The pathogenic process of intervertebral disc degeneration (IDD) is characterized by imbalance in the extracellular matrix (ECM) metabolism. Nucleus pulposus (NP) cells have important roles in maintaining the proper structure and tissue homeostasis of disc ECM. These cells need adequate supply of glucose and oxygen. Islet amyloid polypeptide (IAPP) exerts its biological effects by regulating glucose metabolism. The purpose of this study was to investigate the expression of IAPP in degenerated IVD tissue, and IAPP modulation of ECM metabolism in human NP cells, especially the crosstalk mechanism between apoptosis and autophagy in these cells. We found that the expression of IAPP and Calcr-RAMP decreased considerably during IDD progression, along with the decrease in the expression of AG, BG, and Col2A1. Induction of IAPP in NP cells by transfection with pLV-IAPP enhanced the synthesis of aggrecan and Col2A1 and attenuated the expression of pro-inflammatory factors, tumor necrosis factor (TNF)-α, and interleukin (IL)-1. Upregulation of IAPP also affected the expression of the catabolic markers—matrix metalloproteinases (MMPs) 3, 9 and 13 and ADAMTS 4 and 5. Downregulation of IAPP by siRNA inhibited the expression of anabolic genes but increased the expression of catabolic genes and inflammatory factors. The expressions of autophagic and apoptotic markers in NP cells transfected with pLV-IAPP were upregulated, including BECLIN1, ATG5, ATG7, LC3 II/I and Bcl-2, while significantly increase in the expression of Bax and Caspase-3 in NP cells transfected with pLV-siIAPP. Mechanistically, PI3K/AKT-mTOR and p38/JNK MAPK signal pathways were involved. We propose that IAPP might play a pivotal role in the development of IDD, by regulating ECM metabolism and controlling the crosstalk between apoptosis and autophagy in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.
Islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone, which is involved in the regulation of glucose homeostasis.10 In addition to its function in islet cells, IAPP has also been shown to function as insulin in peripheral tissues. IAPP exerts its biological function through the signaling pathway that includes G-protein-coupled calcitonin receptor (Calcr) and associated receptor activity modifying proteins (RAMPs; RAMP1–3),11 which have been detected in pancreas and many extra-pancreatic tissues, such as central nerve system (CNS), liver, kidney, muscle, and adipose tissue.12,13,14,15,16 Specifically, IAPP acts as a neuroendocrine regulator to control glucose metabolism, or as a growth factor in extra-pancreatic tissues. In addition, previous studies revealed that human IAPP (hIAPP) has the function of promoting the secretion of IL-1β, IL-1α, and TNF-α by activating macrophages in vitro,17,18 all of which have been shown to be closely associated with IDD through degradation of ECM.19,20,21 If IAPP has a predominant role in IVD degeneration, it may be a good target for designing successful therapies.
Autophagy, apoptosis, and senescence of the cells are distinct cellular processes that account for disc degeneration.22,23,24 It is becoming increasingly apparent that apoptosis and autophagy are closely linked but mutually exclusive processes, with complementary pathways involved in in the pathogenesis of IDD.25,26 Apoptosis is closely associated with and shares molecular events with autophagy in the pathogenesis of human diseases.27 Autophagy is an essential intracellular protein degradation system and contributes to intracellular quality control by clearing damaged organelles and damaged proteins.28,29 Therefore, it is essential and necessary to investigate the crosstalk mechanism of autophagy and apoptosis in the IVD cells during the pathogenesis of IDD. The aim of this study was to determine whether IAPP is involved in the development of IDD, especially in the anabolic and catabolic pathways of ECM, and the possible mechanisms.
The degree of degeneration of the IVD tissue was classified using MRI images and confirmed by Alcian blue staining (Figure 1a). Lower expression of IAPP corresponded with higher degenerative degree (Figure 1a). IAPP mRNA expression levels in human IVD tissue, normalized with respect to β-actin, are presented as 2-ΔΔCt (Figure 1b). As can be seen in Figure 1c, IAPP expression in normal IVD tissue (Grade 2) was significantly higher compared with that in degenerated disc tissue. Proteoglycans and collagens are the main components of ECM. During the process of IDD, the gene expression of AG, BG, and collagen (Col)2A1 were decreased (Figures 1d–f), whereas the expression of Col1A2 was increased (Figure 1g), which confirmed disc degeneration. Thus, the results demonstrated that the gene expression of IAPP decreased during the process of disc degeneration.
Detection of IAPP expression in human IVD tissue. (a) Representative images of IAPP protein in human IVD tissue detected by immunofluorescence staining. (b) IAPP, AG, BG, and Col mRNA levels in human IVD tissue samples were determined by RT-qPCR. (c) Statistical analysis showing the relative expression of IAPP mRNA in IVD tissue with varying degrees of degeneration. (d–g) Statistical analysis showing the relative expression of AG (d), BG (e), Col2A1 (f), and Col1A2 (g) mRNAs. Data were shown as means±S.D. *P<0.05, **P<0.01, compared with group 2 (non-degenerated IVD). Grades 3, 4, and 5 signify mild, moderate, and severe degeneration, respectively. (Alcian blue staining ×100; IHC ×200).
RT-PCR analysis was used to detect the mRNA expression of IAPP, Calcr, and RAMP3 (Figure 2a). The results showed that mRNA expression of IAPP and Calcr was significantly lower in degenerated group compared to that in normal group (Figures 2b and c). The gene expressions of RAMP1,2,3 were all lower in the degenerated group, compared to those in the normal group (Figures 2d–f). Cell lysates from both groups were examined by western blot, which also confirmed a significant decrease of Calcr and RAMP3 protein expressions in degenerated NP cells (Figures 2g–i). The results indicated that the protein expression levels of Calcr and RAMP3 were also lower and consistent with the decreased expression of IAPP (Figures 2j–o). Knockout of IAPP gene expression resulted in lower expression of Calcr and RAMP3 receptors. Pramlintide also promoted the expression of Calcr and RAMP3 receptors. Thus, Calcr and RAMP3 receptors are required for secreted IAPP to suppress IVD degeneration.
Detection of the expression of IAPP and its receptors in NP cells. (a) IAPP and Calcr mRNA levels in normal and degenerated IVD NP cells were determined by RT-qPCR. (b) Statistical analysis showing mRNA expression of IAPP in different groups. (c) Statistical analysis showing mRNA expression of Calcr. (d–f) Statistical analysis showing mRNA expression of RAMP1, RAMP2, and RAMP3. (g) Protein expression of Calcr and RAMP3 in NP cells was determined by western blotting. (h) Statistical analysis showing the relative expression of Calcr protein. (i) Statistical analysis showing the relative expression of RAMP3 protein. (j–l) Statistical analysis showing mRNA expression of IAPP in NP cells infected with pLVX-mCMV-Puro, pLVX-mCMV-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2- Puro-IAPP, or treated with pramlintide. (m) Protein expression of Calcr and RAMP3 in NP cells of different groups were determined by western blotting. (n) Statistical analysis showing the relative expression of RAMP3 protein. (o) Statistical analysis showing the relative expression of Calcr protein. Data were shown as means±S.D. *P<0.05, **P<0.01, compared with the normal group.
As shown in Figures 3a and b, the expression of IAPP was enhanced in pLV-IAPP group and decreased in pLV-siIAPP group, compared to that in control group. Upregulation of IAPP resulted in an increase in glucose uptake (Figure 3c) and cell proliferation in cultured NP cells (Figure 3d). Knockdown of IAPP resulted in lower expression of aggrecan and Col2A1 and higher expression of TNF-α and IL-1, whereas upregulation of IAPP resulted in higher expression of aggrecan and Col2A1 and lower expression of TNF-α and IL-1, compared to in thencontrol group (Figures 3e–j). We treated NP cells with pramlintide, a synthetic analog of amylin, which exerted similar effects as pLV-IAPP.
The effects of IAPP on ECM anabolic gene expression and pro-inflammatory factor secretion in NP cells. Immunocytochemistry assay revealed IAPP protein expression in NP cells infected with pLVX-mCMV-Puro, pLVX-mCMV-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2-Puro-IAPP, or treated with pramlintide. (a) Immunocytochemistry assay revealed IAPP protein expression in NP cells after transfection. (b) RT-PCR showed the expression of IAPP in NP cells in different groups. (c) MTT assay showed higher cell viability resulting from the upregulation of IAPP. (d) Glucose uptake measurement. E-H. RT- PCR showed gene expression of aggrecan (e), Col2 (f), and downregulation of TNF-α (g) and IL-1 (h) in different groups. (i and j). ELISA analysis showed protein expression of IL-1 (i) and TNF-α (j) in human NP cells of different groups. *P<0.05, **P<0.01, compared with NC.
To better understand the effects of IAPP on ECM metabolism in NP cells, we knocked down the expression of IAPP using siRNA and examined the catabolic gene expression of MMP-3,9,13, and ADMTS-4 and 5 by western blotting. As expected, expression of the ECM catabolic enzymes MMP-3, 9, 13 and ADAMTS-4 and 5 were strongly increased (Figures 4a–f). Upregulation of IAPP resulted in a significant decrease in the basal protein levels of these catabolic enzymes, and increased levels of Aggrecan and Col2 proteins (Figures 4g and h). These results indicate that IAPP is involved in the in vitro disc degenerative effects mediated by MMPs. Pramlintide also inhibited the expression of MMPs.
Effects of IAPP on the expression ECM catabolic genes in human NP cells. The cells were transfected with pLVX-mCMV-Puro, pLVX-mCMV-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2-Puro-IAPP, or treated with pramlintide. (a) Protein expressions of MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 were determined by western blotting. (b–h) Statistical analysis showing the relative expression of MMP-3 (b), MMP-9 (c), MMP-13 (d), ADAMTS-4 (e), ADAMTS-5 (f), Aggrecan (g), and Col2 (h) proteins. Values represent the means±S.D. *P<0.05, **P<0.01, compared with NC.
Upregulation of IAPP resulted in increased expression of anti-apoptotic Bcl-2 mRNA and decreased expression of pro-apoptotic Bax and caspase-3 mRNAs. Suppression of IAPP expression by pLVX-ShRNA2-Puro-IAPP exerted opposite effects of increasing pro-apoptotic Bax and caspase-3 gene expression (Figures 5a–c). Upregulation of IAPP also increased the expression of autophagy genes, such as BECLIN1, ATG5, and ATG7. Suppression of IAPP expression could inhibit the expression of the autophagy genes (Figures 5d–f). Immunofluorescence assay for the detection of the accumulation of cleave caspase-3 protein in NP cells demonstrated that caspase3-positive NP cells were more apparent in pLV-siIAPP group (Figure 5j). The results of western blot analysis to detect the protein expression of LC-3 indicated that upregulation of IAPP could increase the expression of LC3-2 and the ratio of LC3-2/LC3-1(Figures 5g–i). LC3 is a known autophagy marker and the increased ratio of LC3-2/LC3-1 indicates that autophagy can be intensified by IAPP.
Effects of IAPP on apoptotic and autophagic gene expression in NP cells. NP cells were transfected with pLVX-mCMV-Puro, pLVX-mCMV-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2-Puro-IAPP, or treated with pramlintide. (a–c) RT- PCR analysis was used to detect the mRNA expression of apoptotic genes, including caspase-3 (a), Bcl-2 (b), and Bax (c). (d–f) RT- PCR analysis was used to detect the mRNA expression of autophagic genes, including BECLIN1 (d), ATG5 (e), and ATG7 (f). (g) Protein expression of LC-3 in different groups of NP cells was determined by western blotting. (h and i) Statistical analysis showing the relative expression of LC3-2 (h) protein and the ratio of LC3-2/LC3-1 (i). (j) Representative images of caspase-3 protein expression detected by immunofluorescence staining. Data were shown as means±S.D. *P<0.05, **P<0.01, compared with the NC. (Scale bar magnification: ×400).
To examine the effect of IAPP on intracellular accumulation of free radicals, ROS generation was analyzed by DCFH-DA using flow cytometry (Figure 6a). There was significant decrease in intracellular ROS by the upregulation of IAPP in NP cells transfected with pLV-IAPP, compared to in the NC group. Downregulation of IAPP by siIAPP could induce an obvious ROS burst in NP cells (Figure 6b). The level of apoptosis in NP cells was measured by flow cytometric analysis, which demonstrated that the apoptosis level was much higher in NP cells transfected with pLV-siIAPP, compared with that in NP cells transfected with pLV-IAPP (Figures 6c and d).
ROS content and apoptosis induced by IAPP were determined by Flow Cytometry. (a) Intracellular ROS generation was determined by measuring the fluorescence intensity using flow cytometry in NP cells infected with pLVX-mCMV-Puro, pLVX-mCMV-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2- Puro-IAPP, or treated with pramlintide. (b) Statistical analysis showing results of cell fluorescence for DHE. (c) Flow cytometric measurement for apoptosis incidence. (d). Statistical analysis showing TUNEL-positive rate. For quantitative evaluations, data were shown as means±S.D. *P<0.05, **P<0.01, compared with the NC.
The level of apoptosis in NP cells was confirmed by TUNEL Assay. As shown in Figure 7a, a few apoptotic cells were stained red in normal NP cells, but apoptosis was much higher in NP cells transfected with pLV-siIAPP. TUNEL-positive cell proportion was much lower in NP cells transfected with pLV-IAPP and much higher in NP cells transfected with pLV-siIAPP, compared with in the normal group (Figure 7c). Pramlintide administration could also decrease the apoptotic rate. To detect the regulatory effect of IAPP on cell autophagy, NP cells were infected with pLV-si IAPP or pLV-IApp. Consistent with the expression of autophagic gene, overexpression could induce autophagosome accumulation in NP cells, while downregulation of IAPP could induce a decrease in autophagosome accumulation (Figures 7b and d). Thus, IAPP could mediate autophagy and decrease apoptosis to the rescue NP cells.
Apoptosis and autophage Assay in NP cells induced by IAPP. (a) TUNEL assay to identify apoptotic cells (red) in NP cells transfected with pLVX-mCMV-Puro, pLVX-mCMV-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2- Puro-IAPP, or treated with pramlintide. (Scale bar magnification: ×400) (c). Statistical analysis showing results of apoptosis incidence. (b) EM analysis showed autophagosome formation in NP cells of different groups. Scale bar: 1 μm. (d) Statistical diagram showing autophagosome counts. Values represent the means±S.D. *P<0.05, **P<0.01, compared with NC.
