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Survival Response of Hippocampal Neurons under Low Oxygen Conditions Induced by Hippophae rhamnoides is Associated with JAK/STAT Signaling
Manimaran Manickam,
Affiliation Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Delhi, India
Rajkumar Tulsawani
* E-mail: rktulsawani@yahoo.com
Manimaran Manickam Rajkumar Tulsawani
Janus activated kinase/signal transducers and activators of transcription (JAK/STATs) pathway are associated with various neuronal functions including cell survival and inflammation. In the present study, it is hypothesized that protective action of aqueous extract of Hippophae rhamnoides in hippocampal neurons against hypoxia is mediated via JAK/STATs. Neuronal cells exposed to hypoxia (0.5% O2) display higher reactive oxygen species with compromised antioxidant status compared to unexposed control cells. Further, these cells had elevated levels of pro-inflammatory cytokines; tumor necrosis factor α and interleukin 6 and nuclear factor κappa B. Moreover, the expression of JAK1 was found to be highly expressed with phosphorylation of STAT3 and STAT5. Cells treated with JAK1, STAT3 and STAT5 specific inhibitors resulted in more cell death compared to hypoxic cells. Treatment of cells with extract prevented oxidative stress and inflammatory response associated with hypoxia. The extract treated cells had more cell survival than hypoxic cells with induction of JAK1 and STAT5b. Cells treated with extract having suppressed JAK1 or STAT3 or STAT5 expression showed reduced cell viability than the cell treated with extract alone. Overall, the findings from these studies indicate that the aqueous extract of Hippophae rhamnoides treatment inhibited hypoxia induced oxidative stress by altering cellular JAK1, STAT3 and STAT5 levels thereby enhancing cellular survival response to hypoxia and provide a basis for possible use of aqueous extract of Hippophae rhamnoides in facilitating tolerance to hypoxia.
Citation: Manickam M, Tulsawani R (2014) Survival Response of Hippocampal Neurons under Low Oxygen Conditions Induced by Hippophae rhamnoides is Associated with JAK/STAT Signaling. PLoS ONE 9(2): e87694. https://doi.org/10.1371/journal.pone.0087694
Editor: Thiruma V. Arumugam, National University of Singapore, Singapore
Received: April 17, 2013; Accepted: January 2, 2014; Published: February 6, 2014
Copyright: © 2014 Manickam, Tulsawani. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by: 1. Defence Research and Development Organization, Ministry of Defence, Govt Of India, India. 2. Indian Research and Medical Council (ICMR), Govt. Of India, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
High altitude illnesses is a term that indicates various symptoms of acute mountain sickness comprising headache, nausea, pulmonary edema and cerebral edema occurring during rapid ascent to high altitude by an un-acclimatized sojourn [1], [2]. Impairment of cognitive functions, memory loss, anxiety and depression are some of the nervous system related ill effects of hypoxia [3]. Neuronal damage during hypoxia is well documented and oxidative stress is known to be a cause [4], [5]. Acclimatization to high altitude environment helps alleviate related disorders and hence finding an agent of herbal origin that promotes acclimatization at a faster rate will be promising to tackle the problems during rapid ascent to high altitude.
Hippocampus neurons are the first ones to lose their electrical activity during hypoxia [6]. Protecting hippocampus from hypoxia will prevent many cognitive disorders. Cellular responses including proliferation and survival to many external signaling molecules are executed via Janus kinase-signal transducers and activators of transcription (JAK/STAT) signalling pathway [7]. Janus kinases (JAKs) comprises of four tyrosine kinases namely, JAK1, JAK2, JAK3 and Tyk2. Signal transducers and activators of transcription (STATs) include seven structurally and functionally related proteins namely, STAT1, 2, 3, 4, 5a, 5b and 6. Cytokine binding with its corresponding receptor initiates the signalling cascade of JAK/STAT pathway [8].
Hypoxia alters the cellular redox state which leads to oxidative stress and neuronal damage. Antioxidant defence system of the cell is weakened during hypoxia with an overload of reactive oxygen species [9]. Endogenous antioxidants like reduced glutathione and superoxide dismutase levels reduce during hypoxia. Pro-inflammatory cytokines such as TNFα (Tumour Necrosis Factor α) and IL6 (Interleukin 6) are markers of inflammation and NFκB (Nuclear Factor kappa B) regulates these pro-inflammatory cytokines at transcriptional level [10]. JAK/STAT signalling is responsible for many cellular events and we hypothesised that hypoxia might have an effect on JAK/STAT signalling to elicit its effects.
Hippophae rhamnoides is a high altitude plant, which was once considered as a weed has now proved to have many medicinal properties like antioxidant, anti-inflammatory, cardioprotective activity and cytoprotective activity [11], [12]. Hippophae rhamnoides leaves are rich sources of antioxidants and polyphenols [13]. Therefore, it is hypothesized whether the Hippophae rhamnoides extract treatment induces JAK/STATs signaling to fine tune TNFα mediated responses thereby providing neuronal survival under hypoxia environment.
Chemical Standardization of Sub Critical Aqueous Extract of Hippophae rhamnoides by its Total Phenolics, Flavonoid Content, Antioxidant Activity and Fingerprinting
The % yield of aqueous extract of Hippophae rhamnoides ranges 30.0–33.2%. The phenolic and flavonoid content in aqueous extract was 118.0±0.88 mg/g extract and 96.0±0.36 mg/g extract, respectively (figure 1A). The antioxidant activity of extract was found to be 45.0±0.12 µM TE/g extract (figure 1B). HPLC profile of extract showed the presence of isorhamnetin and kaempferol which are chemical markers compounds reported in Hippophae rhamnoides and are responsible for many pharmacological effects documented.
Figure 1. Depicts total phenols and flavonoid content (A), antioxidant activity (B) and RP-HPLC chromatogram (C) of aqueous extract of Hippophae rhamnoides.
Effect of Hippophae rhamnoides Treatment on Cell Viability, Reactive Oxygen Species, Caspase 3 and Lactate Dehydrogenase Leakage in HT22 Cells Exposed to Hypoxia in the Presence or Absence of Hippophae rhamnoides Extract
Figure 2 depicts protective effects of Hippophae rhamnoides extract in HT22 cells exposed to hypoxia for 24 h in the absence or presence of extract. The cells exposed to hypoxia in absence of extract showed significant cell death with higher levels of reactive oxygen species (figure 2A–B; dark gray bars). Treatment of cells with extract showed dose dependent cyto-protection (figure 2A; light gray bars) and prevented increase in reactive oxygen species (figure 2B; light gray bars). The cells exposed to hypoxia showed shrinkage and changes in the morphology of cell which was prevented by the extract treatment (figure 2C). To understand mode of cell death, the levels of active caspase 3, marker for apoptosis and lactate dehydrogenase leakage, a necrotic marker was evaluated. Cells subjected to hypoxia revealed 1.76 fold increase in active caspase 3 and 1.27 fold leakage of lactate dehydrogenase into the cell culture medium (figures 2D–E; dark gray bars). These cells exhibited both apoptotic and necrotic modes of cells death however; the cell death was predominantly apoptotic. Treatment of cells with extract prevented activation of caspase 3 and lactate dehydrogenase leakage induced by the hypoxia and the effects were dose dependent (figures 2D–E, light gray bars). Overall, Hippophae rhamnoides treatment attenuated cell death and reactive oxygen species induced by hypoxia.
Figure 2. Protective efficacy of Hippophae rhamnoides extract on hypoxia induced cell death.
(A) effect of extract on hypoxia induced on cell death (B) effect of extract on hypoxia induced reactive oxygen species generation (c) photomicrograph of HT22 cells following hypoxia exposure with or without extract, 40x magnification (d) active caspase 3 expression in HT22 cells following hypoxia exposure with or without extract (e) lactate dehydrogenase leakage in HT22 cells following hypoxia exposure with or without extract. *p<0.05; * vs control; # vs hypoxia.
Antioxidant Status of HT22 Cells Exposed to Hypoxia in the Absence or Presence of Hippophae rhamnoides Extract
Figure 3 shows that exposure of HT22 cells to hypoxia resulted in a significant decrease in GSH, GPx and SOD levels in comparison to the unexposed cells (figure 3 A–C; dark gray bars). Treatment with the extract restored the cellular levels/activities of GSH, GPx and SOD which were compromised after hypoxia exposure and the protective effects were in dose dependent manner (figure 3 A–C; light gray bars).
Figure 3. Protective efficacy of Hippophae rhamnoides extract on reduced glutathione levels (GSH; A), glutathione peroxidase (GPx; B) and superoxide dismutase (SOD) in HT22 cells following hypoxia exposure.
The values are mean ± SE of three individual experiments. *p<0.05; * vs control; # vs hypoxia.
NFκB and Inflammatory Cytokines during Hypoxia Exposure in the Absence or Presence of Hippophae rhamnoides Extract
Exposure of cells to hypoxia resulted in a marked increase in NFκB levels (figure 4A; dark gray bar) which is casually associated with the rise in reactive oxygen species. The treatment of these cells with extract attenuated such changes in NFκB induced by hypoxia probably by reducing reactive oxygen species load (figure 4A; light gray bars). To examine the translocation of NFκB into nucleus under hypoxia, cytoplasmic and nuclear fraction were prepared and assayed by Western blotting. Cells treated with hypoxia showed decreased expression of NFκB in cytoplasmic fraction (figure 4B, cytoplasmic; lane 2), with its increase in nuclear fraction (figure 4B, nuclear; lane 2) confirming its translocation into nucleus. The treatment of cells with Hippophae rhamnoides extract prevented NFκB translocation into nucleus observed following hypoxia exposure (figure 4B, cytoplasmic and nuclear; lane 3). Further, an increase in pro-inflammatory cytokines, TNFα and IL6, was recorded following hypoxia exposure relative to control cells (figure 4C–D; dark gray bars). Hippophae rhamnoides treatment attenuated hypoxia induced increase in TNFα and IL6 (figure 4C–D; light gray bars). Moreover, TNFα and IL6 mRNA were found to be higher in cells exposed to hypoxia in the absence of extract (figure 5E–F; lane 2). The cell treated with extract prevented such change in TNFα and IL6 mRNA induced by hypoxia (figure 5E–F; lanes 3–5).
Figure 4. Effects of extract treatment on NFκB (A–B), TNFα (C) and IL6 (D) expression in HT22 cells following hypoxia exposure.
*p<0.05; * vs control; # vs hypoxia.
Figure 5. Immunoblot results of JAK1, STAT3, pSTAT3, STAT5, pSTAT5, STAT5a, STAT5b and β-actin in HT22 cells following hypoxia exposure with or without extract.
Janus Activated Kinase and Signal Transducers and Activators of Transcription during Hypoxia Exposure in the Absence or Presence of Hippophae rhamnoides Extract
