The mechanisms of progressive dopaminergic neuronal loss in Parkinson's disease (PD) remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF) has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated.
In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1) were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS) model of nigral dopaminergic degeneration.
TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ), an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (si)RNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/−) mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the neuroinflammatory LPS model was also observed.
Collectively, these results identify proteolytic activation of PKCδ proapoptotic signaling as a key downstream effector of dopaminergic cell death induced by TNF. These findings also provide a rationale for therapeutically targeting PKCδ to mitigate progressive dopaminergic degeneration resulting from chronic neuroinflammatory processes.
Parkinson's disease (PD) is a debilitating neurodegenerative movement disorder affecting around 2 % of the population over the age of 60 [1, 2]. The key pathological hallmark of the disease is a loss of dopaminergic (DA) neurons in the substantia nigra of the midbrain, resulting in a depletion of striatal dopamine that is clinically manifest as a range of motor and non-motor deficits . Recent studies indicate that a sustained neuroinflammatory response is initiated in the substantia nigra pars compacta (SNpc) during early stages of dopaminergic degeneration, and remains evident in postmortem PD brains with increased microgliosis, dystrophic astrocytes and lymphocyte infiltration [4–6]. Compelling evidence over the last decade from animal models, in vitro studies and PD patients has demonstrated that the protracted neuroinflammation that occurs in the SNpc can exacerbate the degeneration of vulnerable dopaminergic neurons. However, the molecular mediators and mechanisms underlying the neuroinflammatory processes remain to be established.
Tumor necrosis factor α (TNF) has recently emerged as an important neuroinflammatory mediator linked to dopaminergic degeneration in PD. Increased levels of TNF are present in the SNpc and the CSF of postmortem PD patients, and genetic polymorphisms in the TNF gene locus have been linked to the development of PD [7–12]. Increased TNF is also well documented in rodent and nonhuman primate models of PD induced by neurotoxic insults and in transgenic models [13, 14]. Importantly, ablation of TNF signaling using knockout mice for TNFα or its receptors has been shown to attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic degeneration [15, 16]. Furthermore, genetic or pharmacological inhibition of soluble TNF signaling with dominant negative mutants is protective against 6-hydroxydopamine and lipopolysaccharide (LPS)-induced dopaminergic degeneration in vivo [17, 18]. Conversely, the chronic expression of low levels of TNF in the substantia nigra of rats induced by a viral vector causes progressive dopaminergic degeneration with delayed motor deficits . Together, these independent findings strongly implicate TNF in the progressive loss of dopaminergic neurons in PD. However, the signaling mechanisms by which TNF can mediate dopaminergic degeneration have not been characterized.
Protein kinase Cδ (PKCδ), a member of the novel PKC isoform family, is highly expressed in nigral dopaminergic neurons . Recent studies from our laboratory and others have shown that PKCδ is proteolytically activated by caspase-3 during dopaminergic cell death and that genetic or pharmacological targeting of PKCδ can protect against dopaminergic degeneration in PD models [21–24]. In this paradigm, increased oxidative stress in dopaminergic neurons results in proteolytic activation of PKCδ by caspase-3 downstream of the intrinsic mitochondrial apoptotic pathway. The proteolytic cleavage of PKCδ by caspase-3 persistently activates the kinase as an effector of dopaminergic cell death. However, PKCδ activation by extrinsic neuroinflammatory mediators such as TNF has not been studied and would be relevant to the progression of PD by neuroinflammatory mechanisms. Since TNF also activates caspase-3 by signaling through its death receptors, we determined if PKCδ is proteolytically activated during TNF-induced dopaminergic cell death and in the mouse substantia nigra (SN). Our findings herein identify PKCδ as a key downstream target of TNF death receptor signaling in dopaminergic neurons and demonstrate a novel link between neuroinflammatory mechanisms and progressive dopaminergic degeneration.
RPMI, neurobasal medium, B27 supplement, fetal bovine serum, l-glutamine, Sytox assay dye, IR-dye-tagged secondary antibodies, penicillin, streptomycin and other cell culture reagents were purchased from Invitrogen (Gaithersburg, MD, USA). Recombinant rat TNF, LPS (Escherichia coli 0111:B4) and cytosine arabinoside were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant murine TNF and the tumor necrosis factor receptor 1 (TNFR1) neutralizing antibody were from R&D Systems (Minneapolis, MN, USA). Etanercept (Enbrel) was purchased from Amgen, Inc. (Thousand Oaks, CA, USA). Antibodies for rabbit PKCδ and caspase-8 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Tyrosine hydroxylase (TH) antibody was purchased from Chemicon (Temecula, CA, USA) and microtubule-associated protein 2 (MAP-2) antibody from Cell Signaling Technologies (Beverly, MA, USA). 32P-ATP was purchased from Perkin Elmer (Boston, MA, USA) and the AMAXA Nucleofector kit from Lonza (Basel, Switzerland). Caspase assay substrates and inhibitors were purchased from MP Biomedicals (Solon, OH, USA). The DNA fragmentation assay kit was purchased from Roche Applied Science and the Bradford protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
The development and culture conditions of the N27 clonal dopaminergic cell line have been described previously [21, 24, 25]. Similar culture conditions were used in this study. Briefly, cells were cultured in RPMI 1640 medium containing 10 % heat inactivated fetal bovine serum, 2 mM l-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were maintained in a humidified atmosphere of 5 % CO2 at 37°C. RPMI medium containing 2 % fetal bovine serum was used for the TNF treatment. Cells were washed twice in 2 % RPMI serum and then treated with the indicated doses of recombinant rat TNF.
Primary neurons were cultured from ventral mesencephalon tissue of gestational 14-day (E14) mouse embryos, as described previously [21, 26] with some modifications. The ventral mesencephalon was dissected under a microscope and collected in ice-cold Dulbecco's modified Eagle medium F-12 complete medium (DMEM-F12 supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM l-glutamine, 100 μM non-essential amino acids, and 2 mM sodium pyruvate). The tissue was then dissociated using trypsin-ethylenediaminetetra-acetic acid (EDTA) (0.25 %) for 15 minutes at 37°C. Trypsinization was stopped by adding an equal volume of DMEM-F12 complete medium and dissociated tissue was washed in the same medium to remove residual trypsin. The DMEM-F12 medium was aspirated out and the tissue triturated in neurobasal medium containing B-27 antioxidant supplement, 500 μM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. After a single cell suspension was obtained, cells were passed through a 70 μm nylon mesh cell strainer to remove tissue debris and aggregates. Cells were counted using a Beckman Coulter ViCell XR automated cell counter and then plated at an equal density (0.8 × 106 cells per well) in 24 well plates containing coverslips precoated with poly-d-lysine (100 μg/ml). Cultures were maintained in neurobasal medium with B-27 antioxidant supplements and cytosine arabinoside (5 μM) was added to inhibit glial proliferation. Cultures were grown in a humidified CO2 incubator (5 % CO2, 37°C) and the medium was changed every 2 days. Approximately 4-day-old to 5-day-old cultures were used for treatment. The neuronal cultures were verified to be around 98 % free of glial cells at the time of treatment by using immunocytochemistry for MAP-2, glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (iba1) as markers of neurons, astrocytes and microglia, respectively . For TNF treatment, cultures were switched to serum-free neurobasal medium without antioxidant supplements and treated for 48 h. Recombinant murine TNF (30 ng/ml) was added at the beginning of the treatment and re-added again 24 h later. At the end of the treatment, primary cultures were processed for TH immunocytochemistry and neurotransmitter uptake assays.
DNA fragmentation was quantified using the Cell Death Detection ELISA Plus assay kit (Roche Applied Science), as described in our previous publications [21, 22, 24]. This highly sensitive and reliable assay detects and quantifies early changes in apoptosis based on the amounts of histone-associated low molecular weight DNA released into the cytoplasm of cells. Briefly, N27 cells were plated in six-well plates at 0.8 × 106 cells/well and treated the next day with TNF for 16 h. Cells were collected using a cell scraper and centrifuged at 400 × g for 5 minutes. The cells were gently lysed using the lysis buffer provided with the kit. The lysate then was spun down again at 200 g for 10 minutes to pellet the nuclear fraction, and the supernatant was collected and used to measure DNA fragmentation according to the manufacturer's instructions for the ELISA protocol. The absorbance at 405 nm was measured against an 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) solution as a blank (reference wavelength approximately 490 nm) using a Synergy-2 Multi-Mode Microplate Reader (BioTek Instruments, Inc). The absorbance values were normalized to the amount of protein present in the lysates and the data expressed as percent control. The Sytox cytotoxicity assay was performed using the Sytox green dye (Molecular Probes). The assay is based on the principle that live cells with intact plasma membranes can exclude the Sytox dye, which selectively enters cells with a compromised membrane and emits bright green fluorescence on binding to DNA . N27 cells were grown in 24 well plates and 1 μM of the Sytox dye was added at the time of treatment. Cells were treated for 16 h with TNF and pretreated with etanercept. The increase in green fluorescence, as a result of TNF cytotoxicity, was measured using a fluorescence microplate reader (Spectramax Gemini, Molecular Devices) at an excitation of 485 nm and 538 nm emission. Phase contrast and fluorescence images of matching fields were captured on the same cells to visualize the toxicity in N27 cells.
Enzymatic assays for caspase-3 activity were performed as described previously [22, 24] using acetyl-DEVD-amino-4-trifluoromethylcoumarin (Ac-DEVD-AFC, 25 μm) as the fluorometric substrate for the reaction. N27 cells were treated with TNF (30 ng/ml) for 6 h or pretreated with etanercept (5 μg/ml) for 30 minutes. Following treatment, 100 μl of cell extract was incubated with 5 μl of the fluorescence substrate and then incubated at 37°C for 1 h while being protected from light. The fluorescence signal generated upon cleavage of the AFC peptide substrate by caspase-3 was measured at 510 nm with an excitation of 400 nm using a Synergy-2 Multi-Mode microplate reader. Protein concentrations were determined using the Bradford assay. Raw values were normalized using protein concentrations and expressed as percent control.
N27 dopaminergic cells were plated on poly-d-lysine (100 μg/ml) coated coverslips in 24 well plates at 0.2 × 106 cells per well. The next day, the cells were treated with 30 ng/ml of TNF for 6 h in 2 % serum RPMI medium. Cells were fixed in 4 % paraformaldehyde for 20 minutes and washed five times in PBS. The cells were blocked and permeabilized with blocking buffer (5 % goat serum, 0.2 % Triton X, and 0.05 % Tween-20 in PBS) for 1 h and then incubated overnight at 4°C with a rabbit polyclonal PKCδ antibody (1:1,000) that recognizes a C-terminal epitope present in both the native and the proteolytically cleaved protein. The cells were then washed five times in PBS and then incubated with secondary antibody (1:2,000, Alexa 488 goat anti-rabbit) for 1 h at room temperature. Negative controls for non-specific staining that contained secondary antibody alone were used on parallel wells to ensure specificity of the fluorescent signals obtained. The cells were then washed five times in PBS and the nucleus was labeled using the TOPRO 3 dye. The cells were then washed three more times in PBS and coverslips were mounted using the ProLong Gold antifade reagent (Molecular Probes). Confocal images were acquired with a Nikon EZ-C1 confocal system using the 488 nm and 633 nm lasers to visualize PKCδ and TOPRO 3, respectively. Fluorescence spatial intensity plots along the XY plane for PKCδ localization (green channel) and the nucleus (blue channel) were generated using the Nikon EZC1 software to show nuclear translocation.
Lysates from N27 cells and brain tissue were prepared using a modified radio immunoprecipitationassay (RIPA) buffer and normalized for equal amounts of protein using the BCA protein assay (Pierce Biotechnology). Gel loading dye was added to the lysates and stored in aliquots at −80°C. Equal amounts of protein (30 to 60 μg) were loaded for each sample and separated on either 12 % or 15 % SDS-PAGE gels, depending on the molecular weight of the target protein. After separation, the proteins were transferred to nitrocellulose membranes and non-specific binding sites were blocked by incubating the membranes in fluorescent Western blocking buffer (Rockland Immunochemicals) for 1 h, and then they were probed with primary antibodies overnight at 4°C. Primary antibodies used were rabbit polyclonal PKCδ (1:500), mouse monoclonal TH (1:2,000), rabbit polyclonal caspase-8 (1:200). β-Actin (1:5,000) was used as the loading control. After incubation, membranes were washed three times with PBS containing 0.05 % Tween and IR-dye tagged secondary antibodies (1:5,000; Molecular Probes) were added. Membranes were visualized on the Odyssey infra-red imaging system.
The PKCδ enzymatic kinase activity assay was performed as described previously [21, 22, 29]. Briefly, N27 cells or substantia nigra tissues were washed in ice-cold PBS and then resuspended in a mild RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Pierce Biotechnology). The lysates were placed on ice for 20 minutes, sonicated gently and centrifuged at 13,000 rpm for 45 minutes. The supernatant was collected and protein concentration was determined using the Bradford assay . All samples were made up to a concentration of 2 μg/ml, and 500 μg of total protein in a 250 μl volume was immunoprecipitated overnight at 4°C using 5 μg of the PKCδ antibody. The next day, the protein A-agarose beads (Sigma-Aldrich) were added and the samples were incubated for 1 h at room temperature. The protein A-bound antibody complexes were then washed three times in 2 × kinase assay buffer (40 mM Tris, pH 7.4, 20 mM MgCl2, 20 μM ATP, and 2.5 mM CaCl2), and then resuspended in 40 μl of the same buffer. The kinase reaction was started by adding 40 μl of the reaction buffer containing 0.4 mg of histone H1, 50 μg/ml phosphatidylserine, 4 μM dioleoylglycerol, and 10 μCi of [γ-32P] ATP at 3,000 Ci/mM to the immunoprecipitated samples. The samples were incubated for 10 minutes at 30°C. The kinase reaction was stopped by adding 2 × SDS loading buffer and boiling the samples for 5 minutes. The proteins were separated on a 12 % SDS-PAGE gel and the phosphorylated histone H1 bands were scanned using a Fujifilm FLA 5000 imager. Image analysis and band quantification were performed using the Fujifilm Multigauge software package (Fujifilm USA, Stamford, CT).
Design and synthesis of PKCδ siRNA are described in our previous publications [21, 31]. N27 cells were transfected with 40 to 50 nM of either PKCδ or scramble siRNA duplexes using the AMAXA Nucleofector kit, according to the manufacturer's instructions. Transfected cells were counted using a Vi-Cell XR automated cell counter and seeded at equal density (0.5 × 106 cells/well) into six-well plates. The cells were treated 24 h after transfection to allow for optimal knockdown of gene expression. At the end of the treatment, cells were collected and used for the DNA fragmentation assay as described above. For studies with the PKCδ cleavage-resistant mutant (PKCδD327A-CRM), stable N27 cell lines overexpressing either the mutant PKCδD327A protein or the β-galactosidase (Lac Z) protein as the vector control were prepared as described in our publications . Both cell lines were cultured in the presence of blasticidin for three passages before treatment. Expression of the PKCδ CRM-mutant and the Lac Z vector control at the time of treatment was confirmed by staining for the V5-epitope. Cells were plated at equal density (0.7 × 106 cells per well) in six-well plates and allowed to grow overnight. The next day, cells were treated with 30 ng/ml of TNF for 16 h and processed for the DNA fragmentation assay.
Uptake assays for tritiated dopamine on primary EVM cultures were performed according to previously published protocols [17, 33, 34]. After TNF treatment, the primary neuron cultures from PKCδ wild type (+/+) and knockout (−/−) mice were washed three times with 0.5 ml of warm Krebs-Ringer buffer (16 mM sodium phosphate, 120 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4). The cultures were then incubated with 5 μM 3 H]-dopamine (30 Ci/mmol) in Krebs-Ringer buffer at 37°C for 20 minutes. The cells were then washed three times with ice-cold Krebs-Ringer buffer and collected in 1 ml of 1 N NaOH. Scintillation fluid was added to a total volume of 5 ml and radioactivity counts were measured using a Tri-Carb Liquid Scintillation counter (Packard). In parallel wells, the nonspecific uptake of dopamine was determined by incubation with 10 μM mazindol. The nonspecific uptake values were subtracted to obtain the data for high affinity neurotransmitter uptake. Data from dopamine uptake studies were expressed as percent control.
After TNF treatment, primary neuron cultures from PKCδ wild type (+/+) and PKCδ knockout (−/−) mice were fixed in 4 % paraformaldehyde and permeabilized with PBS containing 2 % bovine serum albumin (BSA), 0.2 % Triton X-100 and 0.05 % Tween-20. Blocking buffer (PBS with 2 % BSA) was added for 1 h at room temperature and primary antibodies for TH (1:1,000) and MAP-2 (1:1,000) were diluted in blocking buffer and incubated overnight at 4°C. The next day, cells were washed in PBS four times and incubated with appropriate Alexa-dye conjugated secondary antibodies for 1 h at room temperature. After several washes, the samples were counterstained with Hoechst to label the nucleus, and coverslips were mounted using the Prolong Antifade (Molecular Probes) mounting medium. Images were acquired using a Nikon inverted fluorescence microscope (model TE-2000U) equipped with a SPOT digital camera system (Diagnostic Instruments, Sterling Heights, MI, USA). Image analysis was performed using the Metamorph software package (Universal Imaging Systems, PA, USA).