The results showed that the activation of PI3K/Akt-mTOR was significantly enhanced in NP cells transfected with pLV-IAPP and IAPP siRNA administration could remarkably weaken the effects (Figure 8). MAPK signaling includes p38JNK, which is a key pathway regulating cell proliferation and apoptosis. To further identify the related pathway, we examined the p38/JNK MAPK signaling. The results of western blot analysis showed that IAPP treatment activates p38/JNK MAPK signaling by inhibiting the protein expression of phospho-p38 and phospho-JNK, whereas downregulation of IAPP by siRNA administration could inactivate p38/JNK MAPK signaling, resulting in increased expression of phospho-p38 and phospho-JNK. Therefore, the PI3K/Akt-mTOR signaling and p38/JNK MAPK signaling pathways are coordinately involved in the IAPP mediated regulation of NP cell metabolism.
Effects of IAPP on the expression of PI3K/AKT-mTOR and p38/JNK MAPK signaling. (a) Western blot analysis revealed the phosphorylated and unphosphorylated protein levels of PI3K/AKT-mTOR signaling pathway. (b) Western blot analysis revealed the phosphorylated and unphosphorylated protein levels of P38 and JNK. (c–e) Statistical analysis of the ratio of P-PI3k/PI3K (c), p-AKT/AKT (d), and p-mTOR/mTOR (e) in different groups, including NC, pLV-veh, pLV-IAPP, Ctrl siIAPP, pLV-siIAPP, and Pramlintide. (f–h). Statistical analysis of the ratio of P-P38/P38 (f), p-JNK1/JNK1(g), p-JNK2/JNK2 (h) in different groups. Data are representative of three independent experiments, and P-values are shown: *P<0.05; **P<0.01.
IDD is a progressive phenomenon that begins in the NP with a decrease in proteoglycans.3 Progression of the degeneration in the late stages impacts collagen synthesis and degradation leading eventually to disc collapse,30 and increased mechanical load on the vertebral body.31 Therefore, the treatment of disc degeneration should focus on the homeostasis of the ECM in the disc. The NP cells have a pivotal role in ECM metabolism, providing activated substrates for proteoglycan anabolic and catabolic pathways.32,33,34 The results of our studies showed that the gene expression of AG, BG and Col2A1 were decreased significantly during the process of IDD, accompanied by a decrease in IAPP expression. IAPP exerts its effects in NP cells via calcitonin and RAMP receptors. Decreased expression of IAPP in degenerated NP cells results in the inhibition of Calcr and RAMP3 protein expression, which was confirmed by the transfection of NP with pLV-siIAPP.
Matrix metalloproteinases (MMPs) are a growing family of metalloendopeptidases that degrade some of the extracellular matrix components. Upregulated MMP and ADAMTS cause pathological proteolysis of disc ECM and the subsequent development of IDD.35,36 The inflammatory cytokine IL-1β is a widely used key factor in regulating the expression of MMPs of disc cells, therefore, it is used to mimic the catabolic state of NP cells. Upregulation of IAPP in NP cells transfected with pLV-IAPP resulted in lower expression of MMP-3,9,13 and ADAMTS-4,5. Knocking down IAPP expression in NP cells by transfection with pLV-siIAPP significantly increased the expression of these proteolytic enzymes Thus, IAPP has an important role in MMPs-induced IDD. Similar to chondrocytes, NP cells are sensitive to apoptosis induced by IL-1β. In contrast, IL-1β alone does not induce significant apoptotic changes in AF cells. The results of this study indicated that abnormal expression of IAPP in the NP cellstriggers secretion of IL-1 and TNF-α, which act as pro-inflammatory cytokines that regulate MMP expression and accelerate disc degeneration.
Apoptosis and autophagy are two patterns of programmed cell death, which could decrease the number of viable IVD cells and accelerate the degenerative process.4,24 Apoptosis can be induced in IVD cells in animal models of human disc degeneration, and was increased in diabetic rats.37,38 The NP cells exposed to excessive glucose showed decreased proliferation and increased apoptosis in rat model.39 In this study, IAPP K/D cells were growing in culture without a stimuli (e.g., serum starvation), however, the apoptotic rate was significantly higher in IAPP K/D NP cells, compared with control groups. We found that the downregulation of IAPP in NP cells transfected with pLV-siIAPP resulted in decreased cell proliferation, increased expression of pro-apoptotic Bax and Caspase-3, and intensive TUNEL staining.
Autophagy is a protective mechanism in IVD, degrading excess or damaged proteins and dysfunctional organelles associated with disc degeneration.25 Although the exact mechanism is not clear, some genes have been found to be closely linked to the formation and maturation of autophagosome, such as beclin-1 and microtubule-associated protein 1 light chain 3 (LC3).40,41,42 During autophagy, a cytosolic form of LC3 (LC3-I) is lipidated and converted to LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes.43,44 Thus, LC3-I conversion to LC3-II and accumulation of LC3-II are widely used as reliable autophagosomal markers for monitoring autophagy and autophagy related processes. The increased level of LC3-II/LC3-I, beclin-1, ATG5, and ATG7 indicated the increase of autophagy activity in NP cells transfected with pLV-IAPP. Our studies indicated that autophagy served as an important protective mechanism in human degenerative diseases through the cellular catabolic pathways that included lysosomal degradation and recycling of proteins and organelles, and is regulated by IAPP.
Phosphoinositide-3 kinase (PI3K)/AKT signaling regulates several important functions in cells, such as cell growth, proliferation, migration and apoptosis.45,46,47 Upregulation of IAPP promoted PI3K expression in our experiments, indicating that PI3K/AKT pathway mediated the role of IAPP in regulating apoptosis of NP cells. mTOR is a downstream effector of the PI3K/AKT pathway, and activated mTOR inhibits the process of autophagy.48,49,50 Mitogen-activated protein kinases (MAPK) are activated in response to a variety of extracellular signaling and participate in cell proliferation, differentiation, apoptosis, and cellular stress responses.51 The activation of p38 and JNK MAPK pathway is often correlated with inhibited protein expression of p38 and JNK to prevent apoptosis and induce autophagy.52,53 Our results showed that overexpression of IAPP significantly upregulated the phosphorylation of AKT and mTOR, downregulated the expression of phosphorylated-p38 and JNK and the gene expression of Bcl-2, BECLIN1, ATG5, and ATG7, thereby increasing autophagy and decreasing apoptosis in NP cells. When the expression of IAPP was inhibited by siRNA, PI3K/AKT-mTOR and p38/JNK MAPK signaling was suppressed; expression of caspase-3 and Bax increased; and expression of BECLIN1, ATG5, and ATG7 decreased. Thus, apoptotic effect was enhanced and the autophagic activity was weakened in NP cells transfected with siIAPP. These results confirmed that the PI3K/AKT/mTOR and p38/JNK MAPK signaling was involved in IAPP mediated effects.
The results presented in this study provide new insights and help us understand why disc degeneration is more prevalent in those with inadequate nutrition. IAPP regulates glucose uptake, ECM anabolic and catabolic gene expression, controls the crosstalk between apoptosis and autophagy through PI3K/AKT/mTOR and p38/JNK MAPK signaling in NP cells. During IDD, the expression of IAPP, Calcr, and RAMP3 was decreased, which aggravates IDD through the mechanism described above. Pramlintide, a synthetic analog of amylin, can regulate the secretion of IAPP and improve cell growth, proliferation, autophagy, and apoptosis of NP cells. Therefore, it could be a novel drug candidate for the treatment of IDD.
This study has been approved by the Ethics Committee of Tongji Medical College, and written informed consents have been obtained. Human IVD tissue was obtained at surgery. Patients were selected on the basis of MRI determination of disc degeneration requiring disc excision and spinal fusion surgery for IDD (n=32) or deformity correction surgery for scoliosis (n=8). The IVD cryosections from each group were stained with Alcian blue.
Human tissue samples were obtained with informed consent of the patients and ethical committee approval. The IVD tissues were collected during surgery and transferred to the laboratory within 60 min. Briefly, NP tissues were minced, enzymatically digested in 0.2% type II collagenase and 0.25% trypsin for 3 h, and filtered to obtain single-cell suspensions. The cells were seeded and cultured in DMEM/F-12 containing 20% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37 °C in a humidified (5% CO2, 95% air) incubator. The cells were passaged two to three times before being used for the subsequent experiments.
Sections of 5 μm thickness were cut from paraffin-embedded tissues. The sections were deparaffinized in xylene and rehydrated in a gradient of ethanol. After washing, endogenous peroxidase activity was blocked by immersing the slides in 0.3% H2O2 at room temperature. The sections were incubated with polyclonal anti-IAPP (1 : 100), or control rabbit IgG (2 μg/ml) at 4 °C overnight. The sections were incubated for 1 h at room temperature with secondary antibodies, goat anti-rabbit fluorescein-conjugated antibody (1 : 200). Nuclei were visualized by staining with DAPI (Beyotime Biotechnology (Shanghai, China), 0.5 g/ml) and images were captured using fluorescence microscope (Olympus, Takachiho, Japan).
The NP cells were seeded and cultured at a density of 2×105 cells in six-well dishes and allowed to reach 70–80% confluence before infection. After discarding the medium from each well and making the total volume up to 1 ml, NP cells were infected with pLVX-mCMV-ZsGreen-PGK-Puro, pLVX-mCMV-ZsGreen-PGK- Puro-IAPP, PLVX-ShRNA2-Puro, pLVX-ShRNA2-Puro-IAPP, or treated with pramlintide (peprospec, HOR-300). Homo IAPP Gene ID: 3375, and shRNA plasmids for IAPP: sequence 5′-GCTGCAAGTATTTCTCATT-3′. The cells were infected at a multiplicity of infection of 104 vg/ml. Six hours later, the culture medium was added up to 2 ml. Transgenic expression in NP cells was detected using real-time PCR (RT-PCR) and western blot analysis at 48 h after transfection. The efficiency of transfection was quantified by assessing ZsGreen-positive cells under fluorescence microscope (Abcam, Cambridge, UK).
The MTT assay is a colorimetric assay for assessing cell proliferation. The supernatant was aspirated from each well after 72 h, 10 μl MTT was added to each well and incubated for 4 h at 37 °C. The medium from each well was aspirated, 150 μl DMSO was added to each well and the plates were agitated for 10 min in a shaker at 700 r.p.m. Finally, the absorbance was quantified by measuring OD 568 using a microplate reader.
Glucose Uptake Assay Kit (Ab83426, Abcam, Cambridge, UK) was used to measure the uptake of glucose by cultured NP cells. The assay was performed as per the manufacturer's instructions. The concentration of glucose-6-phosphate in the experimental samples was calculated using the standard curve.
TNF-α and IL-1β are important inflammatory mediators associated with IVD degeneration. Human TNF-α,ELISA Kit(Elabscience, E-EL-H0109c) and Human IL-1β ELISA Kit (Elabscience, E-EL-H0149c) were used. Secreted pro-inflammatory cytokines in NP cells of different groups were detected by ELISA methods according to manufacture's procedures.
Quantitative RT-PCR was carried out using an ABI7900/ illumina eco sequence detection system. PCR products were visualized on an ethidium bromide stained agarose gel. The primers used were as Table 1.The cycle threshold (Ct) data were collected and normalized to β-actin. PCR products were electrophoresed in 6% polyacrylamide gels and visualized by ethidium bromide staining. Bio-Rad image system was used to quantify the results. Analysis of relative gene expression was calculated using the 2 (−Delta Delta C(T)) Method (2−ΔΔC).
Protein extracts were separated by 10–12% SDS-PAGE and transferred to PVDF membranes. After blocked with 5% non-fat milk, the membranes were incubated with primary antibodies (as shown in Table 2) at 4 °C overnight. The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, anti-rabbit (1 : 5000; BA1054, Boster), anti-mouse (1 : 5000; BA1051,Boster), anti-goat (1 : 5000; BA1060, Boster), at room temperature for 1 h. The membranes were then washed in TBST buffer and protein bands were visualized with diaminobenzidine (DAB). The intensity of the bands was quantified and analyzed using Bio-Rad image software. β-Actin was used as loading standard.
The cells were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 25 min, washed thrice in PBS, and analyzed using in situ cell death detection kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. The slides were observed under fluorescence microscope.
Cells attached to slides were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. After washing, the slides were blocked with 2% bovine serum albumin (BSA) in PBS for 2 h at 37 °C and then incubated with primary antibodies against: anti-IAPP (1 : 100),anti-caspase-3 (1 : 150). The cells were then incubated with appropriate secondary antibodies; goat anti-rabbit fluorescein-conjugated antibody (1 : 200) or goat anti-mouse fluorescein-conjugated antibody (1 : 200, Boster) for 1 h at room temperature. Nuclei were visualized by staining with DAPI (Beyotime Biotechnology, 0.5 g/ml). Slides were imaged using a microscope with appropriate filters (Olympus), and the images were analyzed using imaging software.
Apoptosis in cell cultures was quantitated by flow cytometry using Annexin V-APC/7-AAD Apoptosis Detection Kit (KeyGEN BioTECH (Jiangsu, China), KGA108). The fluorescence intensity of apoptotic cells was analyzed with a flow cytometer.
After deprivation, the cells were washed in 0.2% PBS and fixed % osmium tetroxide in water for 1 h. After that,the cells were stained in 2% uranyl acetate in darkness for 1 h. Then, the cells were dehydrated in ethanol against a concentration gradient and embedded in Durcopan ACM resin. Finally, the specimen were cut into thin sections and examined under a transmission electron microscope.
All experiments were performed independently at least in triplicate, and the data are presented as mean±S.E.M. Statistical significance was determined by unpaired Student's t-test or one-way ANOVA using GraphPad IPrism 5 software (La Jolla, CA, USA). P<0.05 was Statistical significance was set to P<0.05.
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This study was supported by grants from the financial support of the National Science Foundation of China (NSFC, U1603121; 81201393, 81541056; 2013YGYL015; Nos 2016-176, 201512).


