF administration significantly rescued the amplitudes of the PhNR in the early stage of COHT, which had recovered by approximately 32.56 ± 5.25% on the third day (n = 22, p = 0.031, <0.05) and by 40.78 ± 7.11% in the second week (n = 15, p = 0.008, <0.01). Collectively, these results demonstrated that MANF effectively attenuated RGC functional impairment in the early stage of COHT, and the protective effect in the early stage of COHT was greater than that in the later stage of COHT (p < 0.05).
To assess whether MANF administration could increase RGC survival under COHT, RGC loss was assessed by retrograde FG labeling (Figure 6). The mean RGC density in the eyes before microbead injection was 2633 ± 422 cells/mm2, and microbead-induced IOP elevation led to a gradual reduction of RGC somas over time (no evident differences during the first 3 days, n = 4, p > 0.01). The number of RGCs decreased by 33.98 ± 5.77% during the 1st week (n = 8, p = 0.0066, <0.01) and by 61.33 ± 8.21% during the 2nd week (n = 8, p = 0.0005, <0.001), while the number of RGCs increased by 20.33 ± 4.55% in the 1st week (n = 7, p = 0.041, <0.05) and by 39.77 ± 6.59% in the 2nd week (n = 7, p = 0.0078, <0.01) in the rats treated with MANF after COHT. These results indicated that MANF reduced the death of the RGCs after COHT and increased the survival rate of the RGCs.
FIGURE 6. (A) Representative regions of FG-labeled RGCs in the flat-mounted retinas in the NC group and COHT with or without MANF groups after 0 (n = 4), 7 (n = 5, 8, and 7, respectively), and 14 (n = 5, 8, and 7, respectively) days after the microbead injections, and all images were captured at the same magnification. Scale bar, 100 μm. (B) Quantitation of the FG-labeled RGCs. The data are presented as the means ± SD, ∗∗p < 0.01, ∗∗∗p < 0.001, the COHT group compared with the NC group. #p < 0.05, ##p < 0.01, the COHT + MANF group compared with the COHT group at the same time point after the microbead injections.
To further confirm the protective effect of MANF on RGCs under COHT, the levels of survivin, a protein that inhibits apoptosis, were measured by immunofluorescence staining and Western blot analysis (Figure 7). Quantitative estimation showed that the expression levels of survivin in the ganglion cell layer (GCL) and inner nuclear layer (INL) were significantly decreased by 0.42 ± 0.09-fold (immunofluorescence analysis, n = 6, p = 0.0042, <0.01) vs. 0.59 ± 0.17-fold (Western blot analysis, n = 6, p = 0.0076, <0.01) after 2 weeks of COHT. However, the survivin expression levels were markedly increased by 0.89 ± 0.21-fold (Western blot analysis, n = 6, p = 0.018, <0.05) in the retinas treated with MANF. Together, these findings strongly suggest that intravitreal delivery of MANF can promote the survival of RGCs in this model of chronic glaucoma.
FIGURE 7. (A) Representative immunohistochemical images of survivin staining (red) in retinas from the three different treatment groups at 2 weeks after the microbead injections. The NC group (top, n = 8), COHT group (middle, n = 8), and COHT + MANF group (bottom, n = 8). Red indicates survivin staining; blue indicates DAPI. Scale bar, 50 μm. (B) Quantitative analysis of survivin-positive regions in the retinas from the different treatment groups. (C,D) Western blot analysis of survivin expression in the three different groups. β-actin was used as an internal control. The level of protein in each group is expressed as the value relative to the NC group. The data are presented as the means ± SD, ∗p < 0.05, ∗∗p < 0.01.
Because MANF is a secreted protein and certain amounts of vitreous proteins originate from the retina (Skeie and Mahajan, 2011), there is a significant possibility that MANF may be present in the vitreous. To determine whether MANF was present in the vitreous, the vitreous of human donors was extracted and used for Western blotting to measure the expression of MANF. We found that MANF was exactly present in human vitreous; specifically, a major protein band of the expected molecular weight, 25 kDa, was clearly identified (Figure 8A). The protein composition and content of the vitreous may change under physiological and pathological conditions of the retina. With the use of ELISA assay kits, we detected MANF in all vitreous fluid samples from different retinopathies, including proliferative diabetic retinopathy (with or without vitreous hemorrhage), macular holes, and retinal detachment (Figure 8B). The mean levels of MANF in the vitreous samples from patients with proliferative diabetic retinopathy (n = 21) (3.44 ± 1.07 ng/ml) and retinal detachments (n = 16) (3.43 ± 1.09 ng/ml) were significantly higher than those in patients with macular holes (n = 18) (1.21 ± 0.28 ng/ml) (p < 0.001). The MANF levels were clearly lower in the vitreous samples from patients with pure proliferative diabetic retinopathy (n = 8) (2.07 ± 0.14 ng/ml) compared with those from the patients with proliferative diabetic retinopathy with vitreous hemorrhage (n = 13) (3.77 ± 0.97 ng/ml) (p < 0.01).
FIGURE 8. (A) Western blot analysis of MANF expression in the vitreous of human donors; a major band at the expected molecular weight (25 kDa) is apparent. (B) MANF concentrations in the vitreous of patients with MH (n = 18), PDR (n = 21), and RD (n = 16). The data are presented as the means ± SD, ∗∗p < 0.01, ∗∗∗p < 0.001. RD, retinal detachment; MH, macular hole; PDR, proliferative diabetic retinopathy; p-PDR, pure proliferative diabetic retinopathy without vitreous hemorrhage, PDR + VH, proliferative diabetic retinopathy with vitreous hemorrhage.
There are three important findings in the current study: (1) MANF was detected in the vitreous humor and highly expressed in the RGCs. (2) Exogenous MANF protected the RGCs from hypoxia-induced cell injury and apoptosis both in vitro and in vivo by preventing ERS-mediated apoptosis. (3) MANF levels in the vitreous humor were significantly higher in patients with proliferative diabetic retinopathy and retinal detachments compared with patients with macular holes. This is the first study to investigate the neuroprotective effects of MANF on RGC function and apoptosis both in vivo and in vitro, and it is the first study to report that MANF is detectable in human vitreous humor and exhibits altered expression under pathological conditions.
Mesencephalic astrocyte-derived neurotrophic factor, with a molecular weight of 25 kDa, is widely expressed in mammalian tissues (Lindholm et al., 2008). Increasing evidence indicates that MANF promotes the survival of dopamine neurons at least partially by regulating the ER stress response, and MANF improves motor function more efficiently than other neurotrophic factors (Voutilainen et al., 2015). New research in the latest issue of Science suggests that MANF promotes tissue repair and successful regeneration in the retina (Neves et al., 2016). In view of these findings, we hypothesized that MANF may be expressed in the retina and protect the retinal tissue through particular mechanisms. To test this hypothesis, we needed to study the expression of MANF in the retinal tissue. Our results verified that MANF was highly expressed in RGCs, which provided a good foundation for our further studies.