A single dose LPS injection model that was previously described  was used to induce delayed, progressive loss of dopaminergic neurons in the substantia nigra. C57/BL/6 mice (8 weeks old, n = 6 per group) were anesthetized with a mixture of ketamine-xylazine (100 mg/kg, 10 mg/kg) and carefully immobilized on a stereotaxic apparatus (Benchmark Angle One, Leica Microsystems). The skin above the skull was prepped with alcohol and an incision was made to expose the skull. A single burr hole was carefully drilled at the injection site for the right SN and ophthalmic gel was used to protect the eyes. The stereotaxic coordinates for the injection site were anteroposterior (AP) -3.3 mm, mediolateral (ML) -1.2 mm, dorsoventral (DV) -4.6 mm from bregma . A stainless steel cannula attached to a 5 μl Hamilton syringe was carefully inserted into the hole drilled at the injection site and a single dose of 5 μg LPS in a 1 μl volume was injected at the rate of 0.5 μl per minute. The needle was left in place for another 5 minutes to prevent retrograde flow of liquid along the needle track. Control mice were injected in an identical manner with equal volumes of saline. After surgery, the skin was sutured and carefully held in place using sterile, non-pyrogenic stainless steel clips (Autoclip Wound Closing System, Braintree Scientific, Inc). Mice were allowed to recover on a heating pad (Braintree Scientific, Inc) and were carefully monitored through recovery from anesthesia. All animal procedures were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC).
Data analysis was performed using the Prism 4.0 software package (GraphPad Software, San Diego, CA, USA). The data were first analyzed using one-way analysis of variance (ANOVA) and then Bonferroni's post-test was performed to compare all treatment groups. Differences of P <0.05 were considered statistically significant. The Student's t test was used when differences between two groups were being compared.
To determine the downstream mechanisms underlying TNF stimulation in dopaminergic neuronal cells, we used the N27 dopaminergic neuronal cell model, which has been widely used by several laboratories to study dopaminergic degeneration related to PD [21, 24, 25]. First we examined the neurotoxic effect of TNF on N27 dopaminergic neuronal cells. The Sytox assay and the sensitive DNA fragmentation ELISA were used to quantify TNF cytotoxicity. TNF (30 ng/ml) induced significant increases in Sytox fluorescence and cell death morphology, which could be blocked using the TNF neutralizing drug etanercept (Figure 1A,B). Since TNF cytotoxicity can proceed through activation of caspases in various cell types, we examined caspase activation downstream of TNF signaling. High levels of the pro-caspase-8 protein were detected in N27 cells, and TNF treatment induced activation of caspase-8 at 3 h, with increased accumulation of the active p20 fragment (Figure 1C,D). Again, the activation of caspase-8 was attenuated by pretreatment with the TNF inhibitor etanercept. Since caspase-3 is an important effector caspase downstream of TNF signaling, and is known to be activated in PD models and in the human PD brain [37, 38], we studied caspase-3 activation in TNF-treated N27 cells. We found that TNF treatment for 6 h caused activation of caspase-3, which could be blocked by pretreatment with etanercept and the caspase-8 inhibitor IETD-fmk (Figure 1E). These results indicate that caspase-3 is activated in a caspase-8 dependent manner downstream of TNF death receptor signaling in dopaminergic neuronal cells. Next we used the sensitive DNA fragmentation ELISA assay to quantify TNF cell-death signaling. Exposure to TNF (30 ng/ml) for 16 h resulted in a threefold increase in DNA fragmentation compared to untreated controls (Figure 1F). Pretreatment with the TNF-neutralizing drug etanercept or a caspase-8 inhibitor (IETD-fmk) almost completely blocked TNF-induced DNA fragmentation, indicating that canonical TNF death receptor signaling through activation of caspase-8 is involved in the dopaminergic neurotoxicity caused by sustained exposure to TNF. In preliminary studies, we found that doses as low as 10 ng/ml TNF could induce detectable toxicity in N27 dopaminergic cells. We chose a 30 ng/ml dose of TNF based on previous in vitro studies  and because this concentration induced a consistent neurotoxic response in vitro. The dose range of TNF used in our mechanistic studies is high compared to the levels of circulating TNF in the serum and CSF of PD patients [39–41] due to the fact that high doses are necessary to elicit a detectable response in cell culture models within a shorter timeframe. Also, the circulating TNF has a short half-life and therefore, it does not reflect the elevated levels of tissue TNF in the substantia nigra. The TNF concentration at 10 to 60 ng/ml used in our study is consistent with doses used in cell culture experiments by other researchers [17, 26].
Tumor necrosis factor (TNF)-induced neurotoxicity and caspase activation in N27 dopaminergic neuronal cells. (A) Visualization of TNF toxicity by Sytox fluorescence cytotoxicity assay. N27 dopaminergic cells treated with TNF (30 ng/ml), or pretreated with etanercept (5 μg/ml) for 30 minutes and were processed for Sytox green imaging at 16 h. Increased green fluorescence is evident with TNF treatment, indicating significant neurotoxicity which was also evident in the phase contrast images. (B) Sytox fluorescence was quantified by measuring the fluorescence intensity as relative fluorescence units (RFU) using a plate reader. (C) Activation of caspase-8 by TNF treatment. Lysates from N27 cells treated with TNF (30 ng/ml) for 3 h were probed using a rabbit polyclonal antibody to caspase-8 that detects the procaspase and the active cleaved form. TNF induced a significant increase in the active caspase-8 (p20) fragment, which was blocked by pretreatment with etanercept (5 μg/ml). (D) Band intensities for cleaved caspase-8 from three blots were quantified using densitometric analysis and normalized to β-actin. (E) TNF induced activation of caspase-3. Enzymatic assays for caspase-3 activity in lysates from N27 cells treated with TNF (30 ng/ml) for 6 h or pretreated with either etanercept (5 μg/ml) or the caspase-8 inhibitor IETD-fmk (25 μM) for 30 minutes. Raw values were normalized using protein concentrations and expressed as percent control. TNF treatment induced significant activation of caspase-3, which was blocked by etanercept and the caspase-8 inhibitor IETD-fmk. (F) TNF-induced DNA fragmentation. N27 cells were treated with TNF (30 ng/ml) for 16 h or pretreated for 30 minutes with either etanercept (5 μg/ml) or the caspase-8 peptide inhibitor IETD-fmk (25 μM), and processed for the DNA fragmentation assay. Raw values were normalized using protein concentration and expressed as percent control. TNF treatment significantly increased DNA fragmentation, which was attenuated by etanercept or caspase-8 inhibition. Data represent the group mean ± SEM; n = 6 to 8 per group and experiments were repeated three times. ** (P <0.01) indicates significant differences compared to control cells; ## (P <0.01) and # (P <0.05) indicates significant differences compared to TNF-treated cells.
We recently showed that PKCδ, a proapoptotic kinase belonging to the novel PKC isoform family, is highly expressed in dopaminergic neurons [21, 42]. Further, we demonstrated that PKCδ is activated by caspase-3 mediated proteolytic cleavage at its hinge region following mitochondrial oxidative stress, resulting in persistent activation of the kinase and amplification of apoptotic signals in dopaminergic neurons [21, 22, 24]. Since PKCδ activation by the extrinsic apoptotic pathway in dopaminergic cells or its role in neuroinflammation-induced dopaminergic neurotoxicity has not been examined, we tested if PKCδ can be activated by caspase-3 during TNF-induced death of dopaminergic neuronal cells and function as a downstream apoptotic effector. Interestingly, TNF treatment caused a time and dose dependent proteolytic activation of PKCδ that was detectable 3 h after treatment and increased at 6 h (Figure 2A,B). Dose response studies at the 6 h time point showed that doses as low as 10 ng/ml of TNF could induce detectable proteolytic activation of PKCδ, which increased substantially with 30 ng/ml TNF. Higher doses of TNF (60 ng/ml) did not significantly increase PKCδ cleavage further (Figure 2C,D). Since caspase-8 and caspase-3 were activated by TNF in these cells, we tested if PKCδ proteolytic activation was mediated by this signaling pathway using the specific caspase inhibitors DEVD-fmk and IETD-fmk. Pretreatment with the caspase-8 and caspase-3 inhibitors suppressed TNF-induced proteolytic activation of protein kinase Cδ (Figure 2E,F). Taken together, these results suggest that the proapoptotic kinase PKCδ could be activated by caspase-8 and caspase-3 dependent proteolytic cleavage during TNF toxicity in dopaminergic neuronal cells. The proteolytically cleaved PKCδ fragment appeared as a doublet band in Western blots in our experiments. Several alternate splice forms of PKCδ have been reported, including a caspase-3 cleavage-resistant form and a truncated form lacking the regulatory domain [43, 44]. Also, the C-terminal catalytic domain of PKCδ contains multiple phosphorylation sites. Because the antibody used in these studies recognizes the C-terminal catalytic domain, it is possible that the doublet band of the cleaved product represents a post-translational modification of the cleavage product or one of the truncated PKCδ alternate splice forms.
Tumor necrosis factor (TNF) induced proteolytic cleavage of protein kinase Cδ (PKCδ) in dopaminergic N27 cells. (A) Time-dependent PKCδ proteolytic activation. N27 dopaminergic cells were treated with recombinant TNF (30 ng/ml) for 3 h and 6 h and lysates were probed by Western blotting with an antibody to the C-terminus that detects both the native protein (78 kDa) and the proteolytically cleaved catalytic fragment (38 kDa). TNF treatment caused a time-dependent increase in PKCδ cleavage, which peaked at 6 h. (B) Band intensities for cleaved PKCδ from three blots quantified using densitometric analysis and normalized to β-actin. (C) Dose-dependent proteolytic activation of PKCδ by TNF treatment. N27 cells were treated with increasing doses of TNF (0 to 60 ng/ml) for 6 h and lysates were probed for PKCδ by Western blotting. TNF induced a dose dependent increase in PKCδ cleavage, starting at 10 ng/ml. (D) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. (E) Caspase-3 and caspase-8 dependent proteolytic cleavage of PKCδ. N27 cells were treated with 30 ng/ml of TNF alone for 6 h or in the presence of peptide inhibitors (25 μM) of caspase-8 (Ac-IETD-fmk) and caspase-3 (Ac-DEVD-fmk). Lysates were probed for PKCδ proteolytic cleavage by Western blotting. Proteolytic activation of PKCδ by TNF treatment was reduced by inhibition of caspase-3 and caspase-8. (F) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. Data is expressed as a fold change over control. * (P <0.05) and ** indicate significant differences compared to control cells; # (P <0.05) indicates significant differences compared to TNF-treated cells.
Unlike other proteins for which proteolytic cleavage can result in loss of function or inactivation, the proteolytic cleavage of PKCδ by caspase-3 during oxidative stress-induced dopaminergic cell death has been shown to persistently activate the kinase [21, 22, 24, 45]. We used IP-kinase assays and Western blotting to determine if TNF-induced proteolytic activation of PKCδ leads to sustained activation of the kinase in N27 dopaminergic cells. As shown in Figure 3A, treatment with TNF for 6 h significantly increased proteolytic cleavage of PKCδ, which was blocked by pretreatment with Etanercept or a TNFR1 receptor-neutralizing antibody. PKCδ IP-kinase assays performed in the absence of lipid cofactors showed a robust increase in PKCδ kinase activity in TNF-treated samples, which could be attenuated by pretreatment with etanercept or by TNFR1 receptor neutralization (Figure 3C). These results demonstrate for the first time that PKCδ can be proteolytically activated in dopaminergic cells by a TNFR1-dependent signaling pathway, resulting in sustained activation of the kinase during TNF-induced cell death.
Proteolytic cleavage and activation of protein kinase Cδ (PKCδ) downstream of tumor necrosis factor receptor 1 (TNFR1) in N27 dopaminergic cells. (A) TNFR1-dependent proteolytic activation of PKCδ. N27 cells were treated with tumor necrosis factor (TNF) (30 ng/ml) for 6 h or pretreated with either a TNFR1 neutralizing antibody (TNFR1-nAb, 20 μg/ml) or etanercept (5 μg/ml) for 30 minutes and lysates were probed for PKCδ proteolytic cleavage by Western blotting. TNF induced strong proteolytic cleavage of PKCδ that could be blocked by neutralizing TNFR1 receptor signaling. (B) Band intensities for cleaved PKCδ were quantified using densitometric analysis and normalized to β-actin levels. (C) Immunoprecipitation (IP)-kinase assay for PKCδ activation. IP-kinase assays were performed on the same cell lysates used for Western blots. PKCδ was immunoprecipitated from 500 μg of total protein from each sample and used for the in vitro kinase activity assay with a histone substrate and radiolabeled [γ-32P] ATP. PKCδ activity was quantified using densitometric analysis of the 32P-histone band intensity and expressed as percent control. The proteolytic cleavage of PKCδ induced by TNF treatment was accompanied by a concomitant increase in kinase activity and was dependent on signaling through the TNFR1 receptor. A representative kinase assay gel is shown. Data represent the group mean ± SEM of densitometric values obtained from three independent experiments. *** (P <0.001) denotes significant differences compared to control cells and ## (P <0.01) denotes significant differences compared to TNF-treated cells.
Nuclear translocation of PKCδ has been shown to occur following proteolytic activation by apoptotic stimuli. A bipartite nuclear localization sequence (NLS) has been identified at the C-terminus (amino acids 611 to 623) of PKCδ [46, 47]. The NLS is required for nuclear localization of both the full-length and proteolytically-cleaved forms of PKCδ. However, previous studies have shown that proteolytic activation of PKCδ by caspase-3 facilitates unmasking of the NLS, resulting in increased nuclear translocation of the catalytic fragment during apoptosis [47–49]. To further characterize the role of PKCδ in dopaminergic cell death induced by TNF, we used confocal microscopy to study the subcellular localization of PKCδ. N27 cells were treated with TNF for 6 h, the time point at which the highest level of cleaved PKCδ was detected, and confocal immunofluorescence was used to visualize the subcellular localization of PKCδ. We detected prominent nuclear localization of PKCδ after 6 h in TNF-treated cells, while in control cells PKCδ localized in the cytoplasm (Figure 4A). Nuclear translocation was also clearly evident in spatial fluorescence signal intensity plots (Figure 4B) for PKCδ (green channel) and the Hoechst nuclear stain (blue channel). Control cells had spatially distinct fluorescence signals for the nucleus and PKCδ, showing that PKCδ is largely localized outside the nucleus. However in TNF-treated cells, the spatial fluorescence signals had the same pattern along the XY plane. Interestingly, in addition to the overall increase in nuclear localization with TNF treatment, we also observed punctuate staining of PKCδ at distinct locations around the perinuclear region (shown in 60 × magnification and labeled with red arrows). The nuclear localization of PKCδ following TNF treatment further points to an important role for this kinase in dopaminergic degeneration during TNF toxicity.
Protein kinase Cδ (PKCδ) translocates to the nucleus during tumor necrosis factor (TNF) treatment. (A) Confocal imaging of PKCδ localization. N27 cells were treated with TNF for 6 h and processed for immunocytochemistry using a rabbit polyclonal antibody that recognizes both the native and cleaved form of PKCδ. An Alexa-488 dye-tagged secondary antibody was used to visualize PKCδ (green), and the nucleus was stained using the TOPRO-3 dye (blue). In untreated controls (top panel), PKCδ was distinctly localized to the cytosol. In TNF-treated cells (lower panel), a distinct localization of PKCδ to the nucleus and the perinuclear region is seen. Scale bars are 20 microns. High magnification images (right panel) of PKCδ immunofluorescence staining in N27 cells showing PKCδ accumulation within the nucleus and at distinct spatial locations at the perinuclear region following TNF treatment (red arrows). Representative images are shown; experiments were repeated three times using different secondary antibodies for validation. (B) Fluorescence spatial intensity plots along a representative XY plane for PKCδ localization (green channel) and the nucleus (blue channel) were obtained using the Nikon EZC1 image analysis software. Control images show a distinct spatial localization of green and blue fluorescence intensities in the XY plane indicative of cytosolic PKCδ localization. In TNF-treated cells a spatial colocalization of green and blue fluorescence signals is evident indicating nuclear translocation of PKCδ.
To determine whether TNF-induced PKCδ activation contributes to the cell death process in dopaminergic cells, we used siRNA suppression of PKCδ and a caspase-3 cleavage-resistant mutant of PKCδ (PKCδD327A-CRM). First, N27 cells were transfected with siRNA to PKCδ or scramble siRNA and incubated for 24 h to allow for optimal suppression of PKCδ protein levels, which we validated by Western blotting (Figure 5B). The transfected cells were then treated with TNF for 16 h and processed for the DNA fragmentation assay. In N27 cells transfected with scramble siRNA, TNF caused a significant increase in DNA fragmentation, while the PKCδ siRNA transfected cells were protected from cell death (Figure 5A). These results indicate that PKCδ is required for TNF-induced apoptosis in dopaminergic cells and that genetic or pharmacological modulation of PKCδ may effectively attenuate TNF-induced neurotoxicity. To evaluate the significance of the PKCδ proteolytic cleavage events during proapoptotic TNF signaling, we used N27 cells overexpressing a mutant PKCδ protein resistant to proteolytic cleavage (PKCδD327A-CRM) by caspase-3 due to a point mutation at the cleavage site. N27 cells that stably express either the cleavage-resistant PKCδ mutant or the LacZ protein (Figure 5D) were established as described previously . The cells were treated with TNF for 16 h and processed for DNA fragmentation. As shown in Figure 5C, TNF treatment caused a 2.5-fold increase in DNA fragmentation in LacZ cells, while cells expressing the PKCδD327A-CRM mutant were protected from TNF toxicity, indicating that proteolytic cleavage of PKCδ is crucial for TNF-induced cell death. Together, these results demonstrate that PKCδ functions as a proapoptotic kinase during TNF-induced dopaminergic neurotoxicity and that proteolytic cleavage of PKCδ by caspase-3 downstream of TNF signaling is required for cell death.