A micro dairy producing small-batch raw milk, aged goat cheese, and unique, fresh goat milk specialties.
OakMoon Farm is a micro dairy producing small-batch raw milk aged goat cheeses and unique fresh goat milk specialties. OakMoon is located in the charming hamlet of Bakersville nestled at the foot of Roan Mountain in the heart of the Blue Ridge Mountains of North Carolina. The farm was born of the love and determination of two people who just never know when to give up, and who believe that things happen the way they are supposed to.
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Ganapati Atharvashirsha Audio Versions of this app apk available with us: Featuring interviews with Neneh Cherry and Creole-influenced Dowdelin.
Repeat Mantra 11 ,21, 51times, lyrics in sync with atyarvashirsha images. Or browse results titled:. It is the most widely recited Sanskrit text among devotees of Ganesha in Maharashtra. If you think there is any mistake in the meaning somewhere, please feel free to email me. Aarti Sangrah Audio in Marathi. Ganapati Atharvashirsha Audio content rating is Everyone.
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English Stories For Kids - Video. Purchasable with gift card. This apk is safe to download. Baal Sanskar - Marathi. After first traveling in India, Tikki Masala was inspired by the deep flavors of authentic Indian classical music.
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Download the app using your favorite browser and click on install to install the app. Kanakadhara Stotram And Maha lakshmi Stotrams.
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Athrvashirsha page gives the meaning of Shri Ganpati Atharvashirsha. If you like Tikki Masala, you may also like: Atharvashirsha avartan carries a great significance on Sankashti.