An in vitro analysis of RGCs is a crucial and nearly indispensable tool for the study of retinal visual physiology and pathophysiology associated with various retinopathies and neuropathies, which cannot easily be realized in animal models. In addition, hypoxia is an important cause of many ocular diseases, including glaucoma (Li et al., 2011). Our in vitro study provided direct evidence of the protective effect of MANF on hypoxia-induced RGC loss. Moreover, CHOP, a key player in ER stress-mediated apoptosis (Oyadomari et al., 2002), and the pro-apoptotic protein cleaved caspase-3 were downregulated by MANF, which suggested that MANF exerts protective effects on RGCs and prevents ERS-mediated apoptosis.
The in vitro environment is relatively simple, and it was unknown whether MANF exerts a similar protective benefit on RGCs in vivo. To further examine the role of MANF in the survival of RGCs under pathological conditions, we used the COHT model, also known as ischemia-reperfusion, which has been widely used to investigate the mechanisms of RGC death (Sandalon et al., 2013). There is compelling evidence that RGC dysfunction occurs earlier and precedes the loss of RGC somas in glaucoma (Shou et al., 2003). Therefore, early functional protection is crucial for slowing the progression of glaucomatous optic neuropathy. The PhNR, which can be measured by a minimally invasive electrophysiological technique, has been reported to originate primarily from the RGCs and their axons (Porciatti, 2015). It provides a direct, objective assessment of the functional changes in the RGCs (Wilsey and Fortune, 2016). In the current study, a significant functional impairment in the RGCs was identified under COHT, while MANF treatment rescued the early PhNR deficit after 3 days of COHT, which indicated that MANF can protect against RGC functional damage in the early stage of COHT, even though RGC function was not returned to normal levels. However, with the progression of COHT, only a small trend of functional improvement was found. It is possible that with prolonged COHT, additional traumatic pathological pathways are activated such that MANF can no longer confer a sufficient protective effect. RGC apoptosis increased following the functional damage after IOP elevation. FG retrograde labeling showed that MANF effectively inhibited RGC apoptosis without decreasing IOP, and the early protection capacity was far greater than the later effects, which is consistent with the results of the functional assessment. Additionally, these results were further verified by quantitative analysis of survivin expression levels. Collectively, we can confirm that MANF plays a protective role in RGC function and survival under hypoxia both in vitro and in vivo.
Mesencephalic astrocyte-derived neurotrophic factor is a secreted protein and is detectable in human serum (Galli et al., 2016). Thus, we questioned whether MANF is detectable in the vitreous and whether vitreous MANF levels are associated with retinopathy. As we expected, MANF was found in the human vitreous, and MANF levels were elevated under pathological conditions, such as proliferative diabetic retinopathy and retinal detachment, compared with macular holes. The MANF expression levels were clearly lower in the vitreous samples from patients with pure proliferative diabetic retinopathy compared with those from patients with proliferative diabetic retinopathy with vitreous hemorrhage, and this finding may be due to the mixing of the vitreous with blood, as extremely concentrations of MANF are present in the serum (Galli et al., 2016). These data suggest that MANF in the human vitreous humor may be an interesting protein biomarker for the indirect detection of retinal disorders, which can provide indirect evidence for retinal pathology.
The limitations of this study include the small number of vitreous samples, which may not have been sufficient. A larger number of samples are needed for future studies. In addition, we used patients with macular holes as a healthy control group due to the lack of normal samples. Unfortunately, we also had no data on whether MANF expression levels are associated with the progression of retinal diseases or the expression of other cytokines in the vitreous. Finally, the origins and mechanisms of increased vitreous MANF have not yet been revealed.
This study provides the first direct evidence to date that MANF is highly expressed in RGCs and that exogenous MANF can protect RGCs from hypoxia-induced cell injury and apoptosis in both a rat model of chronic glaucoma in vivo and hypoxia-induced RGC apoptosis in vitro. The mechanisms of MANF protection depended on the prevention of ERS-mediated apoptosis, not decreased IOP. Additionally, an interesting finding of potential clinical relevance in this study is that MANF protein can be detected in the vitreous. Furthermore, patients with proliferative diabetic retinopathy and retinal detachments showed a significantly higher MANF level compared with patients with macular holes. Thus, our results provide important evidence demonstrating that MANF may be a potential therapeutic protein for the treatment of a range of retinal pathologies either in the preclinical stage or after diagnosis to promote the survival of RGCs. Vitreous MANF may be a promising protein biomarker for the indirect assessment of retinal disorders, which could also provide indirect evidence of retinal pathology. Nevertheless, MANF requires further attention and investigation.
QW designs this work, revise it critically and finally approve the version to be published. J-HW contributed in the experimental design, revised the manuscript, collected and analyzed the data, T-TL and S-SD take part in some of the experimental studies, for example, western blotting, PCR analysis and cell culture. F-JG drafted, revised the manuscript and take part in a majority of the work. All authors read and approved the manuscript.
This work was supported by the National Natural Science Foundation of China (Grant NSFC81470641, NSFC81470624, NSFC81400414).
DeSilva, B., Smith, W., Weiner, R., Kelley, M., Smolec, J., Lee, B., et al. (2003). Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20, 1885–1900.
Gao, F., Li, T., Hu, J., Zhou, X., Wu, J., and Wu, Q. (2016). Comparative analysis of three purification protocols for retinal ganglion cells from rat. Mol. Vis. 22, 387–400.
Jiang, L., He, L., and Fountoulakis, M. (2004). Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J. Chromatogr. A 1023, 317–320.
Petrova, P., Raibekas, A., Pevsner, J., Vigo, N., Anafi, M., Moore, M. K., et al. (2003). MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J. Mol. Neurosci. 20, 173–188.
Shou, T., Liu, J., Wang, W., Zhou, Y., and Zhao, K. (2003). Differential dendritic shrinkage of alpha and beta retinal ganglion cells in cats with chronic glaucoma. Invest. Ophthalmol. Vis. Sci. 44, 3005–3010.
Copyright © 2017 Gao, Wu, Li, Du and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