Knockdown of protein kinase Cδ (PKCδ) with siRNA and overexpression of the PKCδ proteolytic cleavage-resistant mutant (PKCδ D327A -CRM) protects against tumor necrosis factor (TNF) toxicity. (A) DNA fragmentation assay in siRNA transfected cells. N27 cells transfected with either siRNA to PKCδ or scrambled control siRNA were treated with TNF for 16 h and processed for the DNA fragmentation ELISA. Raw values were normalized to protein concentrations and expressed as the fold change over the respective controls. TNF-induced DNA fragmentation was significantly reduced in PKCδ siRNA transfected cells. (B) Suppression of the PKCδ protein levels by siRNA was confirmed by Western blotting. (C) DNA fragmentation assay in N27 cells overexpressing the cleavage-resistant PKCδ mutant protein (PKCδD327A-CRM). N27 cells stably expressing the cleavage-resistant PKCδ mutant or the β-galactosidase (LacZ) control gene were treated with TNF for 16 h and processed for the DNA fragmentation assay. Expression of the mutant PKCδ protein was confirmed by imaging the V5 tag by immunocytochemistry. (D) DNA fragmentation induced by TNF was attenuated in N27 cells expressing the caspase-3 cleavage-resistant mutant protein, indicating the proteolytic cleavage is a necessary event for TNF toxicity in these cells. Data represent the group mean ± SEM, n = 6 to 8 per group and experiments were repeated three times. *** (P <0.001) indicates significant differences with TNF treatment compared to controls in scramble siRNA and LacZ expressing cells; ## (P <0.01) indicates significant differences between the two TNF treatment groups.
In order to extend our results from the previous mechanistic studies with the dopaminergic N27 clonal cell model to dopaminergic neurons, we used primary mouse ventral mesencephalic dopaminergic neuron cultures. We established primary mesencephalic neuronal (>95 % glial-free) cultures from PKCδ wild type (+/+) and knockout (−/−) mice to study the role of PKCδ in mediating TNF-induced dopaminergic neurodegeneration. Dopaminergic neurotoxicity was assessed by dopamine uptake assays and by TH-positive cell counting. As shown (Figure 6B,C), TNF exposure caused around 50 % decrease in dopamine uptake and TH-positive cell counts in cultures from PKCδ wild type (+/+) mice, consistent with previous reports of dopaminergic neurotoxicity in similar model systems [17, 26]. However, in primary neuron cultures obtained from PKCδ knockout (−/−) mice, TNF induced only around a 20 % reduction in dopamine uptake and TH-positive neuron counts, indicating that dopaminergic neurons from PKCδ knockout mice were resistant to TNF toxicity. In addition to functional measurement of TNF neurotoxicity by dopamine uptake, we also performed double labeling immunofluorescence morphometric analysis using TH as a marker for dopaminergic neurons and MAP-2 as a pan-neuronal marker. Untreated cultures from both PKCδ wild type (+/+) and knockout (−/−) mice had similar numbers of dopaminergic neurons and displayed extensive branched neurites and normal cell bodies (Figure 6A). In TNF-treated cultures, fewer TH-positive neurons with smaller cell bodies and a clear loss of neurites were evident in cultures from PKCδ wild type (+/+) mice. In contrast, dopaminergic neurons in cultures from PKCδ knockout (−/−) mice displayed visible branched neurites and more numerous cell bodies with normal morphology (Figure 6A lower panel), demonstrating significant protection against TNF toxicity in these cultures.
Primary dopaminergic neurons in EVM cultures from protein kinase Cδ knockout (PKCδ −/−) mice are protected against tumor necrosis factor (TNF) toxicity. Primary mouse EVM neuronal cultures were treated with recombinant TNF (30 ng/ml) in supplement-free neurobasal medium. TNF was re-added 24 h later for a total treatment time of 48 h. PKCδ wild-type (PKCδ +/+) and knockout (PKCδ −/−) EVM cultures were treated in parallel. (A) Immunocytochemistry for tyrosine hydroxylase (TH)-positive neurons. Dopaminergic neurons were identified by TH staining (green) and microtubule-associated protein 2 (MAP-2) (red) was used as a pan-neuronal marker. Images were acquired at 20 × magnification. Representative fields are shown for each treatment. In the TNF-treated group, extensive loss of neurites and degeneration of dopaminergic neurons is evident in cultures from PKCδ wild-type (+/+) mice (top panel) whereas cultures from PKCδ knockout (−/−) mice had visible branched neurites, more numerous cell bodies and were resistant to degeneration induced by TNF (lower panel). (B,C) TH-positive neuron counts and dopamine uptake assays. (B) Dopaminergic neurons were identified by tyrosine hydroxylase labeling, and the number of TH-positive neurons from 12 random fields per well were counted at 20 × magnification for each treatment group. (C) The uptake of tritiated [3 H] dopamine was determined in cultures from PKCδ wild-type (WT) and knockout mice. The values for non-specific uptake of dopamine obtained in the presence of mazindol were subtracted as background and the data expressed as percent control. Data represent the group mean ± SEM, n = 4 to 6 per group and experiments were repeated three times. ***P <0.001 indicates a significant difference between control and TNF-treated groups in PKCδ wild type cultures. # (P <0.05) and ## (P <0.01) indicate a significant difference between TNF-treated groups in PKCδ wild-type and PKCδ knockout (−/−) cultures.
Next, we sought to extend the significance of our results obtained with the N27 dopaminergic cell culture model and primary neurons to an in vivo model of dopaminergic degeneration relevant to neuroinflammation. We selected the widely used stereotaxic LPS injection model of neuroinflammation-induced dopaminergic degeneration for our experiments [17, 35, 50, 51]. In this model, a single injection of LPS into the rodent substantia nigra elicits a sustained, localized neuroinflammatory response resulting in a delayed loss of dopaminergic neurons in the SNpc. We stereotaxically injected C57bl6 mice with a single dose of 5 μg LPS into the substantia nigra and sacrificed them 14 days later, the time at which significant dopaminergic degeneration occurs in this model . The SN tissue was dissected and probed for proteolytic activation of PKCδ using Western blotting and IP-kinase assays. As shown in Figure 7A, mice that received saline injections did not show proteolytic activation of PKCδ in the SN, while LPS-injected mice showed strong proteolytic cleavage of PKCδ at 14 days. The PKCδ Western blots were reprobed for tyrosine hydroxylase to confirm dissection of the substantia nigra region. As expected, the LPS-injected mice had decreased TH protein levels (Figure 7A), indicative of increased dopaminergic degeneration in this model at 14 days [35, 50]. We also used PKCδ IP-kinase assays with a histone substrate on nigral tissue samples from LPS-injected and saline-injected mice. In nigral tissues from LPS-injected mice, we detected substantially higher PKCδ kinase activity compared to saline injected controls (Figure 7C), demonstrating that the proteolytic cleavage of PKCδ resulted in activation of the kinase in vivo. Equal protein loading and accurate dissection of the nigral tissue was confirmed by Western blotting for β-actin and TH, respectively. Together, these results demonstrate for the first time that protein kinase Cδ can be activated by proteolytic cleavage in the substantia nigra following LPS-induced neuroinflammation.
Proteolytic activation of protein kinase Cδ (PKCδ) in the mouse substantia nigra during LPS-induced dopaminergic degeneration. C57BL/6 mice were stereotaxically injected with a single dose of lipopolysaccharide (LPS) (5 μg) or saline in the substantia nigra (SN) to elicit dopaminergic degeneration. Mice were sacrificed after 14 days. (A) Proteolytic cleavage of PKCδ in mouse substantia nigra tissue. Western blots were performed on nigral tissue lysates obtained from mice stereotaxically injected with either LPS or saline. Strong proteolytic cleavage of PKCδ is seen in LPS-injected mice and was essentially absent in the saline-injected animals. Blots were also probed for tyrosine hydroxylase and β-actin to confirm accurate dissection of the nigral tissue and equal protein loading respectively. (B) PKCδ cleaved band intensity was quantified by densitometric analysis after normalization to β-actin (n = 4 to 6 mice per group). (C) PKCδ kinase activity in mouse substantia nigra tissue. PKCδ IP-kinase assays were performed on nigral tissue lysates. Kinase activity was quantified by densitometric analysis of the 32P-histone bands (n = 4 mice per group). A representative kinase gel is shown. Western blots of tyrosine hydroxylase and β-actin were performed on the same lysates to verify accurate dissection of the nigral tissue. Data represent the group mean ± SEM. ** (P <0.01) indicates a significant difference between saline and LPS-injected mice.
In this study, we identify PKCδ as a novel signaling mediator downstream of TNF-induced toxicity in dopaminergic neurons and demonstrate that proteolytic activation of PKCδ regulates dopaminergic degeneration induced by TNF (Depicted schematically in Figure 8). We also demonstrate for the first time, the proteolytic activation of PKCδ in the mouse substantia nigra by neuroinflammatory mechanisms during LPS-induced dopaminergic degeneration. The proteolytic activation of PKCδ by neuroinflammatory insults via the extrinsic apoptotic pathway has not been studied, therefore it is essential to elucidate the mechanisms underlying progressive degeneration of dopaminergic neurons by chronic neuroinflammatory processes involving TNF and other neurotoxic mediators [52, 53]. Based on the substantial literature supporting a degenerative role for TNF during dopaminergic cell death in PD models, we hypothesized that PKCδ can be proteolytically activated downstream of canonical TNF death receptor signaling by caspase-3 in dopaminergic neurons. Since proteolytic cleavage of PKCδ by caspase-3 typically results in persistent activation of the kinase as an apoptotic effector, we reasoned that blocking proapoptotic PKCδ signaling could protect against TNF toxicity.
Schematic of protein kinase Cδ (PKCδ) activation by proteolytic cleavage downstream of tumor necrosis factor (TNF) signaling in dopaminergic neurons. (1) Sustained TNF signaling leads to caspase-8 activation, presumably by autoproteolytic cleavage at the receptor complex (2). Active caspase-8 leads to downstream activation of caspase-3, which can be blocked by the caspase-8 inhibitor IETD-fmk (3). Caspase-3 activates PKCδ by proteolytic cleavage (4) at the hinge region (shown in red), releasing the catalytic fragment and causing constitutive activation of the kinase (5). The constitutively active PKCδ catalytic fragment mediates proapoptotic signaling by phosphorylation of its downstream substrates, resulting in DNA fragmentation and dopaminergic cell death (6). Blocking the PKCδ signaling pathway using siRNA, targeted gene knockout or a caspase-3 cleavage-resistant mutant can protect against dopaminergic cell death induced by TNF toxicity.
The results from our mechanistic studies using N27 rat dopaminergic cells indeed demonstrate for the first time that soluble TNF induces a time-dependent and dose-dependent proteolytic cleavage of PKCδ by a caspase-8 and caspase-3 signaling pathway, indicative of canonical TNF death receptor signaling via caspase-8. Using the anti-TNF drug Etanercept and neutralizing antibodies to the TNFR1 (p55) death receptor, we show that proteolytic cleavage of PKCδ by soluble TNF is dependent on TNFR1 signaling and is accompanied by a substantial increase in PKCδ kinase activity (Figure 3), consistent with proteolytic activation. Previous reports from our laboratory and other independent research groups have shown that PKCδ proteolytic activation contributes to apoptosis induced by diverse neurotoxic stimuli that converge on the intrinsic mitochondrial apoptotic pathway primarily through intracellular oxidative stress mechanisms [21–24, 31, 32, 54–56]. Taken together, our present results suggest that proteolytic activation of PKCδ could be a common effector of cell death via both intrinsic and extrinsic cascades in dopaminergic neurons.
Rapid and dynamic translocation to distinct subcellular locations following activation is a characteristic feature of PKCδ in different cell types, and typically delineates the unique functional role of this kinase . Various substrates have been identified as targets of PKCδ phosphorylation in different subcellular locations that participate in a range of multiple cellular functions, from apoptosis to cell migration and activation of immune cells . In particular, studies have demonstrated that nuclear translocation of PKCδ is required for induction of apoptosis and is driven by a bipartite nuclear localization sequence at its C-terminus [48, 49, 59]. Our confocal immunofluorescence experiments reveal a distinct localization of PKCδ to the nucleus 6 h after TNF treatment, the time point at which maximum proteolytic cleavage of PKCδ was evident, consistent with its proapoptotic function (Figure 4). While intense PKCδ staining is evident throughout the nucleus in TNF-treated cells, there is also a distinct pattern of accumulation of PKCδ at specific sites around the perinuclear region and within the nucleus. Interestingly, in a previous study, TNF superfamily ligands, such as BAFF (B cell-activating factor belonging to the TNF family), have been shown to prevent nuclear accumulation of full length PKCδ and thus prolong cell survival in B cells . The substrates of PKCδ and the functional significance of nuclear localization of the kinase during neuroinflammation are currently under investigation in our laboratory. However, the kinetics of PKCδ proteolytic activation and its translocation to the nucleus observed in our present study, both indicate of a proapoptotic role for PKCδ downstream of TNF. Indeed, suppressing total PKCδ protein levels using siRNA or blocking proteolytic activation using a PKCδ cleavage-resistant mutant (PKCδD327A-CRM) affords robust protection against TNF neurotoxicity (Figure 5). The results from our mechanistic studies are further supported by our experiments in primary mesencephalic neuronal cultures treated with soluble TNF, which induces degeneration of dopaminergic neurons in these cultures [17, 26]. Although TNF was less toxic to primary dopaminergic neurons than the classical Parkinsonian neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) , we observed a substantial reduction in TH neuron numbers and functional impairment as determined by dopamine uptake studies in cultures from PKCδ wild type (+/+) mice (Figure 6). Importantly, dopaminergic neurons in mesencephalic cultures obtained from PKCδ knockout (−/−) mice were protected against TNF toxicity, indicating that PKCδ signaling is essential for dopaminergic degeneration triggered by TNF.
Evidence from previous studies [17, 26] and our own results here, indicate that TNF can induce dopaminergic neurotoxicity by a direct mechanism, possibly via the TNFR1 (TNFRSF1) death receptor, which has been shown to be highly expressed on these cells. However, additional lines of evidence suggest that TNF can also contribute to dopaminergic degeneration indirectly by potentiating microglial reactive oxygen species (ROS) and other neurotoxic mediators, which can increase local oxidative stress levels in the SNpc [19, 52, 61]. The elevated TNF levels in the SN in both animal models and human PD patients and the concomitant microglial activation indicate that potentiation of microglial neurotoxic responses by TNF likely occurs in vivo, although it may not be the primary mechanism of dopaminergic neurotoxicity, as suggested by McCoy et al. using mixed neuron-glial cultures. A chronic increase in TNF levels in the nigrostriatal system may drive the progressive loss of compromised and vulnerable dopaminergic neurons in the pro-oxidant environment of the SNpc, both by TNF apoptotic signaling and by potentiation of microglial neurotoxic responses, including ROS and RNS production [17, 19, 26, 52, 62]. Nonetheless, our data demonstrating that PKCδ is proteolytically activated in the substantia nigra in the neuroinflammatory LPS model (Figure 7) of PD, implicate proapoptotic PKCδ signaling as a common downstream effector of dopaminergic cell death triggered by convergent neuroinflammatory mechanisms involving TNF and other neurotoxic mediators in the mouse brain.
Despite extensive research into the etiology of PD for several decades, a cogent basis for the vulnerability and extensive loss of SNpc dopaminergic neurons still remains elusive. The emerging consensus is that PD results from complex interactions between environmental, genetic and cellular processes that induce dopaminergic cell death over time [3, 63]. This highlights the multifactorial nature of the disease process and underscores the importance of identifying therapeutic targets that are relevant across these multiple mechanisms of disease pathogenesis. While oxidative stress, mitochondrial dysfunction and proteasomal impairment have been identified as causative factors that can trigger dopaminergic cell death; reactive microgliosis and dysregulated neuroinflammation can sustain this process and result in progressive neurodegeneration [6, 52, 53]. We previously showed that proteasomal impairment, mitochondrial dysfunction and oxidative stress induce proteolytic activation of PKCδ to drive dopaminergic cell death [24, 45, 64]. Our results herein suggest a crucial role for proapoptotic PKCδ signaling during dopaminergic cell death induced by extracellular TNF and neuroinflammatory mechanisms. Pharmacological targeting of PKCδ with kinase inhibitors such as rottlerin has shown efficacy in protecting against dopaminergic degeneration in vitro and in the MPTP mouse model of PD [21, 55]. Our results from this study provide a rationale for pharmacological targeting of PKCδ to mitigate neuroinflammatory events associated with progressive degeneration of dopaminergic neurons in PD. Taken together, our results herein suggest that proteolytic activation of PKCδ could be a common downstream target for multiple mediators of dopaminergic degeneration by both extrinsic and intrinsic cell death mechanisms, making it an attractive therapeutic target during the progressive phase of PD.
This study was supported by grants from the National Institutes of Health NS 65167, NS078247 and NS74443.
RG designed studies, performed experiments and wrote the manuscript. VA assisted with study design and manuscript editing. AGK and AK conceived the study, the coordination of experiments and editing of the manuscript drafts. All authors were involved in editing drafts of the manuscript and approved the final manuscript.