text:
» 3D SERGI TURU
Zilberman | Berlin is delighted to announce the exhibition Recurrence 2 with works by Heba Y. Amin, Alpin Arda Bağcık, Isaac Chong Wai, Pedro Gómez-Egaña, Zeynep Kayan, Sandra del Pilar and Simon Wachsmuth and curated by Lotte Laub. While the first edition in the Recurrence series focused on the reappearance of a 19th theme, namely the tension between nature and technology, Recurrence 2 looks at changes in how history is viewed as well as ruptures and transitions. It is about ambiguity rather than binarity (yes or no, for or against, right or wrong), while our tendency is always to simplify reality, so that we can get a grip on it, but at the same time this is exactly the starting point for conflicts and polarisations of the kind that are again heating up the world's political climate.
In George Orwell's novel 1984 (completed in 1948), the protagonist Winston Smith works for the Ministry of Truth where he is responsible for revising old newspaper articles from The Times and adapting them to the current party line in accordance with the party slogan: "Who controls the past controls the future." The manipulative handling of historiography is clear here.
Isaac Chong Wai's two paintings When are we wrong? When are we right? present both these questions in colourful lettering on complementary monochrome backgrounds; acrylic lettering applied by hand in a sans serif font, as if done by machine. Observers are being addressed as a social collective and our power of judgement is being discussed. The binary opposition of true and false or right and wrong is supported by the complementary colours, which provide an optical illustration of the principle of bivalence. The work is asking about the conditions for truthful judgements or correct decisions. Does how we judge or decide depend on historical circumstances? Can something be wrong that was once right and vice versa? Is it possible to retrospectively assess whether a decision was right or wrong, or to lose sight of earlier judgement or decision-making criteria? Does a society's understanding of history depend on a temporary selection of the events and developments being observed? Or are we being unnoticeably manipulated in our perception of reality? How does a story find its image in the press and later in historiography? These questions are leitmotifs that appear throughout the exhibition.
A roll of paper unrolled on a long table that Simon Wachsmuth has filled with rows of small black squares using a wide calligraphy felt pen may be reminiscent of scrolls or scroll paintings, or of assembly line work. It is about the balance between individual artworks and standalone gestures, and repetition, the principle of the serial on the other hand, economy and efficiency. The title of this work, Of Copying, is taken from the mathematician, philosopher and mechanical engineer Charles Babbage's 1832 book On the Economy of Machinery and Manufactures. Three photographic works by Wachsmuth are exhibited alongside Of Copying and show figurative Byzantine frescos (from Cappadocia) with scratched out faces, references to 8th and 9th century iconoclasm. Both works, Of Copying and In Dialogue Form, show clearly how a simple gesture can create a maximum effect, an increase in impact through repetition or effacement of found material, a binarity of construction and destruction, two movements of historical progression and accordingly of historiography.
The binarity of black and white as well as seriality recur in Zeynep Kayan's work Insulator III, a series of small-format video stills showing a person dressed in black in a bent-over position. The background is formed from two rows of slightly curved cardboard at different brightness levels, four in each row. In each successive image, there are small differences in the bending of the foreground figure as well as in a segment of the background, until the figure disappears in the final image, which is set apart from the rest of the series, and an opening in the background becomes visible. This final image gives the series a destination, a way out, and thus a story. From the end, the series is afforded a new interpretation.
Heba Y. Amin's Non Architectural Renderings take up urban as well as industrial architectures that are reduced to almost abstract drawings. Taking large cities such as Cairo and its surrounding settlements as her starting point, Amin creates an impression of deserted spaces. The falling lines and surfaces, light and shade in a stark black-and-white contrast, are at the same time reminiscent of the film architectures of the 1920s (for example in Metropolis). Not only do they provide a setting: through the reuse of expressionist stylistic devices, they also shape dreamlike images that appear to depict visions and fears of the future that are nourished by the past.
In her interventions, Sandra del Pilar turns to old etchings and woodcuts (dating from the 18th to the 20th century) that awaken present-day impressions in the viewer. By drawing contemporary elements into historical depictions, the artist brings unfamiliar aspects into the interpretation and affords the portrayals a future that was not foreseeable when the original works were produced. She has, for example, drawn the head of a statue of Lenin into the middle of an old etching of a ballroom in the former Imperial Palace of Strasbourg. This palace (today the Palace of the Rhine) was built for Kaiser Wilhelm I to celebrate the annexation of Alsace-Lorraine into the German Empire, a territory that was lost back to France after World War I. The Lenin monument erected in East Berlin in 1968, to celebrate the 51st anniversary of the October Revolution and as a symbol of German-Soviet friendship, was demolished following the collapse of the GDR in 1991, although the head was buried near Berlin-Köpenick, unearthed for exhibition purposes in 2015, and taken to Berlin-Spandau. By drawing the head of this Lenin statue, including the transport cables, into the ballroom, the artist is illustrating the comparability of changing historical evaluations: territories acquired and lost (Alsace-Lorraine for the German Empire, the GDR for the Soviet Union), the glorification and rejection of rulers (the German Kaisers Wilhelm I and II, and Lenin, the founder and head of government of the Soviet Union). In doing so, del Pilar is generating awareness of the constructed quality of historiography and of the conditioning of viewing habits.
Alpin Arda Bağcık investigates the issue of events becoming images and the changing interpretation of these images based on their dissemination in the space. In his artist's book Diazem, he applies an imprint technique that reproduces an original painting, based on a press image, less clearly on each successive page. The more incomplete each image becomes, the more we interpret into it, like with ink-blots tests in psychological assessments or the bridging of patchy news items with conspiracy theories.
In three drawings that from part of a larger series (Mitre Marrows), Pedro Gómez-Egaña deals with the history of automatons. One of the first automatons, Jacques de Vaucanson's Digesting Duck from 1739, is a duck that could seemingly digest food. Although the automaton was able to move independently, the digestion aspect was a hoax, but people loved the idea of a machine having such real-life characteristics. Another drawing shows text that has been extracted from a Grindr dating-app conversation. It is handwritten in the style of an automaton that could write poetry but couldn't create breaks in the lines. It is ironic that such an intimate conversation is intended for an automaton. The third drawing, Brother Sister 2, plays with the idea of attributing family ties to automatons insofar as they imitate biological or human processes. An ironic light is shed on human behaviours that can be imitated by automatons, and on the human aspiration to be represented by automatons.
The recurrence of historical events and their reinterpretation, recurrence in the form of series of similar motifs, the recurrence of stylistic elements to emphasize contexts, or the machine-based imitation of recurring manifestations of life – these are the elements of the exhibition. When societies are on the right track, when they are not – this is a judgement we will leave to the viewers of Recurrence 2 to make.
Text: Lotte Laub
For more inquiries, please contact berlin@zilbermangallery.com.
- Heba Y. Amin
- Alpin Arda Bağcık
- Isaac Chong Wai
- Sandra Del Pilar
- Pedro Gómez-Egaña
- Zeynep Kayan
- Simon Wachsmuth