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Bussey Glacier () is a glacier flowing west from Mount Peary to the head of Waddington Bay on Kyiv Peninsula on the west coast of Graham Land. It was first charted by the French Antarctic Expedition under Jean-Baptiste Charcot, 1908–10, and named by the UK Antarctic Place-Names Committee in 1959 for Group Captain John Bussey of the Directorate of Overseas Surveys.

See also
 List of glaciers in the Antarctic
 Glaciology

References

External links
 SCAR Composite Gazetteer of Antarctica

Glaciers of Graham Coast

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No one who's seen the Sun Painting could ever forget it. Bob Miller created this luminous exhibit during the Exploratorium's early years. A heliostat on the roof, far overhead, tracks the sun's movement and reflects the sunlight off a mirror down into the dark, cavernous space. Bouncing off other mirrors, the light reaches a wall of prisms, breaks into a luminous spectrum of colors, and reflects off mylar panels. These bright-hued scribbles of color shift with moving air currents and whenever someone discovers that the prisms can be set rotating. Like a stained glass window in an ancient cathedral, the Sun Painting prepares those entering the Palace of Fine Arts for a transformative experience.
Bob Miller was also famous for his Light Walk, which (one time I went with him) included a stop outside to look at the tiny images of the sun coming through a simple sheet of peg board. Nearly every time I see one of those tiny pinhole suns—sometimes with the shadow of a branch moving across it—I remember Bob. Now, more than ever.
Bob's piece Sun Painting was the first rebuild I ever worked on at the Exploratorium, as an intern with Eric Dimond.
I didn't know Bob very well, except through his work. But I had a beer with him once, long ago before I knew anything about him, and in a single hour learned a lot through his eyes. "Notice how the light from that neon sign is focused by each of the billion bubbles in the head on that Anchor Steam", he would say. I thought he was looking for some new effect to turn into an exhibit, but really, he was just, always, looking for the pleasure of looking.
A few years ago, Norman Tuck told me how he had seen Bob off on an ocean voyage from the pier in San Francisco, then drove over to the bridge and called his cell to say a last farewell as the ship passed out through Golden Gate. I am still struck by the power of that action – a friend passes over a bridge as a friend passes under. As the ocean's waves draw them apart, light waves connect them, and two hands wave wishes of bon voyage across the void. Artists know how to live, do they not?
Bob Miller's Sun Painting, one of the Exploratorium's first works of art. Photo copyright Exploratorium, www.exploratorium.edu.


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Fining Parents When Children Are Late For School Won't Work, Says Former School Governor
The caller made the comments on Andrew Castle's LBC show after it was reported that parents are facing £60 fines when their children are late for school.
Local councils and schools in West Midlands, Hampshire and Essex have told parents that they will face fines of up to £60 if their children are consistently late for school.
Whilst schools and councils up and down the country already have the power to issue fines to parents who take their children out of school during term time, a number of councils are extending the penalties to cases of lateness.
Read more: Andrew Castle: 'Tories Need Abolish Tuition Fees To Impress The Youth Voters'
Andrew Castle speaks to Eleanor on LBC. Picture | LBC
An exasperated parent and former school governor Eleanor from Birmingham explained to Andrew that the fines are another "typical example of lack of understanding of what is actually happening in our schools."
She said: "Many parents and children didn't get the school of their choice, so they're juggling work, they're juggling taking one child to one school and another child to a different school, and simply what with the traffic and everything else."
Although she admitted that there is a problem with the tardiness of pupils, she stands by the fact that she doesn't believe financial punishments for parents won't fix the problem.
She suggests that if children are arriving late to school because of parents, then "detentions for parents" should be considered.
Eleanor says: "If there is persistent lateness then get the parents in for a detention after school. Get them to come in and help their children with their homework."
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Home --> Sports A to Z --> Sports Beginning With "U"
An alphabetical index to all of the information on this Web site about sports beginning with the letter "U".	Includes information about the sports of ultimate frisbee, underwater hockey, underwater rugby, unicycling, and unihockey. See also Sports A to Z and Pictures_of_Sports.


Do you really need two different moisturizers?
Can your skin really tell the difference between a day cream and a night cream or is it purely psychological, or worse, a marketing ploy?
The truth is, there is a huge difference between the two. If you're really serious about your skin care, then you already know how essential a night cream is to your routine, especially when it comes to whitening and anti-aging treatments.
To know the difference between day cream and night cream, make sure to remember the 3 P's of day cream and the 3 R's of night cream.
Day creams are designed to moisturize and protect you against the sun and its harmful UVA and UVB rays that cause hyperpigmentation, sunburn, premature aging, and even skin cancer, which could be fatal when discovered too late. Not only that, but today's sophisticated day creams contain ingredients like niacinamide and vitamin B3 that help suppress melanin production (melanin causes skin darkening), while protecting your skin from environmental aggressors that can cause skin dullness.
A good day cream knows how to play well with makeup—it helps your skin retain moisture without making your skin oily. Day creams also help prep your skin for your makeup base, whether it be BB cream, liquid or powder foundation.
An oil-free day cream, with the right formulation, can also double as a primer for makeup. It helps smooth out pores and makes your makeup base glide onto skin.
Your body uses your sleeping time to repair itself, produce collagen, and grow new cells. A night cream is the supercharged sister of your complementary day cream; its ingredients target your skin at a deeper, cellular level, and works overtime while you sleep to repair any damage caused by the sun, stress, and environmental impurities.
Night creams work with the skin to hasten cell turnover, leaving you with fresher, newer skin cells. A cream with 12 HSA, for example, helps regulate genes that control melanin production, which, when partnered with a day cream that lightens skin as it protects, will double the effects of your whitening routine.
Most night creams contain a thicker formula and here's why it's okay: they contain higher concentrations and formulations, so your skin can repair itself better while you snooze. This way, you can wake up to a firmer, more elastic, and ever-youthful complexion.