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text:
Upfront Blog
Meghan McCain Twitter photo: online overshare 101
John McCain's daughter got a crash course on what not to do with social media, posting a playful – and some say racy – photo of herself on Twitter Wednesday night.
Jake Turcotte/The Christian Science Monitor
By Andrew Heining
It was supposed to be an innocuous image – a playful snapshot of Meghan McCain's "night in" with Andy Warhol's autobiography.
But the self-taken image of a pouty Meghan McCain, daughter of former presidential candidate Sen. John McCain, posted to TwitPic Wednesday night – and her profanity-laden Tweets in response to other users' comments – has caused an uproar, and serves as an important lesson for the share-happy set.
Twitter can be something different to everyone who uses it. For one thing, there's no manual. We've seen it used as operatic inspiration, as personal shopper, even as interview platform – who can forget the "Twitterview?" It can also be a powerful tool under an oppressive government as we saw in Iran, or a great crowd-sourced fund-raiser.
But one thing that's rarely trumpeted in the headlines is that Twitter can be a great instrument of self-destruction. Sure, celebrities get most of the headlines (see: Miley Cyrus, John Mayer), but too much opining online can lead to serious offline trouble.
A classic example is the experience of "@theconnor." Last March, he turned to Twitter for advice on a job offer: "Cisco just offered me a job! Now I have to weigh the utility of a fatty paycheck against the daily commute to San Jose and hating the work," he tweeted. A short time later a response arrived: "Who is the hiring manager. I'm sure they would love to know that you will hate the work. We here at Cisco are versed in the web." The exchange earned the "Cisco Fatty" nickname, and he's become a veritable Internet meme – with a website, YouTube parodies, and, well, reference in this post.
It's easy to see why publicly badmouthing a potential employer isn't a good idea. But was McCain's deed – posting a provocative photo of herself to TwitPic – so bad? Probably not, but the truth is, it doesn't matter.
It's easy to forget, as McCain likely did, that Twitter's "Update" button could just as easily read "push at your own risk." As a public figure, (a blogger and vocal supporter of her father's campaign), her every move is watched. That doesn't stop online. As we've shown, Twitter is a powerful tool. But without calm and careful attention, the instant communication that it offers – free from the filter of PR minders and image consultants – can be a dangerous one as well.
Update: Meghan McCain posted the following to her Twitter account at 3:45 EST Thursday: "I would like to thank my family and friends for their support. This has been an embarrassing experience but also a learning one. I will not be deleting my twitter account but I will be more careful in the future about my use with the medium."
We're using Twitter, but you won't find any racy photos of us here: @CSMHorizonsBlog.
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redpajama_set_name: RedPajamaCommonCrawl