redpajama_set_name: RedPajamaCommonCrawl


Why we all want children... what is the meaning of Love..?
Discussion in 'Trying to Conceive' started by Anna Marie, Sep 28, 2005.
... the innocence and idealism is wonderful..
year-olds, "What does love mean?" The answers they got were broader and deeper than anyone could have imagined.
said to the neighbour, the little boy said, "Nothing, I just helped him cry"
Awww, how nice are they. I had a lump in my throat reading them!
I think I might try that with the kids I work with and post the answers, it would be interesting if they thought the same.
I feel all warm inside now.
Awwww.... lovely. I want a child like that!!!
That is so sweet what lovely answers. sometimes it teaches you alot listening to children!!
I can remember once when my daughter was just starting her first day at school. Me being worried sick told her I loved her loads and loads, and she replied that it was not as much as she loved me. So I spread my arms as wide as I could and said to her that I loved her 'this much', and she couldn't beat that because her arms were littler than mine. Her response was to instantly put her hands behind her back and said 'but I love you this much'. Confused? I asked her what it meant and she put both hands in front and said 'this much' while making a large circle around her until her arms touched at the back. How could I beat that?! Kids. Love 'em.
I believe we should all be like children instead of trying to be something we arnt just to please people.
Each one inspired me and came at the right time.
Reading those quotes from children really cheered me up. Chrildren are so caring and always have something funny to say with their questions and hillarious answers. My seven year old niece cracks me up and often brings a tear to my eye.


Focusing largely on dramatic production within and about Ireland since the resurgence of political unrest in the late 1960s, this course will consider the ways in which rival visions of community are shaped through a series of different artistic forms (whether generic, aesthetic, or rhetorical). Plays, screenplays, and verse drama by Seamus Heaney, Roddy Doyle, Martin McDonagh, Brian Friel, Frank McGuinness will be generically, historically framed by selected narrative fiction, critical non-fiction, poetic narratives by Eavan Boland, Angela Bourke, Heaney and Doyle. A course film series, and participation in the international conference, "Re-Imagining Ireland", held at UVA (Saturday May 10) will be expected course requirements.


This place has lots of stuff for dancers and clothes just to work out in. My oldest is going on her second pair of pointe shoes. We got her pair in the Orlando store. Did a great job for one yr. Now need to get a second pair.
Very nice people and very helpful. They bought out shoes for my daughter without measure her feet. They fit her perfect . My daughter is 5'7 and half and wears a size 11 in adults her feet are still growing. We will be back in Aug. 2013.


Generally, the following parameters are entered for a program block.
the exception of the straight line/circle).
The tool length offsets become active as soon as the tool is changed.
edge that you would like to use for machining.
that you enter the correct cutting edge number.
with the data and press the "To program" softkey.
The selected tool is accepted into the parameter screen form.


One of the very finest very large growing tree type Crape Myrtles from the National Arboretum breeding program. Tall and not very wide in time this very fast and upright growing tree sports a spreading crown. In late July to October huge panicles of bright lavender flowers remain showy for weeks. Its a soft color but the display is opulent. In time the bark exfoliates to patches of cinnamon red and mahogany. Very showy. Fall color is an intense display of reds/orange/purple. Full sun and virtually any soil- thrives in clay soils and the reflected heat of parking strips. Of all the Crape myrtle cultivars perhaps this free blooming 26′ tall tree is the best for a candidate as a street tree. Excellent cold hardiness as well as disease resistance. Well irrigated trees will easily put on 4′ of growth in a year.


Microsoft may have to pay more than a thousand dollars to a user whose PC was upgraded to Windows 10 without permission. The money covers damage after allegedly breaking the computer.
The claim followed an unwanted update from Windows 8.1 to Windows 10 in March 2016. That's when Microsoft was using the Windows Update service to download the Windows 10 files and then automatically install it on a restart unless the user spotted what was happening and actively opted out.
The move was hugely controversial at the time, not just for the unwanted update, but because the unrequested file downloads affected people on limited data use broadband plans.
The complaint said the man's computer no longer worked after the update and had to be repaired, with Microsoft support staff unable to fix the problem. The update also meant he was no longer able to use remote surveillance of a property.
The man had asked for €3,000 compensation to cover the cost of the repairs, replacement security cameras and the time the man spent fixing the problems.
Microsoft quibbled with the extent of the claim and noted the free customer support it offered, but didn't deny the download had happened without permission.
The panel decided that because Windows is licensed, the updates count as services under local consumer laws. It said Microsoft breached rules that say services must be carried out in a careful and professional way.
In deciding the financial compensation, the panel said Microsoft didn't have to pay for new security cameras as the man hadn't proven its mistake made it necessary to buy them. It also rejected a claim for lost working time as he hadn't proven his estimated figure was valid.
Should this ruling be followed elsewhere in similar cases? Should Microsoft face punitive penalties rather than just paying compensation? How clear do warnings have to be before users can take on the risk and responsibility for installing software updates?
Microsoft generally provides the user the chance to opt out of "automatic" updates. There are novices who may not realize the implications of opting out. The company should make it clear what the likely impact is. Unwanted updates should not happen.
Microsoft is notorious for having little consumer friendly advice. Errors that occur are shown as "Error 8x34x5xx89 happened. You're done." and the user is forced to try and look it up on a machine that isn't running. "Updates" are loaded with software for 3D something or other without a request or acceptance by the user. Microsoft says "Maybe you'll want it in the future so we intend to keep on the system." and doesn't allow the user to remove it. XBox? I have abso;ute;y no need whatsoever for XBox anything and yet it is loaded with the "upgrade" and then I have to remove it again! Microsoft is definitely NOT consumer oriented!