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My first Saturday blog. I promised seven days a week and I'll deliver. To be honest "giving up my weekend" is not as much of a sacrifice as people seem to think. It's not like I'm closing down all the local clubs, hitting the slopes at Tahoe, whatever. A good Friday for me is: Put Gettysburg on and watch Pickett's Charge three or four times. I've been reading Shelby Foote's Dixie-sucking histories again and they piss me off so bad I can't get enough of seeing double loads of canister blast that fence, sending those big babies from ol' Virginny flying up in the air to rethink their position on the constitutionality of secession.
You can feel the war in Libya shifting today. This is what it must be like to go to a stage play and wait around between scenes while they wheel the props off and drag in the new ones. The next act is coming up, and it's pretty clear what it's going to be: PR war, tribal deal-making war.
Whoa, things are heating up in a good way around here. First I want to thank everybody who helped with my puzzlement over that shot of an MBT with its turret popped off, lying in the sand 30 yards away from the chassis.
First, I have to own up: the photo I used isn't the one I was talking about, because I'm new at blogging and haven't been able to load the photos I want. The reason I'm mentioning this is that a reader calling himself "Kingtoots" pointed out in a damn good comment to yesterday's blog that the picture I ended up using shows a blasted self-propelled gun, not an MBT. Ya got me, King T. It was the off-circle turret and too-thick barrel that gave me away, I bet.
Who ever imagined Libya…I mean Libya, with a record of O-and-Forever, with a knockout from Chad on its record…would put on a show like this? Some of the pictures I've seen are so amazing I've been trying to figure them out for days. Like this one of a dead Libyan tank after the French Air Force hit it. Look at that turret! Flew clean off, like the turret-chassis joint's almost intact, and landed 30 yards away. That's what they call a precision air-to-ground munition.
There've been a few interesting assassinations recently. Not around here, unfortunately. Definitely not the one in Tucson. The dork empties a huge magazine into a crowd, kills nine people including a little girl, and doesn't even kill the congresswoman he went there to get. And why her, anyway? She may've been just another plastic blonde, nothing special–but there's way worse, WAY worse, crawling around Arizona. Just one example: I can't believe there's not one Mexican in Phoenix with enough cojones to blast that bigmouth sheriff, Arpaio. Jared the Retard could've done it without leaving his home state. If you expand your search to DC, I can think of dozens…uh, think I'll stop right there.
War Nerd: Georgians Gone Wild Video Clips!


WASHINGTON — President Trump said on Tuesday that he would consider delaying a March 2 deadline to reach a trade deal with China, saying the United States might not impose higher tariffs on Chinese goods if talks with Beijing were going well.
"If we're close to a deal where we think we can make a real deal, I could see myself letting them slide for a little while," Mr. Trump said during remarks at the Oval Office.
Mr. Trump's comments came as American officials were in Beijing for a week of talks with Chinese officials to try to resolve a trade war that has begun to inflict economic damage on both sides of the Pacific. The two countries are trying to work out significant differences ahead of the March 2 deadline, when the United States has said it will increase tariffs on $200 billion worth of Chinese goods to 25 percent from 10 percent.
Both Mr. Trump and his top trade negotiator, Robert Lighthizer, have previously said that the deadline is a firm date and that the United States will not extend the timeline, which Mr. Trump and President Xi Jinping of China agreed upon during a dinner in Buenos Aires last year.
But with many of the biggest issues unresolved and the deadline drawing near, Mr. Trump appeared ready to give both sides more time to negotiate. And he once again suggested that he and Mr. Xi may ultimately need to iron out the remaining differences before a final deal is reached.
"At some point, I expect to meet with Xi and make the parts of the deal that the group is unable to make," Mr. Trump said.
The president had originally suggested that such a meeting could take place ahead of the deadline but reversed course last week, saying he would not meet with Mr. Xi before March 2.
A delegation of top American trade officials, including Mr. Lighthizer and Steven Mnuchin, the Treasury secretary, arrived in Beijing this week to try to negotiate the outlines of a deal.
But steep challenges remain to striking an agreement that addresses the administration's primary concerns, including China's practice of forcing American companies to hand over valuable technology and restricting access to its market. Mr. Lighthizer, who briefed senators last week, told lawmakers that one of the biggest issues was ensuring that China followed through with whatever commitments it makes. Senators told Mr. Lighthizer that they wanted the text of any agreement or memo of understanding with China to be made public.
For now, China primarily appears willing to buy more American goods, like soybeans, but has not indicated how many other concessions it is prepared to make. And while both countries have expressed optimism about bridging their differences, Mr. Xi is facing pressure in China not to agree to a deal that would jeopardize his country's economic or national security. China's economy is growing at its slowest pace in years, in part because of the American tariffs.
Mr. Trump on Tuesday once again portrayed China's economic weakness as the United States' strength, saying it was in China's interest to make a deal. The United States, he said, could benefit by retaining tariffs on $250 billion worth of Chinese goods and once again inaccurately suggested that China was paying the levies.
While the United States is collecting billions in tariffs, that money is not coming directly from China but from companies and customers who buy imported goods.