To examine activation of STAT proteins and dependence on upstream Janus activated kinases, HT22 cells were exposed to hypoxia and expression of JAK/STATs were measured by Western blotting. Exposure of cells to hypoxia resulted in an increased expression of JAK1, STAT3 and STAT5 (figure 6A–B, D; lane 2) which was concomitantly associated with phosphorylation of STAT3 and STAT5 (figure 6C, E; lane 2). An increase in mRNA levels of JAK1 and STAT3 were observed when cells were exposed to hypoxia (figure 5A–B; lane 2) in comparison to the controls (figure 5A–B; lane 1). STAT5a and STAT5b are reported to mediate signals for a broad spectrum of cytokines. In the present study, STAT5a was found to be highly expressive while no significant change was observed in STAT5b expression in cells exposed to hypoxia (figure 6F–G; lane 2). However, mRNA levels of STAT5a and STAT5b were found to be higher (figure 5C–D; lane 2) when compared with respective controls (figure 5C–D; lane 1).
Figure 6. mRNA expressions of JAK1, STAT3, STAT5a, STAT5b, TNFα, IL6 and β-actin in HT22 cells following hypoxia exposure with or without extract.
Cells exposed to hypoxia in the presence of extract revealed augmented JAK1 and STAT5b expression (figure 6A, G; lanes 3–5) along with increased phosphorylated STAT5 (figure 6E; lanes 3–5). Similarly, mRNA expression of JAK1 and STAT5b were found to be enhanced in cells exposed to hypoxia in the presence of extract (figure 5A, D; lanes 3–5). Further, treatment of cells with extract decreased levels of STAT3 and STAT5a (figure 6B, F; lanes 3–5) in comparison to the cells exposed to the hypoxia in the absence of extract (figure 6B, F; lane 2). The mRNA levels of STAT3 and STAT5a were found to be reduced after extract treatment (figure 5B–C; lanes 3–5) in comparison to the hypoxic cells grown in the absence of extract (figure 5B–C; lane 2).
Effects of JAK1, STAT3 and STAT5 Inhibitors on JAK1, pSTAT3, pSTAT5 Expression and Cell Viability under Hypoxia in the absence or Presence of Hippophae rhamnoides Extract
The optimal concentration of JAK1, STAT3 and STAT5 specific inhibitors, that reduces protein expressions of JAK1, STAT3 and STAT5 maximally in HT22 cells, were found to be 25 nM, 5 µM and 50 µM, respectively, when exposed to normoxia or hypoxia (data not shown). Exposure of cells to hypoxia in the presence of specific inhibitor of JAK1 resulted in increased phosphorylation of STAT5 (figure 7C; lane 7) and decreased phosphorylation of STAT3 (figure 7B; lane 5) in comparison to hypoxia suggesting that in absence of functional JAK1 activation of STATs occur through alternate cytokine signaling. Cell treated STAT3 and STAT5 inhibitor, revealed decrease in Jak1 activity (figure 7A, lane 5, 7) in these cells indicating interdependence of STAT3 and STAT5 on JAK1 activity. Interestingly, hypoxic cells treated with JAK1 inhibitors in the presence of extract revealed higher expression of JAK1 comparing to the cells treated with JAK1 inhibitor alone, possibly extract did play role in restoring JAK1 activity in hypoxic cells (figure 7A; lane 4). Cells treated with extract and having suppressed STAT3 showed much higher JAK1 expression (figure 7A; lane 6). The findings suggest that extract augment JAK1 expression probably by decreasing STAT3 under hypoxia. Further, cells treated with extract and having suppressed STAT5 resulted in decreased JAK1 expression (figure 7A; lane 8) indicating phosphorylation of STAT5 was dependent on functionally active JAK1. Moreover, decrease in STAT3 was associated with higher STAT5 levels and vice versa indicating opposing role STAT3 to STAT5 in HT22 cells when exposed to hypoxia (figure 7B–C; lanes 5 and 7). The data suggest that Hippophae rhamnoides extract activated JAK1 activity in hypoxic cells and further changes in the levels of STATs were sequential.
Figure 7. Effect of JAK1, STAT3 and STAT5 selective inhibitors on JAK1, pSTAT3, pSTAT5 expression and cell viability following hypoxia exposure with or without extract.
In another study, the effects of JAK1, STAT3 and STAT5 inhibitors on cell survival under hypoxia in the absence or presence of Hippophae rhamnoides extract was studied. The cells exposed to hypoxia in presence of inhibitors of JAK1, STAT3 and STAT5 showed more cell death by 11.6%, 13.2% and 7.5%, respectively vs cell exposed to hypoxia without these inhibitors (figure 7E; bars 3, 5, 7 vs 2) suggesting that JAK/STAT components are essential for cell survival under hypoxia. Further, HT22 cells treated with these inhibitors in the presence of extract did not offer protection as that provided by the extract at 100 µg/ml concentration alone (figure 7E; bars 2, 4, 6, 8 and figure 2; bar 5). The data suggest that active cellular JAK1, STAT3 and STAT5 were required for the biological activity exhibited by the extract under hypoxia.
Effects of JAK1, STAT3 and STAT5 Inhibitors on NFκB, IL6 and TNFα Expression under Hypoxia in the Absence or Presence of Hippophae rhamnoides Extract
The effect of JAK1, STAT3 and STAT5 inhibitors on hypoxia induced inflammatory response was studied in HT22 in the absence or presence of Hippophae rhamnoides extract using ELISA assay. The levels of NFκB were found to be increased in cell exposed to hypoxia in the presence of JAK1 inhibitor (figure 8A; bar 3). There was no change observed in NFκB expression in hypoxic cells grown in the presence of STAT3 and STAT5 inhibitor (figure 8A; bars 5, 7). Treatment of hypoxic cells with JAK1 inhibitors alone or in the presence of extract revealed higher expression of NFκB than the hypoxia cells evidencing cross talk between JAK1 and NFκB signaling (figure 8A; bar 4). Cells treated with extract and having suppressed STAT3 or STAT5 showed decreased levels NFκB comparing to the hypoxic cells (figure 8A; bars 6 and 8).
Figure 8. Effect of JAK1, STAT3 and STAT5 selective inhibitors on NFκB, IL6 and TNFα expression following hypoxia exposure with or without extract.
Exposure of cells to hypoxia in the presence of specific inhibitor of JAK1 resulted in increased release and accumulation of TNFα into culture medium (figure 8B; bar 3) when compared to hypoxic cells (figure 8B; bar 2) evidencing dependence of TNFα signaling on functional JAK1. The hypoxic cells treated with JAK1 inhibitor and extract revealed lesser release of TNFα into culture medium comparing to the cells treated with JAK1 inhibitor alone than hypoxic cells (figure 8B; bar 4) which might be due increased JAK1 activity in these cells by the extract treatment. TNFα which activates STAT3 was found to be accumulated in culture medium in cells treated with specific inhibitor of STAT3 (figure 8B; bar 5) when compared to hypoxic cells (figure 8B; bar 2). The accumulation of TNFα into culture medium further enhanced in cells treated with STAT3 inhibitor and extract in comparison to hypoxia exposed cells (figure 8B; bar 6 compared to bar 2) probably due to decrease in STAT3 levels by the extract. The cell treated with STAT5 inhibitor alone or with extract had no significant effect on TNFα release (figure 8B; bars 7, 8). This finding suggests that higher TNFα expression under low oxygen environment is responsible for maintaining higher cellular JAK/STATs activity thereby initiating pro-survival signaling pathway.
No significant change in IL6 release into culture medium in cells was observed in cells treated with JAK1 inhibitor alone or in the presence of extract (figure 8C; bars 3, 4) compared to hypoxic cells (figure 8C; bar 2). However, significant accumulation of IL6 was evident in cells treated with either STAT3 inhibitor or STAT5 inhibitor (figure 8C; bars 5, 7). IL6 can activate both STAT3 at lower concentration and STAT5 higher concentration in isolation or parallel and therefore suppression of any of component may lead into accumulation of IL6. The cells co-treated with STAT3 inhibitor and extract showed more accumulation of IL6 (figure 8C; bar 6) than the cells treated with STAT3 inhibitor alone (figure 8C; bar 5) which suggests that extract prevented hypoxia associated increase in STAT3 thereby causing accumulation of IL6 into culture medium. Further, there was no change observed in cell co-treated with STAT5 inhibitor and extract (figure 8C; bar 8) compared to the cell treated with STAT5 inhibitor alone (figure 8C; bar 7) suggesting STAT5 is not primary target of IL6 under low oxygen conditions.
Effects of JAK1, STAT3 and STAT5 Inhibitors on Active Caspase 3 and Lactate Dehydrogenase Leakage under Hypoxia in the Absence or Presence of Hippophae rhamnoides Extract
The effect of JAK1, STAT3 and STAT5 inhibitors on hypoxia induced caspase 3 cleavage and lactate dehydrogenase release into medium was studied in HT22 in the absence or presence of Hippophae rhamnoides extract. The JAK/STAT signaling associated with various cellular events including cell survival, differentiation and death. To confirm whether JAK-STATs offer survival to HT22 cells under hypoxia, we measured levels of caspase 3 and lactate dehydrogenase leakage in cells suppressed of JAK1 or STAT3 or STAT5 in the absence or presence of Hippophae rhamnoides extract using specific inhibitors. The cells treated with JAK1 inhibitor showed statistically non significant increase in active caspase 3 compared to hypoxic cells which was accompanied by significantly leakage of lactate dehydrogenase into culture medium (figure 9A–B; bar 3) compared to hypoxic cells (figure 9A–B; bar 2) evidencing higher necrotic cell death in absence of functionally active JAK1. Further, cells co-treatment with JAK1 inhibitor and extract showed lesser expression of active caspase 3 and prevented leakage of lactate dehydrogenase into culture medium (figure 9A–B; bar 4) which may be due to augmented JAK1 expression by the extract. Cell treated with STAT3 inhibitor revealed reduced active caspase 3 and lactate dehydrogenase leakage into medium (figure 9A–B; bar 5) suggesting pro-apoptotic role of STAT3 in HT22 cells under low oxygen conditions. The treatment of cell with STAT3 inhibitor in the presence of extract further decreased active caspase 3 expression and lactate dehydrogenase leakage into medium compared to hypoxic cells (figure 9A–B; bar 6) indicating extract prevents hypoxia associated apoptosis by decreasing STAT3 activity in these cells. The cells treated with STAT5 inhibitor showed higher active caspase 3 levels (1.15 fold) and lactate dehydrogenase leakage (1.04 fold) into culture medium compared to hypoxic cells (figure 9A–B; bar 7) indicating STAT5 participates in preventing caspase 3 mediated apoptosis in HT22 cells under hypoxia environment. Co-treatment of cells with STAT5 inhibitor and extract revealed further decrease in active caspase 3 expression and prevented leakage of lactate dehydrogenase into culture medium (figure 9A–B; bar 8) which might be due to suppression of STAT3 and increased STAT5 expression by the extract.
Figure 9. Effects of extract treatment on active caspase 3 activity (A) and lactate dehydrogenase leakage into culture medium (B) in HT22 cells following hypoxia exposure.