1/3 of all Retail Fashion sales start with online searches.
This note has come from the contractors, just to let you know where everything is up to.
New Evening Courses For Women in Business.
NABMA and NMTF working with Google to promote digital help for markets and traders.


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Home / QS Community Board / How TAPS Can Help after the Death of a Service Member
Military Life September 26, 2019
How TAPS Can Help after the Death of a Service Member
Erika S., Editor in Chief
Service Member Support
The Tragedy Assistance Program for Survivors (TAPS) is America's support group for those whose lives have been changed by the death of a loved one serving in the Armed Forces. Learn how TAPS can connect survivors to the resources they need.
If you've lost a Service member, navigating military policies and procedures can make the death more difficult to deal with than a nonmilitary loss. Common feelings include:
Frustration if you are unable to know the exact circumstances of the death.
Anger with the military, government agencies, the Service member who died or the circumstances that resulted in the death.
Confusion by the pressure to feel proud of your Service member's military contribution, while feeling sad about the loss.
It might help you to talk about your feelings with others who have experienced a military loss.
Seek Support from Friends and Family
At the funeral, friends and family may have said, "I'm here, whatever you need." Now is the time to consider your needs and assess the people in your life who might provide valuable support. People generally will fit into one of the following three categories of assistance:
Those who will listen. "Listening" friends tell you to call anytime – and mean it. They will listen as you talk about what happened.
Those who can help you with practical tasks. "Doer" friends will clean your house, cut your lawn and cover for you at work. Just remember to ask for help when you need it and accept help when it's offered.
Those who offer relief from the hard work of grieving. Friends who offer a diversion from grieving don't often get the respect they deserve because people have mixed feelings about catching a game or going to a movie during this time. But you will need a short break – a chance to place your focus somewhere else for just a little while.
And remember, there is no right or wrong way to grieve. You are not wrong for grieving for more or less time than someone else or for dealing with your loss in a different way than others. To help navigate through grief, see the QuickSeries®: Grieving Guide: Coping with Grief and Loss.
Turn to TAPS for Support
Connecting with people who have suffered a loss like yours can be very helpful.
TAPS offers compassionate care to those grieving the loss of a loved one who died while serving in the Armed Forces or as a result of his or her service. Since 1994, TAPS has provided comfort and hope through a national peer support network and connection to grief resources, at no cost to surviving families and loved ones.
TAPS provides the following:
Peer-based emotional support
Grief and trauma resources and information
National Military Survivor Helpline
QuickTip: Experiencing grief after a significant loss is normal, and many people are able to move forward and rebuild their lives with the care and support of family and friends. However, if your emotions seem to be growing stronger and interfering with your ability to function, seek the advice and support of a professional.
For more on the supportive services TAPS provides, including their annual Good Grief Camp for kids, visit their website, call the National Military Survivor Helpline at 800-959-TAPS (8277) and see the QuickSeries® guides: TAPS Survivor Guide, TAPS Benefits and Finances, and TAPS Children's Grief Guide.
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We already have 'Eject' in the context menu of CD/DVD drive, which ejects the drive tray. But to insert the tray inside,we've to manually push insert button on CD/DVD drive. There is a workaround which lets you insert the drive tray without touching it physically. Simply by putting the 'Insert' sub-context(as shown in above figure) menu on CD/DVD drives Context menu. For this to happen, we've to call a dll file from our system32 folder.
First of all, download this file CDEject.zip and extract it. There will be two files CDeject.dll and CDeject.reg. Now follow the step by step procedures.
2. CDeject.reg -> execute it. It shows a warning message, accept that and click OK.