Following her recent election to serve as the State Representative for the 29th Legislative District, Melanie Morgan has announced her resignation from the Franklin Pierce Schools Board of Directors. Director Morgan was elected to serve as the District 1 Director in November of 2015 and has been a strong advocate for the interest of Franklin Pierce Schools students and families.
During Director Morgan's three years with the Franklin Pierce School Board of Directors, she was very active in the community, continually soliciting the voices of students, families and community members. She challenged her school board colleagues, district leaders and community members to consider the impact of policies on all students, families and our broader community. During her term of service, district changes included the appointment of a new superintedent, the hiring of a Family Engagement Coordinator and Director of World Languages and Equity, the launch of a process to develop a district equity statement, and a heightened consciousness on the impact of policies and practices on underrepresented students. Director Morgan has also served as a champion in raising awareness of the unique accomplishments of Franklin Pierce Schools across the region and state.
She will undoubtedly bring her experience as a domestic violence advocate, housing commissioner, school board member and community activist to her new role in Olympia, while continually advocating for the interests of her constituents. Through her time on the school board, her community involvement and time spent on the campaign trail, she has a strong pulse on the concerns of members of the 29th Legislative District including housing affordability and homelessness, P-12 education, and the need for community resources and infrastructure in unincorporated Pierce County.
Director Morgan's resignation leaves a vacancy in the Franklin Pierce Schools Board of Directors District 1 position. To learn more about the position and apply to serve in this capacity, please visit www.fpschools.org/boardvacancy.


The 43rd India Gem and Jewellery Awards ceremony presented by the Gem and Jewellery Export Promotion Council (GJEPC) at the Regal Room, Hotel Trident, Mumbai witnessed an incredible attendance of all the prominent personalities of the gems and jewellery industry along with the notable names from the social and political field. The awards ceremony held on 18th March 2017 saw an impressive turnout. The Chief Guests for the function was Devendra Fadnavis, Hon'ble Chief Minister of Maharashtra. Other dignitaries present for the ceremony were Praveenshankar Pandya, Chairman, GJEPC; Kirit Bhansali, Convener – Awards-Sub Committee, Anil Virani, Nirupa Bhatt, MD, GIA – India Middle East along with mining leaders and banking industry leaders.
"India is the largest consumer of gold, accounting for more than 20 per cent of world's total gold consumption. A major exporter of gems and jewellery, Maharashtra is one of the key Indian States with gemstones reserves and mining potential. India is world's largest cutting and polishing centre for diamonds. I congratulate all the winners and wish this event, all success," he added.
Regarding the demands of the industry for exhibition centre, Jewellery Park and university, honourable CM said that the state government would work closely with the industry and try to fulfill these demands. Regarding the university, CM mentioned that this university would be an unconventional one which would be providing all the skill development courses needed for the industry.
The ceremony clasps a very high standing position in the trade as it rewards the best and sets a greater scale, directing the trade towards improvement. The annual awards not only identify the exertions of the exporters of the Indian gem and jewellery industry, but it also rejoices the excellence displayed by the gem and jewellery exporters in India. The industry specialists along with the noble members of the industry who celebrated with the industry for its accomplishment and pliability in the testing times appreciated the industry's efforts.
The ceremony was hosted by none other than Hussain Kuwajerwala, Television actor and achor along with popular TV actress Sakshi Tanwar who with their entertaining ways kept the audiences entertained throughout the evening.
The much-awaited awards ceremony began after much delay of almost one hour. However, the industry patrons and guests waited patiently till the start of the award ceremony. Over a 40 awards were given across various categories. Lifetime Achievement Award was given to Sevantilal Shah of Venus Jewel.
Thank you note was presented by Kirit Bhansali, Convener- Awards Sub-committee, GJEPC. The awards evening further continued with fervour at the dinner party that was hosted by the GJEPC for all the attendees.


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10 New ownership structure for Kelly Racing
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Ekstrom "worried" pay drivers could flood GT3-based DTM
By Rachit Thukral
Published on Saturday January 2nd 2021
DTM RSS feed
Mattias Ekstrom is "worried" and "scared" that the DTM will get flooded with pay drivers when the series moves to GT3 regulations in 2021.
The DTM will undergo a major transformation next year when high-downforce Class One cars are dropped in favour of comparatively slower and easier-to-drive GT3 machines.
This change will also mark the end of manufacturer participation in the DTM, one of the series' hallmarks, with privateer teams now required to raise their own budgets.
The DTM has always been known for attracting top talent, with drivers coming from other championships such as Formula 1 mixing with newcomers from the now-defunct European F3 series to form one of the most competitive grids in any championship.
This was largely made possible by heavy involvement of German manufacturers, who were known to be spending tens of millions of dollars every year in their home series, but an increasing appetite for electric racing in recent years has prompted them to look at other categories and turn DTM into a privateer-led series.
Two-time DTM champion and long-time Audi driver Ekstrom fears the DTM's image as a destination for leading drivers will change quickly in its GT3 era as independent teams will likely favour those who bring a budget along with them.
"I think DTM will always try to position themselves as a championship with the best of the best driving," Ekstrom told Autosport.
"And most of the time the best of the best are also having a higher demand in terms of financial requests and so on.
"For me not having manufacturers or manufacturer support, it will be very difficult to be attractive for drivers and teams who are on a professional level and don't finance themselves with gentleman drivers.
"If DTM would start becoming a platform with gentleman drivers or pay drivers will come, I think it would be very fast changing the image of the series from being the place where best of the best drivers are driving to where one those who can find a budget can drive, and that is one thing I'm worried and I'm also scared about that will end up that way."
Ekstrom first joined the DTM a year after it was revived in 2001 and stayed in the category for 18 seasons, spending his entire tenure with factory Audi squad Abt.
The Swedish driver blasted the DTM for its "too low entertainment value" earlier this year following Audi's dominant start to the season, adding the world no longer needs the long-running German touring car category.
Ekstrom retired from the DTM at a time it featured a healthy three-way rivalry between Audi, BMW and Mercedes, but he feels the manufacturer were overspending on building cars when their high budgets could have been spent on improving the show instead.
"I always felt for many years they would spend a lot of money on very expensive engineering," Ekstrom said.
"I won't say it was wasted money, but it felt like the money that was invested in extreme technical development was just overpricing the product versus having a product that might have been a bit cheaper.
"You could have spent money to do more races, instead of doing fewer races and then overpricing the engineering.
"I'm still convinced the way money was spent in the past wasn't made in the optimum way, and I hope that now in the future they realise that more races is going to help the fans to get racing more continuity and to watch more races."
More DTM News
Revised 2021 DTM calendar finalised The DTM has announced a slightly revised calendar for 2021 after receiving approval from the FIA, with Russia's Igora Drive venue being dropped and Monza named the season opener 1610708528 DTM
Albon set to race a Ferrari in the DTM Alex Albon and Liam Lawson are set to race Ferrari 488 GT3s in the DTM as part of Red Bull's return to the category, Autosport has learned 1610626380 DTM
Frijns: DTM return difficult due to FE clashes Robin Frijns has conceded it might be tricky for him to continue full time in the DTM in 2021 due to potential clashes with Formula E and coronavirus-related uncertainties 1610549396 DTM
Albon to contest DTM in 2021 with Red Bull backing Alex Albon will contest select DTM races in 2021 with backing from Red Bull after being dropped by the energy drink brand's lead Formula 1 squad 1609769412 DTM
Auer: BMW could have closed door to Audi in DTM Lucas Auer believes BMW could have overturned the deficit to Audi in 2021 had the DTM retained Class One regulations for another year 1608973685 DTM
Team Rosberg becomes latest squad to commit to DTM The Team Rosberg squad that powered Rene Rast to his three DTM titles has become the second ex-factory Audi outfit to announce it will remain in the DTM next year 1608738727 DTM