In fact, many neuronal disorders involve oxidative stress and this may occur due to suppression of antioxidants. Several reports have shown that hypoxic stress induces reactive oxygen species production leading to cellular pathophysiology [14]. In the present study, HT22 cells exposed to hypoxia displayed higher reactive oxygen species with significant cell death. Further, cellular antioxidant status was found compromised after hypoxia exposure and treatment of Hippophae rhamnoides extract restored such changes which might be due to high phenolics and flavonoids content and many of the bioactive compounds possessed by the extract thereby preventing cell death and maintained antioxidant status. The leaves extract of Hippophae rhamnoides are reported to be rich in flavonoids [15] and is potent reactive oxygen species scavenger [16]. Cells subject to hypoxia revealed higher expression of active caspase 3 and leakage of lactate dehydrogenase into the culture medium confirming both apoptotic and necrotic cell death however, the cell death was predominantly apoptotic. It is reported that hypoxia exposure results in apoptosis in various cell types and is accompanied by apoptotic necrosis or secondary necrosis [17], [18], [19]. Treatment of cells with extract prevented activation of caspase 3 and lactate dehydrogenase leakage induced by the hypoxia and the effects were dose dependent.
TNFα is a pleiotropic and proinflammatory cytokine that has diverse cellular functions such as apoptosis, cell proliferation depending on the level and duration of the hypoxia exposure. Depending on the levels or duration of stressor, TNFα binds to cell surface receptors such as TNFR1 or TNFR2. A marked increase in TNFα release into culture medium was observed in hypoxic cells along with its transcriptional activation. It is documented that ratio of STATs in central nervous system play an important role in determining apoptotic or anti-apoptotic response [20] and various cytokines are involved in the regulation of the immune system and inflammation through JAK/STAT. In this study, it was observed that exposure of cells to hypoxia in the presence of specific inhibitor of JAK1 resulted in increased release and accumulation of TNFα into culture medium. The hypoxic cells treated with JAK1 inhibitor and extract decreased this TNFα release into culture medium. The cells treated with extract augments JAK1 activity both protein and mRNA levels. Therefore, an increase in JAK1 activity in these cells may have resulted in decreased TNFα release suggesting functional JAK1 is required for the tuning of TNFα. TNFα which activates STAT3 was found to be accumulated in culture medium in cells treated with specific inhibitor of STAT3 when compared to hypoxic cells. The accumulation of TNFα into culture medium further enhanced in cells co-treated with STAT3 inhibitor and extract in comparison to hypoxia exposed cells. The data suggests that extract prevented increase in STAT3 levels induced by hypoxia. Further, induction of JAK1 by the extract harmonizes TNFα to reduce neuronal cell death.
Growing evidence has indicated that cellular redox plays an essential role not only in cell survival, but also in cellular signaling pathways such as NFκB. It is well documented that treatment of cells with oxidants can activate the NFκB pathway [21]. A higher NFκB expression was evident in the cells exposed to hypoxia and treatment of Hippophae rhamnoides extract restored such changes possibly due to reduction in hypoxia-induced oxidative stress by the extract. A cross talk between JAK/STAT and NFκB pathways is well documented which could be mediated by various agents including suppressor of cytokine signaling [22], [23], [24]. In cells exposed to hypoxia in the presence of JAK1 inhibitor NFκB levels were found to be elevated however no change in its expression was observed with STAT3 or STAT5 suppression. It is reported that in JAK1 deficient cells, NFκB activation is observed without activation of STATs through TYK2 dependent pathway [25]. Further, it is also recorded that a strong link between oxidative stress and JAK/STAT signaling exist [26], [27]. The functional loss of JAK1, which regulate many cellular processes, may imbalance cellular homeostasis thereby activating NFκB through alternate channels to initiate survival response. It is documented that activation of cellular NFκB initiate survival response in many cell lines depending on the micro-environment and stimuli [28], [29]. The treatment of cells, lacking functional JAK1, with extract could not reduce NFκB evidencing cross talk between NFκB and JAK1 in HT22 cells under hypoxic micro environment. In cell having suppressed STAT3 or STAT5, treatment with extract reduced NFκB expression indicating changes in NFκB levels under hypoxia is independent of phosphorylation STAT3 or STAT5.
IL6 belongs to a family of cytokines and plays a crucial role in acute inflammatory response, growth and survival signaling. IL6 binds to IL6Rα receptor leading in activation of Janus activated kinase and phosphorylation of signal transducer and activator of transcription (STAT) family members. JAK/STAT signaling plays an important role in executing downstream effects of various cytokines induced by various stress response mechanisms including hypoxia thereby exerting many physiological actions such as cell survival, differentiating, death etc. depending on the levels of exposure and cell type. There are reports stating activation of JAK/STATs by hypoxia [8], [30] and further downstream effect of IL6 is executed through JAK/STAT pathway [31]. Particularly, in central nervous system dysregulation of JAK/STAT signaling is associated with brain inflammation process and many neuronal disorders [32]. In the present study, an increase in expression of JAK1 on exposure to hypoxia was noticed with phosphorylation of STAT3 and STAT5. Our results are in accordance with previous reports showing increase in mRNA levels of JAK1, STAT3 and STAT5 on exposure to hypoxia [8], [33]. Activation of STAT3 via IL6 [34], [35], [36] is associated with the angiogenesis process in many cell types under hypoxia exposure [37], [38], [39]. It is documented that IL6 is pleiotrophic cytokine activating multiple pathways including JAK/STAT3 but at higher concentrations it also activate STAT5 to establish a negative feedback loop to balance STAT3 dependent inflammatory response [40]. In the present study, a significant accumulation of IL6 was observed in cells treated having suppressed STAT3. Further, cells co-treated with STAT3 inhibitor and extract showed excessive accumulation of IL6 than the cells treated with STAT3 inhibitor alone which confirm that extract prevented hypoxia associated increase in STAT3 evidencing interdependence of IL6 and STAT3 under hypoxic environment. Further, accumulation of IL6 was evident in cells treated with STAT5 inhibitor confirming cross talk between IL6 and STAT5 as well. However, no change was observed in cell co-treated with STAT5 inhibitor and extract suggesting STAT5 is not primary target of IL6 under low oxygen conditions. Although, the basal levels of the STAT3 proteins were lowered with the extract treatment, the phosphorylation of STAT3 was evident which was independent of IL6 activation. It is documented that extracts from various plants induce growth factors, angiogenesis and extracellular matrix proteins [41]. Therefore, it is possible that Hippophae rhamnoides promoted growth factors signaling which phosphorylated STAT3 independent of IL6. STAT3 is important in transducing pro-apoptotic signals and along with STAT5 promotes cell survival [32], [42]. In this study, it was observed that suppression of STAT3 decreased active caspase 3 and lactate dehydrogenase leakage into culture medium compared to hypoxia and when STAT5 is inhibited a reversed trend was visible. However, suppression of STAT3 or STAT5 results in more cell death than hypoxic cells evidencing necessity of functional STAT3 and STAT5 for cell survival in low oxygen environment. The data obtained from the study indicate that a balance between STAT3 and STAT5 in HT22 cell under low hypoxia is crucial for cell fate.
Further, STAT5a and STAT5b mediate signals for a broad spectrum of cytokines. In this study, activation of JAK1 was observed accompanied by phosphorylation of STAT3, STAT5a and STAT5b following hypoxia exposure both at protein level and their up-regulations at mRNA levels. It is documented that JAK1 activation is accompanied by the STAT3 and STAT5b phosphorylation in human B cells [34]. It is also reported that phosphorylation of STAT3 is dependent on upregulation of Akt in response to IL10 but independent of Jak1 in cardiomyocyte [35]. The treatment of cells with the extract resulted in upregulation of JAK1 and STAT5b with the phosphorylation of STAT3 and STAT5. Moreover, the up-regulation of IL6, STAT3, STAT5a mRNA levels in cells exposed to hypoxia was minimized after the extract treatment. The findings indicate that extract treatment regulated JAK1/STAT5b in HT22 cells exposed to hypoxia. The JAK1/STAT5b induction after Hippophae rhamnoides extract treatment probably fine tune IL6/STAT3/STAT5a mediated responses to hypoxia thereby regulating angiogenesis and erythropoeisis processes.
Overall, the present study highlights that Hippophae rhamnoides extract treatment renders neuroprotection against hypoxia in HT22 cells via induction of JAK1/STAT5b pathway.
ASE 350 Dionex Corporation (Sunnyvale, CA, USA), Spectrophotometer Bio-Rad. ELISA reader (Molecular Devices, USA), Spectrofluorimeter (Varian, USA).
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Specific antibodies against JAK1, STAT3, pSTAT3, STAT5 and pSTAT5 and HRP conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc., California, USA while NFκB p65 and β-actin antibodies were procured from Abcam Inc, Cambridge, MA, USA. NFκB, TNFα and IL6 Elisa kits were purchased from (R & D Systems, USA). Lowry and Bradford protein assay reagents were purchased from Pierce (Rockford, IL, USA) and St. Louis, MO, USA), respectively. Selective JAK1 inhibitor (Cat no. 420099), STAT3 inhibitor III WP 1066 (Cat no. 573097) and STAT5 inhibitor (Cat no. 573108) was purchased from Merck KGaA, Darmstadt, Germany. Other high quality research grade chemicals were purchased from Sigma (St. Louis, MO, USA).
Hippophae rhamnoides leaves were collected in the month of September from hilly regions of Western Himalayas, India, where the plant grows widely under natural conditions. The Defence Institute of High Altitude Research, Leh, India, where the voucher specimen (DIP-HIP/2012) of the plant material is preserved in the herbarium, carried out the ethanobotanical identification of the plant. Fresh leaves of seabuckthorn were cleaned and washed thoroughly with water and re-washed with distilled water. Washed fresh leaves were dried under shade in a clean, dust free environment and crushed using laboratory blender. The permission for the collection of study material was obtained from Defence Institute of High Altitude Research, Ministry of Defence, Government of India. The field study did not involve endangered or protected species.