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Working Paper Series – Submission Guidelines for Authors
The FCEA Working Paper Series is a platform for expeditious sharing of new and evolving knowledge in the emerging field of climate engineering. Working Paper Series papers disseminate the work of physical scientists, social scientists, legal scholars and policy makers. It seeks to establish a space where the policy, legal, scientific, and academic worlds intersect on climate engineering issues.
Examples of published papers can be found [here].
The format of the Series will vary according to subject and may include brief and longer working papers, policy briefings, literature reviews, original research studies, and reflective essays. Our target audiences include government ministries and agencies, the scientific community, academics, non-governmental and intergovernmental organizations and the interested general public.
Publications in the series will normally grow out of invitations to authors; however, we also welcome proposals that meet our criteria. Both solicited and unsolicited manuscripts will be subject to review on the basis of scholarship, the extent of original research and rigor of analysis. All prospective publications must not have been published previously in any other venue.
Authors are responsible for the content of Working Paper Series publications, and the views and interpretations expressed are not necessarily those of FCEA or American University.
Prospective authors should submit an abstract to Working Paper Series to info@ceassessment.org.
1. Scope of the Series
Any topic pertinent to climate engineering will be considered. This includes scientific and technological assessment of discrete options, ethical considerations, potential governance architectures, and the pertinent scope and architecture for public deliberation.
2. Who May Contribute?
Anyone may submit an original manuscript to be considered for publication in the Working Paper Series, provided that they hold the copyright to the work being submitted or is authorized by the copyright owner or owners to submit the article. Authors are the initial owners of the copyrights to their works.
Submitted manuscripts cannot have been previously published, nor be forthcoming in an archival journal or book (print or electronic). Please note: "publication" in another working-paper series does not constitute prior publication. In addition, by submitting material to the Working Paper Series, the author is stipulating that the material is not currently under review at another journal (electronic or print) and that they will not submit the material to another journal (electronic or print) until the completion of the editorial decision process. Publishing a paper with the Working Paper Series does not prohibit an author from submitting the same paper, or a modified version of the paper, to another journal. If you have questions about the submission terms for the Working Paper Series, please write to info@ceassessment.org.
3. General Submission Guidelines
Papers should be between 2500-5000 words (including footnotes). Pages should be numbered and double-spaced. Footnotes should be prepared in 10-point font size, 100% scale, and normal spacing. Tables, diagrams, and illustrations should be included in the text of the paper (please also submit such materials in a separate file).
The paper title should be italicized.
Direct Air Capture: Potential Regulatory Approaches in the United States
Author Name(s)
The author's name should be placed immediately below the chapter title.
Can Citizen Juries Help Us Ensure Effective Public Deliberation About Climate Engineering?
Thomas T. Smith
Author(s) Affiliation(s):
The author's (authors') affiliation(s) should be placed in a footnote following the author's (authors') name(s).
Thomas G. Smith.[1] This should be the first footnote of the paper.
Paper Subdivisions
An article may be divided into sections, subsections and second-order subsections using an Arabic number-based system. Arabic numbers should be separated by a period. Sections should be formatted in bold type, subsections in bold, and second-order subsections in italics.
1. The Role of Environmental Impact Assessment in Climate Engineering Research and Development
1.1. National Approaches
1.2. International Approaches
1.2.1 The Espoo Convention
Footnotes: Formatting
All citations should be formatted as footnotes, i.e. do not embed citations in the text or use endnotes.
All papers should include an abstract of approximately 50-75 words, to be placed at the outset.
4. Review Procedure
Upon submission, you will receive acknowledgment of receipt. Your post submission will be reviewed by FCEA Faculty and Working Paper Series editors and returned within two weeks.
Editors may accept or reject any Working Paper Series submissions. In all cases, you will get an explanation of the decision. In cases of rejection, we are unable to accept resubmissions.
Include a photo and a short bio of no more than 200 words, which can include links to your work.
Please write to info@ceassessment.org with additional questions.


ANNAPOLIS, Md. (AP) — Maryland House Republicans have re-elected Dels. Nic Kipke and Kathy Szeliga (shuh-LEG-uh) to leadership positions.
Kipke was re-elected Tuesday to be the House minority leader. Szeliga was re-elected to be the House minority whip.
The House Republican Caucus re-elected them a day before the state's 90-day legislative session begins.
Kipke, of Anne Arundel County, has served as the House minority leader since 2013. Szeliga, of Baltimore County, has served as House minority whip since 2013.


Microtargeting gives your organisation the ability to put smart content in front of your perfect audience on social media through targeted ads. It is a cost effective and easy solution that's scalable for every sized group.
Social media is a powerful platform for brands interested in building communities of brand ambassadors and loyal consumers. But how do you find that audience? And how do you know your message is being heard? Microtargeting drives measurable results to give your organisation the data needed to build successful marketing initiatives.
Microtargeting campaigns deliver engaging ads to specific audience demographics based on location, behaviours and interests. In other words, we can deliver the right content to your best prospects.
A new landing page with a specific call-to-action.
A microsite asking participants to take a survey.
A long-form story integrated into a section of your website.
In addition to crafting the content, the EVG team designs digital ads that get noticed and drive clicks. From teasing out smart copy to choosing effective imagery, the ads work hand-in-hand with the new content. The results drive engagement, raise brand awareness or meet another KPI determined by the client.
We know it isn't enough to simply post an ad on social media and sit back, waiting for results. The EVG team monitors and measures the results of each campaign on a day-by-day and week-by-week basis. Paying careful attention to the numbers, we analyse and adjust as needed to ensure you see the best results possible. And since EVG believes in transparency, we share that data with you as we work together to refine and improve the campaign throughout its lifespan.
Let EVG develop a microtargeting campaign for you so you can do what you do best.


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Many health advantages have been attributed to soy isoflavones, which are components of soy-protein foods, yet it is still unclear to what extent the isoflavones themselves are responsible for these benefits. In a new study published in the American Journal of Clinical Nutrition, Jenkins et al. compared the effects of a low-saturated fat control diet, and soy-protein diets with either a high or low isoflavone content in the context of their effects on risk for coronary artery disease. Compared with the control diet, both soy diets significantly improved the subjects' cholesterol profiles and reduced systolic blood pressure in men. There were no major differences between the high- and low-isoflavone soy protein diets in their beneficial effect on blood lipids and blood pressure.
Twenty-three men and 18 postmenopausal women with elevated cholesterol levels participated in the study. The subjects rotated through three 1-month diets that were all very low in saturated fat. In the control diet, the main protein-containing foods such as meats and fish were replaced with low-fat dairy products and egg substitute. Low-fat soymilk and a variety of soy-based meat substitutes such as soy hot dogs and tofu burgers took the place of the usual protein sources in the two soy protein-containing diets. Body weight, blood lipids, and blood pressure were measured before and after each diet. After each soy diet, total cholesterol, the ratio of low-density lipoprotein (LDL) to high-density lipoprotein (HDL) cholesterol, homocysteine concentrations, and estimated overall cardiovascular risk were lower than they were after the control diet. Additionally, serum concentrations of LDL were lower after the high-isoflavone diet. The only significant difference between the sexes was a tendency toward reduced blood pressure in men after the high-soy isoflavone diet.
A wide range of small but beneficial effects were associated with the substitution of soy-protein for animal protein foods in the subjects' diets that did not differ significantly between the high- and low-isoflavone diets, indicating that even low-isoflavone soy foods can produce favorable results. Two unique aspects of the study were that a variety of soy-protein foods were used to substitute for the usual protein sources and that the soy diets continued to improve the subjects' blood lipid profiles even after dietary saturated fat consumption was maximally reduced.