UFO "CRASHING" Caught on Cam over California! Watch the VIDEO! - Anung Bago? What is New? What are the Latest?
UFO "CRASHING" Caught on Cam over California! Watch the VIDEO!
UFO Caught on Cam During Typhoon Ruby! WATCH the Viral Video! Dubstepping Toddler Caught on Cam! What a Talented Kid! WATCH the Viral VIDEO Watch viral video : Dying Girl, Touched by An Angel Caught on Cam! Cook in 3 Seconds! Watch This VIRAL VIDEO to Know How!
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text:
import sys
import time
import kafka
import struct
from common import to_date, to_hex, unpack_ts

def global_header(magic=0xa1b2c3d4L, version_major=2, version_minor=4, zone=0,
                  sigfigs=0, snaplen=65535, network=1):
    return struct.pack("IHHIIII", magic, version_major, version_minor, zone,
                       sigfigs, snaplen, network)

def packet_header(pkt_raw, msg_key):
    epoch_micros = struct.unpack_from(">Q", bytes(msg_key), 0)[0]
    secs = epoch_micros / 1000000
    usec = epoch_micros % 1000000
    caplen = wirelen = len(pkt_raw)
    hdr = struct.pack('IIII', secs, usec, caplen, wirelen)
    return hdr

def consumer(args):
    # connect to kafka
    brokers = args.kafka_brokers.split(",")
    kafka_consumer = kafka.KafkaConsumer(args.topic, bootstrap_servers=brokers)

    # if debug not set, write libpcap global header
    if args.debug == 0:
        sys.stdout.write(global_header())

    # start packet capture
    packet_count = 0
    for msg in kafka_consumer:

        # if debug not set, write the packet header and packet
        if args.debug == 0:
            sys.stdout.write(packet_header(msg.value, msg.key))
            sys.stdout.write(msg.value)

        elif packet_count % args.debug == 0:
            print 'Packet: count=%s dt=%s topic=%s' % (
                packet_count, to_date(unpack_ts(msg.key)), args.topic)
            print to_hex(msg.value)

        packet_count += 1
        if args.packet_count > 0 and packet_count >= args.packet_count:
            break

redpajama_set_name: RedPajamaGithub


It's one thing to struggle to get things into a suitcase, another thing to pay overweight but downright frustrating when your baggage does not arrive, especially with the perishables you so carefully packed!
That's what happend to me last week. Fortunately, thanks to my sister, the tightly, securely and well-encased goodies survived their overnight delay.
At the heart of this frustration lies sloppy service. The agent to whom I had to report my non-baggage arrival could not tell me why my baggage had not arrived the same time with me - he said he did not know and was not informed of any irregularity. Add to that his incompetence of putting the name of my travelling companion on the claim form instead of mine!
Wait, it's not over. The airline supervisor on duty wants me to return the next morning and explain my dilemma to the morning agent because she will not be on duty then (instead of her making a note and leaving it for the morning agent to follow-up). On top of that, she suggests that I write a letter to the Manager because "...my telling him that customers were complaining will not do anything." What is she doing there then?
I am sick of the incompetent and shabby way Caribbean travellers are treated, not just by our own but also by those fighting for market share and not delivering the altitude of service they boast.


Can we relate what you are not evaluating whether our summary is a different tense. Writing courses, like courses in inside higher education system is founded upon a factor in graduate school.
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Having love wallpapers on your computer can really brighten up your day. There are many images to do with love that are guaranteed to make you smile. So many people are familiar with the heart and the bow and arrow as symbols of love towards one another that having these as your background can remind you of that special someone, any time of day. Love wallpapers for your desktop can also sport a loving message or quotation as well as loving images. These words can have powerful meanings or just simply say 'I love you' but one thing is for sure, each message will leave you feeling warm and happy inside. Hearts are a very versatile image, as many people will like to have these as their wallpaper without it being especially about love. They can be used as general images creating a fresh and funky picture.
The cupid with its bow and arrow is a typical type of love backgrounds, although you can now find the design in a range of different forms, such as cartoon or fine art. You can even find some love quotations and pictures with a touch of humour about them. Bashful cartoon teddy bears or a comic strip style wallpapers with a romantic but silly ending can make you laugh. Bringing such humour to a love story is a refreshing alternative to old fashioned pictures of love. Even jokes about love can be appealing to some, wanting a more comedy feel to their love backgrounds.
Valentine's Day is a time when many users of computers will change their desktop backgrounds. Love backgrounds will become most popular around this time as people tend to feel happy and loving and like to think about their loved ones. The different styles of wallpaper you can choose from include cute and cuddly animal pictures, exotic and colourful designs, scenes of natural beauty and well known poems and quotes from over the years.


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Approx. Square Footage: 1,300 sq. ft.
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text:
Q: php field with live evaluation In a content type I need a field where I can put some php code (returning an array) and where I see the render the result clicking on a 'preview' (in a popup or another tab for example)
A typical content of that field could be
$x = mysql_connect(...);
$x = mysql_query("SELECT ...");
$result = array();
while($row = mysql_fetch_assoc($)) {$result[]=...}
return $result;

Editing a new node, I would write this code in that field and click on preview to show the result in a table.