Total population: 36 households (very large).
Taxable units: Taxable value 2 geld units. 9.5 villtax.
Households: 19 villagers. 10 smallholders. 5 slaves. 2 free men.
Ploughland: 6 lord's plough teams. 6 men's plough teams.
Other resources: Meadow 8 acres. Woodland 20 pigs. 1 mill. 1 fishery.
Livestock in 1066: 4 cobs. 7 cattle. 27 pigs. 200 sheep.
Livestock in 1086: 8 cobs. 6 cattle. 27 pigs. 240 sheep. 14 beehives.
Overlords in 1066: Ely (St Etheldreda), abbey of; (Earl) Harold.


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Upon arrest anywhere in Jefferson county, a person must be processed at the Jefferson county jail. In order to be released from jail, a few steps must occur, including being "booked in" and posting bail. When the bail bond amount is decided, a bail bonds agent is sent to post the bail bond at the local jail. Now the release process begins and typically takes anywhere from one to two hours to complete. Although cash bonds are allowed, it is usually not a common practice due to financial limitations.


Revelation 14:5 And no lie was found in their mouths; they are blameless.
And in their mouth no lie was not found; they are blameless.
They never tell lies, and they are innocent.
They have never been known to tell lies; they are faultless.
In their mouth no lie was found. They are blameless.
and no lie was found on their lips; they are blameless.
In their mouth was found no lie; they are blameless.
And there is no falsehood found in their mouths as they are without fault.
They've never told a lie. They are blameless.
And in their mouth was found no guile, for they are without blemish before the throne of God.
And in their mouth there was found no lie; for they are without spot before the throne of God.
And no lie has ever been found upon their lips: they are faultless.
Blessed is the man whose sin the LORD does not count against him, in whose spirit there is no deceit.
True instruction was in his mouth, and nothing false was found on his lips. He walked with Me in peace and uprightness, and he turned many from iniquity.
Strong's Greek 5579: A lie, falsehood, untruth; false religion. From pseudomai; a falsehood.
Verse 5. - And in their mouth was found no guile; no lie (Revised Version). They had not suffered themselves by self deceit (the second beast) to be beguiled into worship of the first beast - the world. Alford very appropriately refers to Psalm 15:1, 2, "Lord, who shall abide in thy tabernacle? who shall dwell in thy holy hill? He that walketh uprightly, and worketh righteousness, and speaketh the truth in his heart." For they are without fault before the throne of God; they are without blemish. The following phrase is omitted by nearly every authority. The word ἀμώμος, "without blemish," reminds us of the "Lamb without blemish" (cf. 1 Peter 1:19; Hebrews 9:14). Thus again they receive appropriate reward. While on earth they kept themselves undefiled; now they are, like the Lamb, free from blemish (see on ver. 4).
14:1-5 Mount Sion is the gospel church. Christ is with his church, and in the midst of her in all her troubles, therefore she is not consumed. His presence secures perseverance. His people appear honourably. They have the name of God written in their foreheads; they make a bold and open profession of their faith in God and Christ, and this is followed by suitable actings. There were persons in the darkest times, who ventured and laid down their lives for the worship and truth of the gospel of Christ. They kept themselves clean from the wicked abominations of the followers of antichrist. Their hearts were right with God; and they were freely pardoned in Christ; he is glorified in them, and they in him. May it be our prayer, our endeavour, our ambition, to be found in this honourable company. Those who are really sanctified and justified are meant here, for no hypocrite, however plausible, can be accounted to be without fault before God.