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Light rain early...then remaining cloudy with showers in the afternoon. A few flurries or snow showers possible. High 43F. Winds NW at 5 to 10 mph. Chance of rain 70%..
A few clouds. Low around 25F. Winds light and variable.
ClubLIfe Health and Fitness owner Alex Rosenstrach, center, at the Back the Blue rally at Kinderhook Village Square on June 18.
Contributed photo
State police execute a search warrent of Club Life owner Alex Rosenstrach's residence in Kinderhook. Lance Wheeler/For Columbia-Greene Media
Police search gym owner's home
Columbia-Greene Media
Jul 8, 2020 Updated Jul 17, 2020
KINDERHOOK — The Columbia County Sheriff's Office and New York State Police have launched a joint investigation into allegations of an assault in Kinderhook this weekend.
"We have an ongoing investigation with reference to an assault that happened in Kinderhook Sunday morning," Columbia County Sheriff David Bartlett said Wednesday.
Bartlett would not provide details regarding the investigation.
"Circumstances are such that I am unable to release any information yet, other than what is already out there," Lt. Louis Bray from the Sheriff's Office.
Bray would not confirm, deny or specifically identify the information that "is already out there."
The Sheriff's Office, with assistance from state police, executed a search warrant at 3193 county Route 21 late Tuesday afternoon, according to the Columbia County District Attorney's office. The home is owned by Alex Rosenstrach, the owner of ClubLife Health and Fitness, according to county property records.
The search warrant was signed by Columbia County Judge Richard Koweek.
State police are assisting the investigation, but the Sheriff's Office is the lead agency, said Beau Duffy, state police public information director in Albany.
No arrests were made as of noon Wednesday, Barlett said.
Rosenstrach kept ClubLife open during the COVID-19 business shutdown, despite Gov. Andrew Cuomo's COVID-19 business shutdown executive order, and reopened May 15. ClubLife also continued to operate after it was shuttered under a cease-and-desist order from the Kinderhook Code Enforcement Department.
"ClubLife closed yesterday (July 7) voluntarily because of the ongoing investigation," Kinderhook Town Supervisor Patsy Leader said Wednesday.
ClubLife was issued a cease-and-desist order from the Town of Kinderhook in mid-May.
"While the Town of Kinderhook sought to work with this business owner to secure voluntary compliance with the law, ClubLife refused to follow the clear mandates set forth in the executive orders," Leader said May 15.
Rosenstrach organized a Back the Blue rally June 18 at Kinderhook Village Square.
"We're going through a period in this country where a lot of law-enforcement officers are in fear of getting injured or killed, or losing their jobs," Rosenstrach said at the rally "Twenty-five officers have been shot and one has been stabbed in the last week; somebody has to speak for them."
Rosenstrach said he chose Kinderhook for the site of the rally because he wanted to keep it local, but due to the success of Thursday's rally, he is hoping to schedule more, in other areas of Columbia County in the near future.
Lovewins143 Jul 11, 2020 5:59pm
Hi, I'm a bit confused by the quote "Twenty-five officers have been shot and one has been stabbed in the last week; somebody has to speak for them." I am looking on the website officer down memorial page and this information is not seeming to be correct. While I understand this is a quote I feel like it should be verified before being used. Perhaps I am missing information and if so, my apologies!
Nourish your body in the New Year with a healthy cooking class


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Home » Conference Report » The British Society for Eighteenth-Century Studies – Annual Conference
The British Society for Eighteenth-Century Studies – Annual Conference
Immediately after attending the RMA Research Students' Conference (see last OBERTO blog post), I boarded a train for Oxford and the British Society for Eighteenth Century Studies conference at St. Hugh's College. The theme of this year's event was 'Pleasures and Entertainments'; the immense scope of ideas and research which the speakers contributed within this theme was astounding, including everything from the elite women athletes of the long eighteenth century to 'spiritual alchemy' and the pleasures which could be gained from writing and hearing sermons. Naturally, opera was a significant form of pleasure-seeking and entertainment in the eighteenth century. Opera, singers, celebrity, theatre design, opera-going and opera criticism in England and abroad were all topics of papers and discussion.
St. Hugh's College, Oxford
The first panel I attended was 'Opera, Court Cases and Scandal'. Olga A. Baird spoke about the late eighteenth-century composer Antonio Casimir Cartellieri and his 'mysterious patron "Count Oborsky"' and their activities in the Habsburg territories. A significant part of this paper concerned Count Michal Kazimierz Oginski, who was a patron of musicians in Vienna. At his palace in Slonim, Belarus, Oginski had his own theatre, a ballet company, and singers for regular opera performances. As a youth Obarsky spent time at the palace at Slonim, and through his association with Oginski he became a 'pseudo-count' and patron of musicians and composers, although he remains an enigmatic figure. Oginski's remarkable theatre at Slonim, however, is evidence of the many places of opera performance based in remote provincial centres, purely for the entertainment of particular aristocratic patrons and their households.
After this, Bruce Alan Brown's 'Opera in France, Italy, and on the Moon, as Viewed by a Frenchman, Financier, and Philosophe' examined another mystery – this time the authorship of the Lettresur le Mechanisme de L'Opera Italien (1756). This Lettre makes comparisons between Italian opera buffa and French tragédie lyrique, and questions whether Parisian audiences wanted Italian opera, if they knew why they wanted it, or if perhaps they feared it. The paper touched on the difficulties inherent in interpreting pseudonyms in opera criticism, and deducing whether this Lettre was written by an Italian or French critic. Bruce Alan Brown's research also provides an alternative perspective on La Querelle des Bouffons, and how debates about operatic styles functioned as a substitute for more direct political debate in 1750s France.
Still in the 1750s, but moving over the Channel, Cheryl Duncan illustrated how records of court cases give us a remarkable insight into London's musical life. The legal disputes between violinist Felice Giardini and John Cox – engraver, publisher, and perhaps also an instrument repairer – demonstrate what varied 'portfolio' careers musicians enjoyed or endured during this period. The main operatic link was through Giardini's involvement with the King's Theatre.
On Thursday morning I took advantage of being at such a diverse interdisciplinary conference by listening to Peter Radford speak about the astounding exploits of elite women athletes during the long eighteenth century, as well as Carolyn D. Williams's paper on 'card games and women's "Intellectual Capacity"', before attending the Mason Lecture. This year's lecturer was Dr Jérôme Brillaud, whose 'Parisian Pleasure Domes: Theatre Architecture and Sensory Pleasures in Eighteenth-Century France' charted the changing design of French theatres between 1634 and 1784. In the seventeenth century, the examples of the Hôtel du Marais and the Hôtel de Bourgogne demonstrated the rectangular arrangement of these seventeenth-century theatres: sightlines (particularly in the loggia boxes) were compromised, and made the theatrical experience confrontational rather than creating the circular flow of energy which an amphitheatre style would encourage. Not until the late 1740s and 50s did French architects study ancient theatres, and the theorists' fascination with Greek theatres stimulated a move towards creating theatres in France which would stimulate all the senses. Cochin's 1765 grand plan for an auditorium with a truncated oval shape, promoting physical comfort for audience and actors alike, was never realised, but his design proved influential.
The amphitheatre design improved acoustics and sightlines, democratising – to an extent – the experience of attending the theatre, and provoking changes in acting technique. Brillaud then showed, with a series of exquisite slides, the private theatre of Mademoiselle Guimard (1772), Victor Louis' theatre at Bordeaux, as well as the new ComédieFrançais. He pointed out that as the external design of theatres became important, their civic significance was consequently enhanced. As a civic monument, the theatre became more politicised, and inside the improved sightlines meant that the entire audience (almost) had a 'unified sovereign perspective' in the absence of a 'royal box'. While Brillaud's lecture did not extend to the experience of opera audiences, he demonstrated the significance of audience experience, and this is equally relevant to studies of opera-going culture. Ideas about energy flow and communication within the auditorium, and the interaction between audience and performers are also pertinent to opera.
Later in the afternoon, after I'd presented my own paper –in a session on 'Salons, Circles and Polite Societies' – I had my first experience of chairing a panel: 'Ballads, Songs and Flute Concertos'. In this session, we were back in the world of London's pleasure gardens and theatres. In Heike Nasritdinova's 'Pleasure and Art: The Vauxhall Songs from John Worgan and T. A. Arne to J.C. Bach', we saw how the composers of the Vauxhall songs used particular techniques to ensure the popularity of their songs, and integrate pleasure with art, rationalism with entertainment. J. C. Bach also adapted re-used arias from his operas in his songs for Vauxhall. Meanwhile Patricia McCann's '"A much better Ballad-maker, than Play-wright": A study of Thomas D'Urfey's song collections' took us back into the world of the seventeenth-century theatre, and the rich musical world of which D'Urfey was a part. It was bad luck, to an extent, that D'Urfey was a contemporary of Purcell, and his songs have been somewhat eclipsed by Purcell's, but this paper demonstrated that D'Urfey's remarkable song collections had a strong following. Heike and Patriciaare PhD students, at Universität Regensburg and Queen's University Belfast respectively, and so this was an opportunity to form links with more graduate students. I was also glad that I'd been to a conference which was not exclusively musicological, and I hope to attend again in 2015. The Society also has an Annual Postgraduate Conference, which this year will be held in Venice, in association with Ca' Foscari, University of Venice.
By OBERTO: Oxford Brookes Exploring Research Trends in Opera in Conference Report, Corrina Connor on January 22, 2014 .
← Royal Musical Association Research Students' Conference 2014	Elitism and the 'opera snobs': a response to Katherine Jenkins by Alexandra Wilson →