Extract Preparation
Aqueous lyophilized extract of Hippophae rhamnoides dried leaves was prepared by maceration method as described earlier [43]. One gram of dried Hippophae rhamnoides leaves produced 0.2 g of lyophilized seabuckthorn aqueous extract powder.
Determination of Total Phenol Content
The total phenolic content in extract was estimated using Folin–Ciocalteu reagent (FCR) based assay [44]. The following were added to a tube; 20 µl of stock solution (1 mg/ml) of the extract, 80 µl of water and 500 µl of FCR. After 5 min incubation in the dark at room temperature, 400 µl of 7.5% sodium carbonate solution was added. The mixture was incubated in the dark for 30 min at room temperature and absorbance of the color developed was read at 765 nm. Total phenols (mg/g) in the extract were expressed as gallic acid equivalent (GAE), using standard curve prepared from gallic acid (0.1 mg/ml) solution.
Determination of Total Flavonoid Content
Total flavonoid content was estimated by the aluminum chloride colorimetric assay as described elsewhere [45] using rutin as a standard. 1 ml of extract (1 mg/ml) was added to 4 ml distilled water and subsequently 0.3 ml of 5% NaNO2 solution was added. After 5 min, 0.3 ml of 10% AlCl3 solution was added and allowed to stand for 5 min, then 0.2 ml of 4% NaOH solution was added to the mixture and the volume was adjusted up to 10 ml with distilled water. Absorbance of the mixture was read at 510 nm. Total flavonoid content (mg/g) in the extract was expressed as rutin equivalent.
FRAP (Ferric Reducing Antioxidant Power) Assay
The FRAP assay was estimated using method described elsewhere [46]. The fresh FRAP working solution was prepared by mixing 25 ml of 300mM acetate buffer (3.1 g C2H3NaO2 3 H2O and 16 ml C2H4O2, pH 3.6), 2.5 ml of 10 mM TPTZ (2, 4, 6- tripyridyl-s-triazine in 40 mM HCl), and 2.5 ml of 20 mM FeCl3.6 H2O solution which was warmed at 37°C prior to use. 150 µl of extract (1 mg/ml) was allowed to react with 2850 µl of the FRAP solution for 30 min in dark and the absorbance of the color developed was read at 593 nm. Results are expressed as mg of Trolox equivalent/g of extract, using standard curve prepared from Trolox solution.
HPLC Fingerprinting
HPLC analysis of the subcritical water extract of Hippophae rhamnoides was performed using Waters HPLC system (Waters Corporation, USA) equipped with Waters 515 HPLC pump, Waters 717 plus autosampler and Waters 2487 UV detector. Separation was performed in a symmetry C18 250 mm×4.7 mm ID; 5 µm column (Waters, USA) by maintaining the isocratic flow rate (1 ml/min) of the mobile phase consisting of 1% acetic acid in water (A) methanol (B), applied as gradient elution. The standard substance isorhamnetin and kaempferol were analyzed in the same conditions. Peaks were assigned by spiking the samples with standard substance and comparison of the retention times and spectral matching.
The cell line HT22 is a subclone of the HT4 hippocampal cell line [47]. HT22 cells (kind gift of Dr. Dave Schubert, Salk Institute, San Diego, CA, USA), a murine immortalized hippocampal neuronal cell line was maintained in DMEM medium containing 10% FBS and 1X antibiotic-antimycotic solution as described elsewhere [48]. Cells were then plated in a density of 1×104 cells/ml in 96 well plates or 1×106 cells/ml in 60 mm plates or 3×106 cells/ml in 100 mm plates and maintained for 24 h following treatments. Untreated cells were cultured in normoxia incubator (Heracell 150i, Thermo Scientific, USA) maintaining 5% CO2 at 37°C in DMEM medium while hypoxic conditions were achieved by culturing cells in an hypoxia incubator (Jouan, Saint-Nazaire, France) maintained at 37°C, 0.5% O2, 5% CO2, and 94% N2 atmosphere. Viability of the cells was determined by MTT assay. Cells were exposed to 0.5% O2 for 24 h in presence or absence of Hippophae rhamnoides extract.
MTT (Methylthiazole Tetrazolium) Cytotoxicity Assay
The MTT assay, which is based on conversion of yellow tetrazolium salt to purple-formazan crystals by metabolically active cells, provides a quantitative determination of viable cells. Cells seeded at the density of 1×104 cells per well in 96 well tissue culture plates were allowed to adhere for 24 h at 37°C. Cells were then treated with various concentration of Hippophae rhamnoides extract dissolved in DMEM media. Cells were exposed to normoxia and hypoxia for 24 h. Cytotoxicity was assessed by MTT assay. 50 µl of MTT (1 mg/ml) was added to each well and incubated for 4 h at 37°C. Formazan crystals were solubilized in 100 µl of DMSO by incubating in shaking condition at room temperature for 5 min. Absorbance was taken at 570 nm with 630 nm as reference filter. Absorbance given by untreated cells was taken as 100% cell survival.
Assay for Intracellular Redox State
Intracellular redox state levels were measured using the fluorescent dye 2,7-dichlorofluorescein diacetate (H2-DCFH-DA). Briefly, cells were washed once with HBSS and incubated in the same buffer containing 5–10 µg of DCFH-DA for 30 min at 37°C. Intracellular fluorescence was detected with excitation at 485 nm and emission at 530 nm using Spectra Max Gemini EM (Molecular Devices, Sunnyvale, CA).
After normoxic and hypoxic exposure, the cells were harvested by trypsinization, washed and sonicated for 10 sec in sterile phosphate buffered saline (PBS; pH 7.4) and then centrifuged (Sigma, Munich, Germany) at 1500 g for 10 min at 4°C. The pellet containing cell debris was discarded and the supernatant was used to determine GSH, GPx and SOD levels using commercial diagnostic kits (Randox, UK). The protein content in the homogenate was determined by the Lowry method as mentioned elsewhere [49].
Lactate Dehydrogenase (LDH) Assay
After normoxic and hypoxic exposure, the culture medium was collected for the estimation of extracellular lactate dehydrogenase. Then the cells were harvested by trypsinization, washed and sonicated for 10 sec in sterile phosphate buffered saline (PBS; pH 7.4) and then centrifuged (Sigma, Munich, Germany) at 1500 g for 10 min at 4°C. The pellet containing cell debris was discarded and the supernatant was used to measure intracellular lactate dehydrogenase. The extra cellular and intra cellular lactate dehydrogenase was measured using commercial diagnostic kit (Randox, UK). Leakage of intracellular enzymes was expressed as percent leakage.
Caspase 3 Assay
After normoxic and hypoxic exposure, cells were analyzed for active Caspase 3 using Caspase 3 fluorometric proteases assay kit (Biovision, USA). Briefly, cells were washed sterile phosphate buffered saline (PBS; pH 7.4), resupended of chilled cell lysis buffer and incubated on ice for 10 minutes. Then a 50 µl of 2X reaction buffer (containing 10 mM DTT) was added followed by addition of 5 µl of the 1 mM DEVD-AFC substrate and incubate mixture at 37°C for 1 hour. The cleavage of substrate was detected at excitation 485 nm and emission 535 nm using Spectra Max Gemini EM (Molecular Devices, Sunnyvale, CA).
Cellular Protein Preparation, Western Blotting and Quantitative ELISA
Following indicated treatments, cells were washed thrice with ice-cold phosphate buffered saline (PBS) and lysed in ice-cold cell lysis buffer (50 mM Tris-HCl, pH 7.5, with 120 mM NaCl, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40 and protease inhibitor cocktail). Then cell lysates were centrifuged at 10,000 rpm for 30 minutes. The pellet containing tissue/cell debris was discarded and the supernatant was used for the further analysis. The samples were appropriately diluted for the estimation of protein using Bradford reagent (Sigma, St. Louis, MO).
Quantitative ELISA kits were used for studying expression of NFκB, TNFα and IL6 following the manufactures instructions (R&D Systems, USA). In general, equalized amounts of protein samples were loaded onto each specific antibody coated wells and incubated. After incubation the ELISA plates were further incubated with horseradish peroxidase-conjugated secondary antibodies following washing. The color was generated by incubating the ELISA wells with TMB substrate and color generation was terminated by stop solution. The yellow color generated for the samples was read at 450 nm against the specific standards provided in the kit. The extracellular and intracellular levels of TNFα and IL6 were measured and data is presented as % leakage.
Western blot analysis was used for the detection of JAK/STATs. Briefly, 40 µg of protein was resolved on 10–12% SDS-polyacrylamide gel which was subsequently transferred onto PVDF membrane. The membranes were probed with respective primary antibodies; JAK1 (1∶200), STAT3 (1∶200), p-STAT3 (1∶200), STAT5 (1∶200), p-STAT5 (1∶200), NFκB (1∶1000), β-actin (1∶2000) followed by HRP conjugated secondary antibodies. Immunoblots were detected by 3, 3′-Diaminobenzidine (DAB). To examine nuclear translocation of NFκB, cytoplasmic and nuclear fractions were prepared using cell and nuclear lysis kit (NXTRACT, Sigma, St. Louis, MO). The subcellular fractions were then assayed by Western blot.
cDNA Synthesis and Reverse Transcription-polymerase Chain Reaction Analysis
Hippocampal neurons were lysed and total RNA was extracted with the TRIZOL reagent. Total RNA was qualified and quantified at 260 nm and 280 nm using Beckman spectrometry (USA). The cDNA was synthesized in a 20 µl reaction mixture, according to the protocol of the manufacturer (Thermo Scientific, USA). Specific cDNA was amplified by 35 PCR cycles at 94°C for 1 min, 55–65°C for 1 min and 72°C for 2 min. The PCR primers used in the study are shown in Table 1.
Table 1. PCR primer sequences and annealing temperature.
Treatment of HT22 Cells with Selective Inhibitors of JAK1, STAT3 and STAT5
For treatment with JAK1, STAT3 and STAT5 inhibitors, the stock solutions were made in DMSO and diluted in cell culture medium to make desired concentrations before treatment. An equal amount of DMSO was added to normoxia and hypoxia cells nullifying its effect in the experiment. The cells were grown in 96-well plates for the cell viability experiments and treated with various concentrations of selective inhibitors of JAK1, STAT3 and STAT5. Then the cells were kept in normoxic and hypoxic conditions as mentioned earlier in cells and culture conditions section for 24 h and at the end of experimental period cell viability was determined by MTT assay. In another experiments, cells were grown in 100 mm cell culture plates under normoxia and hypoxia conditions in the presence of selective inhibitors of JAK1, STAT3, STAT5 and the extract and at the end of experimental duration cell lysate was prepared for the protein expression studies using Western analysis and Elisa.
All the experiments were performed on three different occasions and data are presented as mean ± SE. The data was analyzed by one-way ANOVA followed by Dunnet's test for comparing control or hypoxia and the various groups, using GraphPad software. Statistical significance was estimated at the 5% level.