A: To get a version of the loaded node, you should use node_load() or node_load_multiple()
To achieve that in a module, you can use the following code (using db_select())
$nids = db_select("node", "n")
    ->fields("n", array("nid"))
    ->condition("type", "YOUR_CONTENT_TYPE_MACHINE_NAME", "=")
    ->condition("status", 1, "=")
    ->orderBy("created", "DESC")
    ->fetchCol();
$nodes = node_load_multiple($nids);
print_r($nodes); // or use dsm($nodes); if devel module is enabled.

This code will get the full loaded version of the nodes returned by the query. You can use them the way you want.
Hope this helps... Muhammad.

redpajama_set_name: RedPajamaStackExchange


text:
Il Museum of Edinburgh (Museo di Edimburgo) è un museo situato a Edimburgo in Scozia, che illustra le origini della città, la sua storia e le sue leggende. Tra i reperti viene esposta la copia originale del Patto dei Covenanti firmato presso l'antica chiesa francescana Greyfriars Kirk nel 1638, e una ricostruzione del Quartier Generale del Feldmaresciallo Douglas Haig al Fronte Occidentale durante la prima guerra mondiale, usando reperti donati al Museo.

Situato presso la Casa Huntly del XVI secolo, lungo il Royal Mile, il museo è gestito dal Comune di Edimburgo.

Note

Voci correlate
Dean Gallery
Edimburgo
Museo della Scozia
National Galleries of Scotland
Scottish National Gallery of Modern Art
Scottish National Portrait Gallery
Scozia

Altri progetti

Collegamenti esterni

Sito delle Gallerie nazionali scozzesi (anche in italiano)

Articolo sull'edificio del Museo, di Hugh Pearman

Musei di Edimburgo
Musei di arte contemporanea della Scozia
Musei di arte moderna della Scozia
National Gallery of Scotland


This June, the publisher Vintage will release a set of 20 "Vintage Minis: short, compelling titles on big subjects collected from the world's greatest writers.
Each Vintage Mini will comprise of a complete reading experience and will be around 100 pages long. The titles are to be published in A-Format (the size of most mass-market paperbacks) and will feature a range of fiction, non-fiction and essays.
The cover designs for the series are minimalist but vibrant; a different designer was chosen for each edition. Priced at just £3.50 each, they are sure to please readers who would like to broaden their reading habits with a short dose and perhaps inspire exploration into lengthier works from Vintage's back catalogue.
Learn more about the forthcoming collection from the Vintage website.