Carahsoft Technology has partnered with Radiant Logic to make the latter's RadiantOne identity integration platform available to public sector customers and resellers.
The formalized channel patnership will offer access to RadiantOne through Carahsoft's Solutions for Enterprise-Wide Procurement V contracts, Carahsoft said Monday.
Dieter Schuller, vice president of business development at Radiant Logic, said the identity hub enables government entities and organizations retain their existing infrastructure while accommodating potential future requirements.
"We are pleased to make this innovative identity virtualization and synchronization product available to our customers and reseller partners," noted Steve Jacyna, director of emerging cybersecurity solutions at Carahsoft.
RadiantOne is a plug-and-play directory system designed to secure users' identity-related data without restructuring currently deployed systems.


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Profile: Mike Danko
Aviation-law specialist takes flight from big defense firm to make his mark in plaintiffs' trial law
It would be easy to say one client changed Mike Danko's life. But it wouldn't be entirely accurate. The truth is that one client – a victim in a wrongful-death case – opened a door to a career transformation that, when it was complete, surprised even Danko himself.
During what he refers to as the "first half" of his legal career, Danko had been gliding along as a top-notch defense lawyer at an international firm representing large financial institutions. He was handling complex-fraud cases, and unlike most of his colleagues, he actually enjoyed the work and was thriving in a brutally competitive environment.
Happenstance
After a number of years climbing the so-called corporate ladder, Danko did a favor for a friend of a friend, somehow convincing his bosses at the hourly-based defense firm to let him work on that contingency-based, wrongful-death case. Little did he know that it would trigger a profound shift in the way he thought about the law and used his skills as a lawyer.
"I'd gotten to a point in the (defense) practice that everyone kind of wanted, working directly with the general counsel for a bank, and we had a lot of important cases," Danko recalled. "Instead of calling the senior partner, he would call me direct, and that's a very big deal. So I had become more or less the go-to guy.
"One day, the reception desk called me and said the general counsel was on the phone, and I thought about it," he continued. "My (plaintiff) client was 18 and lost her two-year-old baby in a fire. On Thursdays at 10:30, it was her coffee break at work, and I knew that sometimes she liked to call, just to chat, see what was going on. When the general counsel called, it was 10:28 … I said, 'You know, take a message.' It was unheard of to blow off the general counsel like that, and I realized at that point, you know, something's changed for me – now my priorities have switched.
"That's how it happened. It was total happenstance, not what I intended to do and certainly not what I thought I was cut out to do."
Danko left the plush, high-rise offices of that international firm and took his plaintiff's case with him. He joined up with an established personal injury lawyer, diving headlong into plaintiffs' trial law with virtually no training. But with experience came competence – and then proficiency and, ultimately, expertise.
Becoming a trial lawyer
Danko's work as a trial lawyer, which has been recognized by Consumer Attorneys of California as well as other professional associations, has garnered several multimillion-dollar jury verdicts and settlements in wrongful death, catastrophic injury and product liability cases. He believes his education in trial law came on the job, in the courtroom, trying cases, listening to clients' stories, interacting with witnesses and presenting cases to juries. Ninety percent of his work today is in trial practice, he said, and as it happens, law school at University of Virginia did very little to prepare him for it.
"In law school, there's certainly trial practice classes, and I took one of those," Danko said. "Maybe five percent of what they teach you in law school is relevant to trial practice, and that's basically for anybody. Law school does not prepare you for being a trial lawyer at all.
"Really the only way to become a trial lawyer is to try cases and to develop experience," he continued. "Because you can't predict what's going to happen. You think you can plan it out, but you really can't."
In other words, there's no shortcut, he said. There's no way of predicting how a witness will perform on the stand, unless you've directly examined and cross-examined witnesses under similar circumstances previously. There's no way of knowing how an opening statement will be received or how effective it will be unless you've delivered quite a few and actually have seen jurors' reactions.
"How you think it's going to work out in your mind is not how it works out because it's a dynamic thing," Danko explained. "It's not just you making a presentation. You're getting a response from 12 or 14 people. You have to develop a sense for what you're doing and the plan you've put together and what the response is to that."
Danko became the first in his family to graduate college, majoring in psychology and philosophy at Dartmouth. Although his initial interests were in medicine, he said he chose a career in law because it best suited his personality. "From a very young age, I was always able to see two sides of an issue, kind of go back and forth to try to get the right answer," he explained.
And though a career in medicine wasn't meant to be, Danko did not have to give up his interest in science and engineering entirely. Much of the work he does requires a certain knack for those subjects. "To do what I do, lots of product liability stuff, you really do have to have an interest in science – how things work, how they go together and what makes things tick."
Rising to aviation law
That certainly applies to one of Danko's specialties, aviation law. He has achieved a number of large verdicts and settlements in cases involving airplane and helicopter crashes, and he is especially proud that the results of some of those cases have promoted change in the industry and "made aviation safer for everyone."
In one case that he and partner Kristine Meredith handled a couple of years ago, they took on the largest manufacturer of piston aircraft engines. It involved an engine that had been in production for 30 years – and it was defective, Danko said. Even the National Transportation Safety Board, which had called on the manufacturer in the past for assistance in investigations when one of the engines went down, had been duped, he said.
"Basically, the manufacturer was able to distract the NTSB, and as you look at the record, in all of those crashes, the NTSB never faulted the manufacturer," Danko said. "We were able to prove that the engine was indeed defective, that it had caused the crash at issue and had caused previous crashes. And that brought about changes in how those engines are built and the way that company does business."
Being real at trial
Following his belief that there are no shortcuts at trial, Danko said it's important to him that he be open and candid with jurors about the weaknesses of a case. The strengths are important to project, too, he said, but by conceding that there may be some shaky aspects of a case, it allows jurors to relate better to his client – and it gains their trust.
"My view is jurors can really detect BS," Danko said. "They can also detect good lawyering, and they know you're a good lawyer, and they know you have skills, and they distrust those skills.
"If they see what you're trying to do, and they see what you know, what you don't know, that you're not trying to keep anything from them, they're more likely to accept what the case is really about."
Down the road, Danko said he and Meredith will be spending more of their time and efforts in the area of mass torts against pharmaceutical companies, teaming with another firm, Girard Gibbs. They've already spent the past few years on one case involving a harmful birth control pill made by Bayer, Danko said. They plan on taking other cases involving a diabetes drug that purportedly causes cancer.
"Many people were talking about Big Pharma, and they would talk about these companies' practices being suspect, and I didn't really know what they were talking about," Danko said. "But once I got into it, in all my years, I have not found an industry that does more unnecessary harm to people than the pharmaceutical industry – driven entirely by profit motive."
Knowing right from wrong
Outside the office, Danko is an aviation nut. He is an experienced pilot with more than 3,000 hours logged flying airplanes and helicopters. "My airplane is really a traveling machine that I use for my work. It's a great machine for getting places," he said. "The helicopter is totally impractical and has no use other than basically harassing your friends and neighbors by flying over their houses – and not leaving until they come out and wave."
Danko often advises young lawyers or just talks with them casually about the law, and he is refreshed by their proclivity for doing the right thing. He cited a recent conversation among a diverse group of lawyers – four veteran and two "inexperienced" attorneys – talking about a case.
The experienced lawyers talked about all the problems with the case, Danko recalled, and that's what was driving the conversation. The two younger lawyers just listened, and when they had heard all the facts of the situation, their only response was it didn't seem right that this company would do that to this client.
"I thought, 'that's pretty good,'" Danko said. "Young lawyers I see today, I tell them, 'You've got it in your gut, you know what right and wrong is, and you're willing to pursue that.' My feeling is don't lose that – I guess that's what I would tell them.
"Take what's good – the ability to see what's right and wrong and to feel it in your gut – and let that motivate you, and you'll be fine."
Stephen Ellison is a freelance writer and an award-winning newspaper journalist based in San Jose. Contact him at This email address is being protected from spambots. You need JavaScript enabled to view it..
Danko