Home » Should I Move In With My Partner?
You and your partner may be thinking about the future. What is the next step in your relationship? An unplanned pregnancy or even a "false alarm" pregnancy test may be a great opportunity to look at your relationship and make some decisions about your future together. Ask yourself three big questions as you think through your options.
Is my partner "the one"?
Is this a relationship that you see as a long term life partnership? Does your partner share that same long term view? Ask yourself, "would I completely trust this person with my money, my children, my car keys, my health and well being?" Is your partner helping you achieve your dreams? Does your partner get along with your family? Does he or she help you "shoulder your burdens" financially, emotionally and physically? Are there things you'd change in this person?
Before you start sharing a rent payment, you may want to take a look at your long term goals. Is marriage part of your vision for your future? Living together actually lowers your chances of getting married. 40% of couples living together will never get married. Couples getting married after living together have a 50% higher chance of divorce than couples who did not live together. Are children part of your vision for the future? Three fourths of children born to live-in couples will see their parents split up before reaching age 16. And all studies show that children are safer, stronger, and have less behavioral problems when living with parents who are married. Living together might seem like a way to save money and get to know this person, but cohabiting has been shown to lower levels of happiness, increase sexual unfaithfulness, and increase likelihood of divorce.
If you've been able to answer question #1 with a clear "Yes!" then you and your partner should begin to talk about marriage. Your first thoughts might be "I'm not ready!" "We can't afford it!" or "I want to wait until we are financially set up." But when you've found the right person, it's the right time to start this discussion. Pre-marriage counseling is a great way to explore what is going on in your relationship. And if you feel uncomfortable bringing it up, maybe we can help. Our Life Support program lets you explore your feelings and can help you to share your thoughts with your partner. Let's talk. Call us today at 412-687-7767.
How much does an abortion cost in Pittsburgh?


text:
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Clean Technology,News,R&D
The Research Connection – March 2019: Clean Tech Edition
Featured R&D projects:
Next Generation Power Grid Monitoring
Synthetic Jet and Smart Blades Have the Potential to Expand Wind Energy
Modeling Strategies for Overhauling the Aging Power Grid
Designing a more recyclable plastic
Locally grown renewable energy
Next generation batteries
New York's Largest Battery Storage Project
Horizontal Ribbon Growth of Single-Crystal Silicon Wafers for Solar Cells
Advanced carbon capture materials derived from biomasses
This edition's contributors:
Clarkson University | Rensselaer Polytechnic Institute | SUNY Research Foundation (SUNY ESF, SUNY Poly, UAlbany) | Union College
Project: A team of undergraduate researchers at Union College, led by assistant professor of electrical and computer engineering Luke Dosiek, are analyzing GPS time-synchronized power grid measurements in order to estimate grid stability and detect anomalies such as rogue oscillations. Having real-time awareness of these phenomena is critical for maintaining reliability and resiliency as the smart power grid evolves to include more renewables, distributed generation, electric vehicle charging, etc. The team has designed a low-cost measurement device for the rapid prototyping of their algorithms using real-world data, in addition to using measurements from Union's on-site cogeneration plant. Additionally, research is being performed into the best ways to present these real-time analysis results to grid operators so as to avoid data overload and operator fatigue. This includes the potential for sonification, in which power grid measurements are directly converted to audio signals.
Contact: Phil Wajda, Director of Media Relations, wajdap@union.edu
Potential Applications: Smart Grid, Power grid research, Energy research
Project: Michael Amitay, director of the Center for Flow Physics and Control at Rensselaer Polytechnic Institute, has developed a synthetic jet that is being tested on wind turbines. The jets have the ability to regulate turbine blade vibrations, essentially creating a smart blade. This research has the potential to expand wind turbine use by allowing them to operate more efficiently in low- and high-wind conditions. The synthetic jets will also extend the life of turbine blades by mitigating structural stress.
Contact: Reeve Hamilton, Director of Media Relations, hamilr5@rpi.edu
Potential applications: Wind energy, wind turbines
Project: The ALSETLab at Rensselaer Polytechnic Institute specializes in the modeling and simulating of cyber-physical systems. Led by Luigi Vanfretti, an associate professor of electrical, computer and systems engineering at Rensselaer, the lab's name – ALSET – stands for Analysis Laboratory for Synchrophasor and Electrical Energy Technology. Vanfretti and his team use tools that have not been in widespread use in power electronics to develop models and run simulations to better understand how to overhaul the aging power grid and integrate it with more renewable energy sources.
Potential applications: Cyber-physical systems, renewable energy, smart grid technology
Project: Rich Gross is a professor of chemistry and chemical biology at Rensselaer Polytechnic Institute. He is working on plastics and other materials that have the performance benefits of synthetic materials, but can be broken down by safe and mild processes to their original building blocks for reuse. Gross has developed several approaches to sustainable plastics, such as polymers made from plant-derived oils. He is also working on improving the efficiency of naturally occurring enzymes to break down polyethylene terephthalate (PET), which is used in clear and colored plastic water bottles and many other products.
Potential applications: Sustainable plastics, reusable materials, chemical biology
Project: Driven by the need to develop more sustainable energy and consumer products, enhance the planet's resilience to climate change, and encourage economic development in rural areas, the College of Environmental Science and Forestry is working with university, private and public partners to research and develop shrub willow for bioenergy production and other uses. Shrub willow can be grown on marginal agricultural land to produce large amounts of renewable biomass for energy. Lifecycle assessments show that willow is a climate-neutral fuel that does not increase greenhouse gas emissions in the atmosphere. There are opportunities to collaborate with the research team and purchase licensed willow varieties.
Contact: Dr. Timothy A. Volk, Senior Research Associate, tavolk@esf.edu
Potential applications: Sustainable energy for heat and power, biofuels, ecosystem services, bioremediation, living snow fences, nutrient buffers, wastewater treatment
Project: Despite their many advantages, current lithium ion batteries, which use liquid or organic electrolytes, have met a number of safety and scaling challenges that hinder new technological advancements. A research team at SUNY Polytechnic Institute is exploring an all solid state battery as a possible replacement. Working with industry partners, the team is investigating numerous solid thin film electrolytes for batteries that have theoretically shown competitive ionic conductivity measurements compared to the liquid electrolytes.
Contact: Harry Efstathiadis, Associate Professor of Nanoengineering, hfstat@sunypoly.edu
Potential applications: Biomedical research, diagnostics
Project: As power and transportation converge and the world's energy demands move to large-scale wind and solar projects, large-scale energy storage projects provide the missing piece to keep the electric grid stable. Key Capture Energy, an independent developer of utility-scale battery storage projects, partnered with the University at Albany through the StartUpNY program and is collaborating with faculty experts in electrical and computer engineering to develop next-generation energy storage technology. There are opportunities to collaborate with Key Capture Energy.
Contact: Matt Grattan, Director of Economic Development, mgrattan@albany.edu
Potential applications: Grid modernization, energy diversification
Project: This project investigates a novel process for manufacturing single-crystal silicon wafers used to convert solar energy to electrical energy in high efficiency solar cells. Currently, these wafers are produced by the Czochralski growth process. Compared to Czochralski growth, horizontal ribbon growth has the potential to produce thinner and larger wafers at a faster rate and a lower cost (possibly 75 percent lower). However, commercialization has been difficult because there is a lack of understanding of the physics. Scientists at Clarkson are developing a numerical model of the process that can be used to predict and optimize the process so that it can be successfully commercialized. This could make solar electricity significantly cheaper than any other energy source. The team seeks collaborators in solar energy/silicon wafer and solar cell manufacturers.
Contact: Dr. Brian Helenbrook, bhelenbr@clarkson.edu
Potential applications: Solar energy
Project: Scientists at Clarkson University design and characterize biomass-derived nanoporous carbons for post-combustion CO2 capture from flue gas streams. This work has generated basic understanding of the structure-property relationships of these materials and suggest new strategies for designing advanced porous materials with pre-determined selectivity and capacity for capturing CO2 molecules. It was shown that their low-cost of preparation, combined with the material's unique pore architectures and unique functionalities, will position porous carbons at the forefront of CO2 sorbents for industrial applications. The team is interested in collaborating with industrial partners for pilot testing and large-scale implementation.
Contact: Dr. Mario Wriedt, Assistant Professor of Chemistry & Biomolecular Science, mwriedt@clakson.edu
Potential applications: Small-molecule separation and storage, environmental chemistry
About The Research Connection: The Research Connection is a quarterly feature in the Center for Economic Growth's monthly, online newsletter, The CEG Indicator. This special feature highlights R&D being conducted by researchers at Capital Region colleges and universities and others throughout the SUNY system. The Research Connection spotlights academic R&D in CEG's focus technology sectors: Nanotechnology and Semiconductors, Cleantech/Energy, Biotechnology, Advanced Materials, Population Health Technology and Information Technology.
Each edition of The Research Connection will highlight several research projects in a specific technology sector. The Research Connection will keep CEG investors (2,500+) and CEG Indicator subscribers (9,000+) informed on the cutting-edge R&D that is being conducted by SUNY and other academic researchers that could potentially transform their industries. It will also encourage collaboration, patent, licensing and other opportunities.
Don't miss these insights into the trends that are shaping the Capital Region's economy. Sign up for CEG's e-news and follow us on:
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text:
Q: Spring boot, empty multipart files A request comes from the service, multipart/form-data
http://joxi.ru/D2P5g6Eiq0NjjA
Controller method:
@RequestMapping(value = "/*", params = "create", method = RequestMethod.POST)
ResponseEntity<?> createPost(
        @RequestParam MultiValueMap<String, String> allParameters,
        @RequestParam(value="data", required=false) CommonsMultipartFile locationMapFileData,
        MultipartHttpServletRequest mrequest,
        HttpServletRequestWrapper request,
        @RequestBody MultipartFile[] submissions
) {
   return sapService.createPost(allParameters);
}

Debug: http://joxi.ru/52a7XJySEMwG5r
what am I doing wrong? how can i get the contents of files?
p.s. sorry by translate english

A: At first, the files are not the request body, they are part of it and there is no built-in HttpMessageConverter that can convert the request to an array of MultipartFile.
Thus, you need to specify the name of the part (there may be several parts with the same name), for example, we expect a few files under the "files" part:
@RequestMapping(value = "/*", method = RequestMethod.POST)
ResponseEntity<?> createPost(@RequestParam("files") MultipartFile[] uploadFiles)
{
  ...iterate over the uploadFiles and perform the necessary processing for each file
  return new ResponseEntity<>(HttpStatus.OK);
}

redpajama_set_name: RedPajamaStackExchange


On Wednesday, Tsvangirai was sworn in as the country's president by longtime prime minister and bitter political rival Robert Mugabe. The move altered months of political unrest after Tsvangirai claimed victory in last year's election and was subsequently stonewalled in taking power by Mugabe, the nation's longtime ruler.
The promise to pay teachers was welcome news to the Christian aid organization World Vision as well as UNICEF, an international aid arm of the United Nations.
The crisis may be stemmed to some degree if schools re-open once teachers are paid. Schools are primary distribution centers for food aid. Foreign currency is valuable in the country because domestic monies have been devalued by very high inflation.
Zimbabwe is also entering the height of is "hunger period" where resources are scarce due to agricultural limitations.
"If schools remain closed, the feeding program will not commence, at this time, the height of the hunger period," said World Vision's Stewart Muchapera in Zimbabwe.
Against the backdrop of the political changes, UNICEF released a study Wednesday revealing that 94 per cent of schools in rural Zimbabwe remain closed.
The study came from routine visits across Zimbabwe which showed that 66 of 70 schools were abandoned. In the only fully operational school found during visits, a third of pupils were reporting for classes. Many of the abandoned schools have been vandalized.
The country is still wrestling with a cholera epidemic. Cholera has spread to become a larger issue in rural areas. About 3,000 people have died from cholera and there have been more than 54,000 reported cases.
The water-borne nature of cholera is also heightening concerns that malaria may soon be on the rise. The U.S. Agency for International Development (USAID) said today it was providing $200,000 to boost efforts at indoor malaria spraying and stave off further deterioration of the country's public health system.