Authors are thankful to Director, DIPAS, Delhi and Dr. Kshipra Misra, Head of the Department for their constant support and constructive suggestions and Director, DIHAR, Leh, India for providing study material. Mr. Manimaran M acknowledges the senior research fellowship support from Indian Council of Medical Research (ICMR), Government of India, under SRF-fellowship for conducting research work at DIPAS.
Conceived and designed the experiments: RT. Performed the experiments: MM RT. Analyzed the data: MM RT. Contributed reagents/materials/analysis tools: RT. Wrote the paper: RT MM.
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More info on Atmospheric railway
Atmospheric railway: Map
Arriving at Kingstown, The Illustrated London News, 1844
An atmospheric railway uses air pressure to provide power for propulsion. A pneumatic tube is laid between the rails, with a piston running in it suspended from the train through a sealable slot in the top of the tube. By means of stationary pumping engines along the route, air is exhausted from the tube leaving a vacuum in advance of the piston, and air is admitted to the tube behind the piston so that atmospheric pressure propels it and the train to which it is attached.
Historical applications
In 1799 George Medhurst of London discussed the idea of moving goods pneumatically through cast iron pipes, and in 1812 he proposed, but never implemented, blowing passenger carriages through a tunnel.
In 1835 Henry Pinkus launched a prospectus for the National Pneumatic Railway Association. In 1838, when the gas engineer Samuel Clegg and the marine engineers Jacob and Joseph Samuda jointly took out a patent "for a new improvement in valves" that atmospheric propulsion became possible. The partnership set up a working model at the Samuda Brothers' workshop in Southwark in 1839, and a demonstration track of the Birmingham, Bristol & Thames Junction Railway at Wormwood Scrubs between 1840 and 1843. In 1841 Joseph d'Aguilar Samuda published A Treatise on the Adaptation of Atmospheric Pressure to the Purposes of Locomotion on RailwaysThe Clegg-Samuda system attracted the attention and support of some of the foremost railway engineers of the day, notably William Cubitt, Charles Vignoles and Isambard Kingdom Brunel, each of whom was engaged on the construction of new railway lines. It was also severely criticised by other engineers and railway commentators, notably Robert Stephenson and John Herapath.
Dalkey Atmospheric Railway
The first practical use of the system was on the Dublin and Kingstown Railway's Dalkey Atmospheric Railway between Kingstown (Dún Laoghaire ) and Dalkey , Ireland This line was built by Vignoles and operated between 1844 and 1854.
London and Croydon Railway
Cubbit recommended the system for the London and Croydon Railway between London Bridge station and Croydon . Clegg and Samuda were invited by the directors to supply equipment to operate their trains between London Bridge and Epsom . The first stage of this project (between Croydon and Forest Hill ) opened in January 1846, but many problems with both the pumping equipment and in maintaining air-tight seals in the delivery pipes were encountered. The London and Croydon Railway became a part of the London Brighton and South Coast Railway in July 1846 and the new board of directors invited Samuda to operate the new atmospheric railway on their behalf in return for a fixed fee. Once further propulsion problems became apparent in the second section of line to be equipped, between Forest Hill and New Cross , during 1847 the atmospheric method of propulsion was abandoned and the equipment sold.
South Devon Railway
The extension of Brunel's broad gauge railway westward from Exeter towards Plymouth by the South Devon Railway Company (SDR) was one of his interesting uses of technical innovation. Brunel and others from the GWR travelled to Ireland to view the atmospheric system at Dalkey first hand. Afterwards Brunel's engineer of locomotives for the GWR, Daniel Gooch, calculated that conventional locomotives could work the proposed Plymouth line at lower cost, but Brunel's concerns with the heavy grades led him to try the atmospheric system regardless.
The section from Exeter to Newton (now Newton Abbot ) was completed on the principle, with stationary engines at around intervals. Trains ran at speeds of up to 70 mph (112 km/h), but service speeds were usually around 40 mph (64 km/h). The level portions used pipes and the steeper gradients west of Newton were to have used pipes. It is not clear how the change between the two pipe sizes would have been achieved unless the piston carriages were changed at Newton. It is also unclear how the level crossing at Turf was operated as the pipe projected above the rails.
The harsh environment of the line, which runs next to the sea and is soaked with salt spray in even moderate winds, presented difficulties in maintaining the leather flaps provided to seal the vacuum pipes, which had to be kept supple by being greased with tallow; even so, air leaked in, destroying the vacuum.
Starcross pumping house.
Atmospheric-powered service lasted less than a year, from 1847 (experimental services began in September; operationally from February 1848) to 9 September 1848. The accounts of the SDR for 1848 suggest that the atmospheric traction cost 3s 1d per mile (£0.10/km) compared to 1s 4d (£0.04/km) for conventional steam power. Part of the problem was that the engines had to be run for longer than expected, as they were not initially connected to the telegraph and so had to pump according to the railway timetable until the train passed, which increased pumping costs.
Despite the building of several engine houses, the system never expanded beyond Newton. The proposal to use the same system on the Cornwall Railway was not pursued.
There are remains of several South Devon Railway engine houses, including one at Starcross , on the estuary of the River Exe. It is a striking landmark and a reminder of the atmospheric railway, which the name of the village pub also commemorates. A section of the pipe, without the leather covers, is preserved in Didcot Railway Centre.
Other early applications
The Paris –Saint-Germain railway between Bois de Vésinet and Saint-Germain-en-Laye, France ( ) 1847-60Beach Pneumatic Transit One city block long subway beneath Broadway in New York City , USA 1870 - 1873.The Crystal Palace atmospheric railway of 1864 had seals around the carriage, so (like Rammell's similar GPO Railway) the whole carriage fits in a tube tunnel and was propelled by the large fixed fan.
Recent applications
The Aeromovel Corporation markets an automated people mover that is air driven. The elevated lightweight trains ride on a concrete box girder containing electric motors that drive air inside the girder, creating a constant airflow. Each car has a square plate protruding into the box girder. The plate is rotated into the airflow to catch the wind and accelerate the car.
Systems have been built in Porto Alegre , Brazil (a two-station demonstration line) and in Taman Mini Indonesia Indah, Jakarta, Indonesia (a two-mile six-station loop serving a theme park).
A web page describing a system called 'Whoosh' was available for a time. The system is a single monorail track of the vacuum tube with a track either side. The piston in the tube is connected to the carriage below which is supported by wheels running on the tracks each side of the tube.
Illustration from A Treatise on the Adaptation of Atmospheric Pressure to the Purposes of Locomotion on Railways, Samuda
The supporters of the atmospheric system claimed it had several advantages over traditional motive power by steam locomotive.
Hillclimbing ability. On the two longest-lived applications, at Dalkey and Saint-Germain, this seems to have been vindicated: the system was used on uphill journeys and gravity in the other direction. Brunel assumed that the system would work on the very challenging gradients of up to 1 in 38 on the Plymouth main line if the South Devon application had been extended beyond Newton, probably by increasing the diameter of the tube on the gradients (although this would have involved a complex expanding piston arrangement); however here it was tested only on a relatively flat section.
Operating efficiencies. Atmospheric railways could be operated on cheaper and lighter tracks which did not have to carry the weight of a locomotive, and could take advantage of sharper curves.
Fuel efficiency. It was far cheaper to maintain and operate a few large pumping engines than a large number of individual locomotives.
Cleanliness. The smoke and dirt from the steam engines was kept away from the passengers.
Safety. The system could achieve higher speeds, but it would be impossible to operate two trains on the same stretch of track simultaneously and so collisions would be avoided.
The failure of the system was due to technical problems with the stationary engines and the leather seals on the vacuum pipes. The former were suffered by the London and Croydon Railway but would have been overcome with more experience by the manufacturers and operators. The difficulty of maintaining an air-tight seal in the vacuum pipes was a serious problem, particularly for the South Devon Railway Company, which was never satisfactorily solved using the materials and technology of the 1840s.
The atmospheric system also suffered from a number of operating problems.
Shunting the trains into atmospheric formation was difficult or cumbersome (although this would have seemed less of a problem in an era when much shunting was carried out by horse- or man-power).
A change in traction, with consequent delays, would be necessary if an atmospheric line became part of a through route.
There had to be gaps in the atmospheric tubes at points, with flyovers or similar arrangements at junctions; and special arrangements would have been needed at level crossings.
The atmospheric system foresaw the inherent efficiencies of delivering centrally generated power to the line side rather generating it on individual locomotives, as would ultimately become the normal practice with electrification systems. The use of modern materials and technology would overcome many of the problems of the original systems, but atmospheric railways were ultimately too inflexible for widespread use.
R. A. Buchanan, 'The Atmospheric Railway of I.K. Brunel', Social Studies of Science, Vol. 22, No. 2, Symposium on 'Failed Innovations' (May, 1992), pp. 231-2.
Samuda, J. D'A (1841). A Treatise on the Adaptation of Atmospheric Pressure to the Purposes of Locomotion on Railways. London: John Weale, 59 High Holburn.
* 239-256.
New York Times, Nov 10 1852
The Whoosh: Innovative Public Transport
p.5-8.
Beach Pneumatic Transit
Crystal Palace pneumatic railway
Cable railway - a more successful albeit slow way of overcoming steep grades.
Funicular - a system of overcoming steep grades using the force of gravity on downbound cars to raise upbound cars
Steam catapult - the arrangement of seal and traveller is essentially the same, albeit all steel.
Vactrain - combination of gravity and air power