text:
Home » history » Vietnamese Traditional Clothing
Vietnamese Traditional Clothing
Published on: May 12, 2022 by Cherish
Have you ever wondered what traditional Vietnamese women and men dress like? Or what is their national costume or clothing style? Let's take a look at the Vietnamese culture, especially their traditional clothing.
This article discusses about the traditional costumes of Vietnam. Scroll down and continue reading if you're interested in learning more about Vietnamese traditional costumes.
Trang phục Việt, Việt phục, also known as Vietnamese clothing, refers to the traditional costume worn by Vietnamese people, specifically the áo dài, the country's unofficial traditional dress. Due to the wide and active cultural exchanges in which ancient Vietnam participated, the Vietnamese clothing contains both indigenous and foreign aspects, resulting in the formation of a distinct Vietnamese clothing style.
Vietnamese people usually wear clothes made in cotton. Northern Vietnam and southern Vietnam have different styles. In north Vietnam, many locals wear sandals constructed from discarded tire rubber. Many men and women in south Vietnam dress in western-style attire. The Vietnam traditional dress, ao dai is still worn by some ladies. The majority of women in rural areas wear loose white shirts with skirts. Men in rural areas also dress in western clothing. Vietnamese people prefer to dress in light attire. Rural women dress in loose-fitting dark slacks and shirts with brightly embroidered embroidery. Many girls and women in the cities wear the traditional Vietnam dress, ao dai, a long tunic with loose-fitting leggings.
Dresses and shirts are worn by an increasing number of urban women. Simple shirts and trousers are common among rural and working-class males. The majority of city males dress in Western-style clothing.
Vietnamese people nowadays frequently use western clothing known as Âu phục, but its popular name is simply Quần áo thường ngày.
Ao Dai
One of the traditional Vietnamese dress is the áo dài. Vietnamese men and women can now wear áo dài on formal occasions in addition to suits and gowns. The traditional ao dai has a long, split tunic that's worn over slacks. Ao means shirt in English and Dài refers to "long." The phrase can be used to refer to any outfit that includes a long tunic, such as "nhật bình."
The Nguyễn of Phú Xuân designed the predecessor of the Ao dài in the 18th century. The Áo ngũ thân, a five-paneled popular shirt worn in the 19th and early 20th centuries, derived from this ensemble. In the 1920s and 1930s, Nguyễn Cát Tường and other Hanoi artists recreated the ngũ thân into a modern version clothing, inspired by Paris trends.
The Ao Dai is also the traditional Vietnamese clothing for males. It's a long silk tunic with a traditional snug collar that buttons down on the left side. The male tunic is worn with loose pants underneath and falls below the knees.
During formal occasions, such as weddings and ceremonial events, the men's version of ao dai, or modified ao dai, is also worn. One of the few Vietnamese words found in English-language dictionaries is ao dai. The ao dai goes well with the nón lá and khăn vấn.
Due to its traditional appearance, which resembles Chinese-style clothing Ao ai is easily identifiable.
Non La/Conical Palm-Leaf Hat
Non La is a part of the Vietnamese women clothing. When visiting Vietnam, you may see that Non La is being photographed by local females as they walk down the street. This conical hat is extremely useful in the countryside in a tropical country like Vietnam. It shields farmers from rain and direct sunlight.
Non La, like many other traditional Vietnamese costumes, has its own story, which is based on a mythology about Vietnam's wet rice culture. It is about a beautiful goddess descended from the skies to save humanity from a massive flood. She donned a massive hat composed of four round leaves sewn together with bamboo rods. That hat was so enormous that it protected the people from the rain and restored normalcy to their lives. A temple was built to honor the Goddess as the Rain-Defending Goddess after she passed away. The residents attempted to replicate the Goddess's hat by going into the forest and gathering natural and readily available materials such as palm leaves, Moc tree bark, and bamboo to construct the Non La as we know it today. For rice farmers, boatmen and women on rivers, and local street merchants in big cities, the Non La has become a daily need.
Ao Tu Than
One of the several Vietnamese traditional costumes called Ao tu than, is associated to Áo ngũ thân, which means "five-part dress."
Women wore the Áo tứ thân for generations before they wore the áo dài. As Vietnam extended southern, áo tứ thân began to be linked with women from the north.
The áo tứ thân was a peasant woman's attire in Vietnam, which explains why it was usually made of plain fabric in dark hues, unless it was for special occasions like festivals or weddings.
Despite of its many variations, the basic áo tứ thân is made up of:
A flowing outer tunic that nearly reaches the floor. It has a jacket-like front opening. The tunic separates at the waist into two flaps: a full back flap, which is made up of two sewed-together flaps, and two front flaps that are not sewn together but can be knotted together or left dangling. Under the tunic, a long skirt is worn.
Vietnamese women wore Yếm as undergarments in the old period. It is worn under the skirt and outer tunic and comes in a various fabrics and colors. A silk sash is also worn as a belt around the waist.
In the present-day Vietnam, Ao Tu Than is no longer widely used in the local daily life but can be found in several northern-related festivals and events with more vivid colors.
Ao Ba Ba
Ao Ba Ba is the traditonal clothing of southern Vietnam. The áo (shirt) is the top section of the garment that covers the torso. It is largely connected to rural southern Vietnam, particularly the Mekong Delta. The áo bà ba is a long-sleeved, button-down silk shirt with a scooped neck that is generally worn with silk slacks as a top and bottom combo. The shirt is long and divided down the sides at the waist, generating two flaps with two pockets.
The basic style of traditional Ao Ba Ba has been passed down through the centuries, however it has been updated to reflect current fashion trends. The new Ao Ba Ba for women has been closely stitched to envelop the body and emphasize the exquisite curves of a woman's figure.
This elaborate shirt is easily identifiable since it is frequently worn with a checkered scarf, also known as bandana. While not sophisticated, this bandana can be black and white or brown and serves as a subtle emphasis for the wearer's informal look.
Conical hat and bandana, along with Ao Ba Ba, defined the beauty and tenderness of the people in Southern Vietnam.
Traditional Clothes Of Ethnic Groups In Vietnam
Traditional Dress Of Thai Ethnic Group In Vietnam
In Vietnam, Thai ethnic group's costume is elegant and discreet. The costume is basically a plain blouse and a long skirt with sun, flower, and dragon themes embroidered on it. Belts and Pieu scarves, as well as a few items of silver jewelry, make it perfect. Thai girls wear blouses with an even number of buttons, whereas married women wear blouses with an odd number of buttons.
Traditional Dress of the H'mong Ethnic Group in Vietnam
The H'mong hill group makes up roughly 1.2 percent of the Vietnamese population and is the eighth largest minority ethnic group in the country. They live in the mountainous regions of the north and west.
In Vietnam, the Hmong choose to dress in a more sophisticated, yet lively and colorful style with a bohemian flair. Their costume consists of linen and comes in various colors and embroidered patterns. A black blouse, a colorful skirt, leggings, and headpiece make up the ensemble. Silver beads and jewelry are frequently affixed to the women's clothing to add an identity or specific styles and spirituality.
Traditional Clothing Of The Cham Ethnic Group In Vietnam
The Cham, who have long settled along the central Vietnamese coast, have a complex culture that is heavily inspired by Indian culture. Although Cham's outfit is not as vibrant as other Vietnamese attire, it has its own characteristics and charming beauty.
Long one-piece sarongs or fabric wrappers are worn by both men and women. Females wear long-sleeved pullover blouses, while males wear shirts with buttons down the center. The waistband is wrapped around the waist and over the chest. Because of its outstanding design with golden iridescent hues and meticulous detail, it is the main focus of the outfit. The bright and elegant long costumes are worn by Cham women on important occasions such as the Kate New Year festival, the Ramuwan festival, marriages, and the celebration of females achieving adulthood. The only option for attending religious mass is the white one.
History Of Vietnamese Traditional Costumes
Vietnam's clothing and textile history represents the country's culture and tradition, which traces back to the Bronze Age, when people of various cultures lived in the country, as well as the Chinese's lengthy dominance and accompanying cultural influence, as well as the brief French colonial control. The dynamic cultural exchanges that happened as a result of those foreign cultural influences had a great impact on the history of clothing in Vietnam, eventually leading to the creation of an unique Vietnamese clothing style, such as the Ao dài. Besides that, because Vietnam has a diverse ethnic population, there are numerous various clothing styles that reflect the ethnicity of the wearer.
Barkcloth, silk, and banana fiber cloth have all been used and made in Vietnam since earlier civilizations. Kapok and hemp were also commonly used prior to the introduction of cotton.
For at least a thousand years, the Chinese dominated in the north, while the Champa, an independent monarchy influenced by Indian culture, governed in the south. The clothing designs that emerged in Vietnam during this timeframe included both indigenous and imported foreign features; the upper classes were more easily impacted by foreign cultures than the common people. The higher classes of Vietnam in Northern Vietnam wore attire that matched Chinese fashions, and this style of clothing remained long after Chinese authority ceased under the independent kingdoms of Đại Việt and Champa.
Vietnamese people have been wearing fashionable foreign clothes since the nineteenth century. After the fall of Saigon, the ao dài was temporarily prohibited, but it was quickly reinstated. In Vietnam, high school girls wear it in white as part of their school uniform. Female receptionists and secretaries also wear it. Northern and southern Vietnam have different styles. The áo dài for men and women, and suits or sometimes áo gam (modified version of áo dài) for men, are the most common types of Vietnamese clothing nowadays.
Traditional Vietnamese costumes have been affected by western new styles and have been substituted by more modern and practical clothing styles, yet its charm and essential characteristics have been kept. Vietnamese people are very proud of their distinct wearing style and culture. And travelers continue to be drawn to this magnificent country by its charm.
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