The Lancet is the world's leading independent general medical journal. The journal's coverage is international in focus and extends to all aspects of human health. The Lancet publishes the original primary research and review articles of the highest standard.
Cell: Stem Cell covers primary research, reviews, and commentaries, including issues of stem cell research policy and ethics.
NeuroImage, A Journal of Brain Function, provides a vehicle for communicating important advances, using imaging and modelling techniques to study structure-function relationships in the brain. The journal publishes original research articles, papers on methods or modelling, theory and position papers that describe the use of imaging approaches to studying the brain.
As the leader in its field, the Journal of the American College of Cardiology (JACC) publishes original peer-reviewed clinical and experimental reports on all aspects of cardiovascular disease. Topics covered include coronary artery and valve disease, congenital heart defects, vascular surgery, cardiomyopathy, drug treatment, new diagnostic techniques, findings from the laboratory, and large multicenter studies of new therapies.
Gastroenterology is the most prominent journal in the field of gastrointestinal disease. As the official journal of the AGA Institute, Gastroenterology delivers up-to-date and authoritative coverage of both basic and clinical gastroenterology.
The American Journal of Orthodontics & Dentofacial Orthopedics is the official publication of the American Association of Orthodontists, its constituent societies, the American Board of Orthodontics and the College of Diplomates of the American Board of Orthodontics. Coverage includes successful diagnostic procedures, imaging techniques, bracket and archwire materials, extraction and impaction concerns, orthognathic surgery, TMJ disorders, removable appliances, and adult therapy.
CANCER is a peer-reviewed publication of the American Cancer Society integrating scientific information from worldwide sources for all oncologic specialties. The objective of CANCER is to provide an interdisciplinary forum for the exchange of information among oncologic disciplines concerned with the etiology and course of human cancer.
The Journal of Molecular Biology publishes original scientific research concerning studies of organisms or their components at the molecular level. Published weekly, the journal provides up-to-date and comprehensive coverage of all aspects of molecular biology.
The Lancet Neurology is recognized for providing a global, authoritative and independent forum for the highest quality clinical neurology research and opinion, covering such conditions as stroke, multiple sclerosis, dementia, movement disorders, epilepsy and headache.


text:
Euploea besinensis är en fjärilsart som beskrevs av Hans Fruhstorfer 1899. Euploea besinensis ingår i släktet Euploea och familjen praktfjärilar. Inga underarter finns listade i Catalogue of Life.

Källor

Praktfjärilar
besinensis

redpajama_set_name: RedPajamaWikipedia


Coal. Process Solutions ... We design, manufacture and supply the MD range of gravity separation equipment including spirals, ... Spiral Concentrators. Features.
A study of particle separation in a spiral concentrator - ResearchGate. 19 Dec 2017 ... Therefore, a research project was undertaken to determine the distribution of coal particles across the cross-section of a spiral in a Reichert LD4 coal spiral.
Feb 19, 2017· FL Krebs Coal Spiral Concentrator. Gold. Solutions. Individual Industry Solutions · Material Handling Solutions · Minerals Solutions · Tailings .
and coal industries for treating fine ore. Developments over the years include; ... Spiral concentrators, high capacity spirals, slimes, heavy minerals.
Krebs Spiral Concentrators for Mineral Applications ... hensive range of Spiral units for the minerals, coal, environmental, and related industries.
Ultrafine coal cleaning using spiral concentrators - ScienceDirect. The conventional circuit fine coal circuit typically incorporates spiral concentrators to treat nominal 1 × 0.15 mm material.
Spiral Concentrator For Coal . Coal spiral concentrators from Multotec reduce floor space requirements, have long life, and reduce maintenance costs.
Spiral Concentrator - Outotec. Outotec spiral concentrators are used in most applications, particularly for . Iron ore Chromite Mineral sands Glass sand Gold Coal Mica .
Multotec has developed a range of spiral concentrators specifically engineered and optimised for the Australian coal mining industry.
Multotec has designed and optimised both single (SX4) and double stage (MX7 and SX7) Coal Spiral Concentrators to treat a size range of 2.0 to 0.1 mm.


text:
Antonio Manuel Ruiz Fernández (born 20 February 1977), commonly known as Romerito, is a Spanish retired footballer who played mainly as a defensive midfielder, and is the manager of Xerez Deportivo FC.

Playing career
Born in Seville, Andalusia, Romerito started his career with local side CD Cabecense before moving to Xerez CD in 1997. Initially assigned to the reserves, he was exclusively used in the main squad, making his professional debut on 14 September 1997 by coming on as a half-time substitute for Fernando Román in a 0–0 Segunda División away draw against CD Logroñés.

In 1999, Romerito signed for RCD Mallorca, and was assigned to the B-team in Segunda División B. He made his first team – and La Liga – debut on 17 January 2000, starting and scoring the opener in a 1–2 loss at Real Madrid.

In 2001, Romerito agreed to a deal with Recreativo de Huelva in the second division, but left for Hércules CF in the following January after being rarely used. He would resume his career mainly in the third level in the following years, representing CF Extremadura, SD Compostela, Écija Balompié, Linares CF, Águilas CF, Deportivo Alavés, Lucena CF, Real Balompédica Linense and Atlético Sanluqueño CF.

In August 2013, Romerito joined Xerez Deportivo FC, and helped the club in three consecutive promotions to Primera Andaluza before retiring in June 2016, aged 39.

Coaching career
Immediately after retiring, Romerito started working as a youth manager at his last club Xerez Deportivo. On 4 November 2017, he was named manager of CD Rota in the fifth division, and achieved promotion to Tercera División with the club in 2019.

On 10 March 2020, Romerito left Rota to take over third division side Atlético Sanluqueño, and helped the side to avoid relegation. The following 7 April, however, he was sacked.

On 20 May 2021, Romerito was appointed at the helm of another club he represented as a player, Linense.

Managerial statistics

References

External links

1977 births
Living people
Footballers from Seville
Spanish footballers
Association football midfielders
La Liga players
Segunda División players
Segunda División B players
Tercera División players
Xerez CD footballers
RCD Mallorca B players
Recreativo de Huelva players
Hércules CF players
CF Extremadura footballers
SD Compostela footballers
Écija Balompié players
Linares CF players
Águilas CF players
Deportivo Alavés players
Lucena CF players
Real Balompédica Linense footballers
Atlético Sanluqueño CF players
Xerez Deportivo FC footballers
Spanish football managers
Primera Federación managers
Segunda División B managers
Tercera División managers


text:
Q: MSSQL inserts strings with random spaces inside words to table I have an ASP support application that gets a string from the user and inserts it into the table, making a new record. The field is VARCHAR(4000) in case there are long sql commands or code snippets to record. The problem is, MSSQL puts random spaces in these strings. In the string I see \r\n characters whilst debugging the program but no spaces within random words, like i see in the database. I must eliminate these spaces in the insert and also, read the strings from database with the correct spaces and newlines. Here is my code. The string is the "detay" field. The code seems fine, is the problem in MSSQL or my sql statement? The variables are half turkish, ask in case you need a translation so i will edit.
  protected void btnkaydet_Click(object sender, EventArgs e)
    {
        if (txtbaslik.Text == "")
        {
            baslikErr.Text = "ERROR";
            baslikErr.Visible = true;
        }
        else
        {
            baslikErr.Visible = false;
        }
        if (txtdetay.Text == "")
        {
            detayErr.Text = "ERROR";
            detayErr.Visible = true;
        }
        else
        {
            detayErr.Visible = false;
        }
        if (txtdetay.Text != "" && txtbaslik.Text != "")
        {
            OdbcConnection baglanti = new OdbcConnection();
            StringBuilder sorgu = new StringBuilder();
            OdbcCommand komut = new OdbcCommand();

            string veritabani = null;
            veritabani = ;//cant give the db string for reasons :)
            baglanti.ConnectionString = veritabani;
            baglanti.Open();
            veritabani = null;

            komut = baglanti.CreateCommand();

            if (string.IsNullOrEmpty(hdnFilename.Value))
            {
                sorgu.Append(" INSERT INTO DESTEK (baslik, detay, tarih) VALUES (?,?,?) ");
                komut.CommandText = sorgu.ToString();

                komut.Parameters.Add("@baslik", OdbcType.VarChar).Value = this.txtbaslik.Text;
                komut.Parameters.Add("@detay", OdbcType.VarChar).Value = this.txtdetay.Text;
                komut.Parameters.Add("@tarih", OdbcType.DateTime).Value = DateTime.Now.ToString();
            }
            else
            {
                sorgu.Append(" INSERT INTO DESTEK (baslik, detay, tarih, dosya) VALUES (?,?,?,?) ");
                komut.CommandText = sorgu.ToString();

                komut.Parameters.Add("@baslik", OdbcType.VarChar).Value = this.txtbaslik.Text;
                komut.Parameters.Add("@detay", OdbcType.VarChar).Value = this.txtdetay.Text;
                komut.Parameters.Add("@tarih", OdbcType.DateTime).Value = DateTime.Now.ToString();
                komut.Parameters.Add("@dosya", OdbcType.VarChar).Value = hdnFilename.Value;
            }
            komut.ExecuteNonQuery();

            komut = null;

            baglanti.Close();
            baglanti = null;

        }
    }

A: I have found a solution to my answer.
        string detay = this.txtdetay.Text;
        detay = detay.Replace(@"\r\n", "");
        detay = detay.Replace((char)13, '£');
        detay = detay.Replace((char)10, '£');
        detay = detay.Replace("£", "");

Apparently i had to replace the escape characters with spaces. Leaving this here, in case anyone else has same issue.