redpajama_set_name: RedPajamaCommonCrawl


Cow bone hairpipes, two (2) sections with 45 pieces each. Strung vertically through tanned hide strips through dark, heavy tanned hide pieces - two (2) per section for a total of 4. Two sections are separated vertically by 43 rows of brass and heishi beads - on same leather strips. Two (2) cobalt blue glass beads are tied on the perimeter of each side of every leather strip and heavy tanned hide section. Three (3) leather strips around neck are strung with intermixed cobalt blue glass and brass beads. Ornament at top portion/center of: abalone disk strung with 2 elk teeth, 2 quill wrapped leather strips with 4 copper colored metal cones and yellow dyed horsehair, and commercial made red, white and blue vertically striped ribbon. Attached half way down, center is 3" L carved bone or tooth. Added to bottom is hide bound, quillwork strip with base of rawhide in red, cream and light purple in geometric design. Finished on bottom with 21 metal cones with red dyed horse hair - may be later addition.


Have you ever just felt like a snack, but wanted something that was savoury and not sweet? I think this happens to many of us, and there are many people nowadays who are not supposed to eat sugar laden foods.
These "biscuits" are one such food, which as well as being a snack, could be served as an entre, with pre-dinner drinks, or just nibbled on while watching television at night!
You can else serve them as a starter with vegetable sticks, and you will find that they keep for ages in the fridge.
Firstly you will need to grate the Parmesan cheese - I always like to buy Parmesan in block form and grate myself as this avoids chemicals which are put in to stop the cheese sticking together.
Next you place individual tablespoons of grated Parmesan on a baking tray lined with baking paper. Then press each circle down with your fingers to form a disc - easy!
Cook them in oven for about 2 minutes.
Then remove from oven and leave for 1 minute before removing with a slicer, and placing on paper towel.
When they are cool, these discs of Parmesan will harden to a salty, chewy crunchiness, and you may not be able to stop eating them till they are gone!


You can customize any of our reclaimed wood nightstands to be exactly the size and color you need. Our customer in North Carolina has asked to have theirs finished in our federal blue color with a very rustic top in our fruitwood color. A salvaged wood end table or nightstand can be used in any home where there is a need to have a small cabinet. Design your own drawer/shelf/or door combination. We can work with you to maximize the use you get on your salvaged wood nightstand. Size in image: 16″x16″ x 28″. Priced as seen $350 each. Scroll down to see additional images.


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L'isola di Feklistov (in russo Остров Феклистова, ostrova Felikstova) è un'isola della Russia nel mare di Okhotsk che fa parte dell'arcipelago delle isole Šantar. Amministrativamente appartiene al kraj di Chabarovsk del Circondario federale dell'Estremo Oriente.

Geografia

L'isola con la sua superficie di 372 km² è la seconda isola dell'arcipelago in ordine di grandezza, dopo Bol'šoj Šantar; è lunga circa 40 km, il suo punto più alto è il monte Povorotnaja (г. Поворотная) con 485 m s.l.m. Si trova circa 20 km ad ovest di Bol'šoj Šantar, al di là dello stretto Severnyj (Северный проливом).

Nella parte settentrionale, separato dal mare da una sottile striscia di terra, c'è il lago Lis'e (озеро Лисье), in cui si getta il fiume omonimo. Sulla costa meridionale dell'isola c'è la grande baia Lebjaž'ja (губа Лебяжья) che si suddivide in tre baie minori: baia di Ėnegel'm, di Sobolev e di Rosset (Энегельма, Соболева, Россета). Nella baia di Ėnegel'm si trova la piccola isola Šilova (остров Шилова, ) e a sud di quella di Rosset c'è l'isola di Suchotin.

La maggior parte dell'isola è ricoperta dalla taiga.

Sull'isola sono stati scoperti giacimenti di metalli del gruppo del platino e l'isola fa parte di una delle maggiori fasce metallogeniche dell'Asia nord-orientale (la Kondyor-Feklistov metallogenic belt)

Note

Collegamenti esterni
 Mappa N53

Isole del mare di Ochotsk
Geografia del Territorio di Chabarovsk

redpajama_set_name: RedPajamaWikipedia


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Vegas, Bambino! The Metropolitan Opera's 2019 production of Rigoletto. Marty Sohl/Metropolitan Opera hide caption
Marty Sohl/Metropolitan Opera
'Rigoletto' In Vegas And The Pleasures Of The Metropolitan Opera
July 17, 2019 • A recent Pop Culture Happy Hour trip to New York took the team to see Rigoletto at the Metropolitan Opera. A jester in a rat pack? We saw it.
In Fairview, a respectable African American family prepares for a big dinner. But we slowly learn there's something off about Keisha (MaYaa Boateng) and her relatives. Julieta Cervantes/Soho Rep hide caption
Julieta Cervantes/Soho Rep
The Pulitzer-Winning Play 'Fairview' Is About Being Watched While Black
July 14, 2019 • It starts with a perfect black family in a model household, like a '70s sitcom. Then it gets weird. Playwright Jackie Sibblies Drury says she was inspired by an experience with police surveillance.
Kim Davey (left), a passenger in Menzi Mngoma's Uber, made a video of his in-car opera singing that went viral. Kim Davey/Facebook hide caption
Kim Davey/Facebook
Video Of An Uber Driver In South Africa Singing Opera Goes Viral
July 13, 2019 • Menzi Mngoma is hoping that the exposure will help him further his dream of becoming an international singing sensation who doesn't have to drive for Uber.
LaChanze (left) and Elizabeth Teeter star in the Atlantic Theater Company's off-Broadway production of the musical adaptation of The Secret Life of Bees. Ahron R. Foster/Courtesy of The Secret Life of Bees hide caption
Ahron R. Foster/Courtesy of The Secret Life of Bees
'The Secret Life Of Bees' Takes On A Second Life As Musical Theater
July 13, 2019 • Sue Monk Kidd's bestselling novel tells the story of a white girl taken in by a family of black beekeepers. The premise posed a welcome challenge for black Pulitzer-winning playwright Lynn Nottage.
Indya Moore walks the runway as Angel in the FX series Pose. Michael Parmelee/FX hide caption
Michael Parmelee/FX
'Pose' Choreographer Creates A Safe Space — On The Runway
July 11, 2019 • Twiggy Pucci Garçon got her start competing at drag balls — now, she's the runway routine choreographer for the FX show Pose, which celebrates ball culture in New York City in the '80s and '90s.
Author Mo Willems supervises a rehearsal of the musical adaptation of Don't Let the Pigeon Drive the Bus! at the Kennedy Center in Washington, D.C. As the very first Education Artist-in-Residence, "I can say at this point that I am the greatest ... " he laughs, "I'm certainly in the top five." Chip Somodevilla/Getty Images hide caption
Kids' Author Mo Willems Has A New Creative Challenge (And So Should You)
July 2, 2019 • The creator of the Pigeon series, Knuffle Bunny, and Elephant & Piggie is currently an Artist-in-Residence at the Kennedy Center. He says if you want kids to draw, you should let them see you drawing.
Drag Queen Cloud performs at Wigstock in New York in 1994. Paul Hurschmann/Associated Press hide caption
Paul Hurschmann/Associated Press
50 Years After Stonewall
How Drag Queens Have Sashayed Their Way Through History
June 27, 2019 • Drag is experiencing a golden age, thanks to TV's RuPaul's Drag Race. But the tradition of drag performance has its roots as far back as Greece and has gone through a radical evolution in the U.S.
Jordan Weatherston Pitts (center) sings the role of Renata, the drag queen, with the company of New York City Opera's Stonewall. Sarah Shatz/Courtesy of New York City Opera hide caption
Sarah Shatz/Courtesy of New York City Opera
'Stonewall' Opera Marks Uprising's 50th Anniversary
June 22, 2019 • New York City Opera has commissioned Stonewall, a new opera premiering one week before the 50th anniversary of the riots that sparked the modern gay-rights movement.
Franco Zeffirelli pictured above in New York in October 1974. Jerry Mosey/AP hide caption
Jerry Mosey/AP
Franco Zeffirelli, Creator Of Lavish Productions On Screen And Stage, Dies At 96
June 15, 2019 • The Italian director staged sumptuously visualized operas, which often resembled Renaissance paintings brought to life. On film, he brought Shakespearean and biblical tales to the big screen.
Tony winner Ali Stroker performs "I Can't Say No" from Rodgers & Hammerstein's Oklahoma! onstage during the 2019 Tony Awards. Theo Wargo/Getty Images for Tony Awards Productions hide caption
Theo Wargo/Getty Images for Tony Awards Productions
The 2019 Tony Awards
June 10, 2019 • We are recapping this year's Tony Awards. James Corden was back as the host, and it was a big night for the musical Hadestown and the Irish play The Ferryman. The musicals Tootsie and Oklahoma! also picked up multiple awards.
The cast of Hadestown performed at the Tony Awards on Sunday night, as the show won eight awards. John P. Filo/CBS hide caption
John P. Filo/CBS
'Hadestown' Finds Bliss At The Tony Awards
June 10, 2019 • Anaïs Mitchell's long-developing musical won eight awards, including best musical, while The Ferryman won best play.
Brian Drye plays trombone with Tony-nominated actress Amber Grey onstage in Hadestown. Matthew Murphy/Courtesy of Hadestown, The Musical hide caption
Matthew Murphy/Courtesy of Hadestown, The Musical
Into The Spotlight: Recognizing Broadway Musicians This Tony Awards Season
June 9, 2019 • Musicals get Tony Awards. Scores get Tony Awards. Orchestrators get Tony Awards. But this year, a number of Broadway musicians are stepping out of the pit and onto the stage.
Stephanie Hsu and Will Roland in the Broadway production of Be More Chill. Maria Baranova/Courtesy of Be More Chill hide caption
Maria Baranova/Courtesy of Be More Chill
Our Great Big Theater Roundup
May 29, 2019 • We recently packed up and road-tripped to New York to check out some selections from the current Broadway theater season. We went to musicals and plays, revivals and new works, and we have a lot of thoughts.
Jason Moran and Alicia Hall Moran think deeply about how the past is preserved and kept vital through music and culture. Dawound Bey/Courtesy of the artist hide caption
Dawound Bey/Courtesy of the artist
'Two Wings: The Music Of Black America In Migration' Celebrates Journey Millions Took
May 24, 2019 • Singer Alicia Hall Moran and pianist Jason Moran mix original music works with 20th century spoken word to reflect on The Great Migration, when African Americans moved from the South to the North.
Whitney Houston's estate announced a slew of new projects featuring the beloved singer, including a hologram tour, a new album or previously unreleased songs, a possible Broadway musical and a Vegas show. Matt Sayles/AP hide caption
Whitney Houston Hologram Tour And New Album In The Works 7 Years After Her Death
May 20, 2019 • The mega-star sold tens of millions of records and had 11 No. 1 hits throughout her career. And in less than year, Houston, or at least a light-projected version of her, could be at it again.
Anukul Ghosh (left), 63, and Rabin Kumar Majhi, 51, pose as a newly married couple. Ghosh used to play female characters in the 1970s and 1980s. He has now retired from Jatra. Soumya Sankar Bose hide caption
Soumya Sankar Bose
A Photographer's Search For The Vanishing Stars Of Bengali Folk Theater
May 19, 2019 • Ten years after leaving his childhood home in West Bengal, photographer Soumya Sankar Bose returned to find that stars of Jatra, a popular local form of folk theater, were fading from public view.
The Latin American Library at Tulane University is digitizing and archiving a selection of Spanish language soap operas from the 1960s. The illustration above appeared in a brochure titled "What Is and How to Produce a Radionovela," published by America's Productions, Inc. Courtesy of the Latin American Library, Tulane University hide caption
Courtesy of the Latin American Library, Tulane University
Massive Digitization Effort Is The Latest Plot Twist For Cuban Radio Soap Operas
WWNO - New Orleans Public Radio
May 18, 2019 • The Latin American Library at Tulane University is digitizing a whopping collection of Cold War-era, must-hear entertainment — Spanish language radionovelas made by Cuban emigrés in Miami.
In recent years, Dave Chappelle returned to stand-up comedy in force with a series of specials, released on Netflix. He will receive the Mark Twain Prize on Oct. 27, 2019. Lester Cohen/Netflix hide caption
Lester Cohen/Netflix
Dave Chappelle To Be Awarded Mark Twain Prize For American Humor
May 7, 2019 • The comic and actor, known for his boundary-pushing stand-up routines and his sketch comedy TV series Chappelle's Show, will receive the award in a gala at the Kennedy Center.
Remembering Jo Sullivan Loesser, Wife Of 'Guys And Dolls' Creator Frank Loesser
May 3, 2019 • Jo Sullivan Loesser, who died April 28, married Frank Loesser after starring in his Broadway show, The Most Happy Fella. After Frank died in 1969, she helped preserve his legacy.
Isabelle McCalla (left) and Caitlin Kinnunen star as Alyssa and Emma in The Prom. Deen van Meer/Polk & Co. hide caption
Deen van Meer/Polk & Co.
At This Tony-Nominated 'Prom,' The Theme Is Acceptance
May 3, 2019 • In the Broadway musical The Prom, a high school PTA cancels the dance rather than allowing two girls to attend together. The show was just nominated for seven Tony Awards.
Hadestown was nominated for 14 Tony Awards, including one for best musical and another for best leading actress in a musical (Eva Noblezada, center). The awards ceremony is scheduled for June 9. Matthew Murphy/Courtesy of Hadestown, The Musical hide caption
Tony Awards Nominations 2019: 'Hadestown' And 'Ain't Too Proud' Lead The Count
April 30, 2019 • Tootsie, Beetlejuice and The Prom round up the Tony category for best musical, while The Ferryman, Gary, Ink, Choir Boy and What the Constitution Means to Me are all up for best play.
Bertie Carvel (left) plays a young Rupert Murdoch in the play Ink, which chronicles the media mogul's early attempts to upend the insular world of British newspaper publishing. Also pictured: Bill Buell, Jonny Lee Miller, Robert Stanton and Eden Marryshow. Joan Marcus/Boneau/Bryan-Brown hide caption
Joan Marcus/Boneau/Bryan-Brown
'Ink' Goes Back In Time To Remember Murdoch Before He Was A Mogul
April 28, 2019 • The play chronicles Rupert Murdoch's attempts to upend the insular world of British newspaper publishing — how he bought a struggling paper called The Sun and turned it into a widely read tabloid.
Margaret Trudeau, once in the limelight as a socialite and Canada's first lady, is now set to present her own life story in the one-person show Certain Woman of an Age. Kirsten Miccoli/Courtesy of Second City hide caption
Kirsten Miccoli/Courtesy of Second City
Margaret Trudeau Takes Center Stage — This Time, On Purpose
April 27, 2019 • A wife and then a mother to Canadian prime ministers, she addresses her time in the public eye and her battles with mental illness in an upcoming one-woman show called Certain Woman Of An Age.


GB/T 7714 Xu, Fu-Liu,Yang, Chen,He, Wei,et al. Bias and association of sediment organic matter source apportionment indicators: A case study in a eutrophic Lake Chaohu, China[J]. SCIENCE OF THE TOTAL ENVIRONMENT,2017,581:874-884.
APA Xu, Fu-Liu.,Yang, Chen.,He, Wei.,He, Qi-Shuang.,Li, Yi-Long.,...Ξng, Baoshan.(2017).Bias and association of sediment organic matter source apportionment indicators: A case study in a eutrophic Lake Chaohu, China.SCIENCE OF THE TOTAL ENVIRONMENT,581,874-884.
MLA Xu, Fu-Liu,et al."Bias and association of sediment organic matter source apportionment indicators: A case study in a eutrophic Lake Chaohu, China".SCIENCE OF THE TOTAL ENVIRONMENT 581(2017):874-884.


Are you gluten intolerant? Want to learn more about gluten free products?
Pantry Plus is hosting a Gluten Free Day this Saturday, May 23, from 11:00 am to 4:00 pm.
Stop by to receive 10% off all gluten free products!
There will be demo's and tastings with 8 vendors, including: Pete's Gluten Free, CeleeakNak, Doci, Zangarry Cheese, Genuine Health, Baldwin St. Hot Dogs, and RND Bakery.


As Rota College, our English education goal is to prepare our students for the global world and make them the individuals who have a word to speak in every field. With the studies we perform in this context and our perspective on English education, our students practice over daily life requirements, technological innovation, artistic activities, sports activities and project-based work. Our English lessons are being maintained in the quality of learning by "doing and experiencing" with the studies prepared by domestic and foreign teachers who are experts in their field.
- can express themselves well before community.
With "Smart Board" application in all classes, the course topics are presented to our students through "i -tool" and "e -book" options, the learning environment is enriched in terms of visual and auditory aspect .
On the specific date of the period, our students present their experiences in English lessons and the issues they want to share within a portfolio. Portfolio presentations that support making individual speak and gaining self-confidence are also the studies prepared by the students with pleasure at the same time.
Debate events are the activities that the students performed into two groups of three students before the jury and other students, in which the different aspects of a topic are defended, and English communication is provided effectively. In English debates, the students perform many skills such as making interpretation in foreign language, expressing opposing comments, asking in English. These activities contributing to social aspects of the individuals contributing to the development of the social aspects are monitored each year with growing interest.
Quiz Shows are the competitions in which the students fused in terms of social things, they increase their intellectual knowledge and develop their English skills by enjoying.
Three different quiz shows each year as elementary, middle and high school have both the contestants and the audiences spend pleasant moments and update their knowledge.
In our school, three different English Drama Clubs as elementary, middle and high school are performed separately. The plays of which the preparations are begun at the beginning of Education and Training year are performed at the end of the year, and the importance of the art for society is described by our students by experiencing.
Important works of world literature exhibited in English contributes our students to practice using gestures-mimes, adjusting the tone of their voice, and emphasizing in the right place and at the right time.
Scrabble Club which contributes the cognitive process and the multiplication of English vocabulary as well as allowing our students to have a fun and deductive activities through the tournaments held both within the school and in the province, district and country.
In Conversation Club, our students share the topics in the agenda with a presentation and they improve their communicative capabilities. They prepare presentations on the special days and celebrations and organize special programs in cooperation with other social clubs.
Comenius Project is one of the European Union Lifelong Education Programs. The program allows the improvement of the interrelations between the European cultures, the elimination of certain prejudices and the application of the multi-partnered projects ensuring the expansion of the participants' perspective thoroughly. Within this scope, 5 project partners (Germany, Spain, Portugal, Romania and Lithuania) from Europe have visited Rota Anatolian High School for the Comenius Project with the title of "Are we ready for the climate change?". Students and teachers all together have participated in the project's preparation process and the Climate Change and Its Possible Effects have been discussed within the various perspectives of different cultures.
E-twinning projects which are among of the European Projects allow students to practice on-line. And it's a program which consists of multi-partnered project studies within the Europe. Students make their presentations in English during the class through Skype so they have a chance to use the latest technological devices.
This project allows our students to share their culture, life styles, classes and the books they're interested by means of the established communication with the other schools from all around the world. The aim of the project is to ensure a constant communication between the participants and to be able to follow the current agenda of the world.
Our school is in contact with a school from Canada within the e-pals project. And our students are currently able to share their knowledge together with their friends in another part of the world.
DI is a project giving the students from all around the world the opportunities to share the approaches and ideas which can be useful in their studies within the aspect of their creativity and their practice of 21st century. The project is composed of 30 different countries including 48 states of United States and Canada, Turkey, Korea, Guatemala, Poland, China, India, Great Britain, Brazil and Singapore. Within the program, the teams of students conduct the missions consisting of open-ended problems and they make a presentation about the solutions to the set problems in tournaments. The teams trying to describe their missions have a chance to gain life-long experiences and talents such as the management abilities, a sense of cooperative study, problem solving as well as creative and critical thinking. Our school applies the related studies and preparations under the club activities and our student teams represent our school successfully.
The Model United Nations program that is a simulation of real United Nations provide our students to discuss the world agenda issues over another country's citizenship In this program in which the discussions are made in English, the students progress in both social and academic sense. The students participating in the conferences held both in Turkey and in different countries have the opportunity to compare their own country with different countries. Our students prepare with enthusiasm for Model United Nations program implemented in our school as a club activity, and they interest this activity extremely.
Abroad experiences that are the most effective way of improving foreign language is carried out with winter and summer abroad program organized by our institution. In these schools that teach with students of different nationalities, our students find the opportunity to practice English and have long lasting friendships.
In this scope, our school has conducted programs in Bell Bedgebury International and Brighton Newlands Language Schools in the UK. Our abroad language schools programs are organized during semester and summer holidays each year.
Our school is one of the test centers of Cambridge ESOL exams which is valid worldwide. Our each student is regularly given Starters, Movers, Flyers, Ket, Pet and FCE exam training from 3rd class of Primary School to 11th class of High School from the beginning of the year. Our students who want to attend these exams can enroll to the exams through our school.


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Léon Robin (Nantes, 17 January 1866July 1947) was a French philosopher and scholar of Greek philosophy, professor of history of ancient philosophy at the Sorbonne from 1924 to 1936.

Robin, the son of a merchant, began teaching in the Faculty of Letters at Paris in 1913. In 1924 he took up the chair of history of ancient philosophy, which had lapsed after the death of Louis Rodier in 1913. In 1927 he was visiting professor at the University of Pennsylvania. On his retirement from the Paris chair, his successor was Pierre-Maxime Schuhl. Robin subsequently served as Director of the International Institute of Philosophy.

Léon Robin translated the dialogues of Plato into French.

Works
La théorie platonicienne des idées et des nombres d'après Aristote; étude historique et critique, 1908
La théorie platonicienne de l'amour, 1908
(transl. and ed.) Plato, Oeuvres complètes, 1920
La pensée grecque et les origines de l'esprit scientifique, 1923. English translation, Greek thought and the origins of the scientific spirit, 1928
La morale antique, 1938
Pyrrhon et le scepticisme grec, 1944

References

External links

1866 births
1947 deaths
French philosophers
French historians of philosophy
French male non-fiction writers
French translators


BOSTON, MA--(Marketwired - March 21, 2016) - Burst, the mobile video technology platform that enables broadcasters, media companies, rights holders and brands to leverage the power of user-generated content (UGC), today announced that FOX SPORTS Australia is using the Burst platform to acquire, curate and deliver fan-created content for the network's studio shows and live broadcasts of sporting events.
Through its partnership with Burst, FOX SPORTS will now be able to build stronger fan engagement and a better viewer experience in live sports broadcasting. Burst allows FOX SPORTS -- and other broadcasters -- to easily integrate UGC into their existing production workflows with minimal disruption. This capability gives FOX SPORTS the power to be first in the Australian sports market with engaging fan-produced content that can be quickly turned around during live broadcasts.
In pre-game and in-game promotions, FOX SPORTS will reach out to fans with incentive messaging that encourages fans to record unique, authentic video content from their mobile phones. That content is then easily uploaded via the Burst platform and injected into the broadcast feed or displayed in the venue. Uploads are simple and accomplished without fans needing to download an app to participate. As a result, fans can now play a personal role in the broadcast, studio show or in-venue production.
In the U.S., live sporting events are the one area that appears to be unaffected by the rise of streaming video on demand, according to the Nielsen "Year in Sports Media 2015" report. In fact, 95% of total sports program viewing happened live in Q4 2015, the report says. In a TV environment in which consumers have literally thousands of viewing options on multiple screens, Burst and its customers understand the opportunity live TV offers in building strong audience engagement through UGC.
Burst's platform allows customers to build a rich inventory of new video that can be deployed and monetized immediately. Burst has offered a solution to the problem of content rights ownership for broadcasters by creating a process by which the fan assigns a right to the broadcaster to use his or her video as part of its terms of service. Because all owned content stays on the broadcaster's Web and mobile properties, Burst helps broadcasters, rights holders and brands minimize viewer and revenue loss due to "leakage" to social media networks.
"The passion that user generated mobile video brings to a live TV broadcast production is unprecedented," said Bryant McBride, Founder and Chief Executive Officer, Burst, Inc. "Burst helps customers convey this passion in an authentic and energizing way and gives broadcasters, rights holders and venues an innovative method to create a new category of compelling content -- real time fan video."
Founded in 2011, Burst is a leading mobile video technology company that is transforming how broadcasters, publishers, rights holders, brands and venues create, manage and monetize user-generated and controlled video content. Burst's proprietary technology solves the challenges of creating, managing, publishing and monetizing mobile video across all screens with its innovative, mobile-to-broadcast cloud platform. Burst is headquartered in Boston and serves customers around the world including Sinclair Broadcast Group, FOX SPORTS Australia, Horizon Media, NESN and Accuweather.


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Series: Your Brain on Music
Music, emotions, and imagination have gone together ever since the first cave dweller banged on a rock. Modern brain research is helping us understand why.
The Story Behind The Song: Yankee Doodle
"Yankee Doodle" may be a popular kids' tune today, but it got its start as a song of war.
Article: Why Use Primary Sources?
When it comes to telling the true account of an event or an historical time period, there's nothing more real or more exciting than a primary source
The Story Behind The Picture: White Angel Breadline
In White Angel Breadline, her first documentary photograph, Dorothea Lange enabled Americans to see the tragic effects of the Great Depression. The image evoked national sympathy, rather than scorn, for the hungry and homeless.
Article: What It Takes to Become a Professional Singer
So, you want to be a singer. What's next? A beginner's guide to starting a vocal career
The Story Behind The Song: We Shall Overcome
In this story, find out how a song helped steel the courage of African Americans as they struggled for civil rights
Article: Viva la Diva!
A look at some of opera's most demanding female roles
Article: Understanding Different Voice Types
Ever wonder what makes one voice sound different from the next? Check out this look at (and listen to) vocal types and the various colors and textures of the human voice
The Story Behind The Song: This Land is Your Land
Folk singer Woody Guthrie wrote this song to celebrate America's bounty and to protest that not all Americans were getting their fair share
Series: The Story Behind The Song
Through the history of popular songs, discover how history is full of surprising stories of people doing amazing things
The Story Behind The Song: The Star-Spangled Banner
In this story, find out how a captured poet penned the song that became the country's national anthem
Your Brain On Music: The Sound System Between Your Ears
The amazing sound system in the human brain helps explain why people everywhere fill their lives with music
Article: The Skeleton of a Scary Story
Have you ever wanted to scare your friends around the campfire? This article will tell you how!
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They were rebels, they were American, and they dared to be different: the makers of modern dance
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A mini-course in starting a singing career
The Story Behind The Song: The Marines' Hymn
This article gives you the stories behind the words—"From the Halls of Montezuma, To the shores of Tripoli"
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Pointers for your next singing audition
Gear: The Ellipsoidal Reflector Spotlight
One of the jobs of a lighting designer is to be an illusionist; to convince the audience they're somewhere special
The Story Behind The Song: The Battle Hymn of the Republic
Discover how Julia Howe's hymn helped inspire the North in its fight to reunite the country and free African-American slaves
Article: The Pas De Deux: It Takes Two
The pas de deux is not just a dance of love
Your Brain On Music: Tearjerkers
Putting the "sad" in sad songs
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You've been assigned to a chorus. Now what? Some helpful hints for the young singer
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What does it really take to become a professional singer?
Article: Shakespeare in Love
Shakespeare made the pursuit of love just as difficult as leading men to war, or solving your father's murder
Article: Romeo and Juliet
Find out why William Shakespeare's Romeo and Juliet is such an enduring love story. Learn more about Act II's balcony scene and the tragedy's most well-known adaptations
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The Bible warns us that in the final days deception would become so rampant and powerful that many would abandon biblical Christianity for a world-wide system based on Humanism, elevating the thinking of mankind and dismissing God and His Word. Over the past 20-30 years the face of media, public education, and even the Church in America has changed so radically that it looks nothing like to used to. And the sad truth is in the here and now, Secular Humanism is winning the hearts and minds of many youth–youth that will one day lead this nation and our local churches.
Our guest today is Don Veinot of Midwest Christian Outreach as we take a sobering look at the decay of American culture and thought, along with the many problems facing the Christian Church in America.
NextLiberal Theology & Thought: A Cancer Spreading in the Professing Church?
Is 'Transgenderism' Here to Stay?